The biochemical and genetic characterization of the TCA cycle
in Sinorhizobium meliloti
David Meek
Microbiology Unit
Department of Natural Resource Sciences
McGill University, Montreal
August, 2013
A thesis submitted to McGill University in partial fulfillment of the requirements for
the degree of PhD
© David Meek 2013
1
Table of Contents
Abstract ...........................................................................................................................6 Résumé ............................................................................................................................7 Acknowledgments...........................................................................................................8 Preface and Contribution to Knowledge .........................................................................9 Abbreviations used in this thesis...................................................................................12 List of Figures ...............................................................................................................14 List of Tables ................................................................................................................16 General Introduction .....................................................................................................17 Chapter 1: Literature review .........................................................................................19 1.1 Preamble .........................................................................................................19 1.2 Sinorhizobium meliloti ........................................................................................21 1.3 The TCA cycle and malic enzymes in S. meliloti ...............................................24 1.4 Bacterial promoter recognition and transcriptional regulation ...........................28 1.5 Transcription start sites and σ factors of S. meliloti ............................................31 Chapter 2: Materials and Methods ................................................................................34 2.1 Bacterial strains and growth media .....................................................................34 2.2 Bacterial manipulations .......................................................................................34 2.2.1 Conjugation ..................................................................................................34 2.2.2 Transduction .................................................................................................35 2.2.3 Competent cell preparation and transformation ...........................................35 2.3 Biochemical manipulations .................................................................................36 2.3.1 Crude cell extracts and protein quantification ..............................................36 2.3.2 Enzyme assays ..............................................................................................37 2.3.2.1 Malate dehydrogenase ..........................................................................37 2.3.2.2 2-oxoglutarate dehydrogenase ..............................................................38 2.3.2.3 Succinyl-CoA synthetase ......................................................................38 2.3.2.4 Isocitrate dehydrogenase.......................................................................38 2.3.2.5 Pyruvate dehydrogenase .......................................................................39 2.3.2.6 Branched-chained keto-acid dehydrogenase.........................................39 2
2.3.3 ß-galactosidase and gfp-fusion assays ..........................................................39 2.3.4 Purification of MDH.....................................................................................41 2.3.4.1 Purification by HPLC method ..............................................................41 2.3.4.2 Purification by HIS-tag method ............................................................41 2.3.5 Determination of molecular mass of MDH ..................................................41 2.3.6 Optimal pH determination ............................................................................42 2.3.7 Michaelis-Menten enzyme kinetics ..............................................................42 2.3.8 Inhibition and product inhibition assays.......................................................43 2.4 Genetic manipulations .........................................................................................43 2.4.1 Plasmid, genomic DNA and total RNA extraction.......................................43 2.4.2 DNA restrictions and ligations .....................................................................44 2.4.3 MDH N-terminal sequencing .......................................................................44 2.4.4 PCR...............................................................................................................44 2.4.4.1 Primers ..................................................................................................45 2.4.5 RT-PCR and Transcriptional start site .........................................................45 2.4.6 Plasmid construction.....................................................................................46 2.4.6.1 Construction of plasmid pDM52 ..........................................................46 2.4.6.2 Plasmids used for strains Rm30230, Rm30267 and Rm30275
construction .......................................................................................................47 2.4.6.3 Plasmids used for strains Rm30232 (lpdA3-), Rm30282 (lpdA1-) and
Rm30309 (lpdA2-) construction .......................................................................48 2.5 Plant growth conditions, nodule microscopy and photography ..........................49 2.6 Computer programs used ....................................................................................49 Chapter 3: The biochemical characterization of malate dehydrogenase (MDH) .........58 3.1 Introduction .........................................................................................................58 3.2 Results and discussion .........................................................................................59 3.2.1 Malate dehydrogenase purification ..............................................................59 3.2.2 Optimal pH and molecular mass determination ...........................................59 3.2.3 Kinetic properties .........................................................................................60 3.2.4 Product inhibition .........................................................................................61 3.2.5 Inhibition analysis.........................................................................................63 3
3.3 Conclusion...........................................................................................................64 3.4 Figures and Tables ..............................................................................................66 Connecting statement 1 .................................................................................................82 Chapter 4: The genetic characterization of the mdh-sucCDAB operon and promoter
region ............................................................................................................................83 4.1 Introduction .........................................................................................................83 4.2 Results and discussion .........................................................................................84 4.2.1 N-terminal sequencing..................................................................................84 4.2.2 Transcriptional start site determination ........................................................84 4.2.3 Determination of a functional promoter for mdh..........................................85 4.2.4 The role of σ54 in regulation of mdh .............................................................86 4.2.5 RT-PCR ........................................................................................................87 4.2.6 Transcriptional lacZ gene fusion assays .......................................................88 4.3 Conclusions .........................................................................................................90 4.4 Figures and Tables ..............................................................................................93 Connecting statement 2 ...............................................................................................100 Chapter 5: The characterization of two putative sucB alleles encoding for the
dihydrolipoyl succinyltransferase (E2) component of the 2-oxoglutatrate
dehydrogenase complex ..............................................................................................101 5.1 Introduction .......................................................................................................101 5.2 Results and discussion .......................................................................................102 5.2.1 Homology comparison between SMc02483 and SMb20019 ......................102 5.2.2 Construction of S. meliloti strains Rm30230, Rm30267 and Rm30275 ....103 5.2.3 Enzyme assays ............................................................................................104 5.2.4 Plant assays, symbiotic phenotype and microscopy ...................................105 5.3 Conclusions .......................................................................................................106 5.4 Figures and Tables ............................................................................................109 Connecting statement 3 ...............................................................................................115 Chapter 6: The role of three dihydrolipoamide dehydrogenases (LpdA) as functional
subunits of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branchedchain alpha-ketoacid dehydrogenase ..........................................................................116 6.1 Introduction .......................................................................................................116 4
6.2 Results and discussion .......................................................................................118 6.2.1 Percent homology comparison of the three lpdA genes .............................118 6.2.2 Construction of S. meliloti strains Rm30232, Rm30282 and Rm30309 ....118 6.2.3 Enzyme assays ............................................................................................119 6.2.4 Plant assays, symbiotic phenotype and microscopy ...................................120 6.3 Conclusions .......................................................................................................121 6.4 Figures and Tables ............................................................................................125 Summary and general conclusions..............................................................................133 References ...................................................................................................................136 5
Abstract
In the symbiotic association between S. meliloti and its plant host, the
nutrient exchange from plant to bacteroid is in the form of C4-dicarboxylic acids such
as malate. These compounds directly enter the TCA cycle and the derived energy is
used for the nitrogen-fixation process. Malate dehydrogenase (mdh), the two subunits
of succinyl-CoA synthetase (sucCD), and two of the three subunits of 2-oxoglutarate
dehydrogenase (sucAB) are encoded as an operon with the order mdh-sucCDAB. The
expression of this operon is controlled by a single promoter found directly upstream of
mdh. The transcrptional start site was mapped (a guanine residue at postion -63) and
RT-PCR demonstrated that expression is as one polycistronic message in cells grown
in LBmc. Transcriptional lacZ-gene fusions to mdh, sucD and sucA demonstrated that
the mdh promoter is under catabolite control as evidenced by the change in ßgalactosidase expression depending on the carbon-source. Expression was highest
with acetate followed closely by arabinose and glutamate but lowest with pyruvate as
sole carbon-source. MDH was purified, N-terminal sequenced and kinetic assays were
performed to determine Km, Vmax. A pH 10 was found to be the optimal for MDH. 2oxoglutarate exhibited competitive inhibition on MDH. The annotated genome of S.
meliloti contains two alleles (sucB), one chromosomal and one on megaplasmid
pSymB, which putatively encode the E2 subunit of 2-oxoglutarate dehydrogenase.
Only the chromosomally-borne allele was found to be a functional sucB. Lastly,
results of a study of the E3 subunits of pyruvate dehydrogenase, 2-oxoglutarate
dehydrogenase and branched-chained keto-acid dehydrogenase were presented.
6
Résumé
Dans la relation symbiotique entre S. meliloti et la plante M. sativa, l’apport en
nutriments de la plante vers les bacteroids se fait sous la forme d’acides C4dicarboxiliques tel que le malate. Ces composés entrent directement dans le cycle de
Krebs et l’énergie produite est utilisée pour la fixation d’azote. Malate déhydrogénase
(mdh), les deux composantes de succinyl-CoA synthèse (sucCD), et deux des trois
composantes de 2-oxoglutarate déhydrogénase (sucAB), sont inscrites en un opéron
dans l’ordre qui suit ; mdh-sucCDAB. L’expression de cet opéron est contrôlée par un
seul promoteur situé directement en amont de mdh. Le site de début de transcription a
été identifié et RT-PCR a démontré la nature polycistronique de l’expression dans les
cellules provenant de bactéries cultivées avec LBmc. Fusions transcriptionelles aux
gene-lacZ de mdh, sucD et sucA ont été utilisées pour déterminer les niveaux relatifs
d’expression dans des cultures provenant de médiums minimaux contenant des
sources de carbone spécifiques. MDH a été purifié, le N-terminal séquencé, et des
dosages cinétiques ont été performés pour déterminer Km, Vmax, le pH optimal, et
l’effet des inhibiteurs allostériques. Le génome annoté de S. meliloti contient deux
allèles qui encodent supposément les composantes E2 (sucB) de 2-oxoglutarate
déhydrogénase. Un seul des allèles a démontré être une version fonctionnelle de sucB.
Finalement, les résultats d’une étude sur les composantes E3 de pyruvate
déhydrogénase, sur 2-oxoglutarate déhydrogénase et sur déhydrogénase de kéto-acide
de chaines ramifiées seront présentés.
7
Acknowledgments
I would foremost like to thank my supervisor Dr Brian Driscoll for all his time,
expertise and editorial reviews for without it this project and the results described in
this manuscript would be sorely lacking. I would also like to thank NSERC for the
funding of this project. Thanks also go to all the friends made at McGill without
whom, time spent on this project would have seemed much longer. Students who
assisted with obtaining and duplicating some of the results of this project include
Blaire Stevens, Olivier Trottier and Branislav Babic, a big thank-you. Thank-you to
Patricia Goerner-Potvin for translating the abstract.
On a more personal note I would like to thank my Mom for her support both
by encouragement and monetarily, and lastly I would like to thank my wife Dina for
her love and encouragement throughout.
8
Preface and Contribution to Knowledge
Preface
All experimental design, work, data preparation and analysis in Chapter 3 was
carried out by David Meek in collaboration with the student’s PhD supervisor Dr
Brian T. Driscoll except the molecular size determination of MDH which was
performed by Dr Armando Jardim’s lab.
All experimental design, data preparation and analysis in Chapter 4 was carried
out by David Meek in collaboration with the student’s PhD supervisor Dr Brian T.
Driscoll with the following exceptions: Rateb Yousef assisted in the building of the
plasmids used to demonstrate mdh promoter functionality and Blaire Steven perform
one of the ß-gal gene-fusion assay replicates.
All experimental design, data preparation and analysis in Chapter 5 was carried
out by David Meek in collaboration with the student’s PhD supervisor Dr Brian T.
Driscoll with the following exceptions: Oliver Trottier assisted in plasmid construction
and did the mutagenesis of S. meliloti and Michelle Poilly carried out the nodule
microscopy.
All experimental design, data preparation and analysis in Chapter 6 was carried
out by David Meek in collaboration with the student’s PhD supervisor Dr Brian T.
Driscoll with the following exceptions: Branislav Babic assisted in plasmid
construction and did the mutagenesis of S. meliloti and Michelle Poilly carried out the
nodule microscopy.
9
Contributions to knowledge
The work carried out in the first part of this thesis focused on the biochemical
and genetic properties of the TCA cycle enzyme malate dehydrogenase (MDH) while
the second part was to elucidate the functionality of the multiple alleles of specific
enzyme subunits. Contributions to knowledge include the findings that for S. meliloti:
•
MDH functions optimally at pH10
•
MDH functions as a dimer and has a molecular weight of 66 kDa
•
the determined Michaelis-Menten kinetic properties of MDH indicates that the
flow of malate at the critical junction between MDH and NAD+-dependent
malic enzyme DME would preferentially be in the direction of MDH
•
MDH has a steady-state ordered Bi-Bi mechanism
•
MDH is competitively inhibited by 2-oxoglutarate
•
immediately upstream of mdh is its promoter
•
the TSS of mdh is a guanine residue at the -64 nt position relative to the
translational start site
•
the level of expression from the mdh promoter is under catabolic control
•
the mdh-sucCDAB operon is expressed as one polycistronic message in LB
grown cells
•
ß-gal gene-fusions suggest the possibility that sucA may be differentially
expressed from a self promoter under certain growth conditions
10
•
the chromosomal allele SMc02483 encodes for the functional E2 subunit of 2oxoglutarate dehydrogenase (OGD) not the megaplasmid-borne allele
SMb20019
•
strains of S. meliloti deficient in ODG activity due to a disruption in the E2
subunit can induce root nodule formation (Nod+); however, the bacteroids
cannot fix nitrogen (Fix-)
•
An lpdA1 mutant showed that LpdA1 is essential for a functional pyruvate
dehydrogenase (PDH) enzyme
•
An lpdA1 mutant showed LpdA1 has no role in OGD or branched-chained
alpha-keto acid dehydrogenase (BKD) functionality
•
An lpdA2 mutant showed that LpdA2 is essential for a functional OGD
enzyme complex
•
An lpdA2 mutant showed LpdA2 plays no role in PDH or BKD functionality
•
An lpdA3 mutant showed that LpdA3 is essential for a functional BKD
•
An lpdA3 mutant showed LpdA3 plays no role in PDH or OGD functionality
11
Abbreviations used in this thesis
AXXX = absorbance at XXX nm
ACN = aconitase
αCTD = alpha C-terminal domain of the RNA polymerase holoenzyme
αNTD = alpha N-terminal domain of the RNA polymerase holoenzyme
ATP = adenosine triphosphate
BKD = branched-chained keto-acid dehydrogenase
BNF = biological nitrogen fixation
bp = base pair
BSA = bovine serum albumin
CS = citrate synthase
DME = NAD+-dependent malic enzyme
DTT = dithiothreitol
GABA = gamma aminobutyrate
GFP = green fluorescence protein
h = hours
ICD = isocitrate dehydrogenase
IPTG = isopropyl β-D-1-thiogalactopyranoside
kb = kilobase
kg = kilogram
Mb = megabase
MCS = multiple cloning site
MDH = malate dehydrogenase
min = minute
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NAD+ = nicotinamide adenine dinucleotide (oxidized form)
NADH = nicotinamide adenine dinucleotide (reduced form)
NADP+ = nicotinamide adenine dinucleotide phosphate (oxidized form)
NADPH = nicotinamide adenine dinucleotide phosphate (reduced form)
nt = nucleotide
OAA = oxaloacetate
ODXXX = optical density at XXX nm
OGD = 2-oxoglutarate dehydrogenase
ONPG = o-nitrophenyl-ß-galactopyranoside
O/N = overnight
ORF = open reading frames
PDH = pyruvate dehydrogenase
RFU = relative fluorescence units
RNAP = RNA polymerase
RPM = revolutions per minute
RT = room temperature
SA = specific activity
SCS = succinyl-CoA synthetase
SE = standard error
s = seconds
SNF = symbiotic nitrogen fixation
TCA = tricarboxylic acid
TPP = thiamine pyrophosphate
TSS = transcriptional start site
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List of Figures
Figure 1: Diagram of the TCA cycle and selected anaplerotic pathways. Pathways
found in S. meliloti are in black and those not found are in red. ..................................33 Figure 2: The optimal pH curve for the oxidation of malate by MDH from S. meliloti
.......................................................................................................................................68 Figure 3: The optimal pH curve for the reduction of OAA by MDH from S. meliloti .68 Figure 4: Size exclusion chromatograph of the elution of MDH relative to markers of
know size ......................................................................................................................69 Figure 5: Product inhibition by NADH with malate constant and NAD+ varied .........72 Figure 6: Product inhibition by NADH with NAD+ constant and malate varied .........73 Figure 7: Product inhibition by OAA with malate constant and NAD+ varied ............74 Figure 8: Product inhibition by OAA with NAD+ constant and malate varied ............75 Figure 9: Product inhibition by NAD+ with OAA constant and NADH varied ...........76 Figure 10: Product inhibition by NAD+ with NADH constant and OAA varied .........77 Figure 11: Product inhibition by malate with OAA constant and NADH varied .........78 Figure 12: Product inhibition by malate with NADH constant and OAA varied .........79 Figure 13: Dixon plot of the oxidation of malate reaction by MDH with malate at 4
different concentrations and 2-oxoglutarate as inhibitor at 0.1, 2, 5, and 10 mM
concentrations ...............................................................................................................80 Figure 14: Dixon plot of the reduction of OAA reaction by MDH with OAA at 3
different concentrations and 2-oxoglutarate as inhibitor at 0, 5, 10 mM concentrations
.......................................................................................................................................81 Figure 15: The nucleotide sequence of the promoter region of mdh. ...........................94 Figure 16: Fluorescence of S. meliloti cells containing mdh promoter and putative
sucA promoter transcriptional gfp-gene fusion plasmids ..............................................95 Figure 17: RT-PCR of the mdh-sucCDAB operon. .......................................................97 Figure 18: The genetic arrangement of the mdh-sucCDAB operon showing transposon
Tn5-B20 insertion sites and orientation. .......................................................................98 Figure 19: The effect of carbon source on gene expression from plasmid-borne lacZ
fusions ...........................................................................................................................99 Figure 20: Medicago sativa plants that had been inoculated* with S. meliloti wild-type
and sucB mutant strains. .............................................................................................112 Figure 21: Microscopic cross sections of nodules containing bacteroids of strains
RmG212, Rm30230 (sucB mutant), Rm30267 (SMb20019 mutant) and Rm30275
(double mutant) stained with nucleic acid binding dye syto-13. ................................114 Figure 22: The genetic arrangement of the three lpdA genes relative to the operons
encoding subunits of PDH, OGD and BKD. ..............................................................126 Figure 23: Multiple alignment of the three LpdA amino acid sequences. ..................127 14
Figure 24: The symbiotic phenotype of alfalfa plants* inoculated with one of the three
LpdA mutants..............................................................................................................130 Figure 25: Microscopic cross sections of nodules containing bacteroids of strains
RmG212, Rm30282 (lpdA1 mutant), Rm30309 (lpdA2 mutant) and Rm302325 (lpdA3
mutant) stained with nucleic acid binding dye syto-13. .............................................132 15
List of Tables
Table 1: Bacterial strains, plasmids, transposons and phage used in this study ...........51 Table 2: Primers used in this study ...............................................................................56 Table 3: Purification of malate dehydrogenase from S. meliloti by HPLC ..................67 Table 4: The Vmax and Km values calculated for both the oxidation of malate and the
reduction of OAA reactions of malate dehydrogenase at optimum and physiological
pH..................................................................................................................................70 Table 5: Experimental conditions and product inhibition patterns for the oxidation of
malate and reduction of OAA reactions of malate dehydrogenase and the apparent Kis
.......................................................................................................................................71 Table 6: Expression of mdh in an rpoN mutant of S. meliloti.......................................96 Table 7: The percent homology (nucleotide/amino acid) between the two S. meliloti
alleles SMc02483 and SMb20019 with sucB from other related rhizobia. .................110 Table 8: The enzyme specific activities of S. meliloti cells harvested from M9succinate grown media................................................................................................111 Table 9: The shoot dry-weight (SDW) of Medicago sativa (alfalfa) inoculated with S.
meliloti mutant strains. ................................................................................................113 Table 10: The nucleotide and amino acid percent homology between the three lpdA
genes. ..........................................................................................................................128 Table 11: Specific enzymatic activities from crude cell extracts of cells grown in M9
minimal media with succinate, arabinose, and 1% LB together as the carbon source.
.....................................................................................................................................129 Table 12: The average shoot dry weight (SDW) and phenotype of alfalfa plants
inoculated with one of the three LpdA mutant strains harvested 28 days postinoculation...................................................................................................................131 16
General Introduction
Sinorhizobium meliloti is a soil microorganism in the rhizobiaceae family in
the class α-proteobacteria. When S. meliloti is in the rhizosphere of an alfalfa plant, a
series of specific steps occur that culminate in the formation of plant-derived
indeterminant root nodules containing terminally-differentiated bacteroids. A
symbiotic association is thus established, and as in any such association, there are
mutual benefits to the two participants. The bacteroids make available to the plant a
nitrogen source by the conversion of atmospheric dinitrogen to ammonium or alanine
(Waters et al. 1998). In turn, the plant supplies the bacteroids with a microaerobic
environment and an energy source in the form of C4-dicarboxylic acids, such as
succinate and malate (Day and Copeland 1991; McDermott et al. 1989). These
compounds enter directly into the tricarboxylic acid (TCA) cycle where the energy
from their metabolism can be used for the energy-intensive nitrogen fixation process.
16 molecules of adenosine triphosphate (ATP) are required by the enzyme
nitrogenase to convert one molecule of atmospheric dinitrogen to ammonium (Dixon
and Kahn 2004). The ATP and the reducing equivalents needed for this conversion
come directly from the tricarboxylic acid (TCA) cycle. Due to the great importance of
the TCA cycle to the symbiotic relationship between bacteriods and plant, the physical
and genetic properties of the enzymes involved need to be better understood.
This thesis is divided into two parts; the first part is focused on the TCA cycle
enzyme malate dehydrogenase, both its biochemical properties and its genetic
regulation, the second part is focused on two alleles encoding putative dihydrolipoyl
17
succinyltransferase (E2) subunits of the 2-oxoglutarate dehydrogenase (OGD)
complex, and three alleles encoding dihydrolipoamide dehydrogenase (E3) subunits of
the pyruvate dehydrogenase (PDH), OGD and branched-chained alpha-keto acid
dehydrogenase (BKD) complexes.
18
Chapter 1: Literature review
1.1 Preamble
Nitrogen is an essential element for all life. Nitrogen is an abundant element;
however, almost all of it is in the form of N2 gas, unavailable to most organisms. For
plants to grow and animals to thrive, they need nitrogen in a “fixed” form of organic
nitrogen compounds such as amino acids, ammonium (NH4), or nitrate (NO3-). In
many agricultural settings, fertilizer is added to the soil to compensate for the lack of
available nitrogen. It is predicted that worldwide nitrogen-based fertilizer demand will
increase from present levels to a total of 1.13 x 1011 kg by 2015, an annual increase of
1.7% (da Silva 2011). The bulk of the nitrogen in fertilizer is found as ammonia
produced by the Haber-Bosch process in which natural gas is relied on heavily, not
only for the tremendous amount of energy consumed during production but also as a
source of hydrogen to reduce the N2 (Smil 2001). Not only is the production of
ammonia energy expensive, its use can be environmentally costly via pollution of
water resources and the atmosphere. Excess ammonia in the soil is converted to nitrate
by nitrifying bacteria which can then end up as runoff into lakes and rivers or be
denitrified to N2O, a major greenhouse gas. Another implication of over fertilizing is
the decline of soil organic carbon caused by the promotion of carbon utilization and
nitrogen mineralization by microbes that can result in decreasing field yields
(Mulvaney et al. 2009). Clearly, an alternative needs to be found.
Is biological nitrogen fixation (BNF) the answer? Although numbers are
difficult to estimate, it has been estimated that global BNF from all sources can be as
19
high as 3 x 1011 kg fixed nitrogen per year (Galloway 1995) or roughly double that of
man-made fixed nitrogen. The major benefit of BNF is that there are limited economic
costs or environmental ramifications. Free-living aerobic nitrogen fixers, such as
Azotobacter vinlandii, are found widely distributed in soil but the quantity of fixed
nitrogen produced is low due to the lack of abundant substrates to support growth and
vigorous fixation rates (Burris 2001). The vast majority of fixed nitrogen found
worldwide comes from symbiotic nitrogen fixation (SNF). Examples include Frankia,
which forms an association with actinorhizal plants, and the rhizobia/legume
relationship which comprises the largest percentage of the SNF group. The drawback
of this SNF is that it is mostly restricted to plants of the Leguminosae or Fabaceae
family. Although legumes are agriculturally and economically important, when
speaking of worldwide food production it is the grain plants that predominate. The
ultimate goal would be if science could unlock the key(s) to SNF and instill that ability
onto the wheat, rice and corn of the world thus eliminating the need for artificial
fertilization.
Many of the biological aspects of SNF have been elucidated, from both the
plant and microorganism perspectives (for reviews see: (Fischer 1994; Jones et al.
2007; Murray 2011; Terpolilli et al. 2012; van Rhijn and Vanderleyden 1995));
however, much more needs to be done to truly be able to produce genetically-modified
organisms (bacteria/plant) that can have a functioning symbiotic association and fix
nitrogen. One of the critical stages in SNF in bacteria is the generation of energy and
reducing equivalents needed for the nitrogenase enzyme. This energy generation is
20
accomplished via the TCA cycle for which a thorough knowledge would be necessary
for a more complete understanding of SNF.
1.2 Sinorhizobium meliloti
Members of the Rhizobiales order are Gram-negative rods that belong to the αProteobacteria class of the domain Bacteria. Included in this order are the
agriculturally-important nitrogen-fixing bacteria collectively known as rhizobia such
as the genera Bradyrhizobium, Rhizobium, Mesorhizobium and Sinorhizobium (Bergey
et al. 1984). S. meliloti is a free-living microorganism in the soil. Motility occurs by
two to six peritrichous flagella and is important for chemotaxtic response to plant root
secreted flavonoids (Burris 2001). The legume plants that S. meliloti symbiotically
associates with are exclusively of, but not all of, the genera Melilotus (sweet clover),
Medicago (alfalfa) and Trigonella (fenugreek) which all form indeterminate root
nodules.
The genome of S. meliloti totals 6.68 Mb and is comprised of one chromosome
3.65 Mb in size and two megaplasmids, 1.35 Mb pSymA and 1.68 Mb pSymB
(Galibert et al. 2001). This is of comparable size compared to other rhizobial genomes
(including megaplasmids) such as Bradyrhizobium japonicum (9.1 Mb) (Kaneko et al.
2002), Rhizobium leguminosarum (7.8 Mb) (Young et al. 2006), Mesorhizobium loti
(7.5 Mb) (Kaneko et al. 2000) and Rhizobium etli (6.5 Mb) (Gonzalez et al. 2006).
Detailed analysis of the annotated S. meliloti chromosome revealed that this replicon
not only contains most of the housekeeping genes needed for nucleic acid and protein
metabolism and other biosynthetic pathways, but also the genetic information for
21
motility and chemotaxis processes, plant interaction (putative virulence genes) and
stress responses (Capela et al. 2001). The pSymA megaplasmid encodes for many of
the genes necessary for SNF such as those involved in Nod factor biosynthesis (nod)
and those for nitrogenase and nitrogen fixation (nif/fix) (Barnett et al. 2001). To
demonstrate how important pSymA is to the SNF process, deletion studies on pSymA
resulted in plants with either no nodules (Fix-), wild-type numbers of fix+ nodules or
hypernodulated plants which were Fix- (Yurgel et al. 2013). S. meliloti strains that
have been cured of this plasmid cannot induce nodule formation on potential host
plant roots (Oresnik et al. 2000). Alternatively, transforming Agrobacterium
tumefaciens, also of the Rhizobiaceae family, with plasmids that carry the nod and nif
genes of pSymA results in a strain that not only induces root hair curling and the
formation of nodules but are able to invade the host plant-derived infection threads
and into the nodules themselves, albeit at a greatly reduced frequency than S. meliloti,
and nitrogen fixation does not occur (Hirsch et al. 1984). These plasmids, when in
Escherichia coli, can induce alfalfa plants to produce root hair curling and
pseudonodules; however, no bacteria enter any infection threads. More recently, the
author of this paper (Ann Hirsch) is distancing herself from this last result and
attributing the E. coli induced plant phenotype to spontaneous nodulation. The third
replicon, pSymB, carries genes mostly related to solute uptake systems such as those
necessary for C4-dicarboxylic acid (dctA) and phosphate transport (phoCDET), as well
as for exopolysaccharide synthesis (Finan et al. 2001; Watson et al. 1988).
Additionally, genes for transcriptional regulation, cell protection and other catabolic
roles essential for bacterial viability are found on this megaplasmid. Since some of
22
these genes are required for viability and gene density is similar to what is found on
the bacterial chromosome, pSymB could be thought of as a “second chromosome”.
As a free-living soil microbe, S. meliloti has access to a wide variety of
compounds available as carbon sources. Studies have shown that the preferred carbonsource of S. meliloti is succinate over other compounds such as glucose, fructose,
galactose, lactose, and myo-inositol (Hornez et al. 1994; Jelesko and Leigh 1994;
Poole et al. 1994; Ucker and Signer 1978). Carbohydrate metabolism occurs mainly
through the Entner-Doudoroff and pentose phosphate pathways (Martinez-De Drets
and Arias 1972; Mulongoy and Elkan 1977). Through these pathways the resulting
pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase (PDH). Acetyl-CoA
then enters the TCA cycle through condensation with oxaloacetate (OAA) catalyzed
by the enzyme citrate synthase (Fig. 1). L-arabinose, a component of plant cells walls,
is found in the rhizosphere and can support the grown of rhizobia (Stowers 1985).
Arabinose is metabolized in a multistep nonphosphorylative pathway into the TCA
cycle intermediate 2-oxoglutarate (Duncan and Fraenkel 1979). In S. meliloti, many of
the genes necessary for arabinose catabolism are found as an operon (araABCDEF)
located on megaplasmid pSymB (Poysti et al. 2007). Arabinose uptake is carried out
by proteins encoded by araA and araB. The catabolism of arabinose is carried out via
a hypothesized five-step pathway involving a semi-aldehyde dehydrogenase and two
dehydratases encoded by araDEF. The genes encoding the two remaining catabolic
pathway enzymes, arabinose dehydrogenase and a lactonase, are not part of this
operon and are unknown. Poysti et al. (2007) found that plants inoculated with strains
23
of S. meliloti mutated in any one of the arabinose operon genes were Fix+ indicating
that this compound is not important for nitrogen fixation.
Once S. meliloti has differentiated into bacteroids, the plant must provide a
constant and abundant flow of energy in the form of reduced carbon compounds for
the nitrogen fixation process. High concentrations of sugars are found inside nodules;
however, the transport of these compounds appears to only occur via diffusion, and the
rate of uptake of these sugars is not enough to support nitrogen fixation (Glenn et al.
1984a; Glenn and Dilworth 1981a; Glenn and Dilworth 1981b; Hudman and Glenn
1980; Streeter 1980; Udvardi et al. 1990; Vance 2008). In support of this, studies
demonstrated that mutations in sugar uptake or carbohydrate metabolism resulted in
Fix+ plants (Glenn et al. 1984b; McKay et al. 1985; Ronson and Primrose 1979). It has
been shown that plant photosynthates are reduced to the C4-dicarboxylic acid
compounds succinate and malate (Day and Copeland 1991; Fougere et al. 1991).
These compounds must then be actively transported across the peribacteroid
membrane as well as by the dicarboxylate transport (Dct) system on the bacteroid’s
cytoplasmic membrane. The Dct system, composed of a membrane transporter (DctA)
encoded by dctA, is induced by a two-component sensor-regulatory system (DctBD)
that responds to C4-dicarboxylic acids (Yurgel and Kahn 2004). S. meliloti strains
with a defective Dct system can enter nodules and differentiate into bacteroids but are
unable to fix nitrogen (Finan et al. 1988).
1.3 S. meliloti TCA cycle and malic enzymes
24
The TCA cycle is central to carbon metabolism (Fig. 1). Oxidation of TCA
cycle intermediates is coupled to the generation of energy and reducing power in the
form of ATP and NAD(P)H, respectively. Eight enzymes make up the TCA cycle:
citrate synthase (CS, EC 4.1.3.7), aconitase (ACN, EC 4.2.1.3), isocitrate
dehydrogenase (ICD, EC 1.1.1.42), 2-oxoglutarate dehydrogenase (OGD, EC 1.2.4.2),
succinyl-CoA synthetase (SCS, EC 6.2.1.6), succinate dehydrogenase (SDH, EC
1.3.99.1), fumarase (FUM, EC 4.2.1.2), and malate dehydrogenase (MDH, EC
1.1.1.37). Disruption of any TCA cycle enzyme in S. meliloti results in plants with a
Fix- phenotype (Kahn et al. 1995; Koziol et al. 2009; Mortimer et al. 1999); (Duncan
and Fraenkel 1979; Dymov et al. 2004; McDermott and Kahn 1992). This,
presumably, is due to the loss of ATP production needed for the nitrogenase enzyme
emphasizing the importance of this cycle. To date, there have been no reported S.
meliloti SCS or FUM mutants; however, plants inoculated with R. leguminosarum
SCS (Walshaw et al. 1997) and B. japonicum FUM (Acuna et al. 1991) mutants both
were described as conferring a Fix- phenotype .
In S. meliloti, two main operons encode genes of the TCA cycle. MDH is the
first gene in an operon that also encodes the two subunits of SCS (sucCD) and two of
the three subunits of OGD (sucAB) and, thus, has the structure mdh-sucCDAB. This is
the same gene order reported for R. leguminosarum (Poole et al. 1999; Walshaw et al.
1997), A. tumefaciens (Goodner et al. 2001) and M. loti (Kaneko et al. 2000). It is
interesting to note, M. loti also encodes second copies of the sucCD genes; however,
translated amino acid sequence homology between the two sucC and sucD alleles is
only 46% and 48%, respectively, opening the question of which allele encodes for the
25
functional protein of each subunit. The gene order mdh-sucCDAB is conserved in B.
japonicum; however, a study showed that mdh is expressed monocitronically and
sucAB is expressed from its own upstream promoter (Green et al. 2003). The authors
did not determine where the transcription of sucCD originated.
The second TCA cycle operon found in S. meliloti encodes for the four
subunits of SDH and is structured sdhCDAB. This arrangement is conserved across
many rhizobia and is always located upstream of the mdh-sucCDAB operon.
Depending on the rhizobia, these two operons are separated by 2 ORFs in R. etli
(Gonzalez et al. 2006), 3 ORFs in R. leguminosarum (Young et al. 2006) and 8 ORFs
in S. meliloti (Galibert et al. 2001) and M. loti (Kaneko et al. 2000). B. japonicum
exhibits a major difference in genetic arrangement from the other described rhizobia
as the sdhCDAB operon is transcribed divergently from mdh-sucCDAB and is
separated by 55 ORFs, of which one is the gene encoding for another TCA cycle
enzyme ACN (Kaneko et al. 2002), reflecting the phylogenetic divergence of this
genus from other rhizobia (Peter et al. 1996).
At the malate junction of the TCA cycle, the flow of carbon can either continue
to MDH or can be directed out of the cycle though malic enzyme and PDH resulting in
acetyl-CoA which can re-enter the cycle by condensing with OAA (Fig. 1). In
bacteroids, the plant-derived carbon-source is succinate, fumarate and malate.
Succinate and fumarate will ultimately be converted to malate by succinate
dehydrogenase and fumarase. If malate only continued through the TCA cycle via
MDH to form OAA, then the pool of acetyl-CoA would soon be depleted, thus, halting
the cycle. Therefore, the balance of flow though MDH and malic enzyme is critical to
26
maintain the equimolar concentration of OAA and acetyl-CoA. S. meliloti has two
distinct malic enzymes (Driscoll and Finan 1993). One malic enzyme primarily uses
NAD+ as cofactor and is called diphosphopyridine-nucleotide-dependent malic
enzyme (DME). This enzyme can also use NADP+ as a cofactor but with reduced
activity. The second malic enzyme only uses NADP+ as cofactor and is called
triphosphopyridine-nucleotide-dependent malic enzyme (TME). Both of these
enzymes convert malate to pyruvate and CO2 concomitantly with the reduction of
NAD+ or NADP+ to NADH or NADPH (Fig. 1). DME and TME are constitutively
expressed in free-living cells; however, TME is repressed in bacteroids (Driscoll and
Finan 1996; Driscoll and Finan 1997). Strains of S. meliloti lacking DME activity
have a wild-type carbon-utilization phenotype and are able to induce nodule
formation; however, nitrogen fixation does not occur (Driscoll and Finan 1993).
Conversely, strains of S. meliloti lacking TME activity have a wild-type growth
phenotype and are Fix+ (Driscoll and Finan 1996). Bacteroid DME concentrations
were determined to be approximately ten times that of TME (Mitsch et al. 2007). To
determine if higher concentrations of TME would result in a Fix+ phenotype in the
DME mutant, a strain was constructed in this background in which the dme promoter
controlled the expression of the tme genes. Bacteroids of this strain had elevated levels
of TME but were still Fix-. To determine how much DME was required for nitrogen
fixation, a second strain was made in the same Dme- background that had the tme
promoter controlling expression of the dme gene. Even though bacteroids formed by
this strain had a large reduction in DME activity, it was still sufficient to support
27
significant nitrogen fixation, suggesting that sufficient flow of malate to pyruvate was
maintained.
In contrast, dme mutants generated in B. japonicum (Dao et al. 2008), R.
leguminosarum (Mulley et al. 2010) and M. loti (Thapanapongworakul et al. 2010)
formed Fix+ nodules on their respective host plants. Dme- mutants of the broad host
range Sinorhizobium sp. strain NGR234 formed bacteroids with fixation capabilities
ranging from 27 to 83% of wild-type levels depending on the host plant that was
inoculated (Zhang et al. 2012). Clearly different plant hosts may supply different
metabolites that can bypass the lack of DME, resulting in the Fix+ phenotype. This can
also be achieved by the convertion of OAA to pyruvate by the enzymes
phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK) in R.
leguminosarum (Mulley et al. 2010). Therefore, the Fix- phenotype observed in the S.
meliloti DME mutants is unusual and may be unique to the S. meliloti-alfalfa
symbiosis.
The role DME plays in bacteroid metabolism has still not been definitively
established. DME together with PDH forms a pathway where malate is converted to
acetyl-CoA (Fig. 1); however, it is possible DME generates pyruvate which can then
be used as a precursor for some other pathway, such as the generation of reductants for
nitrogenase (Scott and Ludwig 2004).
1.4 Bacterial promoter recognition and transcriptional regulation
The main promoter elements that facilitate specific transcription initiation by
RNA polymerase (RNAP) are the UP (upstream) element, the -35 element, the
28
extended -10 element, the -10 element, and the discriminator element (Haugen et al.
2008). In bacteria, the UP element is an approximately 20 nt A + T rich region located
from -59 to -38 with the consensus 5’-NNAAAWWTWTTTTNNNAAANNN (W = A
or T, N = any base) and seems to be particularly associated with strong promoters
(Estrem et al. 1998; Gourse et al. 2000). The -35 element has a consensus sequence 5’TTGACA in E. coli and runs from -35 to -30 (Harley and Reynolds 1987). The
extended -10 element is located at -17 to -14 and has a consensus sequence 5’-TGTG
(Mitchell et al. 2003). The -10 element is positioned from -12 to -7 and has a
consensus sequence 5’-TATAAT in E. coli (Harley and Reynolds 1987). The
discriminator element is located from position -6 to -4 and has a consensus sequence
5’-GGG (Haugen et al. 2006).
The RNAP core in bacteria is typically composed of 5 subunits, ß ß´ ω α2, and
is catalytically competent but can only recognize DNA nonspecifically (Campbell et
al. 2008). This RNAP core is structurally similar across the three domains of life
(Cramer et al. 2001; Hirata et al. 2008; Vassylyev et al. 2002). The ß and ß´ subunits
are separated by a deep catalytic cleft where the synthesis of the RNA phosphodiester
bonds takes place. The small ω subunit is associated primarily with the ß´ subunit and
instills structural stability. Each of the two α subunits are composed of two domains;
an N-terminal domain (αNTD) and a C-terminal domain (αCTD) joined by a flexible
linker (Blatter et al. 1994). The αNTD dimerizes and forms a scaffold on which the
large ß and ß´ subunits assemble and the αCTDs, located on the opposite side of the
complex from where DNA enters and exits the catalytic pocket, binds DNA of the UP
element.
29
The key component to promoter recognition and transcription initiation is
another subunit factor known as σ (Burgess et al. 1969). The principal sigma factor
responsible for transcription of most of the genes essential for viability is known as the
housekeeping sigma factor. This sigma factor is composed of four helical domains (σ1,
σ2, σ3 and σ4) connected by flexible linkers with each respective domain functionally
distinct and responsible for the recognition and binding of conserved bases in each of
the aforementioned promoter elements (σ1 with the discriminator element, σ2 with the
-10 element, σ3 with the extended element and σ4 with the -35 element) (Campbell et
al. 2002; Malhotra et al. 1996), RNAP core binding and DNA melting (Haugen et al.
2008). Other alternative sigma factors confer upon RNAP the ability to control
transcription in response to several major adaptive situations (Wigneshweraraj et al.
2008). The alternative factor σ54 is unique in that it binds DNA at the -24, -12 region
of the promoter and requires as activator to initiate transcription (Lee et al. 2012).
In conjunction with the sigma factor, other aspects (i.e., transcription factors)
are involved in transcriptional regulation. These include activator molecules that can
interact with promoter DNA to induce a conformational change, thus, improving
promoter quality or can interact directly with the RNAP to compensate for defects in
the promoter (Lee et al. 2012). Repressors prevent binding of RNAP by covering the
promoter in the vicinity of the -35, -10 region (Browning and Busby 2004). Anti-σ
factors are proteins that attach to one or more areas of the σ surface blocking binding
to the promoter DNA or to the RNAP core (Helmann 1999). Transcriptional
interference is when transcriptional activities interfere with one another in cis by the
collision of RNAPs bound to, or initiated from, different convergence, tandem or
30
overlapping promoters (Shearwin et al. 2005). Lastly, DNA methylation can both
repress and activate gene expression by either enhancing or blocking the binding of
repressors or activators at promoters (Low et al. 2001). Gene expression can also be
regulated at the post-transcriptional level through mRNA degradation, by regulatory
domains known as riboswitches that reside in the noncoding regions of mRNA where
metabolites bind and control gene expression and by short noncoding RNAs (cisantisense RNA) that complementarily bind mRNA thereby affecting the translation or
stability of the message (Masse et al. 2003; Winkler and Breaker 2005).
1.5 Transcription start sites and σ factors of S. meliloti
The genome of S. meliloti contains 15 ORFs that encode different sigma
factors (Galibert et al. 2001). The housekeeping sigma factor, σA, from S. meliloti is
684 amino acids in size (Rushing and Long 1995) which is 70 amino acids larger than
that from E. coli (Gribskov and Burgess 1983). The alternative sigma factor σ54,
encoded by rpoN, is responsible for the regulation of genes involved in SNF and C4dicarboxylic acid transport (Ronson et al. 1987). Two RpoH σ factors are found with a
high sequence homology to the E. coli heat-shock factor σ32 (Oke et al. 2001). At least
9 genes are annotated as encoding extracytoplasmic function (ECF) σ factors (rpoE1rpoE9) which are usually regulated by anti-σ factors (Helmann 2002). It has been
found that RpoE2 is the regulator of at least 44 genes, including rpoH2 and rpoE5, and
was named a global regulator of general stress adaptation and to the hyperosmotic
stress response (Flechard et al. 2010; Sauviac et al. 2007).
31
MacLellan et al. (2006) looked at the consensus sequence of 25 experimentally
verified non-σ54 controlled promoter regions in S. meliloti. Based on sequence
conservation and mutational analysis of promoter activity they found the -35 and -10
RNAP binding site to have the consensus structure 5-CTTGAC-N17-CTATAT. A
recent study using RNAseq mapped transcription start sites (TSS) from S. meliloti
grown under 16 different growth and stress conditions (Schluter et al. 2013). The over
17,000 TSS grouped into six categories based on the genomic context of their
transcripts: (1) 4,430 mRNA TSS were assigned to 2,657 protein-coding genes, (2)
171 TSS were assigned to leaderless mRNA, (3) 425 TSS were listed as putative, (4)
7,650 sense TSS represent internal transcripts in the same orientation as, and located
within, protein-coding genes, (5) 3,720 TSS of cis-encoded antisense RNAs and (6)
605 TSS of trans-encoded sense RNA.
In this thesis a couple of the main goals is the better understanding of the
enzyme MDH and the large promoter region controlling expression of the mdhsucCDAB operon.
32
Figure 1: Diagram of the TCA cycle and selected anaplerotic pathways. Pathways found in S. meliloti are in black and those
not found are in red.
Figure modified from (Dunn 1998)
33
Chapter 2: Materials and Methods
2.1 Bacterial strains and growth media
All bacterial strains, plasmids, phage and transposons used in this thesis are
listed in Table 1. Luria-Bertani (LB) media consisted of (per liter): 10 g tryptone, 5 g
yeast extract and 5 g NaCl. For S. meliloti this was supplemented with 2.5 mM MgSO4
and 2.5 mM CaCl2 (LBmc). M9 consisted of (per liter): 2.9 g Na2HPO4, 1.5 g
KH2PO4, 0.25 g NaCl, and 0.5 g NH4Cl. After sterilization, M9 was supplemented
with sterile 0.25 mM CaCl2, 1.0 mM MgSO4 and 0.15 mg/L biotin. Filter-sterilized
(0.45 µm filters) carbon sources (L-arabinose, Na-L-glutamate, D-glucose, L-leucine,
L-malate, Na-pyruvate, or Na-succinate) were added to M9 to a final concentration of
15 mM. M9/LB media was used for selection of transductants (50% 2x LB and 50%
2x M9). Media was solidified with 1.5% agar. E. coli cultures were incubated at 37°C
and S. meliloti at 30°C.
Antibiotics were filter-sterilized (0.45 µm) and used at the following final
concentrations (µg·mL-1): ampicillin 100, chloramphenicol 10, kanamycin 20,
gentamycin 20, neomycin 200, streptomycin 200 and tetracycline 10.
2.2 Bacterial manipulations
2.2.1 Conjugation
To mobilize plasmids, cultures of the recipient, donor and mobilizer strain
(MT616) grown overnight (O/N) in LB with appropriate antibiotic then washed 2x in
34
sterile saline then the three cultures mixed in a 1:1:1 ratio and spotted onto LB agar
plates. Controls were the pure cultures. Following O/N incubation, the spots were
scraped with a sterile stick, suspended in saline and 100 µL spread onto LB agar
containing the appropriate selective antibiotics.
2.2.2 Transduction
Transducing phage ΦM12 was used to transfer the mini-Tn5 insertion in
SMb20019 (Pobigaylo et al. 2006) from Rm2011 to Rm1021 using the protocol
previously outlined (Glazebrook and Walker 1991). Briefly, lysates were made by
diluting 0.5 mL of O/N culture of strain Rm30208 (SMb20019-) in 4.5 mL LBmc.
Two drops of wild-type phage was added and incubated overnight on a rotation wheel
at 30°C. Two-three drops of chloroform was added, vortexed lightly and allowed to
settle O/N at 4°C. The top of the lysate was recovered, centrifuged (2000 g, 5 min,
4°C) and stored at 4°C. This lysate was diluted to a multiplicity of infection (MOI) of
0.5 of which 1 ml was added to 1 mL of recipient Rm1021 culture followed by gentle
mixing and RT incubation for 20 min without shaking. This was washed two times
with 2.5 mL sterile saline, suspended in 1 mL saline, and 100 µL plated on LBM9
supplemented with streptomycin and neomycin.
2.2.3 Competent cell preparation and transformation
Competent cells were prepared by the CaCl2 method previously described
(Sambrook et al. 1989). Briefly, 10 mL of an O/N liquid culture of DH5α was used to
inoculate 250 mL LB supplemented with 15 mM glucose. This was grown to an OD600
0.5 then placed on ice for 10 min followed by centrifugation (4200 g, 5 min, 4°C). The
35
pellet was gently suspended in 100 mL of solution A (100 mM CaCl2) and left on ice
for 1 h. Following centrifugation (2700 g, 10 min, 4°C) the pellet was gently
suspended in 10 mL solution B (100 mM CaCl2, 10% glycerol), 100 µL aliquots
placed into cold tubes and stored at -80°C.
To transform cells, frozen competent cells were allowed to thaw on ice then
2.5 µL of plasmid DNA was added, gently mixed and left on ice for 30 min. Cells
were then heat shocked by placing tubes in a heating block (42°C) for 90 s then cold
shocked on ice for 2 min. To this, 1 mL prewarmed (37°C) LB was added and to
compensate for phenotypic lag, tubes were places in an incubator (37°C) with shaking
for 45 min. Following centrifugation (1675 g, 10 min, RT) the pellet was suspended in
100 µL LB and then spread onto LB agar plates with appropriate antibiotics.
2.3 Biochemical manipulations
2.3.1 Crude cell extracts and protein quantification
Liquid cell cultures were grown overnight in LBmc supplemented with
antibiotics if needed for plasmid maintenance. Cultures were washed 2-3x in washing
buffer (20 mM TRIS-HCl pH 7.8, 1 mM MgCl2) then suspended (4 mL·g-1 cell pellet
wet weight) in ice cold sonication buffer (20 mM TRIS-HCl pH 7.8, 1 mM MgCl2, 10
mM ß-mercaptoethanol, 10% glycerol). Cells were disrupted by sonication (Sonifier
Cell Disruptor, Heat systems – Ultrasonics Inc, Plainview, NY, USA); 8-10 cycles of
10 s at 90 Watts with 5 min cooling on ice between cycles. Extracts were centrifuged
(18000 g, 20 min, 4°C) to pellet the cell debris. Aliquots of the supernatant were
frozen at -20°C until needed.
36
The Bradford method was utilized to quantify protein concentration (Bradford
1976) using reagents and protocols from Bio-Rad (Bio-Rad Laboratories, Hercules,
CA. USA). A standard curve was generated using dilutions of BSA in ddH2O.
2.3.2 Enzyme assays
Enzyme activities were determined in triplicate using an Ultrospec 2000
spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). The initial slopes of the
activity curves were used to calculate the specific activity (SA) using the following
formula:
SA = Δ absorbance x reaction volume (mL)
time (min) x protein (mg) x ε
where Δ absorbance is the calculated initial slope and ε is the extinction coefficient of
the compound being monitored spectrophotometrically adjusted to convert absorbance
values to nanomoles. Units for SA are nmol·min-1·mg protein-1. Standard errors (SE)
were calculated as follows:
SE = SD
√n
where S is the statistical average of SA and n is the number of replicates.
2.3.2.1 Malate dehydrogenase
Malate dehydrogenase (EC1.1.1.37) assay was performed as described
previously (Englard and Siegal 1969). For the oxidation of malate reaction, each
cuvette contained 100 mM glycine-NaOH (pH 10), 85 mM L-malate, 2.5 mM NAD+
37
and ddH2O to 1 mL. For the reduction of oxaloacetate, each cuvette contained 100
mM glycine-NaOH (pH 10), 200 µM NADH, 3 mM oxaloacetate and ddH2O to 1 mL.
Reactions were monitored spectrophotometrically at wavelength 340 nm. Reactions
were initiated by the addition of 0.1 mg crude cell extract. ε = 6.22 x 10-3 nmol-1
2.3.2.2 2-oxoglutarate dehydrogenase
2-oxoglutarate dehydrogenase (EC1.2.4.2) assays were performed as
previously described (Reed and Mukherjee 1969). To a cuvette was added 50 mM
phosphate buffer (pH 8), 1 mM MgCl2, 2 mM NAD+, 3 mM cysteine-HCl, 0.2 mM
TPP, 0.1 mg crude cell extract and ddH2O to 1 mL. Reactions were initiated by the
addition of 60 µM Na-CoA and 1 mM 2-oxoglutarate and monitored at 340 nm. ε =
6.22 x 10-3 nmol-1
2.3.2.3 Succinyl-CoA synthetase
Succinyl-CoA synthetase (EC6.2.1.6) activities were done as previously
described (Bridger et al. 1969). Reactions were done in a quartz cuvette and contained
50 mM TRIS-HCl (pH7.2), 0.1 mM KCl, 10 mM MgCl2, 10 mM Na-succinate, 0.4
mM ATP, 0.1 mM coenzyme A and ddH2O to 1 mL. Reactions were initiated with the
addition of 0.1 mg crude cell extract and monitored at 230 nm. ε = 4.5 x 10-3 nmol-1
2.3.2.4 Isocitrate dehydrogenase
Isocitrate dehydrogenase (EC1.1.1.42) enzyme assays were done as described
previously (Reeves et al. 1971). Each reaction contained 20 mM TRIS-HCl (pH7.5), 2
mM MnCl2, 0.5 mM NADP+, 0.1 mg crude cell extract and ddH2O to 1 mL. To start
38
the reactions 0.5 mM DL-Na isocitrate was added and monitored at 340 nm. ε = 6.22 x
10-3 nmol-1
2.3.2.5 Pyruvate dehydrogenase
Assays for pyruvate dehydrogenase (EC1.2.4.1) activity was done as
previously described for 2-oxtoglutarate dehydrogenase (Reed and Mukherjee 1969)
by substituting 2-oxoglutarate with Na-pyruvate.
2.3.2.6 Branched-chained keto-acid dehydrogenase
Assays for branched-chained keto-acid dehydrogenase (EC1.2.4.4) were done
using the modified method described previously (Harris and Sokatch 1988). To a
cuvette was added 30 mM potassium phosphate buffer (pH 8), 2 mM MgSO4, 2 mM
DTT, 0.1% Triton X-100, 0.56 mM TPP, 0.56 mM CoA, and 1.4 mM NAD+, 0.1 mg
of crude cell extract and ddH2O up to the final volume of 1 ml. Reactions were started
with the addition of 0.28 µM keto-leucine and followed spectrophotometrically at
wavelength 340 nm. ε = 6.22 x 10-3 nmol-1
2.3.3 ß-galactosidase and gfp-fusion assays
ß-galactosidase activity was adapted from a previously described method
(Miller 1972). Cultures were grown O/N in liquid M9-minimal media with selected
sole-carbon source then suspended in Z buffer (60 mM Na2HPO4·7H2O, 40 mM
Na2HPO4·H2O, 10 mM KCl, 1 mM MgSO2·7H2O and 50 mM ß-metcaptoethanol) to a
final volume of 4 mL and an OD675 of approximately 0.1 which was then dispensed as
1 mL aliquots into 4 tubes. One tube was used as an OD675 reference (with a tube of Z
39
buffer as a blank). To the three remaining tubes (plus a fourth containing just Z buffer
for a blank) 10 µL chloroform and 5 µL 0.1% SDS was added followed by vortexing.
These four tubes were equilibrated in a 30°C water bath for 10 min.
The assays were initiated by the addition of 200 µL ONPG (4 mg·mL-1 in Z
buffer) to the tubes, lightly vortexed, a timer started and the tubes replaced into the
30°C water bath. Reactions were stopped, when the yellow colour was between A420
0.2-0.6, by the addition of 1 M Na2CO3. Tubes were centrifuged (1000 g, 10 min, RT)
and the A420 of the supernatant determined. Miller units were calculated as follows:
Miller Units =
A420 x 1000
time (min) x OD675
For the gfp-transcriptional promoter fusion assays, cultures were grown in
triplicate, O/N in liquid M9-media with selected carbon sources and gentamycin for
plasmid maintenance. Aliquots of 200 µL were put into 96 well flat bottom black
walled ploystrol mircoplates (Greiner Bio-one, VWR, Mississauga, Ont) and placed in
a microplate reader (Tecan Infinite 200, Mannedorf, Switzerland). First the OD600 was
measured followed by the GFP fluorescence unit (excitation 390 nm, emission 510 nm
(Karunakaran et al. 2005)). Relative fluorescence (RFU) was determined by the
formula:
RFU = fluorescence at 510 nm – background
OD600
40
2.3.4 Purification of MDH
2.3.4.1 Purification by HPLC method
Crude cell extracts were prepared from a 4 L culture of Rm1021. MDH
enzyme assays were carried out at pH 10 to eliminate possible extraneous activity due
to DME from the host E. coli which is inactive in alkali conditions (Driscoll and Finan
1997). Protein purity was achieved by first separating the extract using (NH4)2SO4
precipitation and keeping the 40-75 % fraction. Using a GradiFrac system (Amersham
Pharmacia Biotech, Baie D’Urfe, Que.) the extract was sequentially passed through a
size exclusion column (HiPrep 26/60, Sephacryl S-300), NAD+ cofactor utilizing
affinity column (HiTrap Blue) and an ion exchange column (Resource Q). Fractions
from each step were loaded onto a SDS-PAGE (4 % stacking gel, 12.5 % running gel),
run at 50 mA, and visualized by staining with Coomassie Blue R250.
2.3.4.2 Purification by HIS-tag method
A 1 L culture of Ec10495 was grown and induced with IPTG as per the
protocols for the pQE80Lvector (QIAGEN, Mississsauga, Ont.). Cells were harvested
and the resultant crude cell extract passed through a Ni-affinity column (His-select
cartridge, Sigma-Aldrich, Oakville, Ont.) with purified MDH eluted as per the
manufacturer’s instructions. SDS-PAGE was run and stained as described in previous
section.
2.3.5 Determination of molecular mass of MDH
41
The HPLC-purified MDH was used for size exclusion chromatography
performed on a Beckman-Coulter system Gold equipped with a Superdex 200 column
(GE HealthCare, Baie d’Urfe, Que.) equilibrated with 50 mM phosphate pH 7.5, 150
mM NaCl, 5 mM ß-mercaptoethanol and 20 µl sample containing 50-200 µg of
protein incubated with 10 mM ß-mercaptoethanol for 20 h at 4 0C. The column was
developed at 0.5 mL/min and the eluate was monitored at 280 nm. The Superdex 200
column was calibrated using thyroglobulin (670 kDa), bovine IgG (160 kDa),
ovalbumen (45 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.3 kDa) (BioRad, Hercules, Calif.).
2.3.6 Optimal pH determination
The specific activity of MDH was determined at values ranging from pH 5.5 to
11.5 for the oxidation of malate and the reduction of OAA reactions. The buffers (100
mM) used: MES for pH 5.5 and 6, PIPES for pH 6.5 and 7, HEPES for pH 7.5 and 8,
TRIS for pH 8.5 and 9, Glycine for pH 9.5, 10, 10.5 and 11.
2.3.7 Michaelis-Menten enzyme kinetics
The HIS-tagged MDH was used to determine the maximum velocity (Vmax) and
the binding constant (Km) at both its optimum and physiological pH. Assay conditions
for each reaction was set up as described in section 2.3.2.1 with the concentration of
one substrate held constant and the other varied. Concentrations of each substrate for
the oxidation of malate reaction varied between 0.0425 – 42.5 mM for malate and 0.1
– 2.5 mM for NAD+. Concentrations of each substrate for the reduction of OAA
reaction varied between 0.01 – 3 mM for OAA and 15 - 500 µM for NADH.
42
2.3.8 Inhibition and product inhibition assays
The HIS-tagged MDH was used to determine any allosteric inhibition caused
by other TCA cycle intermediates and the malate analog maleic acid. The Vmax and Km
was determined for MDH for the oxidation and reduction reactions as described in
section 2.3.2.1 with the addition of 10 mM potential inhibitor (citrate, isocitrate, 2oxoglutarate, succinate, fumarate, pyruvate and malice acid). The concentration of 2oxoglutarate used to build Dixon plots was 0.1, 2, 5 to 10 mM for the oxidation
reaction and 1, 5, and 10 mM for the reverse reaction.
Product inhibition by all four substrates (malate, NAD+, OAA and NADH) was
carried out by varying the concentrations of both substrates independently at a fixed
concentration of product inhibitor. The HIS-tagged MDH was used for these assays.
Assay conditions were set up as described in section 2.3.2.1. All the concentrations
used for the constant and varied substrates and the product inhibitor are listed in Table
5. Double reciprocal plots (Vmax-1 vs [substrate]-1) were graphed of all eight conditions
and from each of these replots were constructed of the slopes and axis intercepts vs
product inhibitor concentrations.
2.4 Genetic manipulations
2.4.1 Plasmid, genomic DNA and total RNA extraction
Plasmid DNA was isolated from O/N cultures of E. coli grown with selective
antibiotics using protocols and mini-prep purification columns supplied by QIAGEN
(Mississauga, Ont.). Total genomic DNA isolation from S. meliloti was carried out as
43
previously described (Meade et al. 1982). S. meliloti genome sequence data was
accessed
via
the
genome
project
(http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi).
web
Total
RNA
site
was
extracted from O/N cultures of LBmc grown S. meliloti using the RNeasy mini kit
from Qiagen (Mississauga, Ont) following provided protocols. RNA samples were
further treated to DNAse (Invitrogen, Mississauga, Ont) digestion as per provided
protocols.
2.4.2 DNA restrictions and ligations
All DNA manipulations were carried out using enzymes from Invitrogen
(Mississauga, Ont) following their provided protocols.
2.4.3 MDH N-terminal sequencing
HPLC-purified MDH was used for sequencing to avoid the 6 histidine residues
on the N-terminal of the tagged protein. After SDS-PAGE visualization, MDH was
transferred to polyvinylidene difluoride (PVDF) sequencing membrane (Westran
PVDF membrane, Bio-Rad, Mississauga, Ont.) by electroblotting following
manufacturer’s instructions (Trans-Blot Cell, Bio-Rad, Mississauga, Ont.) which was
then sent to Genome Quebec (Montreal, Que.) for sequencing.
2.4.4 PCR
PCR conditions were essentially all set up the same way following
manufacturer’s suggested component concentrations. If different the conditions will be
specifically stated in the relevant section. Standard TAQ polymerase was purchased
44
either from Invitrogen (Mississauga, Ont) or Bioshop Canada (Burlington, Ont). High
fidelity Crimson TAQ polymerase was purchased from New England Biolabs
(Whitby, Ont). The denaturation step was always at 95°C for 45 s, anneal temperatures
varied depending on the primers, and extension temperatures were either 72°C for the
standard TAQ or 68°C for the Crimson TAQ. The number of cycles varied from 25 if
the DNA concentration was high to 35 if the concentration was low.
2.4.4.1 Primers
All primers in this study are listed in Table 2 and were designed using
MacVector 7.2 software (Oxford Molecular Ltd., Genetics Computer Group, Madison,
WI) and purchased from Eurofins MWG Operon (Huntsville, AL). Sequences are
listed in Table 2.
2.4.5 RT-PCR and Transcriptional start site
Total RNA was harvested from S. meliloti cells that had been grown O/N in
LBmc. RT-PCR was carried out using the QIAGEN (Mississauga, Ont) one-step RTPCR kit and the reagent concentrations as per manufacture’s protocols. The primers
are listed in Table 2. The reaction conditions were as follows: reverse transcription at
50°C for 30 min; reverse transcription inactivation and DNA polymerase activation at
95°C for 15 min. The reactions were cycled 20 times as follows; denaturation at 94°C
for 45 s; annealing at 60°C for 10 s; extension at 72°C for 1.5 min. A final extension at
72°C for 10 min was done.
45
The transcriptional start site of mdh was determined by using the 5’-RACE
system (version 2.0) from Invitrogen (Mississauga, Ont) following manufacturer’s
protocols. The gene specific primers (GSP I & II) used are listed in Table 2. cDNA
was made using the primer GSPI. PCR of the dC-tailed cDNA was performed using
the primer GSP II and the kit included abridged anchor primer (AAP). The
thermocycler was set up as follows: reactions were cycled 35 times with denaturation
at 940C for 30 s, annealing at 550C for 30 s and extension at 720C for 1.5 min. After
cycling a final extension at 720C for 5 min was done. Resultant PCR fragments were
sequenced (Genome Quebec, Montreal, Qc) using primer Dave18.
2.4.6 Plasmid construction
2.4.6.1 Construction of plasmid pDM52
Primers were designed to amplify mdh from plasmid pDS15 and to facilitate
ligation into pQE80L restriction sites for BamH1 or HindIII were incorporated on the
5’-ends. PCR amplification conditions were; 10 µL 10x buffer, 1.5 µL 10 mM dNTP
mix, 1 µL 50 mM MgSO4, 1.5 µL 10 µM each primer, 2 µL pBS1, 1 µL platinum Pfx
polymerase (Invitrogen, Mississauga, Ont.), 10 µL 10x PCR enhancer solution, 21.5
µL water. Cycling was carried out 30x with denaturing for 45 sec at 94 0C, annealing
for 30 sec at 62 0C, and extension for 1.5 min at 72 0C. DNA polymerase Pfx leaves
blunt-ended PCR products; therefore, to ligate them into vector pGEM-T easy
(Promega, Madison, WI) adenine over-hangs had to be added to the 3’-ends; 5 µL
PCR product, 1 µL 10x Taq buffer, 0.2 µL 10 µM dATP, 1 µL Taq polymerase
(Invitrogen, Mississauga, Ont.), 2.8 µL water. This was incubated at 70 0C for 30 min.
46
The PCR products with 3’-dATP overhangs were ligated to pGEM-T easy as per
manufacturer’s protocols resulting in pDM51. The mdh fragment was excised from a 1
% agarose gel after double BamH1 and HindIII (Invitrogen, Mississauga, Ont.)
restriction digest using a gel extraction kit (QIAGEN, Mississauga, Ont.) followed by
ligation to pQE80L (QIAGEN, Mississauga, Ont.) resulting in pDM52.
2.4.6.2 Plasmids used for strains Rm30230, Rm30267 and Rm30275 construction
Plasmid pDS15, which carries the complete mdh-sucCDAB operon, was
digested with restriction enzyme XhoI and resolved in a 1% agarose gel. The 1100 kb
fragment of SMc02483 (sucB) was cut out and purified using Qiagen QIAquick Gel
Extraction Kit (Mississauga, Ont). This was ligated into XhoI digested cloning vector
pBluescript KS+ (Fermentas Thermo Fisher, Whitby, Ont) resulting in plasmid
pDM100. This plasmid was double digested using EcoRI and KpnI and resultant
fragment ligated into similarly digested suicide vector pVIK112 (Kalogeraki and
Winans 1997) resulting in plasmid pTR7. Triparental mating mobilized pTR7 into S.
meliloti strain RmG212 where a single homologous recombination event resulted in a
strain with a transcriptional lacZ-fusion to, and gene knock-out of SMc02483
designated Rm30230.
A previously published mini-Tn5 transposon insert in SMb20019 in S. meliloti
strain Rm2011 was obtained (Pobigaylo et al. 2006). It was preferable to have the
transposon in the same strain background as Rm30230 so the marker was transduced
into RmG212 resulting in strain Rm30267. Plasmid pTR7 and the mini-Tn5
transposon both carry kanamycin/neomycin resistance markers so to facilitate
47
selection of a double mutant the resistance marker of pTR7 was changed to
trimethoprim using Epicenter EZ-Tn5 kit (Madison, WI) resulting in plasmid pTR10.
Triparental mating introduced pTR10 into Rm30267 where a single homologous
recombination event resulted in a strain with a transcriptional lacZ-fusion to, and gene
knock-out of SMc02483.This double mutant strain was named Rm30275.
2.4.6.3 Plasmids used for strains Rm30232 (lpdA3-), Rm30282 (lpdA1-) and
Rm30309 (lpdA2-) construction
To construct the three mutant strains, internal regions of each lpdA gene were
PCR amplified. Primers lpdA1forfrag with incorporated EcoR1 restriction site on the
5’-end and lpdA1revfrag with a 5’ XhoI restriction site were used to amplify an
internal 263 bp fragment of lpdA1. This PCR fragment was ligated to pGEMT-easy
(Promega Corp., Madison, WI) where it was cut out by double digest using EcoR1 and
XhoI and ligated to similarly digested suicide vector pVIK112 (Kalogeraki and
Winans 1997) resulting in plasmid pDM48. To facilitate future work in making a
single strain with multiple lpdA deletions, the resistance marker of plasmid pDM48
was changed by inserting EZ-Tn5 <TET> (Epicentre Biotechnologies, Madison, WI)
into nptII following manufacturer’s protocols and selecting for tetracycline resistance,
kanamycin sensitivity resulting in plasmid pBB1. Primers lpdA2for and lpdA2rev
were used to amplify a 382 bp internal fragment of lpdA2 and ligated to the cloning
vector pGEM-T easy (pJM01). The fragment from a double digest using EcoR1 and
KpnI was ligated to similarly digested pVIK112 resulting in pBB2. Plasmid pDM49
was constructed in the same manner as for pDM48 using primers lpdA3forfrag and
lpdA3revfrag which amplified an internal 492 bp fragment of lpdA3. Conjugal mating
48
mobilized suicide plasmids pBB1, pBB2 and pDM49 into the S. meliloti Lac- strain
RmG212 where a single homologous recombination event will result in a strain with a
transcriptional lacZ-fusion to, and gene knock-out of lpdA1 (Rm30282), lpdA2
(Rm30309) or lpdA3 (Rm30232).
2.5 Plant growth conditions, nodule microscopy and photography
Alfalfa (Medicago sativa cultivar Iroquios) seedling growth conditions, S.
meliloti inoculation, and SDW determination was carried out as previously described
(Dymov et al. 2004). After excision from the roots, nodules were placed in a solution
of 4% formaldehyde in PIPES buffer (50 mM, pH 7) and stored at 4oC. Nodules were
place directly onto a microscope slide and immersed in 20 µL of 40 µM SYTO-13
stain (Invitrogen, Mississauga, ON) in 50 mM PIPES buffer (pH 7) then thin sliced
longitudinally. Slices were then covered with a glass coverslip and sealed. Images
were obtained on a Eclipse E800 confocal laser scanning microscope (Nikon Canada,
Mississauga, Ont) equipped with a Radiance 2100 Laser Scanning System and
LaserSharp 2000 v. 6.0 software (BioRad, Mississauga, Ont) with excitation at 488
nm and emission at 515 to 530 nm.
2.6 Computer programs used
Nucleotide and amino acid consensus sequences were analyzed using
MacVector 7.2 software (Oxford Molecular Ltd., Genetics Computer Group, Madison,
WI) running the CLUSTAL W algorithm (Thompson et al. 1994). All graphs were
visualized using Kaleidagraph (Synergy Software, Reading, PA) and calculations for
49
kinetic constants were carried out using the OriginPro 8 software (Origin Labs,
Northampton, MA).
50
Table 1: Bacterial strains, plasmids, transposons and phage used in this study
Strains, plasmids, transposons or
phage
Characteristics
Source or
reference
Rm1021
SU47 str-21, Smr
Meade et al. 1982
RmG212
Rm1021 Lac-, Smr
J. Glazebrooke,
MIT, USA
Rm5422
Rm1021 ntrA75::Tn5
Finan et al. 1988
Rm30041
Rm1021 pDS15
This study
Rm30049
Rm1021
mdh448::Tn5tac1, Smr,
Nmr
Dymov et al. 2004
Rm30062
Rm30049 pDS15
This study
Rm30096
RmG212 pSI33
This study
Rm30107
RmG212 pSI32
This study
Rm30108
RmG212 pSI34
This study
Rm30110
RmG212 pSI38
This study
Rm30129
Rm1021 pDS15par
This study
Rm30131
Rm30049 pDS15par
This study
Rm30208
Rm2011 SMb20019360::mTn5; Nxr, Smr,
Nmr, GUS+
Pobigaylo et al.
2006
Rm30230
RmG212
SMc02483Ω::pTR7; Smr,
Nmr
This study
Rm30232
RmG212 lpdA3::pDM49,
Smr, Nmr, Lac-
This study
Sinorhizobium
meliloti
51
Rm30233
Rm1021 pDM55
This study
Rm30234
Rm1021 pDM56
This study
Rm30267
RmG212 SMb20019360::mTn5; Smr, Nmr,
GUS+
This study
Rm30271
Rm1021 pDM63
This study
Rm30272
Rm1021 pDM64
This study
Rm30275
Rm30267 pTR10
SMb20019-360::mTn5;
Smr, Nmr, Tpr, GUS+
This study
Rm30282
RmG212 lpdA1::pBB1,
Smr, Tcr, Lac-
This study
Rm30309
RmG212 lpdA2::pBB2,
Gmr, Nmr, Lac-
This study
Rm30344
Rm30230 pDS15
This study
Rm30345
Rm30267 pDS15
This study
Rm30346
Rm30275 pDS15
This study
DH5α
F- endA1 hsdR17 (rk-mk-)
supE44 thi1 recA1 gyrA96
relA1 Δ(argF-lacZYA)
U169 φ80dlacZ ΔM15λ-
BRL Inc
DH5α λpir
DH5α pir+
Laboratory strain
MT607
Pro-82 thi-1 endA esdR17
supE44 recA56
Finan 1986
MT616
MT607 pRK600
Finan 1986
Ec10251
DH5α λpir pVIK112
Kalogeraki and
Winans, 1997
Ec10484
DH5α λpir pDM48
This study
Escherichia coli
52
Ec10485
DH5α λpir pDM49
This study
Ec10495
DH5α pDM52
This study
Ec10591
DH5α λpir pBB1
This study
Ec10499
DH5α λpir pTR7
This study
Ec10514
DH5α λpir pTR10
This study
Ec10592
DH5α λpir pBB2
This study
pLAFR1
IncP cosmid cloning
vector, Tcr
Friedman et al. 1982
pRK600
In trans mobilizing vector,
Cmr
Finan 1986
pRK2013
ColE1 replicon with RK2
transfer region; Kmr/Nmr
Figurski and
Helinski 1979
pRK7813
IncP cloning vector, Tcr
Jones and Gutterson
1987
pBluescript KS+
High copy number cloning
vector, Ampr
Fermentas
pGEM-T easy
PCR cloning vector, Ampr
Promega
pQE80L
His-tag cis-repressed
vector, Ampr
QIAGEN
pVIK112
R6K oriV, suicide cloning
vector, lacZ transcriptional
fusion, Kmr
Kalogeraki and
Winans, 1997
pOT1
Broad-host range promoter
probe vector, Gmr
Allaway et al. 2001
pBB1
pDM48 nptII::EZ-Tn5,
Kms / Tcr
This study
pBB2
pVIK112 carrying 363 bp
lpdA2 fragment, Kmr
This study
Plasmids
53
pDM48
pVIK112 carrying 263 bp
lpdA1 fragment, Kmr
This study
pDM49
pVIK112 carrying 492 bp
lpdA3 fragment, Kmr
This study
pDM51
pGEM-T easy carrying
mdh, Ampr
This study
pDM52
pQE80L carrying mdh,
Ampr
This study
pDM55
pOT1 carrying partial mdh
and promoter area-gfp
fusion, forward
orientation
This study
pDM56
pOT1 carrying partial
sucA and putative
promoter area-gfp fusion,
forward orientation
This study
pDM63
pOT1 carrying partial mdh
and promoter area-gfp
fusion, reverse orientation
This study
pDM64
pOT1 carrying partial
sucA and putative
promoter area-gfp fusion,
reverse orientation
This study
pDM100
pBluescript carrying
1100bp XhoI fragment of
SMc02483 from pDS15,
Ampr
This study
pDS15
pLAFR1 carrying 23 kb
partial EcoR1 genomic
digest of S. meliloti
Rm1021, Tcr
Dymov et al. 2004
pDS15par
pDS15 with a Tn3par
insertion in the Tc
resistance gene, Kmr/Nmr
This study
pJM01
pGEM-T easy carrying
PCR fragement of lpdA2,
This study
54
Ampr
pSI32
pDS15, sucA162::Tn5B20 (in reverse)
This study
pSI33
pDS15, mdh383::Tn5-B20
This study
pSI34
pDS15, sucA1479::Tn5B20
This study
pSI38
pDS15, sucD111::Tn5B20
This study
pTR7
pVIK112 carrying
EcoR1/KpnI fragment of
pDM100; Kmr/Nmr
This study
pTR10
pTR7 npt::EZ-Tn5;
Kms/Nms, Tpr
This study
EZ-Tn5 <TET-1>
In vitro transposable
element, Tcr
Epicentre Biotech.
EZ-Tn5 <DHFR-1>
In vitro transposable
element, Tpr
Epicentre Biotech.
Tn5-B20
Promoterless lacZ
Simon et al. 1989
φM12
Generalized transducing
phage
Finan et al. 1984
Transposons
Phage
Amp =ampicillin, Cm = chloramphenicol, Gm = gentamycin, GUS = ß-glucuronidase, Km =
kanamycin, Neo = neomycin, Nx = nalidixic acid, Tc = tetracycline, Tp = trimethoprim
55
Table 2: Primers used in this study
Primer
name
Application
MDH-start
MDH HIS-tag
ATG CGG ATC CAT GGC
GCG CAA CAA CGAT CG
MDH-end
MDH HIS-tag
ACT GAA GCT TTT ACT TCA
GGC TGG GTG CGA TG
GSP I
5’-RACE
AGA GCC GGA AGC GCG
AAG AG
NA2
GSP II
5’-RACE
CGT CGA GCG GGT TGG
TGA TG
NA
Dave18
5’-RACE
CCG GCG GTG ACG ATG
CAG AC
NA
mdh-sucC
RT-PCR
GCG CTT CCG GCT CTT CCT
CG
sucC-mdh
RT-PCR
GCG CAT TGC CGT CGA
AGG AG
sucC-sucD
RT-PCR
CGC GAC ACG ACG GAA
GAG GA
sucD-sucC
RT-PCR
CCG GCG GGA CAT CGC
TCA CG
sucD-sucA
RT-PCR
GCT CGG TCG GCG TGC TTT
CG
sucA-sucD
RT-PCR
CGG GTC GAG CTT GGC
GTG CA
sucA fwd
RT-PCR
CGG CCT TCT CGC GTT CCT
CG
sucA rev
RT-PCR
CGA GAC CGC CCG CTT
GTG AC
lpdA1-int
forward
lpdA1
mutagenesis
1
Sequence (5’ → 3’)
TTC TGA ATT CTT TCC GCC
TTC GCC CGT AAG
Amplicon size
(nt)
1003
1221
702
1165
1201
263
56
1
lpdA1-int
reverse
lpdA1
mutagenesis
AAT GTC TAG ACG ACA
TTG GTC TTT CCG TAG CC
lpdA2-int
forward
lpdA2
mutagenesis
TAT TGA ATT CTC TCA TCG
TTA TCG GAA GCG G
lpdA2-int
reverse
lpdA2
mutagenesis
TTA TGG TAC CGC TCC TCA
CCC TTT TCG TTC G
lpdA3-int
forward
lpdA3
mutagenesis
TCT AGA ATT CTT CGT AGA
CGG CAA GAC GGT GG
lpdA3-int
reverse
lpdA3
mutagenesis
TCA ATC TAG ACG ATT TCC
TCC AAC CCC CAG
lpdA1 fwd
lpdA1
mutagenesis
CGA AGA CAG CAG AAA
ACA CGA CTG
lpdA1 rev
lpdA1
mutagenesis
TGA GAA CCT CCC CGC
ATT GTA G
lpdA2 fwd
lpdA2
mutagenesis
TCC GAC AAG GCG ACT
TAC G
lpdA2 rev
lpdA2
mutagenesis
AAT GCG GGG TTC AGT
TGG
lpdA3 fwd
lpdA3
mutagenesis
GGC GCT GAT TTT CGT TGA
AGG A
lpdA3 rev
lpdA3
mutagenesis
CGG TGA ATC CGG GAT
TCA GTT
363
492
1445
1406
1394
= Underlined nucleotides correspond to restriction enzyme recognition site, 2 = not applicable
57
Chapter 3: The biochemical
dehydrogenase (MDH)
characterization
of
malate
3.1 Introduction
The TCA cycle starts with the condensation of acetyl-CoA to oxaloacetate
(OAA) forming citrate. Through one turn of the cycle, sequential enzymatic steps
metabolize citrate to malate. The final step in the cycle is the oxidation of malate to
OAA (oxidative reaction) with the concomitant reduction of NAD+ to NADH
catalyzed by the enzyme malate dehydrogenase (MDH, Ec 1.1.1.37). The resulting
OAA is now available to condense with acetyl-CoA forming citrate to start the cycle
again. MDH can also catalyze the reverse reaction reducing OAA to malate (reductive
reaction) with the simultaneous oxidation of NADH to NAD+.
Since C4-dicarboxylates, such as succinate or malate, are the sole energy
source supplied to bacteroids (Day and Copeland 1991), acetyl-CoA must be
synthesized from these substrates to maintain the TCA cycle. Thus, malate is diverted
from the cycle and in a successive two step oxidative decarboxylation, performed by
NAD+-dependent malic enzyme (DME) and pyruvate dehydrogenase, acetyl-CoA is
formed which can be condensed with OAA to form citrate (Driscoll and Finan 1993).
To maintain efficient functioning of the TCA cycle, the concentrations of malate and
acetyl-CoA must be kept at approximately the same molar ratio. Controlling the
conversion rate of malate to OAA (or to pyruvate) can be achieved by the biochemical
regulation of MDH and DME, the regulation of the expression of dme and mdh, or by
a combination of the two mechanisms.
58
DME from S.meliloti has been biochemically characterized as have MDH
from other organisms (Courtright and Henning 1970; Driscoll and Finan 1997;
Murphey et al. 1967; Plancarte et al. 2009; van der Rest et al. 2000; Wise et al. 1997).
However, to date no information has been published for this symbiotically important
enzyme from S. meliloti. This chapter focuses on the biochemical properties of MDH
with the aim of answering the hypothesis that the flow of malate through MDH and
DME is controlled by their respective biochemical properties.
3.2 Results and discussion
3.2.1 Malate dehydrogenase purification
Crude cell extract from LBmc-grown Rm1021 was precipitated by ammonium
sulphate followed by purification through various HPLC columns resulting in a 217fold purification (Table 3). SDS-PAGE confirmed the presence of one band after final
column purification (Not shown). A His-tagged MDH was also purified by passing
crude cell extract from Ec10495 through a Ni-affinity column. Both HPLC-purified
MDH and His-tagged MDH were assayed at pH 10 and their specific activities
compared. No difference was found between the two activities, and since it was easier
to obtain larger quantities of the pure His-tagged MDH protein, it was decided to use
this form of the protein for all subsequent experiments except for the N-terminal
sequencing, as the six histidine residues would be needlessly sequenced, and the
molecular mass determination for which they would add unnecessary mass.
3.2.2 Optimal pH and molecular mass determination
59
The specific activity of purified MDH was determined at values ranging from
pH 5.5 to 11.5 for the oxidation and reduction reactions. Optimal activity was obtained
at pH 10 for both reactions (Figs. 2 and 3). MDH enzymatic activity became difficult
to calculate as the initial velocity slopes became very erratic below pH 7.5 and above
pH 11. The mass of denatured MDH was calculated from an SDS-PAGE and
determined to be 33 kDa relative to protein markers (data not shown), which is the
mass predicted from the translated nucleotide sequence (Dymov et al. 2004).
However, when MDH was eluted from a size exclusion chromatography column, the
mass was determined to be approximately 66 kDa (Fig. 4). This value is what would
be expected for a homodimer enzyme. Several other researchers have reported a
homodimer structure for MDH from a variety of other organisms including the
bacteria Haemophilus influenza (Yoon and Anderson 1988) and E. coli (Zaitseva et al.
2009), Archaea (Mevarech et al. 1977), algae (Miyatake et al. 1986), and fungi
(Teague and Henney 1976). Interestingly the only published data from another
member of the order rhizobiales shows that MDH from B. japonicum bacteroids has a
molecular mass of 138.6 kDa but with a subunit mass of only 36 ± 2 kDa indicating
that this enzyme functions as a homotetramer (Waters et al. 1986). In addition, MDH
from B. japonicum functions optimally at pH 8, several fold less than MDH from S.
meliloti.
3.2.3 Kinetic properties
The maximum substrate turnover rate (Vmax) and substrate affinity (MichaelisMenten constant Km) was determined at optimal pH 10; however, as the cytoplasm of
S. meliloti has been shown to be approximately pH 7.6 (O'Hara et al. 1989), assays
60
were also done at close to physiological pH values (Table 4). A slightly higher value
of pH 7.8 was opted for these experiments because of the analytical difficulties at
close to pH 7.5 mentioned in section 3.2.2. At pH 10, inhibitory effects of substrates
and cofactors were observed at concentrations greater than 40 mM for malate and 1.5
mM for NAD+ for the oxidative reaction and 3 mM OAA and 0.5 mM NADH for the
reductive reaction. At pH 7.8, inhibition occurred at concentrations greater than 15
mM malate and 1.5 mM NAD+ for the forward reaction and 0.15 mM OAA and 0.1
mM NADH for the reverse reaction. As would be expected, the Vmax and Km values are
higher when calculated from the optimal pH conditions versus the physiological pH
conditions.
The kinetic values obtained for the oxidation of malate by MDH at
physiological pH were compared to those obtained for DME (Driscoll and Finan
1997). The Michaelis-Menten constants (Km) of MDH for malate and NAD+ were 9fold and 2-fold higher than for DME, respectively. The maximum Vmax values for
MDH and NAD+ were 40-fold and 63-fold higher than for DME, respectively. The Km
value for MDH indicates the ability of this enzyme to bind malate at a much lower
concentration than DME can and the higher Vmax value means MDH oxidizes malate at
a faster rate than DME. Thus, in free-living S. meliloti, the flow of malate is
preferentially via MDH to OAA.
3.2.4 Product inhibition
Product inhibition studies using initial velocities can verify the enzyme kinetic
mechanism and, thus, establish the order of substrate binding and product release
61
(Cleland 1963a; Cleland 1963b; Cleland 1963c). Product inhibition assays were
conducted at pH 7.8 on both the oxidation of malate and reduction of OAA reactions
for MDH (Table 5).
In the oxidation of malate reaction, with NAD+ as the variable substrate and an
unvaried malate concentration, linear competitive inhibition was observed with
NADH (Fig. 5). With malate as the variable substrate and an unvaried NAD+
concentration, linear non-competitive inhibition was observed with NADH (Fig. 6).
With NAD+ as the variable substrate and an unvaried malate concentration, linear noncompetitive inhibition was observed with OAA (Fig. 7). With malate as the variable
substrate and an unvaried NAD+ concentration, linear non-competitive inhibition was
observed with OAA (Fig. 8). Replots of the slope and intercept values versus product
concentration for all four of the above reactions were linear (Figs. 5-8, inserts)
indicative of a simple reactive system.
Assays for the reduction of OAA reaction, with NADH as the variable
substrate and an unvaried OAA concentration, linear competitive inhibition was
observed with NAD+ (Fig. 9). With OAA as the variable substrate and an unvaried
NADH concentration, linear non-competitive inhibition was observed with NAD+
(Fig. 10). When NADH was the variable substrate and an unvaried OAA
concentration, linear non-competitive inhibition was observed with malate (Fig. 11).
With OAA as the variable substrate and an unvaried NADH concentration, linear noncompetitive inhibition was observed with malate (Fig. 12). Replots of the slope and
intercept values versus product concentration for all four of the above reactions were
linear (Figs. 9-12, inserts).
62
The above results are not consistent with either a Ping-Pong-type or random
mechanisms, if a rapid equilibrium condition did exist. A Theorell Chance mechanism
is not possible as two competitive inhibition results must be obtained in each reaction
direction, specifically in this case between malate and OAA and between NAD+ and
NADH. The product inhibition pattern observed, with only one competitive inhibition
result obtained in each reaction direction between NAD+ and NADH, and with all the
replot graphs being linear, is consistent with a steady-state ordered Bi Bi mechanism
(Leskovac et al. 2006). In this type of mechanism, the binding of substrates is ordered
on both sides of the reaction thus cofactor (A) binds the enzyme (E) first followed by
the substrate (B) and the product (P) is released before the cofactor (Q) and can be
represented by the schematic:
E ↔ EA ↔ EAB ↔ EPQ ↔ EQ ↔ E
This observed mechanism for MDH from S. meliloti is consistent with other
bacterial MDH studies published to date such as from E. coli (Heyde and Ainsworth
1968; Wright et al. 1995), Pseudomonas stutzeri (Labrou and Clonis 1997),
Haemophilus influenza (Yoon and Anderson 1988) and H. parasuis (Wise et al. 1997).
3.2.5 Inhibition analysis
The oxidative and the reductive reactions were tested at physiological pH 7.8
with citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, pyruvate, and maleic acid
as potential inhibitors. The only compound found to have any inhibitory effects on the
kinetic values of MDH was 2-oxoglutarate. The apparent Km value of MDH increased
but the Vmax remained unchaining in the presence of 2-oxoglutarate. This effect on the
63
binding constant but not on the maximum turnover rate is consistent with competitive
inhibition where the inhibitor reversibly binds the enzyme at its active site, thus
blocking it. Dixon plots confirmed competitive inhibition and gave an inhibition
constant Ki of 5 mM in the oxidative reaction and a Ki of 13 mM in the reductive
reaction (Figs. 13 and 14). Similar values have been reported for the MDH of the
archaebacterium Sulfolobus acidocaldarius; 5 mM of 2-oxoglutarate inhibited MDH
to 44% activity levels (Harti et al. 1987). In Cornybacterium glutamicum, 100 mM of
2-oxoglutarate decreased MDH activity 51% in the oxidation of malate reaction and to
30% in the reverse reaction (Genda et al. 2003).
The link between carbon and nitrogen metabolism is a close one and centered
around 2-oxoglutarate (for review see (Commichau et al. 2006)). Nitrogen
assimilation begins with the formation of glutamate and glutamine from the
condensation of 2-oxoglutarate and ammonium via the GS/GOGAT system or by
glutamate dehydrogenase. The nitrogen needed for the biosynthesis of other nitrogenbearing compounds is derived via secondary transfer from glutamate and glutamine.
When 2-oxoglutarate levels rise under N-deficient conditions, MDH will become
increasingly inhibited and malate can then be diverted out of the TCA cycle via DME
to pyruvate and/or acetyl-CoA which can then be converted into other compounds or
stored as poly-hydroxybutyrate. This regulation of MDH would then allow the cell to
avoid a build-up of citrate that has been shown to be lethal in S. meliloti (Koziol et al.
2009).
3.3 Conclusion
64
In this study, the physical properties of MDH from S. meliloti were
determined. MDH has a molecular mass of 66 kDa and functions as a homodimer
optimally at pH 10 in both oxidative and reductive directions. The reaction was shown
to have a steady state ordered Bi Bi mechanism. These physical attributes are similar
to other previously described MDHs.
The Michaelis-Menten kinetic properties for MDH, when compared to DME
values, showed that malate is more readily oxidized via MDH. In free-living cells, this
is understandable as acetyl-CoA is available via a number of other pathways and can
condense with OAA to form citrate making possible the efficient continuation of the
TCA cycle. For free-living cells, the biochemical properties of MDH do control the
flow of malate and the optimal turning of the TCA cycle. However, in bacteroids the
plant-provided carbon compounds all flow to malate. Based on the biochemical
properties of MDH, malate could preferentially flow in the oxidation direction to OAA
resulting in an imbalance between OAA and acetyl-CoA and an impediment to
efficient energy generation via the TCA cycle. To compensate for this, the control
mechanism can simply be based on inhibition. The insufficient supply of acetyl-CoA
will cause a slowing down of the TCA cycle and thus an increase in 2-oxoglutarate
and OAA concentrations which will inhibit MDH forcing the malate through DME.
Inhibition of DME also occurs by high concentrations of acetyl-CoA forcing malate
through MDH, thus, controlling and correcting for any imbalances in the cycle
(Driscoll and Finan 1997).
65
3.4 Figures and Tables
66
Table 3: Purification of malate dehydrogenase from S. meliloti by HPLC
Step
Volume
Protein
Specific activity
Total activity
Yield
Purification
(mL)
(mg)
(µmol·min-1·mg protein-1)
(µmol·min-1)
(%)
(fold)
50
1469
1.29
1895
100
1
20.3
371
1.78
660
35
1.38
Sephacryl S300 / Hi-Trap Blue
11
7.1
21.8
155
8
16.9
Resource Q
1
0.15
280
42
2
217
Crude extract
(NH4)2SO4 40-75%
67
Figure 2: The optimal pH curve for the oxidation of malate by MDH from S. meliloti
1200
1000
(nmol/min/mg protein)
Specific activity
800
600
400
200
0
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
11.5
pH
Figure 3: The optimal pH curve for the reduction of OAA by MDH from S. meliloti
900
800
700
(nmol/min/mg protein)
Specific activity
600
500
400
300
200
100
0
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
pH
68
Figure 4: Size exclusion chromatograph of the elution of MDH relative to
markers of know size
MDH was run through the column at pH 7.5. Markers: 1.35 kDa Vitamin B12; 17 kDa Equine
myoglobin; 44 kDa Ovalbumen; 158 kDa Bovine IgG; 670 kDa Thryglobulin.
69
Table 4: The Vmax and Km values calculated for both the oxidation of malate and the reduction of OAA reactions of malate
dehydrogenase at optimum and physiological pH
pH 10
pH 7.8
Vmax (nmol·min-1·mg protein-1)
Km (mM)
Vmax (nmol·min-1·mg protein-1)
Km (mM)
Malate
5.1 x 105 ± 3.8 x 104
9.1 ± 2.2
2.2 x 104 ± 1.7 x 103
0.96 ± 0.25
NAD+
5.5 x 105 ± 5.3 x 104
0.41 ± 0.13
3.0 x 104 ± 3.4 x 103
7.0 x 10-2 ± 1.9 x 10-2
Oxaloacetate
1.3 x 106 ± 1.2 x 105
0.53 ± 0.12
2.7 x 105 ± 5.2 x 103
2.8 x 10-2 ± 1.1 x 10-3
NADH
9.1 x 105 ± 1.1 x 105
7.2 x 10-2 ± 1.1 x 10-2
4.9 x 104 ± 2.0 x 103
1.3 x 10-2 ± 1.8 x 10-3
Variable substrate
Malate oxidation
Oxaloacetate reduction
Results are the average of triple assays ± standard error
70
Table 5: Experimental conditions and product inhibition patterns for the oxidation of malate and reduction of OAA reactions
of malate dehydrogenase and the apparent Kis
Product inhibitor (mM)
Varied substrate range (mM)
Constant substrate (mM)
Observed pattern
Apparent Kis (mM)
NADH 0.0042, 0.042
NAD+ 0.2 – 0.6
Malate 15
C
0.019 ± 0.003
NADH 0.0042, 0.042
Malate 0.5 - 15
NAD+ 0.5
NC
0.034 ± 0.007
OAA 0.01, 0.1
NAD+ 0.2 – 0.6
Malate 15
NC
0.018 ± 0.001
OAA 0.01, 0.1
Malate 0.5 - 15
NAD+ 0.5
NC
0.034 ± 0.006
NAD+ 0.1, 0.6
NADH 0.005 – 0.05
OAA 0.03
C
1.45 ± 0.34
NAD+ 0.1, 0.6
OAA 0.003 – 0.03
NADH 0.05
NC
0.18 ± 0.054
Malate 1, 15
NADH 0.005 – 0.05
OAA 0.03
NC
4.64 ± 0.80
Malate 1, 15
OAA 0.003 – 0.03
NADH 0.05
NC
3.50 ± 0.74
C = competitive inhibition
NC = noncompetitive inhibition
71
Figure 5: Product inhibition by NADH with malate constant and NAD+ varied
Reciprocal initial velocities are plotted vs reciprocal NAD+ concentrations at the NADH concentrations
indicated in the legend. Results are the average of triple assays ± standard error. The insert graph is the
replot of the slopes of the primary double-reciprocal plots.
72
Figure 6: Product inhibition by NADH with NAD+ constant and malate varied
Reciprocal initial velocities are plotted vs reciprocal malate concentrations at the NADH concentrations
indicated in the legend. Results are the average of triple assays ± standard error. The insert graph is the
replot of the slopes and the intercepts of the primary double-reciprocal plots.
73
Figure 7: Product inhibition by OAA with malate constant and NAD+ varied
Reciprocal initial velocities are plotted vs reciprocal NAD+ concentrations at the oxaloacetate
concentrations indicated in the legend. Results are the average of triple assays ± standard error. The
insert graph is the replot of the slopes and intercepts of the primary double-reciprocal plots.
74
Figure 8: Product inhibition by OAA with NAD+ constant and malate varied
Reciprocal initial velocities are plotted vs reciprocal malate concentrations at the oxaloacetate
concentrations indicated in the legend. Results are the average of triple assays ± standard error. The
insert graph is the replot of the slopes and the intercepts of the primary double-reciprocal plots.
75
Figure 9: Product inhibition by NAD+ with OAA constant and NADH varied
Reciprocal initial velocities are plotted vs reciprocal NADH concentrations at the NAD+ concentrations
indicated in the legend. Results are the average of triple assays ± standard error. The insert graph is the
replot of the slopes of the primary double-reciprocal plots.
76
Figure 10: Product inhibition by NAD+ with NADH constant and OAA varied
Reciprocal initial velocities are plotted vs reciprocal oxaloacetate concentrations at the NAD+
concentrations indicated in the legend. Results are the average of triple assays ± standard error. The
insert graph is the replot of the slopes and the intercepts of the primary double-reciprocal plots.
77
Figure 11: Product inhibition by malate with OAA constant and NADH varied
Reciprocal initial velocities are plotted vs reciprocal NADH concentrations at the malate concentrations
indicated in the legend. Results are the average of triple assays ± standard error. The insert graph is the
replot of the slopes and the intercepts of the primary double-reciprocal plots.
78
Figure 12: Product inhibition by malate with NADH constant and OAA varied
Reciprocal initial velocities are plotted vs reciprocal oxaloacetate concentrations at the malate
concentrations indicated in the legend. Results are the average of triple assays ± standard error. The
insert graph is the replot of the slopes and the intercepts of the primary double-reciprocal plots.
79
Figure 13: Dixon plot of the oxidation of malate reaction by MDH with malate at 4
different concentrations and 2-oxoglutarate as inhibitor at 0.1, 2, 5, and 10 mM
concentrations
Results are the average of triple assays ± standard error.
80
Figure 14: Dixon plot of the reduction of OAA reaction by MDH with OAA at 3
different concentrations and 2-oxoglutarate as inhibitor at 0, 5, 10 mM concentrations
Results are the average of triple assays ± standard error.
81
Connecting statement 1
It was hypothesized that the flow of malate through MDH and DME was
controlled by their biochemical properties. In free-living cells, this would seem to be
true; however, in bacteroids, the answer is not clear cut. The control of flow could be
simply due to product inhibition, the effect of OAA on MDH, or there could be
genetic elements involved. This leads to the hypothesis that the flow of malate through
MDH and DME is genetically controlled. To answer this a closer investigation of the
mdh promoter is needed.
82
Chapter 4: The genetic characterization of the mdh-sucCDAB operon
and promoter region
4.1 Introduction
The intergenic region between mdh and the upstream ORF SMc02478 is 183
nucleotides. This region is rich in A/T residues with numerous stretches of four or five
contiguous A and T nucleotides, particularly in the region immediately upstream of
the putative -35 RNAP binding site (Fig. 15). This region may correspond to the
RNAP subunit αCTD recognition upstream element (Ross et al. 1993). The promoter
regions of 99 S. meliloti non- σ54 controlled genes have been analyzed and a conserved
-35/-10 motif (5-CTTGAC-N -CTATAT-3’) seems to be common to all (MacLellan
17
et al. 2006). The -35 part of this motif is present, albeit in a slightly altered form, in
the promoter area of mdh in S. meliloti suggesting the use of this sigma factor in mdh
expression. The amino acid sequence of SigA is very similar to that of RpoD from E.
coli (Rushing and Long 1995); however, the conserved -35/-10 motif is shifted one
nucleotide perhaps explaining why some S. meliloti genes are poorly expressed in E.
coli (Bae et al. 1989; Dusha et al. 1986).
In R. leguminosarum, mdh is the first gene in an operon that also encodes the
two subunits of succinyl-CoA synthetase (sucCD) and two of the three subunits of 2oxoglutarate dehydrogenase (sucAB) (Walshaw et al. 1997). S. meliloti has the same
mdh-sucCDAB genetic arrangement and that mdh and sucC are co-transcribed (Dymov
et al. 2004). Scrutiny of the S. meliloti genome sequence reveals no promoter
consensus elements upstream of sucC and sucD and, thus, expression of these genes is
83
probably regulated from the mdh promoter. Poole et al. (1999) showed that strains of
R. leguminosarum carrying mutations in the suc genes have elevated expression from
the mdh promoter, which suggests that the mdh-sucCDAB operon is expressed as a
single mRNA from the mdh promoter.
In the preceding chapter it was shown how the biochemical properties of MDH
could probably control the flow of malate through MDH and DME. This chapter is to
investigate the hypothesis whether the flow of malate through MDH is genetically
controlled.
4.2 Results and discussion
4.2.1 N-terminal sequencing
The nucleotide sequence of the region upstream of mdh reveals a ShineDelgarno consensus sequence (Shine and Dalgarno 1974) immediately upstream of the
putative ATG translational start site (Fig. 15). To confirm that the translational start
site was indeed a methionine residue as predicted from the nucleotide sequence, as
well as to verify that no post-translational modifications have occurred, purified MDH
was N-terminally sequenced. The results are in agreement with the predicted
translated nucleotide sequence (M-A-R-K-I-A-L-I-G-S-G-M-I-G…….) and verify the
translational start site is an ATG codon with no further post-translational modification
on this end of MDH. This stretch of amino acids is identical to all rhizobia MDHs
submitted to the GenBank data base.
4.2.2 Transcriptional start site (TSS) determination
84
To determine the TSS of mdh, total RNA was harvested from S. meliloti cells
that had been grown in LBmc. The resulting PCR product from the 5’-RACE reaction
was sequenced and revealed that the TSS is a guanine nucleotide located 64 bp
upstream of the ATG translational start site of mdh (Fig. 15).
4.2.3 Determination of a functional promoter for mdh
To ascertain whether the area immediately upstream of mdh is the functional
promoter for the mdh-sucCDAB operon, a transcriptional gfp-gene fusion was made. A
500 nt fragment comprising approximate 300 nt upstream of mdh and 200 nt of mdh
was cloned into vector pOT1 in the forward orientation resulting in plasmid pDM55.
As a control, the same fragment was ligated in the reverse orientation relative to gfp
resulting in plasmid pDM63. Plasmids were mobilized into the Rm1021 background
by triparental mating resulting in strains Rm30233 and Rm30271. Cultures were
grown in LBmc and M9-minimal media containing arabinose, acetate, glucose
glutamate, malate, pyruvate, succinate or GABA as sole-carbon sources and the
fluorescence measured. Strain Rm30233 had fluorescence units significantly (p<0.05)
higher than those obtained for strain Rm30271 in all test conditions indicating the
promoter for mdh is immediately upstream of the operon (Fig. 16).
This experiment also included S. meliloti strains carrying plasmids that had a
gfp-gene fusion to sucA to determine if there was a functioning promoter upstream of
it. In this case, a 600 nt fragment comprising the 281 nt intergenic region between
sucD and sucA and approximately 300 nt of sucA were ligated into vector pOT1 in
both the forward and reverse orientation relative to gfp resulting in plasmids pDM56
85
and pDM64 respectively. Transformed S. meliloti strains Rm30234 and Rm30272
were grown under the same conditions as above.
No difference in RFUs between strains Rm30234 and Rm30272 was observed
when grown on any of the sole carbon sources. When grown in LB liquid media, there
was a significant (p<0.05) RFU elevation for strain Rm30234 relative to strain
Rm30272 (Fig. 16). Whether this indicated that sucA has a functioning promoter under
this condition needs further testing. R. leguminosarum, like S. meliloti, expresses the
mdh-sucCDAB operon from a promoter upstream of mdh (Walshaw et al. 1997). This
differs with what was found for B. japonicum for which evidence suggests sucA
expression is initiated from its own upstream promoter (Green et al. 2003). However,
there is still the possibility that once S. meliloti has differentiated into bacteroids, the
putative sucA promoter may be functional in planta. Unfortunately to answer this, new
plasmids would have to be constructed as the plasmids made in this experiment may
be lost without selective antibiotic pressure in planta and is beyond the scope of this
thesis; however, our lab is presently analyzing the transcriptome of S. meliloti, both
free-living and bacteroids, which may help answer this question in the future.
4.2.4 The role of σ54 in regulation of mdh
The sigma factor primarily responsible for the regulation of C4-dicarboxylic
acid transport and nitrogen fixation genes in S. meliloti is σ54 (Ronson et al. 1987). To
test if this transcription factor plays a role in the regulation of mdh, plasmid pSI33,
which carries mdh with a Tn5-B20 insertion, was introduced into ntrA mutant strain
Rm5422 (Finan et al. 1988). Results showed that σ54 played no role in transcription
86
regulation as there was no observable difference in lacZ expression when compared to
Rm1021 in cells that had been grown in M9 media containing either glucose or
arabinose as the sole-carbon source (Table 6).
4.2.5 RT-PCR
Reverse transcriptase-PCR (RT-PCR) was used to determine if the genes in
mdh-sucCDAB were cotranscribed as one polycistronic message in cells that have
been grown in LBmc. The positive control primer set successfully amplified a region
complementary to a region within the sucA encoding sequence (1201 bp, lane 4).
Amplification of the mdh to sucC intergenic region resulted in a fragment of 1221 bp
(lane 5), the sucC to sucD intergenic fragment was 702 bp (lane 6), and the sucD to
sucA intergenic fragment was 1165 bp (lane 7) (Fig. 17). Several attempts were made
to amplify the sucA to sucB intergenic region using many different primer
combinations but with no better results than those shown in Figure 17 (lane 8).
Analysis of the genome sequence in the sucA-sucB intergenic region and sucB
revealed the presence of a palindromic RIME element (Østerås et al. 1995), which
may contribute to the difficulties in amplifying this region also encountered with PCR
from genomic DNA.
In S. meliloti, the mdh-sucCDAB genes are expressed as an operon (Fig. 17).
This has also been shown to be the case for R. leguminosarum (Poole et al. 1999;
Walshaw et al. 1997).
In B. japonicum, mdh is transcribed as a monocistronic
message, and the sucAB genes are transcribed from a promoter upstream of sucA in an
operon that also includes the genes scdA and lpdA (Green et al. 2003). It is uncertain
87
where transcription of sucCD initiates in B. japonicum. The uncoupling of mdh
transcription in B. japonicum may reflect a key difference in the TCA cycle regulation
in this organism. In R. leguminosarum, it has been hypothesized that co-transcription
of the mdh and suc genes allows coordinate expression of the TCA cycle, and that
pools of amino acids, especially glutamate, which are by-products of the TCA cycle,
are important for its regulation (Poole et al. 1999; Walshaw et al. 1997). A model for
amino acid cycling in the R. leguminosarum-pea symbiosis has been proposed
(Lodwig et al. 2003) and their findings are highly relevant to the study of S. meliloti
physiology as well.
4.2.6 Transcriptional lacZ gene fusion assays
To get a better picture of the regulation of the mdh-sucCDAB operon than
could be obtained from analysis of enzyme assay data alone, Dymov et al. (2004)
preformed transposon Tn5-B20 mutagenesis of plasmid pDS15, which carries the
entire operon. Tn5-B20 itself carries a promoterless lacZ reporter gene and can
generate transcriptional lacZ fusions to genes into which it is inserted if in the correct
forward orientation (Simon et al. 1989). As shown in the previous section, he genes in
the mdh-sucCDAB operon are co-transcribed, therefore, there is a possibility that
intergenic regions within the operon could affect gene expression, and indeed the
sucD-sucA intergenic region is large enough to possibly encode a promoter (Dymov et
al. 2004), so the key genes mdh, sucD, and sucA were targeted (Fig. 18). Tn5-B20
insertions yielding lacZ gene fusions were isolated in mdh, sucD, and sucA and also
reversed in sucA to be used as a control. Isolation of strains in which the insertions
had been recombined into the genome, generating TCA cycle mutant strains, was
88
attempted but such strains could not be recovered (Dymov et al. 2004). Thus, the
plasmid-borne fusions were conjugated into the Lac- strain RmG212 and assayed for
expression of β-galactosidase activity under different sole carbon-source conditions.
The mdh operon is under catabolic control, as evidenced by the approximately
4-fold differences in expression depending on the carbon source provided for growth
(Fig. 19). The mdh-sucCDAB operon showed highest expression on acetate followed
closely by arabinose and glutamate.
The metabolism of acetate involves the
conversion of acetate to acetyl-CoA, which is then condensed with oxaloacetate (the
product of MDH) to form citrate.
In addition, biosynthesis from TCA cycle
intermediates flow through OGD and SCS, to form precursors for amino acid and
nucleotide synthesis. In this respect, MDH, SCS and OGD activities need to be
maintained to allow for continuous cycling of the TCA cycle and to allow for
biosynthesis of large molecules from a two-carbon precursor. In E. coli, metabolism
of acetate requires the glyoxylate pathway, and while the activities of the glyoxylate
enzymes isocitrate lyase and malate synthase can be detected in free-living cells of S.
meliloti, B. japonicum and R. leguminosarum, it was shown that R. leguminosarum
encodes malate synthase G, not malate synthase A of the glyoxylate cycle (Duncan
and Fraenkel 1979; García-de los Santos et al. 2002; Green et al. 1998). Analysis of
the S. meliloti genome indicated the presence of a gene for malate synthase G (glcB)
but not for a malate synthase A homologue (aceB). Clearly this pathway needs more
attention in rhizobia as it does not necessarily follow the E. coli model.
When arabinose or glutamate is the sole-carbon source, the expression of sucA
was significantly (p<0.05) higher than sucD. These results contradict those obtained
89
using the transcriptional gfp-gene fusions where sucA expression was found to be at
the same levels as those of the negative controls (except possibly when grown in LB).
Arabinose, via a multi-enzymatic pathway, and glutamate, via glutamate
dehydrogenase, are catabolized to 2-oxoglutarate which can directly enter the TCA
cycle (Fig. 1). It would be under these growth conditions that one would expect a
higher need of OGD and so a functional promoter upstream of sucA could be
upregulated to meet that need.
4.3 Conclusions
The ATG translational start codon, as predicted from the genome sequencing
project, was confirmed by N-terminal sequencing of MDH. The transcriptional start
site was found to be a guanine residue located 64 nt upstream of the ATG codon and
the region immediately upstream of mdh was shown to be the functional promoter for
mdh with expression of the mdh-sucCDAB operon as one polycistronic message in
LBmc-grown cells. An examination of the S. meliloti genome revealed a 281
nucleotide intergenic region immediately upstream of sucA large enough for a
promoter. Transcriptional lacZ-gene fusions suggested the possibility of a second
conditional promoter upstream of sucA as expression of sucA was higher than for sucD
in arabinose and glutamate grown cells. Opposed to this, promoter fusions to gfp did
not show an elevation of expression in sucA relative to control samples in cells grown
with arabinose or glutamate. Recently, RNAseq was used to map all TSS in the S.
meliloti genome and the authors determined that sucA has a TSS 73 nt upstream of the
ATG translational start site (Schluter et al. 2013); however, as this was only a
90
genome-wide survey they did not verify each TSS using 5’-RACE or other molecular
techniques to determine if the putative TSS were functional or not. The TSS global
survey confirmed the results of the 5’-RACE that the TSS for mdh was indeed a
guanine residue at the -64 position. Currently, we are carrying out transcriptomic
analysis of RNA harvested from free-living S. meliloti cultures grown with different
carbon sources and from bacteroids which should answer whether sucA does have a
functional promoter, and if so, under what conditions is it operative.
The purpose in this section was to answer the hypothesis that the flow of
malate through MDH was controlled genetically. Presently a complete picture is not
conclusive. To further our studies of the genetic regulation of mdh, two approaches are
being taken. Preliminary studies in our lab using qRT-PCR showed constitutive MDH
expression under all test conditions. Expression was higher, but not significantly, with
acetate, arabinose or succinate as the sole-carbon source but it was not significant and
may be due to the fact cells are growing faster under these growth conditions. To
further analyze the promoter region, twenty-two plasmids were constructed each
carrying a different fragment length of the mdh promoter region that had been ligated
into the MCS of plasmid pOT1 (Allaway et al. 2001) thereby creating a transcriptional
gene fusion to gfp (Fig. 15). Three of the 22 plasmids are controls, one containing the
complete mdh promoter region, a second containing a fragment where the 5’ end is 4
nucleotides downstream from the guanine TSS and the third containing a fragment
with the ‘5 fragment end is 10 nucleotides upstream from the putative Shine-Delgarno
binding site. The remaining 19 plasmids each carry a promoter fragment length
varying from nucleotide -85 to -3, relative to the TSS, and each approximately 5
91
nucleotides shorter than the preceding one. Presently, these plasmids are being
mobilized into the S. meliloti background where the individual strains will be grown in
M9-minimal media with different sole-carbon sources and also used to inoculate
alfalfa plants. Cells and bacteroids will be harvested, fluorescence measured and
differences in fluorescence from separate plasmids may indicate a variation in the
RNAP holoenzyme binding site.
92
4.4 Figures and Tables
93
Figure 15: The nucleotide sequence of the promoter region of mdh.
Translational start site green, Shine-Delgarno consensus sequence blue, transcriptional start site red, putative -10 -35 RNA polymerase binding site underlined, *
indicate 5’ end of PCR fragments for RNA holoenzyme polymerase binding site assays.
*
*
*
*
*
CCCGCAAAATTCAGCGGCGGAATGACGGTCCGGCGACATCAGTTCTTACGTTTACGTAAGAATTTTACGATCTAAC
-100
-90
-80
-70
*
*
* *
*
*
*
*
*
*
*
* *
*
*
*
CGATTGAAATTGCTCTGCCAAAAAGTATCGATTGATATTTTTCCCTTTGCCGTTTAAGGGCTTTCGCGAAGGTTTGG
-60
-50
-40
-30
-20
-10
*
CGTTTGCCGCGTTTCAGGCCTTGATCATTTTGGGATCACAAAGGAAGCACTTTCATGGCGCGCA
+10
+20
+30
+40
+50
+60
+70
94
Figure 16: Fluorescence of S. meliloti cells containing mdh promoter and putative
sucA promoter transcriptional gfp-gene fusion plasmids
Results are the average of triple assays ± 95% confidence interval. Letters (a, b, c) represent ANOVA
results where treatments with different letters were found to be significantly different (p < 0.05).
95
Table 6: Expression of mdh in an rpoN mutant of S. meliloti.
Expression of ß-galactosidase in an rpoN mutant relative to wild-type from cells
grown in M9 minimal media with either glucose or arabinose as the sole-carbon
source.
C-source
Glucose
Arabinose
ß-galactosidase activity*
Rm1021 pSI33
Rm5422 pSI33
42.6 ± 3.3
36.8 ± 1.2
68.5 ± 1.8
71.6 ± 2.5
* Values represent the mean of triplicate measurements (Miller Units) ± standard error
96
Figure 17: RT-PCR of the mdh-sucCDAB operon.
Primer pairs span the five intergenic regions between the four genes.Total mRNA was extracted from
LBmc-grown cells. Lanes 1 and 9, 100 bp ladder; lanes 2, control A (no mRNA); lane 3, control B (no
RT); lane 4, control C (primer pair in sucA only); lane 5, mdh-sucC; lane 6, sucC-sucD; lane 7, sucDsucA; lane 8, sucA-sucB
1
2
3
4
5
6
7
8
9
97
Figure 18: The genetic arrangement of the mdh-sucCDAB operon showing transposon Tn5-B20 insertion sites and orientation.
Black arrow heads indicate orientation of the lacZ-gene fusion.
98
Figure 19: The effect of carbon source on gene expression from plasmid-borne lacZ
fusions
The strains used in this study were: RmG212 (Lac-), Rm30096 (mdh383::Tn5B20), Rm30107
(sucA162::Tn5B20 reversed), Rm30110 (sucD111::Tn5B20), Rm30108 (sucA1479::Tn5B20). Values
are the combined means of triplicate assays from two replicate experiments ± 95% confidence interval.
Letters (a, b, c) represent ANOVA results where treatments with different letters were found to be
significantly different (p < 0.05).
99
Connecting statement 2
The focus now turns to the second part of this thesis and the role of multiple
alleles encoding for specific subunits of enzymes of the TCA cycle. The annotated
genome of S. meliloti carries two alleles encoding for the second subunit (E2) of
OGD. The hypothesis is the functional dihydrolipoyl succinyltransferase (E2) subunit
of 2-oxoglutarate dehydrogenase is encoded by SMc02483 (aka sucB) not SMb20019
100
Chapter 5: The characterization of two putative sucB alleles encoding
for the dihydrolipoyl succinyltransferase (E2) component of the 2oxoglutatrate dehydrogenase complex
5.1 Introduction
The holoenzyme 2-oxoglutarate dehydrogenase (OGD), that catalyzes the
conversion of 2-oxoglutarate to succinyl-CoA, is composed of multiple copies of each
of three enzymes; oxoglutarate dehydrogenase (E1) encoded by sucA, dihydrolipoyl
succinyltransferase (E2) encoded by sucB and dihydrolipoyl dehydrogenase (E3)
encoded by lpdA. The oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA is
a complex multistep reaction. In the first step, 2-oxoglutarate is decarboxylated in a
reaction with the E1 subunit-bound cofactor thiamine pyrophosphate (TPP) resulting
in a hydroxysuccinyl derivative. Two electrons from an oxidized E2-bound
lipoyllysine group are exchanged for the succinyl group from TPP forming a succinyl
thioester with the reduced lipoyl group. Transesterification with coenzyme A results in
a free succinyl-CoA molecule and a fully reduced dithiol form of the lipoyl group. The
lipoyllysine group is reoxidized by transfer of electrons from E3-bound FADH2
which, in turn, is reoxidized by transfer of electrons from NAD+. The overall reaction
is:
2-oxoglutarate + CoA-SH + NAD+
→
succinyl-CoA + CO2 + NADH
In S. meliloti, the putative genes for the two subunits E1 (sucA) and E2 (sucB,
SMc02483) are the final two genes in the mdh-sucCDAB operon (Dymov et al. 2004).
The upstream genes of this operon encode for the TCA cycle enzymes malate
101
dehydrogenase (mdh) and the two subunits of succinyl-CoA synthetase (sucC and
sucD). The S. meliloti genome sequence also has a gene SMb20019 on megaplasmid
pSymB annotated as putatively encoding a sucB allele (Capela et al. 2001). SMb20019
appears to be the second of a two gene operon. The upstream gene of this operon is
annotated as a putative second pdhA allele encoding for the E1 subunit of PDH. OGD
and PDH are closely related holoenzymes with similar multimeric subunit structure,
coenzymes and cofactors.
To determine whether the gene product of SMc02483, SMb20019 or both are
involved in OGD functionality, a mutation was created in each, in separate strains
(Rm30230 and Rm30265, respectively). The disrupted gene from one was then
transduced into the other resulting in a double mutant (Rm30275). All three strains
were tested for carbon-source growth phenotype and plant symbiotic phenotype, in
addition to enzyme assays. In this thesis, the allele SMc02483 is referred to as sucB
while the megaplasmid-borne allele remains SMb20019.
5.2 Results and discussion
5.2.1 Homology comparison between SMc02483 and SMb20019
Sequences were retrieved from the S. meliloti genome project web site. The
nucleotide sequence of sucB was 1251 bases long whereas SMb20019 had a length of
1137 bases, a difference of 114 nucleotides. The nucleotide homology to each other
was 46% and the predicted amino acid sequence homology was 31% (Table 7). The
nucleotide and amino acid sequences of sucB from other organisms of the Order
Rhizobiales were retrieved from the NCBI GenBank and compared to those of sucB
102
and SMb20019 (Table 7). Comparing strains related by Order, the percent nucleotide
homology was between 74-77% between chromosomal borne alleles. Homology was
even higher (82-90%) between strains from the same Family. Amino acid homology
runs from 70-77% of strains from the same Order and 84-96% of strains from the
same Family. Interestingly, the two megaplasmid-borne alleles of sucB from S.
meliloti and Rhizobium sp. NGR234 were more homologous to each other than to each
respective chromosomal allele.
5.2.2 Construction of S. meliloti strains Rm30230, Rm30267 and Rm30275
Suicide vector pVIK112 (Kalogeraki and Winans 1997) carrying an internal
PCR fragment of sucB was isolated (plasmid pTR7). Triparental mating mobilized
pTR7 into S. meliloti strain RmG212 where a single recombination event resulted in a
strain with a transcriptional lacZ-fusion to, and gene knock-out of, sucB designated
Rm30230. A previously published mini-Tn5 transposon insert in SMb20019 in S.
meliloti strain Rm2011 was obtained (Pobigaylo et al. 2006). It was preferable to have
the transposon in the same strain background as Rm30230 so the marker was
transduced into RmG212 resulting in strain Rm30267. Plasmid pTR7 and the miniTn5 transposon both carry kanamycin/neomycin resistance markers, thus, it was
necessary to change the resistance marker of pTR7 to trimethoprim to select for a
double mutant by using Epicenter’s EZ-Tn5 kit. This resulted in plasmid pTR10.
Triparental mating introduced pTR10 into Rm30267 resulting in double mutant strain
Rm30275. Mutation of all target genes were confirmed in all new constructs via PCR
and Southern blot (data not shown).
103
5.2.3 Enzyme assays
In S. meliloti, arabinose enters the TCA cycle at 2-oxoglutarate; therefore, it
was hypothesized that a strain with a non-functioning OGD should have difficulty
growing with that particular compound as a carbon source. We had previously found
that OGD assays of crude cell extracts from cells grown in LB had a very high level of
background activity in control samples containing no 2-oxglutarate substrate. This
was, perhaps, due to the presence of non-specific dehydrogenases (Charles et al. 1990).
To minimize the background activity, enzyme assays were performed on sonicated
crude cell extracts from M9-succinate grown cells. Isocitrate dehydrogenase (ICD), an
enzyme of the TCA cycle, was chosen as control because its encoding gene icd is not
part of the mdh-sucCDAB operon. ICD activity was the same for all tested samples.
Both Rm30230 and the double mutant Rm30275 had greatly diminished OGD
activity while MDH and SCS activities were elevated 4 fold relative to Rm1021
(Table 8). This observation is consistent with what has been observed in R.
leguminosarum suc mutants (Poole et al. 1999; Walshaw et al. 1997), which have an
identically structured mdh-sucCDAB operon as S. meliloti, as well as in a chemicallyinduced S. meliloti OGD mutant (Duncan and Fraenkel 1979). In the latter mutant,
MDH activity increased 3.7-fold and succinyl-CoA synthetase (expressed from
sucCD) increased 4.7-fold, compared to the wild-type strain. The higher activity of
MDH might be a response to the increased levels of 2-oxoglutarate, or to some other
related compound or metabolite, that accumulates in OGD blocked cells (Walshaw et
al. 1997) in an attempt to remove the blockage by increasing the transcription of the
OGD genes. As a consequence mdh would be upregulated because it is cotranscribed
104
from the same promoter as the sucAB genes in both R. leguminosarum and S. meliloti.
However, this was not observed in B. japonicum (Green and Emerich 1997a) which
expresses monocistronic mdh from a separate promoter (Green et al. 2003).
All enzyme activities in strain Rm30267 were similar to Rm1021 (Table 8).
Plasmid pDS15 complemented strains Rm30230 and Rm30275 and restored all lost
OGD activity. All strains carrying this plasmid also had increased MDH and SCS
activities relative to Rm1021 due to the multiple copies of mdh and sucCD. However,
this plasmid does not carry a complete lpdA2 gene, which encodes for the third subunit
of OGD; therefore, when complementing Rm30230 and Rm30275 enzyme activities
are only restored to wild-type levels. Complemented Rm30267, with multiple copies
of sucAB but only one allele of lpdA2, had only slightly elevated OGD activity.
5.2.4 Plant assays, symbiotic phenotype and microscopy
Mutant strains Rm30230, Rm30267, and Rm30275 plus wild-type Rm1021
were tested on alfalfa (Medicago sativa) plants for their symbiotic phenotype. Plants
inoculated with Rm30230 and Rm30275 were stunted and chlorotic (Fig. 20), had
small white root nodules and showed shoot dry-weights comparable to that of the
uninoculated plants (Table 9) indicating an inability to fix nitrogen (Fix-). Plants
inoculated with strain Rm30267 and wild-type Rm1021 were lush green, had large
pink root nodules and comparable shoot dry-weights (Fix+).
Roots and nodules of plants from each treatment were collected, thin sliced,
stained with the fluorescent nucleic acid binding dye Syto-13 and photographed
microscopically (Fig. 21). Plants treated with the wild-type or Rm30267 strains had
105
fully mature infection threads loaded with bacteria and nodules fully occupied with
bacteroids. However, plants inoculated with strains Rm30230 and Rm30275 had some
incomplete infection threads, and if complete, the nodules were not fully occupied.
The same phenomena was observed with plants inoculated with B. japonicum sucA
mutants where nodules from plants that had been inoculated with the mutants
contained significantly fewer bacteroids than nodules from plants inoculated with
wild-type strains (Green and Emerich 1997b).
5.3 Conclusions
The two genes annotated as sucB by the S. meliloti genome sequencing group
shared less than 50% nucleotide and amino acid homology. The homology between
sucB and sucB from other rhizobial species is very high, especially when compared to
sucB from organisms of the same family.
The previously reported S. meliloti OGD mutant displays a Fix- phenotype
(Duncan and Fraenkel 1979) as do R. leguminosarum OGD mutants (Walshaw et al.
1997). These reported mutants have disruptions in their sucA gene encoding the E1
subunit of OGD. In contrast to this a B. japonicum sucA mutant was described as
having a Fix+ phenotype (Green and Emerich 1997a; Green and Emerich 1997b). This
was likely due to a metabolic bypass around OGD via the GABA shunt. While the
plants were labeled as Fix+, they appeared yellow and smaller and the number of
nodules per plant were fewer than from plants inoculated with wild-type B. japonicum.
Acetylene reduction assay results showed a marked decrease in nitrogenase activity in
the plants inoculated with the mutant strain. In terms of a GABA shunt, enzyme assays
106
showed that B. japonicum do not have any glutamate decarboxylase (Ec4.1.1.15)
activity. This enzyme, which converts glutamate to GABA, is the only metabolic
source for GABA and the the major precursor of the GABA shunt (Fig. 1), without
which the bypass is inoperable. In addition, no homologues of known glutamate
decarboxylase can be found by searching the B. japonicum genome sequence database.
Taken together, it seems that an argument for a GABA shunt bypass leading to
functioning bacteroids in ODG deficient cells may be flawed.
The reason given for the Fix+ phenotype was that after 32 days post inoculation
nitrogenase activity approached wild-type levels. The sucA mutation in the B.
japonicum strain was created by homologous recombination into the genome of a
plasmid carrying a fragment of sucA that has a transposon insertion in it. The nature of
the plasmid integration into the chromosome, which was not detailed in the article,
could occur in two ways. If the insertion was caused by a double homologous
recombination event, the loss of plasmid would result in a stable insert; however, if
there was only a single cross-over event, the entire plasmid would incorporate into the
genome. Both events would instill a sucA- genotype in free-living cells grown with
antibiotics selecting for maintenance of the transposon resistance marker. However,
with loss of the antibiotic selective pressure in planta, the plasmid could recombine
out of the genome and be lost, resulting in reversion to a wild-type phenotype. This
would take time and several generations to manifest itself in slow-growing bacteria
such as B. japonicum. If plasmid loss did occur, the accumulation of revertants could
occur within 32 days.
107
S. meliloti and R. leguminosarum seem to be lacking glutamate decarboxylase
as no homologue can be found in their respective annotated genome (Capela et al.
2001; Young et al. 2006). Any disruption of sucA or sucB would render the cells
incapable of bypassing the blockage at OGD leading to nodules poorly occupied with
Fix- bacteroids indicating the importance of a functioning OGD for proper nodule
invasion and nitrogen fixation.
Disruption of sucB eliminated most OGD activity in the single mutant,
Rm30230, as well as the double mutant. Deletion of SMb20019 had no effect on OGD
activity. Plants inoculated with Rm30230 and Rm30275 were nitrogen-deficient (Fix-)
and as such, grew poorly; whereas the SMb20019 mutant Rm30267 grew to wild-type
proportions. These findings clearly show that the chromosomal allele SMc02483, and
not SMb20019, is the gene that encodes for the functional E2 subunit of OGD, thus,
supporting the original hypothesis.
108
5.4 Figures and Tables
109
Table 7: The percent homology (nucleotide/amino acid) between the two S. meliloti alleles SMc02483 and SMb20019 with sucB from
other related rhizobia.
Microorganism
Taxonomic
Relatedness
To Rm1021
Strain
Sinorhizobium meliloti
Rhizobium etli
Rhizobium leguminosarum
Rhizobium sp.
1021
CFN42
bv. viciae 3841
NGR234*
Bradyrhizobium japonicum
Mesorhizobium loti
USDA110
MAFF303099
SMc02483
Chromosomal
Megaplasmid pNGR234b
Family
Family
Family
Order
Order
Percent homology (nt/aa)
sucB
SMb20019
100/100
82/86
82/84
90/93
48/31
74/70
77/77
46/31
45/30
45/30
48/31
73/72
46/33
45/30
*Rhizobium sp. NGR234 has two alleles annotated sucB, one chromosomal and one on megaplasmid pNGR234b
110
Table 8: The enzyme specific activities of S. meliloti cells harvested from M9-succinate grown media.
Strain
Characteristics
Rm1021
Rm30230
Rm30344
Rm30267
Rm30345
Rm30275
Rm30346
Wild-type
sucB
Rm30230 pDS15
SMb20019
Rm30267 pDS15
Double mutant
Rm30275 pDS15
2-oxoglutarate
dehydrogenase
48.4 ± 4.5
0.9 ± 0.2
33.7 ± 3.8
66.0 ± 4.8
74.5 ± 10.8
0.9 ± 0.4
35.1 ± 5.3
Specific activity*
Malate
Succinyl-CoA
dehydrogenase
synthetase
1226 ± 35
120 ± 1
5167 ± 556
470 ± 14
10049 ± 681
709 ± 72
2504 ± 197
205 ± 17
3661 ± 192
331 ± 14
2895 ± 99
346 ± 9
13607 ± 996
766 ± 23
* Specific activity (nmol·min-1·mg protein-1) expressed as the mean of triplicate assays ± standard error
Isocitrate
dehydrogenase
343 ± 13
408 ± 9
360 ± 18
432 ± 17
332 ± 13
247 ± 7
385 ± 11
111
Figure 20: Medicago sativa plants that had been inoculated* with S. meliloti wild-type and sucB mutant strains.
* = Pictures were taken 28 day post inoculation
112
Table 9: The shoot dry-weight (SDW) of Medicago sativa (alfalfa) inoculated with S.
meliloti mutant strains.
Inoculating
strain
Characteristic
SDW*
% of wildtype
Symbiotic
phenotype
-
8.1 ± 1.3
15
-
Rm1021
Wild-type
54.0 ± 5.5
100
Nod+ Fix+
Rm30230
sucB-
6.9 ± 0.4
13
Nod+ Fix-
Rm30267
SMb20019-
51.6 ± 8.8
96
Nod+ Fix+
Rm30275
sucB-/SMb20019-
9.8 ± 1.2
18
Nod+ Fix-
Uninoculated
* = SDW expressed in milligrams per plant as mean of triplicate samples ± standard error. Each sample
consisted of 8 to 12 shoots (29 to 31 total) harvested 28 days post-inoculation.
113
Figure 21: Microscopic cross sections of nodules containing bacteroids of strains
RmG212, Rm30230 (sucB mutant), Rm30267 (SMb20019 mutant) and Rm30275
(double mutant) stained with nucleic acid binding dye syto-13.
RmG212
Rm30230
Rm30267
Rm30275
114
Connecting statement 3
Three genes are annotated in the S. meliloti genome sequence as
dihydrolipoamide dehydrogenase (lpdA1, lpdA2 and lpdA3). This protein is the third
of three subunits of OGD and also of PDH and BKD. The lpdA1 and lpdA2 alleles lie
a couple of ORFs downstream of operons encoding the E1 and E2 subunits of PDH
and OGD respectively, whereas lpdA3 is part of the BKD operon. The hypothesis is
that each respective LpdA gene product forms part of the dehydrogenase complex that
the allele is found in proximity to and are not interchangeable.
115
Chapter 6: The role of three dihydrolipoamide dehydrogenases
(LpdA) as functional subunits of pyruvate dehydrogenase, 2oxoglutarate dehydrogenase and branched-chain alpha-ketoacid
dehydrogenase
6.1 Introduction
The multimeric enzymes pyruvate dehydrogenase (PDH), 2-oxoglutarate
dehydrogenase (OGD) and branched-chain α-ketoacid dehydrogenase (BKD), are all
similar in that they are composed of three subunits, and each corresponding subunit
performs the same basic function, as detailed in Chapter 5. In E. coli the core of the
PDH complex, to which the E1 and E3 subunits are noncovalently attached, comprises
twenty-four E2 subunits. To these are bound 12 copies of two identical subunits of E1,
and 6 copies of two identical subunits of E3 (Eley et al. 1972). This large molecular
complex is approximately 45 nm in diameter making it visible under an electron
microscope.
Only one gene encodes for the E3 subunit dihydrolipoamide dehydrogenase
(lpdA) in E. coli (Guest et al. 1981). Located immediately downstream of the genes
encoding for the E1 (aceE) and E2 (aceF) subunits of the PDH complex, lpdA can be
expressed with aceEF as one polycistronic message from a promoter (Pace) upstream
of aceE or as a single transcript from its own promoter (Plpd). Expression via two
promoters explains how a single lpdA gene product can be used in more than one
enzyme complex. Having one gene for the E3 subunit of multiple enzymes appears to
be quite common in the published literature with examples including the bacteria
Azotobacter vinelandii (Westphal and de Kok 1988) and Bacillus subtilis (Lowe et al.
116
1983), the fungi Saccharomyces cerevisiae (Dickinson et al. 1986), and Homo sapiens
(Otulakowski et al. 1988), all of which have one lpdA allele expressed from its own
promoter.
Members of the order Rhizobiales seem to be an exception; many species of
this order have multiple copies of lpdA. S. meliloti has three lpdA alleles in its genome
(Capela et al. 2001) with each gene found in close proximity to, or as part of, an
operon with the other subunits of PDH, OGD, or BKD (Fig. 22). The locus for lpdA1
is two ORFs downstream of an operon encoding for the E1 and E2 subunits of PDH
(pdhABC) while lpdA2 is three ORFs downstream of the operon for the other subunits
of OGD (sucAB). The operon for the E1 and E2 subunits of BKD includes, and
terminates with lpdA3. The hypothesis is that each lpdA gene product is specific to the
complex to which it is in proximity and are not interchangeable.
The generation of S. meliloti lpdA mutant strains are discussed in the results
section. Subsequent to their generation, growth curves and ß-galactosidase gene fusion
assays were performed (Babic 2010) and, in turn, offer support to the conclusion of
our hypothesis. The growth rate constants (k) were calculated, relative to wild-type,
when cells were grown in M9-media with arabinose, glutamate, glucose, leucine,
malate, pyruvate or succinate as the sole-carbon source. Most strains grew at a similar
rate to wild-type, exceptions included the lpdA1 mutant strain Rm30282 which could
not grow with pyruvate as sole-carbon source and had a decreased k with succinate,
and malate as the sole-carbon source. Also, the lpdA2 mutant strain Rm30309 showed
a k value of 29 and 34% of wild-type, when grown with arabinose or glutamate as the
sole-carbon source, respectively. Similarly, the lpdA3 mutant strain Rm30232 had a
117
low k at 14% of wild-type when leucine was the sole-carbon source. Transcriptional
gene fusions to lpdA1 and lpdA2 indicate that expression is up-regulated when
arabinose, malate or succinate was the sole-carbon source. Fusions to lpdA3 showed
up-regulation with leucine as the sole-carbon source.
6.2 Results and discussion
6.2.1 Percent homology comparison of the three lpdA genes
The nucleotide and amino acid sequences of the three lpdA genes were aligned
(Fig. 23) and the percent homology of the consensus sequences compared (Table 10).
Comparing pairs of nucleotide sequences resulted in consensus sequences with
homologies ranging from 50 to 55%. Nucleotide translation consensus sequences
resulted in homologies ranging from 31 to 42% of identical amino acids and 17 to
22% of amino acids with similar function. A nucleotide consensus sequence of the
three lpdA genes had a homology of 36%. Comparison of the three translated lpdA
nucleotide sequences resulted in a consensus in which 23% of the amino acids were
identical and 20% were of similar function. Although having a low overall amino acid
homology, there were several short areas of conserved identities comprising known
catalytic regions. These included residues C-41 and C-45, which are involved in the
disulfide centre and FAD interaction (Hopkins and Williams 1995; Toyoda et al.
1998); active site essential H-450, P-451 and E-455 (Benen et al. 1991); and
structurally-important residues, such as I-51 (Kim 2006).
6.2.2 Construction of S. meliloti strains Rm30232, Rm30282 and Rm30309
118
To explore the hypothesis that each lpdA allele encodes a functional enzyme
specific to PDH, OGD, or BKD, the three lpdA alleles were each independently
disrupted by a single cross-over (replicon fusion) with suicide vector pVIK112
carrying an internal PCR fragment from the respective lpdA gene. Integration of the
plasmids also created transcriptional lpdA-lacZ gene fusions. All constructs were
confirmed by PCR and Southern blot (data not shown).
6.2.3 Enzyme assays
Crude cell extracts were made from cultures of Rm30282, Rm30309,
Rm30232 and wild-type RmG212 all of which had grown to late log phase in M9minimal media with succinate/arabinose/1% LB as the carbon source. Specific enzyme
activities were then determined for OGD, PDH, and BKD. Malate dehydrogenase
(MDH) activities were used as positive controls.
LpdA1 mutant Rm30282 had no detectable PDH activity (Table 11). This is
similar to a previously obtained S. meliloti strain with a Tn5 transposon insertion in
SMc01033 located two ORFs upstream of lpdA1 (Soto et al. 2001). The Soto et al.
(2001) study shows that although the transposon was not inserted in lpdA1
specifically, polar effects cause an approximate 16-fold reduction in assays that
measured total PDH activity relative to wild-type. Interestingly, they also did a set of
enzyme assays specifically for the dihydrolipoamide dehydrogenase (E3) subunit. In
this case, they only saw a reduction of approximately 4-fold relative to wild-type.
They hypothesized that this was probably because LpdA2 and LpdA3 were also being
measured and augmented the results.
119
We found the OGD activity in strain Rm30282 was decreased to 39% of wildtype. Unfortunately in the Soto et al. (2001) study of the S. meliloti polar effected
LpdA1 strain, OGD activity was not measured so it is unknown if the same decrease
in activity would have been observed. We observed BKD activity in strain Rm30282
was the same as that seen in the wild-type.
In the LpdA2 mutant Rm30309, PDH and BKD activity levels were
comparable to wild-type while MDH activity was 3.5 times higher (Table 11). This
increase in MDH levels is similar to the increase seen in the sucB mutants and the R.
leguminosarum sucA mutants, as discussed in Chapter 5. OGD activity in the LpdA2
mutant was reduced by 97%. LpdA3 mutant strain Rm30232 had no detectable BKD
activity whereas MDH, PDH and OGD activity were comparable to wild-type levels.
6.2.4 Plant assays, symbiotic phenotype and microscopy
Varying degrees of growth phenotypes among the three LpdA mutant strains
were observed (Fig. 24). Plants inoculated with strain Rm30282 were stunted and had
a SDW approximately double to those of uninoculated plants (Table 12). Portions of
individual plants that had been inoculated with Rm30282 were both chlorotic and
green. Most root nodules were small and white but there were a few larger pink ones.
Soto et al. (2001) found that plants inoculated with the S. meliloti strain where lpdA1
is affected by an upstream (polar) insertion, had nodules that were occupied by
bacteroids but the plants were Fix-. Plants inoculated with Rm30309 grew to about
half the height of the wild-type (57% SDW relative to wild-type) and were all green
indicating a Fix+ phenotype. This finding contradicts the results reported for the sucB
120
mutant discussed in the prior chapter and other OGD mutants obtained by others
researchers (Duncan and Fraenkel 1979; Walshaw et al. 1997). The plants inoculated
with Rm30232 were healthy looking and green, indicating a Fix+ phenotype, and had a
SDW comparable to wild-type.
Dissection and microscopic observation of stained nodules from representative
plants of each strain showed plant cells occupied with differentiated bacteroids (Fig.
25). Plants inoculated with Rm30282 had some incomplete infection threads but
where they did reach into the nodules they were fully occupied by bacteroids. Plant
cell vacuoles appeared perturbed in nodules carrying Rm30282 whereas the vacuoles
in cells carrying the other test strains were well defined. Dissected nodules from plants
that had been inoculated with Rm30309 were full of nitrogen-fixing bacteroids which
also contradict results obtained from other OGD mutants. There was no visible
difference between Rm20232 carrying nodules to those of wild-type.
6.3 Conclusions
Homology comparisons showed little similarity between the three lpdA alleles
with overall homology well below 40%. Common to all three protein sequences were
the important catalytic regions and a few stretches of conserved amino acids that may
be involved with attachment and interaction with the E1 and E2 subunits of the
functional enzyme complex.
The LpdA1 mutant had no detectable PDH activity and reduced OGD activity
indicating that the deletion of the LpdA1 gene product completely inactivates PDH
and that none of the other LpdA subunits can replace it. ODG activity is possibly
121
affected by the loss of LpdA1 because citrate, an upstream TCA cycle precursor
compound of 2-oxoglutarate, cannot be made due to a lack of acetyl-CoA from PDH.
Enzyme activity from the LpdA2 mutant had greatly reduced OGD activity and an
increase in MDH activity. OGD activity may not have been completely abolished in
this strain due to background activity from the non-specific dehydrogenase present in
all crude extracts (Charles et al. 1990). The observed increase in MDH activity was
expected as the same phenomena was seen in the sucB mutants detailed in Chapter 5
and in sucA mutants described by other researchers (Duncan and Fraenkel 1979;
Walshaw et al. 1997). Having a blockage in the TCA cycle caused by a disruption in
OGD will build-up 2-oxoglutarate in the cell. To compensate for this, OGD expression
may be up-regulated in order to try and remove the excess 2-oxoglutarate. Since the
promoter for sucA and sucB is located upstream of mdh, an increased expression of
mdh and sucCD would result because they are all part of the same operon. As OGD
activity was significantly abolished in the LpdA2 mutant strain, it seems that neither
LpdA1 nor LpdA3 can replace it. The disruption in LpdA3 abolished BKD activity but
had no effect on PDH or OGD indicating that LpdA1 or LpdA2 cannot compensate for
the loss of that subunit.
Plants inoculated with the LpdA1 mutant displayed a variable nitrogen fixation
phenotype. Parts of the same plant were chlorotic and other parts lush while some root
nodules were small and white and still others plump and pink. The Fix+ phenotype
observed in the plants inoculated with the lpdA2 mutant was unexpected since a loss
of OGD caused by a disruption in sucB resulted in a Fix- phenotype. The loss of
LpdA2 resulted in OGD enzymatic activity being reduced to 3% of wild-type levels
122
which is not expected to be sufficient to support nitrogen fixation in these cells. The
BKD enzyme plays no role in energy metabolism and, therefore, should have no effect
on the nitrogen fixation process. This is reflected in the results obtained for strain
Rm30232 for which all plants were lush green, SDWs were comparable to wild-type
and nodules were fully occupied with bacteroids.
The results of the enzyme assays showed that the LpdA1 subunit is distinct to
PDH, LpdA2 is distinct to OGD and the LpdA3 subunit is distinct to BKD. The plant
assay experiment revealed a problem. The variable growth phenotype for the LpdA1carrying plants and the unexpected Fix+ phenotype for the plants inoculated with the
LpdA2 mutants may both be due to plasmid instability. All three mutants were
constructed by the single cross-over recombination of a plasmid bearing a fragment of
the target gene. Antibiotic maintenance is necessary in liquid culture to avoid the
possibility of the plasmid recombining out of the chromosome and being lost thereby
reverting the mutant strain to wild-type. The observed variability in nitrogen-fixation
would depend on the stability of the plasmid and when plasmid loss occurred during
plant invasion. If the plasmid was very stable without antibiotic maintenance and loss
occurred late in the invasion process, then it could be that most of the terminally
differentiated bacteroids retained the plasmid insert and would be Fix-. However, if the
plasmid was very unstable and loss occurred early in the invasion when bacteria are
actively dividing down the infection thread, then most of the bacteroids could have
lost the plasmid and would thus have a Fix+ phenotype as was observed for the plants
inoculated with the LpdA2 mutant strain. A plasmid with intermediate stability could
be lost early, late or even lost in some infection threads and not in others. This could
123
result in some nodules containing bacteroids with revertants, and some with mutants,
thereby explaining the variable fixation phenotype observed for the plants inoculated
with LpdA1 mutants.
Due to the uncertainty of plasmid stability in planta causing the variation in
phenotype, it is not clear whether the three LpdA subunits are interchangeable or
discrete. Results of the enzyme assays suggest that the LpdA subunits are unique to
their respective enzyme complexes. To confirm this, strains with stable insert
mutations in the three lpdA genes are being constructed. At present our lab is using
transposon mutagenesis to make single, double and triple mutants and once obtained,
these questions can be readdressed.
124
6.4 Figures and Tables
125
Figure 22: The genetic arrangement of the three lpdA genes relative to the operons encoding subunits of PDH, OGD and
BKD.
Genes in similar colour are putatively co-transcribed from a promoter upstream of pdhA, mdh (not shown) or bkdAα.
126
Figure 23: Multiple alignment of the three LpdA amino acid sequences.
Cysteine amino acids involved in the disulfide centre and interaction with FAD are indicated in red.
Structurally important isoleucine indicated in blue. Major active site indicated in green. Identical amino
acids indicated by asterisks, similar amino acids indicated by dots.
LpdA1
LpdA2
LpdA3
1 MAENYDVIVVGSGPGGYVTAIRSAQLGLKTAIVER-EHLGGICLNWGCI
1
MAYDLIVIGSGPGGYVCAIKAAQLGMKVAVVEKRSTYGGTCLNVGCI
1 MKEISCKLLVLGAGPGGYVAAIRAGQLGVNTVIVEK-AKAGGTCLNVGCI
..*.*.******.**.. ***.
.**.
** *** ***
48
47
49
LpdA1
LpdA2
LpdA3
49 PTKALLRSAEILD---HANHAKNYGLTLEGKITANVKDVVARSRGVSARL
48 PSKALLHASEMFH---QAQHGLEALGVEVANPKLNLQKMMAHKDATVKSN
50 PSKALIHAADEYHRLRAAASGKGPLGLSLSAPAIDLRRTIAWKDGIVGRL
*.***.....
*
.. .*
95
94
99
LpdA1
LpdA2
LpdA3
96 NGGVAFLMKKNKVDVIWGEAKLTKPGEIVVGAPSKPAVQPQNPVPKGVKG 145
95 VDGVSFLFKKNKIDGFQGTGKVLGQGKVSVTN------------EKGE-- 130
100 NGGVTGLLKKAGVKAVIGEGRFVDGKTVDVET------------ETG--- 134
**. * ** .
* .
. *
*
LpdA1
LpdA2
LpdA3
146 EGTYTAKHIIVATGARPRALPGIEPDG--KLIWTYFEAMKPEEFPKSLLV 193
131 EQVLEAKNVVIATGSDVAGIPGVEVAFDEKTIVSSTGALALEKVPASMIV 180
135 LQRIRAEAIVIATGSAPVELPDLPFGG---SVISSTQALALTDVPQTLAV 181
* ...***.
.* .
. .
*.
* .. *
LpdA1
LpdA2
LpdA3
194 MGSGAIGIEFASFYRSMGVDVTVVELLPQIMPVEDAEISAFARKQLEKRG 243
181 VGGGVIGLELGSVWARLGAKVTVVEFLDTILGGMDGEVAKQLQRMLTKQG 230
182 IGGGYIGLELGTAFAKLGSKVTVLEALDRILPQYDADLSKPVMKRLGELG 231
.* * **.* .
.* ***.* * *.
* ...
. *
*
LpdA1
LpdA2
LpdA3
244 LKIITDAKVTKVEKGANDVTAHVETKD-GKVTPIKADRLISAVGVQGNIE 292
231 IDFKLGAKVTGAVKSGDGAKVTFEPVKGGEATTLDAEVVLIATGRKPSTD 280
232 VEVFT--RTAAKRLSADRRGLLAEEN--GRAFEVPAEKVLVTVGRRPVTD 277
.
. .
*
*
. *. .. . * .
.
LpdA1
LpdA2
LpdA3
293 NLGLEALGVKTDRG-CIVTDGYGKTNVAGIYAIGDVAGPPMLAHKAEHEG 341
281 GLGLAKAGVVLDSRGRVEIDRHFQTSIAGVYAIGDVVRGPMLAHKAEDEG 330
278 GWGLEEIDLDHSGR-FIRIDDQCRTSMRGVYAIGDVTGEPMLAHRAMAQG 326
. **
.
. *
.* . *.******
*****.* .*
LpdA1
LpdA2
LpdA3
342 VICVEKIAGVPGVHALDKGKIPGCTYCDPQVASVGLTEAKAKELGRDIRV 391
331 VAVAEIIAGQAG--HVNYDVIPGVVYTQPEVASVGKTEEELKAAGVAYKI 378
327 EMVAEIVAGHKR--SWDKRCIPAVCFTDPEIVGAGLSPEEARAAGIDVKI 374
* .**
**
.. *..
* .
. *
..
LpdA1
LpdA2
LpdA3
392 GRYSFGANGKAIALGEDQGLIKTIFDKKTGELIGAHMVGAEVTELIQGFV 441
379 GKFPFTANGRARAMLQTDGFVKILADKETDRVLGGHIIGFGAGEMIHEIA 428
375 GQFPFQANGRAMTTLSEDGFVRVIARADNHLVLGIQAVGHGVSELSATFA 424
*.. * ***.* .
* .. .
. ..* . .*
*.
LpdA1
LpdA2
LpdA3
442 VAMNLETTEEELMHTVFPHPTLSEMMKESVLDAYGRVLNA* 482
429 VLMEFGGSSEDLGRTCHAHPTMSEAVKEAALSTFFKPIHM* 469
425 LAIEMGARLEDIAGTIHAHPTQSEAFQEAALKTLGHALHI* 465
. .
*.. *
*** ** .*. * . . .. *
127
Table 10: The nucleotide and amino acid percent homology between the three lpdA
genes.
Alignment
LpdA1 + LpdA2
LpdA1 + LpdA3
LpdA2 + LpdA3
LpdA1 + LpdA2 + LpdA3
Nucleotide
53
50
55
36
Amino acid
Identical Similar
36
17
31
22
42
18
23
20
128
Table 11: Specific enzymatic activities from crude cell extracts of cells grown in M9 minimal media with succinate, arabinose,
and 1% LB together as the carbon source.
Strain
RmG212
Rm30282
Rm30309
Rm30232
Characteristics
LacLpdA1LpdA2LpdA3-
MDH
1585 ± 77
1252 ± 35
5635 ± 26
1734 ± 26
OGD
97.5 ± 4.4
37.9 ± 1.3
2.9 ± 0.3
90.2 ± 1.1
PDH
26.2 ± 0.2
0±0
31.3 ± 0.4
33.9 ± 0.3
BKD
16.8 ± 0.4
17.0 ± 0.3
19.2 ± 0.2
0±0
Specific activity is the mean of triplicate assays expressed as nmol·min-1·mg protein-1 ± standard error
129
Figure 24: The symbiotic phenotype of alfalfa plants* inoculated with one of the three LpdA mutants.
* = Plants were photographed 28 days post inoculation
130
Table 12: The average shoot dry weight (SDW) and phenotype of alfalfa plants inoculated with one of the three LpdA mutant
strains harvested 28 days post-inoculation.
Strain
Uninoculated
RmG212
Rm30282
Rm30309
Rm30232
Characteristics
LacLpdA1LpdA2LpdA3-
SDWa
5.60 ± 0.3
42.5 ± 1.7
10.0 ± 0.8
24.2 ± 0.9
37.9 ± 2.2
%WTb
13
100
24
57
89
Plant phenotype
Nod-/FixNod+/Fix+
Nod+/Fix+/Nod+/Fix+
Nod+/Fix+
a = Shoot dry weight expressed as milligrams per plant as mean ± standard error of triplicate samples.
b = Percentage of the wild-type RmG212 value.
131
Figure 25: Microscopic cross sections of nodules containing bacteroids of strains
RmG212, Rm30282 (lpdA1 mutant), Rm30309 (lpdA2 mutant) and Rm302325 (lpdA3
mutant) stained with nucleic acid binding dye syto-13.
RmG212
Rm30282
Rm30309
Rm30232
132
Summary and general conclusions
The first part of this thesis was an analysis of the biochemical properties of
MDH and the genetic regulation of the mdh-sucCDAB operon. The TCA cycle enzyme
MDH functioned optimally at pH10 as a homodimer and had a molecular mass of 66
kDa. The kinetic properties and the enzymatic mechanism determined for MDH from
S. meliloti were very similar to those found for other MDHs from organisms across the
three domains of life. The Michaelis/Menten binding constant (Km) was lower than
that of DME and the turnover rate (Vmax) was faster for MDH than for DME indicating
that the flow of malate at the critical MDH/DME junction would preferentially be in
the direction of MDH. An eqimolar distribution of malate between MDH and DME in
bacteroids could be achieved by the build-up of OAA, a product of the MDH reaction,
which would inhibit MDH, forcing malate through DME thereby replenishing the
acetyl-CoA pool. The TCA cycle intermediate, 2-oxoglutarate, exhibited competitive
inhibition on MDH which would be relevant under free-living, nitrogen-deficient
conditions during which the build-up of 2-oxoglutarate would slow down MDH
thereby forcing the pooling malate out of the TCA cycle.
The promoter for mdh controlled transcription of the whole mdh-sucCDAB
operon. The alternative transcription factor σ54 played no role in the RNAP
holoenzyme binding of the mdh promoter. The TSS for mdh was found to be a guanine
residue located 64 nt upstream of the ATG translational start site. Transcriptional
fusions to selected genes of the mdh-sucCDAB operon showed that the promoter is
under catabolic control due to the differences in gene expression in cells grown with
different carbon-sources. Fusion results also showed that expression of sucA is higher
133
than that of sucD in cells grown with arabinose or glutamate as the sole-carbon source
thereby providing evidence for a functional promoter upstream of sucA. Continuing
work, such as RNAseq, is being carried out to confirm this.
The second part of this thesis was to ascertain whether the multiple genes
encoding dihydrolipoyl succinyltransferase (2 alleles) and dihydrolypoamide
dehydrogenase (3 alleles) were functional and interchangeable. The chromosomal
allele SMc02483 (sucB) had a much higher sequence homology to sucB alleles from
other rhizobia than does the megaplasmid pSymB-borne allele SMb20019. Mutations
were induced in the two annotated sucB alleles and a double mutant was also created.
Strains carrying a disruption in sucB or in sucB plus SMb20019 lacked OGD activity
and both strains induced nodule formation on alfalfa roots but were Fix-. In contrast,
strains of S. meliloti with a SMb20019 disruption retained wild-type levels of OGD
activity and were Nod+ Fix+. Results indicated that the chromosomal-borne allele
SMc02483, not the megaplasmid-borne allele SMb200189, encodes for the functional
SucB of the OGD complex.
Homology was less than 40% between the three LpdA amino acid sequences;
however, they all retained catalytically-important residues. Three strains of S. meliloti
were constructed with each respective strain carrying a mutation in one of the lpdA
genes. The LpdA1 mutant strain had no PDH activity, diminished OGD activity and
wild-type BKD activity levels thus indicating that LpdA1 is the sole enzyme used as
the E3 subunit of the PDH complex and has no involvement with OGD or BKD. The
LpdA2 mutated strain had greatly diminished OGD activity and wild-type levels of
PDH and BKD. This indicated that the LpdA2 is the sole enzyme for the OGD
134
complex and had no role in the PDH or BKD enzyme complex. Strains with an LpdA3
mutation had no BKD activity but retained wild-type PDH and OGD levels indicating
that this enzyme only interacts with BKD and not the other two. Unfortunately,
possibly due to plasmid instability and, thus, the potential loss of the chromosomally
integrated plasmid resulting in the mutants reverting to a wild-type phenotype, the
plant assay results were inconclusive. Our lab continues research on this subject and is
currently constructing stable lpdA mutants with which to further investigate the
phenotypic effect on inoculated plants.
135
References
Acuna, G., Ebeling, S. and Hennecke, H., 1991. Cloning, sequencing, and mutational analysis of the Bradyrhizobium japonicum fumC‐like gene: evidence for the existence of two different fumarases. J Gen Microbiol. 137: 991‐1000. Allaway, D., Schofield, N.A., Leonard, M.E., Gilardoni, L., Finan, T.M. and Poole, P.S., 2001. Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes. Environ. Microbiol. 3: 397‐406. Babic, B., 2010. Genetic and biochemical characterization of dihydrolipoamide dehydrogenases (LPD) in Sinorhizobium meliloti. Masters of Science thesis, McGill University. Bae, Y.M., Holmgren, E. and Crawford, I.P., 1989. Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli. J Bacteriol. 171: 3471‐3478. Barnett, M.J., Fisher, R.F., Jones, T., Komp, C., Abola, A.P., Barloy‐Hubler, F., Bowser, L., Capela, D., Galibert, F., Gouzy, J., Gurjal, M., Hong, A., Huizar, L., Hyman, R.W., Kahn, D., Kahn, M.L., Kalman, S., Keating, D.H., Palm, C., Peck, M.C., Surzycki, R., Wells, D.H., Yeh, K.C., Davis, R.W., Federspiel, N.A. and Long, S.R., 2001. Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci U S A. 98: 9883‐9888. Benen, J., van Berkel, W., Zak, Z., Visser, T., Veeger, C. and de Kok, A., 1991. Lipoamide dehydrogenase from Azotobacter vinelandii: site‐directed mutagenesis of the His450‐Glu455 diad. Spectral properties of wild type and mutated enzymes. Eur. J. Biochem. 202: 863‐872. Bergey, D.H., Krieg, N.R. and Holt, J.G., 1984. Bergey's manual of systematic bacteriology. Williams & Wilkins, Baltimore, MD. 4 v. (xxvii, 2648 p. ). Blatter, E.E., Ross, W., Tang, H., Gourse, R.L. and Ebright, R.H., 1994. Domain organization of RNA polymerase alpha subunit: C‐terminal 85 amino acids constitute a domain capable of dimerization and DNA binding. Cell. 78: 889‐896. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal. Biochem. 72: 248‐
254. Bridger, W.A., Ramaley, R.F. and Boyer, P.D., 1969. Succinyl coenzyme A synthetase from Escherichia coli. Methods Enzymol. 13: 70‐75. Browning, D.F. and Busby, S.J., 2004. The regulation of bacterial transcription initiation. Nat Rev Microbiol. 2: 57‐65. Burgess, R.R., Travers, A.A., Dunn, J.J. and Bautz, E.K., 1969. Factor stimulating transcription by RNA polymerase. Nature. 221: 43‐46. 136
Burris, R.H., 2001. Nitrogen fixation. Encyclopedia of life sciences. John Wiley & Sons, Ltd. Hoboken, NJ. Campbell, E.A., Muzzin, O., Chlenov, M., Sun, J.L., Olson, C.A., Weinman, O., Trester‐Zedlitz, M.L. and Darst, S.A., 2002. Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol Cell. 9: 527‐539. Campbell, E.A., Westblade, L.F. and Darst, S.A., 2008. Regulation of bacterial RNA polymerase sigma factor activity: a structural perspective. Curr Opin Microbiol. 11: 121‐127. Capela, D., Barloy‐Hubler, F., Gouzy, J., Bothe, G., Ampe, F., Batut, J., Boistard, P., Becker, A., Boutry, M., Cadieu, E., Dreano, S., Gloux, S., Godrie, T., Goffeau, A., Kahn, D., Kiss, E., Lelaure, V., Masuy, D., Pohl, T., Portetelle, D., Puhler, A., Purnelle, B., Ramsperger, U., Renard, C., Thebault, P., Vandenbol, M., Weidner, S. and Galibert, F., 2001. Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc. Natl. Acad. Sci. U S A. 98: 9877‐9882. Charles, T.C., Singh, R.S. and Finan, T.M., 1990. Lactose utilization and enzymes encoded by megaplasmids in Rhizobium meliloti SU47: implications for population studies. J. Gen. Microbiol. 136: 2497‐2502. Cleland, W.W., 1963a. The kinetics of enzyme‐catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim. Biophys. Acta. 67: 104‐
137. Cleland, W.W., 1963b. The kinetics of enzyme‐catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory. Biochim. Biophys. Acta. 67: 173‐187. Cleland, W.W., 1963c. The kinetics of enzyme‐catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection. Biochim. Biophys. Acta. 67: 188‐196. Commichau, F.M., Forchhammer, K. and Stulke, J., 2006. Regulatory links between carbon and nitrogen metabolism. Curr. Opin. Microbiol. 9: 167‐172. Courtright, J.B. and Henning, U., 1970. Malate dehydrogenase mutants in Escherichia coli K‐
12. J. Bacteriol. 102: 722‐728. Cramer, P., Bushnell, D.A. and Kornberg, R.D., 2001. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science. 292: 1863‐1876. da Silva, J.G., 2011. Current world fertilizer trends and outlooks to 2015. Food and agriculture organization of the U. N., Rome, Italy. Dao, T.V., Nomura, M., Hamaguchi, R., Kato, K., Itakura, M., Minamisawa, K., Sinsuwongwat, S., Le, H.T., Kaneko, T., Tabata, S. and Tajima, S., 2008. NAD‐Malic Enzyme Affects Nitrogen 137
Fixing Activity of Bradyrhizobium japonicum USDA 110 Bacteroids in Soybean Nodules. Microbes Environ. 23: 215‐220. Day, D.A. and Copeland, L., 1991. Carbon metabolism and compartmentation in nitrogen‐
fixing legume nodules. Plant Physiol. Biochem. 29: 185‐201. Dickinson, J.R., Roy, D.J. and Dawes, I.W., 1986. A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae. Mol. Gen. Genet. 204: 103‐107. Dixon, R. and Kahn, D., 2004. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol. 2: 621‐631. Driscoll, B.T. and Finan, T.M., 1993. NAD(+)‐dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation. Mol. Microbiol. 7: 865‐873. Driscoll, B.T. and Finan, T.M., 1996. NADP+ ‐dependent malic enzyme of Rhizobium meliloti. J. Bacteriol. 178: 2224‐2231. Driscoll, B.T. and Finan, T.M., 1997. Properties of NAD(+)‐ and NADP(+)‐dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and differential expression of their genes in nitrogen‐fixing bacteroids. Microbiology. 143 ( Pt 2): 489‐498. Duncan, M.J. and Fraenkel, D.G., 1979. Alpha‐ketoglutarate dehydrogenase mutant of Rhizobium meliloti. J. Bacteriol. 137: 415‐419. Dunn, M.F., 1998. Tricarboxylic acid cycle and anaplerotic enzymes in rhizobia. FEMS Microbiol. Rev. 22: 105‐123. Dusha, I., Schroder, J., Putnoky, P., Banfalvi, Z. and Kondorosi, A., 1986. A cell‐free system from Rhizobium meliloti to study the specific expression of nodulation genes. Eur J Biochem. 160: 69‐75. Dymov, S.I., Meek, D.J., Steven, B. and Driscoll, B.T., 2004. Insertion of transposon Tn5tac1 in the Sinorhizobium meliloti malate dehydrogenase (mdh) gene results in conditional polar effects on downstream TCA cycle genes. Mol. Plant Microbe Interact. 17: 1318‐1327. Eley, M.H., Namihira, G., Hamilton, L., Munk, P. and Reed, L.J., 1972. Alpha‐keto acid dehydrogenase complexes. 18. Subunit composition of the Escherichia coli pyruvate dehydrogenase complex. Arch. Biochem. Biophys. 152: 655‐669. Englard, S. and Siegal, L., 1969. Mitochondrial L‐malate dehydrogenase of beef heart. Methods Enzymol. 13: 99‐100. Estrem, S.T., Gaal, T., Ross, W. and Gourse, R.L., 1998. Identification of an UP element consensus sequence for bacterial promoters. Proc Natl Acad Sci U S A. 95: 9761‐9766. 138
Finan, T.M., Oresnik, I. and Bottacin, A., 1988. Mutants of Rhizobium meliloti defective in succinate metabolism. J. Bacteriol. 170: 3396‐3403. Finan, T.M., Weidner, S., Wong, K., Buhrmester, J., Chain, P., Vorholter, F.J., Hernandez‐
Lucas, I., Becker, A., Cowie, A., Gouzy, J., Golding, B. and Puhler, A., 2001. The complete sequence of the 1,683‐kb pSymB megaplasmid from the N2‐fixing endosymbiont Sinorhizobium meliloti. Proc Natl Acad Sci U S A. 98: 9889‐9894. Fischer, H.M., 1994. Genetic regulation of nitrogen fixation in rhizobia. Microbiol. Rev. 58: 352‐386. Flechard, M., Fontenelle, C., Blanco, C., Goude, R., Ermel, G. and Trautwetter, A., 2010. RpoE2 of Sinorhizobium meliloti is necessary for trehalose synthesis and growth in hyperosmotic media. Microbiology. 156: 1708‐1718. Fougere, F., Le Rudulier, D. and Streeter, J.G., 1991. Effects of Salt Stress on Amino Acid, Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of Alfalfa (Medicago sativa L.). Plant Physiol. 96: 1228‐1236. Galibert, F., Finan, T.M., Long, S.R., Puhler, A., Abola, P., Ampe, F., Barloy‐Hubler, F., Barnett, M.J., Becker, A., Boistard, P., Bothe, G., Boutry, M., Bowser, L., Buhrmester, J., Cadieu, E., Capela, D., Chain, P., Cowie, A., Davis, R.W., Dreano, S., Federspiel, N.A., Fisher, R.F., Gloux, S., Godrie, T., Goffeau, A., Golding, B., Gouzy, J., Gurjal, M., Hernandez‐Lucas, I., Hong, A., Huizar, L., Hyman, R.W., Jones, T., Kahn, D., Kahn, M.L., Kalman, S., Keating, D.H., Kiss, E., Komp, C., Lelaure, V., Masuy, D., Palm, C., Peck, M.C., Pohl, T.M., Portetelle, D., Purnelle, B., Ramsperger, U., Surzycki, R., Thebault, P., Vandenbol, M., Vorholter, F.J., Weidner, S., Wells, D.H., Wong, K., Yeh, K.C. and Batut, J., 2001. The composite genome of the legume symbiont Sinorhizobium meliloti. Science. 293: 668‐672. Galloway, J.N., 1995. Nitrogen fixation: anthropogenic enhancement environmental response. Global Biogeo‐chemical Cycles. 9: 235‐252. García‐de los Santos, A., Morales, A., Baldomá, L., Clark, S.R., Brom, S., Yost, C.K., Hernández‐
Lucas, I., Aguilar, J. and Hynes, M.F., 2002. The glcB locus of Rhizobium leguminosarum VF39 encodes an arabinose‐inducible malate synthase. Canadian Journal of Microbiology. 48: 922‐
932. Genda, T., Nakamatsu, T. and Ozak, H., 2003. Purification and characterization of malate dehydrogenase from Corynebacterium glutamicum. J Biosci Bioeng. 95: 562‐566. Glazebrook, J. and Walker, G.C., 1991. Genetic techniques in Rhizobium meliloti. Methods Enzymol. 204: 398‐418. Glenn, A.R., Arwas, R., McKay, I.A. and Dilworth, M.J., 1984a. Fructose metabolism in wild‐
type, fructokinase negative revertant strains of Rhizobium leguminosarum. J. Gen. Microbiol. 130: 231‐237. 139
Glenn, A.R. and Dilworth, M.J., 1981a. Oxidation of substrates by isolated bacteroids and free‐living cells of Rhizobium leguminosarum. J. Gen. Microbiol. 126: 243‐247. Glenn, A.R. and Dilworth, M.J., 1981b. The uptake and hydrolysis of disaccharides by fast‐ and slow‐growing species of Rhizobium leguminosarum. Arch. Microbiol. 129: 233‐239. Glenn, A.R., McKay, I.A., Arwas, R. and Dilworth, M.J., 1984b. Sugar metabolism and the symbiotic properties of carbohydrate mutants of Rhizobium leguminosarum. J. Gen. Microbiol. 130: 239‐245. Gonzalez, V., Santamaria, R.I., Bustos, P., Hernandez‐Gonzalez, I., Medrano‐Soto, A., Moreno‐
Hagelsieb, G., Janga, S.C., Ramirez, M.A., Jimenez‐Jacinto, V., Collado‐Vides, J. and Davila, G., 2006. The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons. Proc. Natl. Acad. Sci. U S A. 103: 3834‐3839. Goodner, B., Hinkle, G., Gattung, S., Miller, N., Blanchard, M., Qurollo, B., Goldman, B.S., Cao, Y., Askenazi, M., Halling, C., Mullin, L., Houmiel, K., Gordon, J., Vaudin, M., Iartchouk, O., Epp, A., Liu, F., Wollam, C., Allinger, M., Doughty, D., Scott, C., Lappas, C., Markelz, B., Flanagan, C., Crowell, C., Gurson, J., Lomo, C., Sear, C., Strub, G., Cielo, C. and Slater, S., 2001. Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science. 294: 2323‐2328. Gourse, R.L., Ross, W. and Gaal, T., 2000. UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition. Mol Microbiol. 37: 687‐695. Green, L.S. and Emerich, D.W., 1997a. Bradyrhizobium japonicum does not require alpha‐
ketoglutarate dehydrogenase for growth on succinate or malate. J. Bacteriol. 179: 194‐201. Green, L.S. and Emerich, D.W., 1997b. The formation of nitrogen‐fixing bacteroids is delayed but not abolished in soybean infected by an [alpha]‐ketoglutarate dehydrogenase‐deficient mutant of Bradyrhizobium japonicum. Plant Physiol. 114: 1359‐1368. Green, L.S., Karr, D.B. and Emerich, D.W., 1998. Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions. Archives of Microbiology. 169: 445‐451. Green, L.S., Waters, J.K., Ko, S. and Emerich, D.W., 2003. Comparative analysis of the Bradyrhizobium japonicum sucA region. Canadian Journal of Microbiology. 49: 237‐243. Gribskov, M. and Burgess, R.R., 1983. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene. 26: 109‐118. Guest, J.R., Cole, S.T. and Jeyaseelan, K., 1981. Organization and expression of the pyruvate dehydrogenase complex genes of Escherichia coli K12. J. Gen. Microbiol. 127: 65‐79. Harley, C.B. and Reynolds, R.P., 1987. Analysis of E. coli promoter sequences. Nucleic Acids Res. 15: 2343‐2361. 140
Harris, R.A. and Sokatch, J.R., 1988. Branched‐chain amino acids. Methods Enzymol. 166: 181‐182. Harti, T., Grossebueter, W., Goerisch, H. and Stezowski, J.J., 1987. Crystalline NAD/NADP‐
dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biol. Chem. Hoppe‐Seyler. 368: 259‐267. Haugen, S.P., Berkmen, M.B., Ross, W., Gaal, T., Ward, C. and Gourse, R.L., 2006. rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase. Cell. 125: 1069‐1082. Haugen, S.P., Ross, W. and Gourse, R.L., 2008. Advances in bacterial promoter recognition and its control by factors that do not bind DNA. Nat Rev Microbiol. 6: 507‐519. Helmann, J.D., 1999. Anti‐sigma factors. Curr Opin Microbiol. 2: 135‐141. Helmann, J.D., 2002. The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol. 46: 47‐110. Heyde, E. and Ainsworth, S., 1968. Kinetic studies on the mechanism of the malate dehydrogenase reaction. J. Biol. Chem. 243: 2413‐2423. Hirata, A., Klein, B.J. and Murakami, K.S., 2008. The X‐ray crystal structure of RNA polymerase from Archaea. Nature. 451: 851‐854. Hirsch, A.M., Wilson, K.J., Jones, J.D., Bang, M., Walker, V.V. and Ausubel, F.M., 1984. Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa. J Bacteriol. 158: 1133‐1143. Hopkins, N. and Williams, C.H., Jr., 1995. Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Biochemistry. 34: 11757‐
11765. Hornez, J., Timinouni, M., Defives, C. and Derieux, J., 1994. Unaffected nodulation and nitrogen fixation in carbohydrate pleiotropic mutants of Rhizobium meliloti. Curr. Microbiol. 28: 225‐229. Hudman, J.F. and Glenn, A.R., 1980. Glucose uptake by free‐living and bacteroid forms of Rhizobium leguminosarum. Arch. Microbiol. 128: 72‐77. Jelesko, J.G. and Leigh, J.A., 1994. Genetic characterization of a Rhizobium meliloti lactose utilization locus. Mol Microbiol. 11: 165‐173. Jones, K.M., Kobayashi, H., Davies, B.W., Taga, M.E. and Walker, G.C., 2007. How rhizobial symbionts invade plants: the Sinorhizobium‐Medicago model. Nat. Rev. Microbiol. 5: 619‐
633. 141
Kahn, M.L., Mortimer, M.W., Park, K.S. and Zhang, W., 1995. Carbon metabolism in Rhizobium‐legume symbiosis. In: Nitrogen fixation: fundamentals and applications. Tikhonovich, I. A., Provorov, N. A., Romanov, V. I., and Newton, W. E. (Eds.). pp. 525‐532. Kluwer Academic. Dordrecht. Kalogeraki, V.S. and Winans, S.C., 1997. Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene. 188: 69‐75. Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T., Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M., Matsuno, A., Mochizuki, Y., Nakayama, S., Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, C., Yamada, M. and Tabata, S., 2000. Complete genome structure of the nitrogen‐
fixing symbiotic bacterium Mesorhizobium loti. DNA Res. 7: 331‐338. Kaneko, T., Nakamura, Y., Sato, S., Minamisawa, K., Uchiumi, T., Sasamoto, S., Watanabe, A., Idesawa, K., Iriguchi, M., Kawashima, K., Kohara, M., Matsumoto, M., Shimpo, S., Tsuruoka, H., Wada, T., Yamada, M. and Tabata, S., 2002. Complete genomic sequence of nitrogen‐
fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res. 9: 189‐197. Karunakaran, R., Mauchline, T.H., Hosie, A.H. and Poole, P.S., 2005. A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram‐negative bacteria. Microbiology. 151: 3249‐3256. Kim, H., 2006. Activity of human dihydrolipoamide dehydrogenase is largely reduced by mutation at isoleucine‐51 to alanine. J. Biochem. Mol. Biol. 39: 223‐227. Koziol, U., Hannibal, L., Rodriguez, M.C., Fabiano, E., Kahn, M.L. and Noya, F., 2009. Deletion of citrate synthase restores growth in Sinorhizobium meliloti 1021 aconitase mutants. J. Bacteriol. 191: 7581‐7586. Labrou, N.E. and Clonis, Y.D., 1997. L‐Malate dehydrogenase from Pseudomonas stutzeri: purification and characterization. Arch. Biochem. Biophys. 337: 103‐114. Lee, D.J., Minchin, S.D. and Busby, S.J., 2012. Activating transcription in bacteria. Annu Rev Microbiol. 66: 125‐152. Leskovac, V., Trivic, S., Pericin, D. and Kandrac, J., 2006. Deriving the rate equations for product inhibition patterns in bisubstrate enzyme reactions. J. Enzyme Inhib. Med. Chem. 21: 617‐634. Lodwig, E.M., Hosie, A.H., Bourdès, A., Findlay, K., Allaway, D., Karunakaran, R., Downie, J.A. and Poole, P.S., 2003. Amino‐acid cycling drives nitrogen fixation in the legume‐Rhizobium symbiosis. Nature. 422: 722‐726. Low, D.A., Weyand, N.J. and Mahan, M.J., 2001. Roles of DNA adenine methylation in regulating bacterial gene expression and virulence. Infect Immun. 69: 7197‐7204. 142
Lowe, P.N., Hodgson, J.A. and Perham, R.N., 1983. Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched‐chain 2‐oxo acids in Bacillus subtilis. Biochem. J. 215: 133‐140. MacLellan, S.R., MacLean, A.M. and Finan, T.M., 2006. Promoter prediction in the rhizobia. Microbiology. 152: 1751‐1763. Malhotra, A., Severinova, E. and Darst, S.A., 1996. Crystal structure of a sigma 70 subunit fragment from E. coli RNA polymerase. Cell. 87: 127‐136. Martinez‐De Drets, G. and Arias, A., 1972. Enzymatic basis for differentiation of Rhizobium into fast‐ and slow‐growing groups. J Bacteriol. 109: 467‐470. Masse, E., Majdalani, N. and Gottesman, S., 2003. Regulatory roles for small RNAs in bacteria. Curr Opin Microbiol. 6: 120‐124. McDermott, T.R., Griffith, S.M., Vance, C.P. and Graham, P.H., 1989. Carbon metabolism in Bradyrhizobium japonicum bacteroids. FEMS Microbiol. Rev. 63: 327‐340. McDermott, T.R. and Kahn, M.L., 1992. Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene. J. Bacteriol. 174: 4790‐4797. McKay, I.A., Glenn, A.R. and Dilworth, M.J., 1985. Gluconeogenesis in Rhizobium leguminosarum MNF3841. J. Gen. Microbiol. 131: 2067‐2073. Meade, H.M., Long, S.R., RuvKin, G.B., Brown, S.E. and Ausubel, F.M., 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J. Bacteriol. 149: 114‐122. Mevarech, M., Eisenberg, H. and Neumann, E., 1977. Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization. Biochemistry. 16: 3781‐3785. Miller, J.H., 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 352‐359. Mitchell, J.E., Zheng, D., Busby, S.J. and Minchin, S.D., 2003. Identification and analysis of 'extended ‐10' promoters in Escherichia coli. Nucleic Acids Res. 31: 4689‐4695. Mitsch, M.J., Cowie, A. and Finan, T.M., 2007. Malic enzyme cofactor and domain requirements for symbiotic N2 fixation by Sinorhizobium meliloti. J Bacteriol. 189: 160‐168. Miyatake, K., Washio, K., Yokota, A., Nakano, Y. and Kitaoka, S., 1986. Purification and immunological properties of NAD+‐linked malate dehydrogenase isozymes from Euglena gracilis. Agric. Biol. Chem. 50: 2651‐2653. 143
Mortimer, M.W., McDermott, T.R., York, G.M., Walker, G.C. and Kahn, M.L., 1999. Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides. J. Bacteriol. 181: 7608‐7613. Mulley, G., Lopez‐Gomez, M., Zhang, Y., Terpolilli, J., Prell, J., Finan, T. and Poole, P., 2010. Pyruvate is synthesized by two pathways in pea bacteroids with different efficiencies for nitrogen fixation. J Bacteriol. 192: 4944‐4953. Mulongoy, K. and Elkan, G.H., 1977. The role of 6‐phosphogluconate dehydrogenase in Rhizobium. Can J Microbiol. 23: 1293‐1298. Mulvaney, R.L., Khan, S.A. and Ellsworth, T.R., 2009. Synthetic nitrogen fertilizers deplete soil nitrogen: a global dilemma for sustainable cereal production. J Environ Qual. 38: 2295‐2314. Murphey, W.H., Barnaby, C., Lin, F.J. and Kaplan, N.O., 1967. Malate dehydrogenases. II. Purification and properties of Bacillus subtilis, Bacillus stearothermophilus, and Escherichia coli malate dehydrogenases. J. Biol. Chem. 242: 1548‐1559. Murray, J.D., 2011. Invasion by invitation: rhizobial infection in legumes. Mol Plant Microbe Interact. 24: 631‐639. O'Hara, G.W., Goss, T.J., Dilworth, M.J. and Glenn, A.R., 1989. Maintenance of Intracellular pH and Acid Tolerance in Rhizobium meliloti. Appl. Environ. Microbiol. 55: 1870‐1876. Oke, V., Rushing, B.G., Fisher, E.J., Moghadam‐Tabrizi, M. and Long, S.R., 2001. Identification of the heat‐shock sigma factor RpoH and a second RpoH‐like protein in Sinorhizobium meliloti. Microbiology. 147: 2399‐2408. Oresnik, I.J., Liu, S.L., Yost, C.K. and Hynes, M.F., 2000. Megaplasmid pRme2011a of Sinorhizobium meliloti is not required for viability. J Bacteriol. 182: 3582‐3586. Østerås, M., Stanley, J. and Finan, T.M., 1995. Identification of Rhizobium‐specific intergenic mosaic elements within an essential two‐component regulatory system of Rhizobium species. J. Bacteriol. 177: 5485‐5494. Otulakowski, G., Robinson, B.H. and Willard, H.F., 1988. Gene for lipoamide dehydrogenase maps to human chromosome 7. Somat. Cell Mol. Genet. 14: 411‐414. Peter, J., Young, W. and Haukka, K.E., 1996. Diversity and phylogeny of rhizobia. New Phytologist. 133: 87‐94. Plancarte, A., Nava, G. and Mendoza‐Hernandez, G., 2009. Purification, properties, and kinetic studies of cytoplasmic malate dehydrogenase from Taenia solium cysticerci. Parasitol. Res. 105: 175‐183. Pobigaylo, N., Wetter, D., Szymczak, S., Schiller, U., Kurtz, S., Meyer, F., Nattkemper, T.W. and Becker, A., 2006. Construction of a large signature‐tagged mini‐Tn5 transposon library 144
and its application to mutagenesis of Sinorhizobium meliloti. Appl. Environ. Microbiol. 72: 4329‐4337. Poole, P., Reid, C., East, A., Allaway, D., Day, M. and Leonard, M., 1999. Regulation of the mdh‐sucCDAB operon in Rhizobium leguminosarum. FEMS Microbiol. Lett. 176: 247‐255. Poole, P.S., Blyth, A., Reid, C.J. and Walters, K., 1994. myo‐inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv. viciae. Microbiology. 140: 2787‐2795. Poysti, N.J., Loewen, E.D., Wang, Z. and Oresnik, I.J., 2007. Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism. Microbiology. 153: 727‐736. Reed, L.J. and Mukherjee, B.B., 1969. Alpha‐ketoglutarate dehydrogenase complex from Escherichia coli. Methods Enzymol. 13: 55‐61. Reeves, H.C., Rabib, R., Wegener, W.S. and Ajl, S.J., 1971. Assay of enzymes of the tricarboxylic acid and glyoxylate cycles. Methods Enzymol. 6A: 435‐436. Ronson, C.W., Nixon, B.T., Albright, L.M. and Ausubel, F.M., 1987. Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol. 169: 2424‐2431. Ronson, C.W. and Primrose, S.B., 1979. Carbohydrate metabolism in Rhizobium trifolii: identification and symbiotic properties of mutants. J. Gen. Microbiol. 112: 77‐88. Ross, W., Gosink, K.K., Salomon, J., Igarashi, K., Zou, C., Ishihama, A., Severinov, K. and Gourse, R.L., 1993. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 262: 1407‐1413. Rushing, B.G. and Long, S.R., 1995. Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti. J. Bacteriol. 177: 6952‐6957. Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold spring Harbor, N. Y. Sauviac, L., Philippe, H., Phok, K. and Bruand, C., 2007. An extracytoplasmic function sigma factor acts as a general stress response regulator in Sinorhizobium meliloti. J Bacteriol. 189: 4204‐4216. Schluter, J.P., Reinkensmeier, J., Barnett, M.J., Lang, C., Krol, E., Giegerich, R., Long, S.R. and Becker, A., 2013. Global mapping of transcription start sites and promoter motifs in the symbiotic alpha‐proteobacterium Sinorhizobium meliloti 1021. BMC Genomics. 14: 156. Scott, J.D. and Ludwig, R.A., 2004. Azorhizobium caulinodans electron‐transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation. Microbiology. 150: 117‐126. Shearwin, K.E., Callen, B.P. and Egan, J.B., 2005. Transcriptional interference‐‐a crash course. Trends Genet. 21: 339‐345. 145
Shine, J. and Dalgarno, L., 1974. The 3'‐terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 71: 1342‐1346. Simon, R., Quandt, J. and Klipp, W., 1989. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram‐
negative bacteria. Gene. 80: 161‐169. Smil, V., 2001. Enriching the earth. Fritz Haber and Carl Bosch, and the transformation of world food production. MIT press, Cambridge, MA. Soto, M.J., Sanjuan, J. and Olivares, J., 2001. The disruption of a gene encoding a putative arylesterase impairs pyruvate dehydrogenase complex activity and nitrogen fixation in Sinorhizobium meliloti. Mol Plant Microbe Interact. 14: 811‐815. Stowers, M.D., 1985. Carbon metabolism in Rhizobium species. Annu Rev Microbiol. 39: 89‐
108. Streeter, J.G., 1980. Carbohydrates in Soybean Nodules: II. Distribution of Compounds in Seedlings During the Onset of Nitrogen Fixation. Plant Physiol. 66: 471‐476. Teague, W.M. and Henney, H.R., Jr., 1976. Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum. Biochim. Biophys. Acta. 434: 118‐125. Terpolilli, J.J., Hood, G.A. and Poole, P.S., 2012. What determines the efficiency of N(2)‐fixing Rhizobium‐legume symbioses? Adv Microb Physiol. 60: 325‐389. Thapanapongworakul, N., Nomura, M., Shimoda, Y., Sato, S., Tabata, S. and Tajima, S., 2010. NAD+‐malic enzyme affects nitrogenase activity of Mesorhizobium loti bacteroids in Lotus japonicus nodules. Plant Biotechnol. 27: 311‐316 Thompson, J.D., Higgins, D.G. and Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position‐specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673‐4680. Toyoda, T., Suzuki, K., Sekiguchi, T., Reed, L.J. and Takenaka, A., 1998. Crystal structure of eucaryotic E3, lipoamide dehydrogenase from yeast. J. Biochem. 123: 668‐674. Ucker, D.S. and Signer, E.R., 1978. Catabolite‐repression‐like phenomenon in Rhizobium meliloti. J Bacteriol. 136: 1197‐1200. Udvardi, M.K., Yang, L.O., Young, S. and Day, D.A., 1990. Sugar and amino acid transport accross symbiotic membranes from soybean nodules. Mol. Plant Microbe Interact. 3: 334‐
340. 146
van der Rest, M.E., Frank, C. and Molenaar, D., 2000. Functions of the membrane‐associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Escherichia coli. J. Bacteriol. 182: 6892‐6899. van Rhijn, P. and Vanderleyden, J., 1995. The Rhizobium‐plant symbiosis. Microbiol Rev. 59: 124‐142. Vance, C.P., 2008. Carbon and nitrogen metabolism in legume nodules. In M. J. Dilworth, E. K. James, J. I. Sprent, & W. E. Newton (Eds.), Nitrogen‐Fixing Legumious Symbiosws (pp. 293‐
320). Dordrecht: Springer. Vassylyev, D.G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M.N., Borukhov, S. and Yokoyama, S., 2002. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution. Nature. 417: 712‐719. Walshaw, D.L., Wilkinson, A., Mundy, M., Smith, M. and Poole, P.S., 1997. Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum. Microbiology. 143: 2209‐2221. Waters, J.K., Hughes, B.L., 2nd, Purcell, L.C., Gerhardt, K.O., Mawhinney, T.P. and Emerich, D.W., 1998. Alanine, not ammonia, is excreted from N2‐fixing soybean nodule bacteroids. Proc. Natl. Acad. Sci. U S A. 95: 12038‐12042. Waters, J.K., Karr, D.B. and Emerich, D.W., 1986. Malate dehydrogenase from Rhizobium japonicum 3Ib‐143 bacteroids and Glycine max root‐nodule mitochondria. Biochemistry. 24: 6479‐6486. Watson, R.J., Chan, Y.K., Wheatcroft, R., Yang, A.F. and Han, S.H., 1988. Rhizobium meliloti genes required for C4‐dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid. J Bacteriol. 170: 927‐934. Westphal, A.H. and de Kok, A., 1988. Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene. Eur. J. Biochem. 172: 299‐
305. Wigneshweraraj, S., Bose, D., Burrows, P.C., Joly, N., Schumacher, J., Rappas, M., Pape, T., Zhang, X., Stockley, P., Severinov, K. and Buck, M., 2008. Modus operandi of the bacterial RNA polymerase containing the sigma54 promoter‐specificity factor. Mol Microbiol. 68: 538‐
546. Winkler, W.C. and Breaker, R.R., 2005. Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol. 59: 487‐517. Wise, D.J., Anderson, C.D. and Anderson, B.M., 1997. Purification and kinetic characterization of Haemophilus parasuis malate dehydrogenase. Arch. Biochem. Biophys. 344: 176‐183. 147
Wright, S.K., Zhao, F.J., Rardin, J., Milbrandt, J., Helton, M. and Furumo, N.C., 1995. Mechanistic studies on malate dehydrogenase from Escherichia coli. Arch. Biochem. Biophys. 321: 289‐296. Yoon, H. and Anderson, B.M., 1988. Kinetic studies of Haemophilus influenza malate dehydrogenase. Biochem. Biophys. Acta. 955: 10‐18. Young, J.P., Crossman, L.C., Johnston, A.W., Thomson, N.R., Ghazoui, Z.F., Hull, K.H., Wexler, M., Curson, A.R., Todd, J.D., Poole, P.S., Mauchline, T.H., East, A.K., Quail, M.A., Churcher, C., Arrowsmith, C., Cherevach, I., Chillingworth, T., Clarke, K., Cronin, A., Davis, P., Fraser, A., Hance, Z., Hauser, H., Jagels, K., Moule, S., Mungall, K., Norbertczak, H., Rabbinowitsch, E., Sanders, M., Simmonds, M., Whitehead, S. and Parkhill, J., 2006. The genome of Rhizobium leguminosarum has recognizable core and accessory components. Genome Biol. 7: R34. Yurgel, S.N. and Kahn, M.L., 2004. Dicarboxylate transport by rhizobia. FEMS Microbiol Rev. 28: 489‐501. Yurgel, S.N., Mortimer, M.W., Rice, J.T., Humann, J.L. and Kahn, M.L., 2013. Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library. Applied and environmental microbiology. 79: 2081‐2087. Zaitseva, J., Meneely, K.M. and Lamb, A.L., 2009. Structure of Escherichia coli malate dehydrogenase at 1.45 A resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun. 65: 866‐869. Zhang, Y., Aono, T., Poole, P. and Finan, T.M., 2012. NAD(P)+‐malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities. Applied and environmental microbiology. 78: 2803‐2812. 148