Oxygen-dependent
regulation of key
components in microbial
chlorate respiration
Miriam Hellberg Lindqvist
Faculty of Health, Science and Technology
Chemistry
DOCTORIAL THESIS | Karlstad University Studies | 2016:13
Oxygen-dependent
regulation of key
components in microbial
chlorate respiration
Miriam Hellberg Lindqvist
DOCTORIAL THESIS | Karlstad University Studies | 2016:13
Oxygen-dependent regulation of key components in microbial chlorate respiration
Miriam Hellberg Lindqvist
DOCTORIAL THESIS
Karlstad University Studies | 2016:13
urn:nbn:se:kau:diva-40698
ISSN 1403-8099
ISBN 978-91-7063-692-9
©
The author
Distribution:
Karlstad University
Faculty of Health, Science and Technology
Department of Engineering and Chemical Sciences
SE-651 88 Karlstad, Sweden
+46 54 700 10 00
Print: Universitetstryckeriet, Karlstad 2016
WWW.KAU.SE
Abstract
Contamination of perchlorate and chlorate in nature is primarily the
result of various industrial processes. The microbial respiration of
these oxyanions of chlorine plays a major role in reducing the
society’s impact on the environment. The focus with this thesis is to
investigate the oxygen-dependent regulation of key components
involved in the chlorate respiration in the gram-negative bacterium
Ideonella dechloratans. Chlorate metabolism is based on the action of
the enzymes chlorate reductase and chlorite dismutase and results in
the end products molecular oxygen and chloride ion. Up-regulation of
chlorite dismutase activity in the absence of oxygen is demonstrated
to occur at the transcriptional level, with the participation of the
transcriptional fumarate and nitrate reduction regulator (FNR). Also,
the chlorate reductase enzyme was shown to be regulated at the
transcriptional level with the possible involvement of additional
regulating mechanisms as well. Interestingly, the corresponding
chlorate reductase operon was found to be part of a polycistronic
mRNA which also comprises the gene for a cytochrome c and a
putative transcriptional regulator protein.
1
2
Tack
Jag är så oerhört tacksam för att jag har haft förmånen att arbeta vid
Karlstads universitet med så många härliga och inspirerande
människor. Det har varit en spännande och lärorik resa och jag har
många att tacka för arbetet som ligger till grund för denna
avhandling.
Ett stort tack till min handledare Thomas Nilsson för att du alltid tagit
dig tid att lyssna, kommentera och ge vägledning vad gäller både
forskning och olika uppdrag som jag har haft under min
doktorandtid. Ditt stöd och engagemang har varit väldigt värdefullt
för mig.
Jag vill även tacka min biträdande handledare Maria Rova som har
bidragit med så mycket kunskap till detta projekt. Tack för alla goda
råd och ditt stöd i mitt arbete på lab.
Tack Anna för all din hjälp och support genom åren! Jag är så oerhört
tacksam över att jag haft dig som doktorandkollega. Vad hade jag
gjort utan dig?
Jag vill också tacka alla övriga kollegor på min avdelning! Ett speciellt
tack till Micke för att du alltid fixar allt och ger så kloka råd. Mina
forna och nutida doktorandkollegor (och möjligtvis framtida
doktorander) Hannah, Ola, Emelie, Christina, Anna-Sofia, Natasja,
Erik, Alex, Sara och Maria, att ha fått dela doktorandtiden och dess
olika vedermödor med er har varit en ynnest. Tack för all peppning
när jag varit lite extra emotionell, alla trevliga fikastunder och djupa
forskningsdiskussioner (och ibland inte så vetenskapliga samtal)
under väntetider på lab.
Slutligen, ett stort tack till min familj! Mamma och pappa för att ni
alltid trott på mig. Er oerhörda kärlek betyder så mycket. Mina bröder
Gustaf och Christian med familj, det betyder så mycket att veta att ni
finns där i vått och torrt. Jag hoppas att jag inte alltid är en för bossig
storasyster.
3
Mina älskade små busfrön Siri och Milla. Tack för all den glädje och
värme ni sprider omkring er. Ni är det finaste som finns!
Per, min älskade livskamrat och bästa vän. Tack för att du finns vid
min sida. Jag älskar dig!
Miriam Hellberg Lindqvist
4
Table of contents
LIST OF APPENDED PAPERS ................................................................... 6
LIST OF ABBREVIATIONS......................................................................... 8
1. INTRODUCTION ..................................................................................... 9
1.1.
OXYANIONS OF CHLORINE ................................................................ 9
1.2.
BIOLOGICAL WASTEWATER TREATMENT ........................................... 10
1.3.
MICROBIAL ENERGY METABOLISM ................................................... 11
1.3.1. Aerobic and anaerobic bacteria .............................................. 11
1.4.
CHLORATE- AND PERCHLORATE-RESPIRING BACTERIA ...................... 12
1.4.1. The biochemical pathway of (per)chlorate reduction .............. 12
1.4.2. Genes involved in (per)chlorate reduction .............................. 15
1.5.
AIM OF PRESENT STUDY ................................................................. 18
2. THEORY ................................................................................................ 19
2.1. FROM DNA TO PROTEIN ..................................................................... 19
2.2. REGULATION OF GENE EXPRESSION ..................................................... 21
2.3. MOBILE GENETIC ELEMENTS................................................................ 23
3. METHODS USED IN PRESENT STUDY............................................... 24
3.1. POLYMERASE CHAIN REACTION ........................................................... 24
3.2. EXPRESSION ANALYSIS ....................................................................... 24
3.2.1. Reverse transcription polymerase chain reaction ..................... 24
3.2.2. Quantitative polymerase chain reaction ................................... 25
3.3. ENZYME ACTIVITY MEASUREMENTS ...................................................... 28
3.4. CREATION AND CLONING OF RECOMBINANT DNA .................................. 29
3.4.1. Reporter gene technology ........................................................ 30
4. RESULTS AND DISCUSSION .............................................................. 32
PAPER I ................................................................................................... 32
PAPER II .................................................................................................. 35
PAPER III ................................................................................................. 38
PAPER IV ................................................................................................. 44
5. CONCLUDING REMARKS .................................................................... 47
6. FUTURE PERSPECTIVE ...................................................................... 48
7. POPULÄRVETENSKAPLIG SAMMANFATTNING............................... 49
8. REFERENCES ...................................................................................... 54
5
List of Appended Papers
I.
Hellberg Lindqvist, M., Johansson, N., Nilsson, T., Rova, M.
2012. Expression of chlorite dismutase and chlorate reductase
in the presence of oxygen and/or chlorate as the terminal
electron acceptor in Ideonella dechloratans. Applied and
Environmental Microbiology. 78:4380-4385.
II.
Hellberg Lindqvist, M., Nilsson, T., Sundin, P., Rova, M.
2015. Chlorate reductase is co-transcribed with cytochrome c
and other downstream genes in the gene cluster for chlorate
respiration of Ideonella dechloratans. FEMS Microbiology
Letters. 362:fnv019
III.
Hellberg Lindqvist, M., Goetelen, T., Fors E., Nilsson T.,
Rova M. Anoxic induction of the chlorite dismutase gene of
Ideonella dechloratans is dependent of the fumarate and
nitrate reduction regulator, FNR. Submitted to Applied and
Environmental Microbiology
IV.
Hellberg Lindqvist, M., Nilsson, T., Rova M. An Fnr-type
transcriptional regulator is responsible for anoxic up-regulation
of chlorite dismutase in Ideonella dechloratans. Manuscript
Reprints of Paper I and Paper III were made with permission from
American Society for Microbiology. Reprint of Paper II was made with
permission from Oxford University Press.
6
My contribution to work in appended papers:
Paper I: I performed the final experimental work published in this
paper. Planning of experiments and preliminary results were
performed by M. Rova and N. Johansson. I wrote parts of the
manuscript.
Paper II: I did all the experimental work in this paper. I wrote parts
of the article.
Paper III: I performed all the experimental work in this paper except
for the mutagenesis and construction of plasmids pBBR1MCS-4lacZ_cld(mut1), pBBR1MCS-4-lacZ_cld(mut2) and pBBR1MCS-2lacZ_cld(82), which were created by T. Goetelen and E. Fors. I also
wrote the first version of the manuscript.
Paper IV: I performed all the planning and experimental work in
this manuscript and part of the writing.
7
List of abbreviations
ArcA
Regulator protein, part of the two-component anoxic
redox control system, ArcBA
ATP
Adenosine triphosphate
cDNA
Complementary deoxyribonucleic acid
Cld
Chlorite dismutase
Clr
Chlorate reductase
CRB
Chlorate reducing bacteria
DNA
Deoxyribonucleic acid
FNR
Regulator protein for the fumarate and nitrate reduction
gDNA
Genomic deoxyribonucleic acid
mRNA
Messenger ribonucleic acid
PCR
Polymerase chain reaction
Pcr
Perchlorate reductase
qPCR
Quantitative polymerase chain reaction
PRB
Perchlorate reducing bacteria
qRT-PCR Quantitative real-time polymerase chain reaction
RNA
Ribonucleic acid
RNAP
RNA polymerase
RT-PCR
Real-time polymerase chain reaction
TSS
Transcription start site
8
1. Introduction
The use of potentially harmful chemicals also includes the
responsibility of appropriate management of these substances. Many
human activities such as agriculture, transport and industry
contribute to pollution of the biosphere. Whether the contaminants
are placed in the water, air or soil, they can cause damage to
ecosystems and can persist in nature for long time. It is very
important to develop sustainable wastewater management systems to
reduce the society’s impact on the environment.
1.1.
Oxyanions of chlorine
The present work deals with oxyanions of chlorine. Chlorine can
combine with oxygen and form four different oxyanions; perchlorate
−
−
−
(ClO−
4 ), chlorate (ClO3 ), chlorite (ClO2 ) and hypochlorite (ClO ). The
term (per)chlorate is used here for perchlorate and chlorate.
Occurrence of these oxyanions in the environment is in general of
anthropogenic origin such as by-products from industrial processes,
disinfectants and herbicides.
The main source of chlorate in the environment originates from the
pulp- and paper industries when bleaching pulp with chlorine dioxide
(Bergnor et al., 1987). Chlorate has historically been used as an
herbicide and is also produced as a by-product in drinking water
disinfection with hypochlorite. Chlorate is toxic to aquatic organisms,
such as brown algae and marine microalgae (Rosemarin et al., 1994;
van Wijk and Hutchinson, 1995), and can cause oxidative damage to
red blood cells in mammals when continuously exposed (Condie,
1986; Couri et al., 1982).
The oxyanion perchlorate has emerged as a contaminant in nature
due to the use of perchlorate salts in products such as rocket fuel,
missiles, airbags and fireworks. Perchlorate has been widely studied
due to its inhibitory effect on iodine uptake in the thyroid gland in
humans (Wolff, 1998).
9
Both chlorate and perchlorate are chemically stable in water and
occurrence in nature has become a problem due to their toxicity and
long range aqueous migration capacity. Since the presence of
oxyanions of chlorine in nature poses a threat to the environment and
is a potential risk to human health (Coates et al., 1999; Renner, 2003;
Urbansky, 2002; WHO, 2005), it is important with efficient
wastewater treatment when using these compounds.
Until recently, the only known natural occurrence of perchlorate in
nature was as minor component in saltpeter deposits in Chile
(Urbansky, 2002). However, it seems like both chlorate and
perchlorate are produced during atmospheric processes (Kounaves et
al., 2010; Rao et al., 2010), which can explain the ubiquitous presence
of microorganisms metabolizing these chemicals on Earth.
1.2.
Biological wastewater treatment
For the removal of (per)chlorates different approaches have been
tried, one of which is biological wastewater treatment (Malmqvist and
Welander, 1992). This method has been shown to be efficient and
simply involves confining naturally occurring microorganisms, mainly
bacteria, which feed on chemicals in the wastewater. Although the
process has been known for almost a century (Åslander, 1928) it is
only in the recent decades that researchers have studied this process
in more detail. Already in 1954 Bryan and Rohlich showed that
microorganisms can reduce chlorate (Bryan and Rohlich, 1954), and
today over 50 bacterial species capable of growth using chlorate or
perchlorate have been identified (Achenbach et al., 2001; Bruce et al.,
1999; Coates and Achenbach, 2004; Coates et al., 1999; Malmqvist et
al., 1994; Rikken et al., 1996; Wallace et al., 1996; Waller et al., 2004;
Wolterink et al., 2002; Wolterink et al., 2005).
A major drawback with this technique is the conflict between effective
removal of organic matter and effective removal of (per)chlorates in
the wastewater. The efficient removal of organic matter requires
oxygen. However, if the wastewater contains large amounts of oxygen,
the microorganisms able to perform (per)chlorate reduction will use
the oxygen instead of reducing chlorate (Yu and Welander, 1994).
10
Different approaches are used in pulp mills today to generate
oxygen-limited conditions for chlorate remediation.
1.3.
Microbial energy metabolism
Microorganisms need energy to support anabolic processes, such as
the synthesis of biomolecules, and growth. Energy is obtained from
catabolic processes where complex molecules are decomposed into
smaller units. The energy provided from the catabolic processes can
be stored as chemical energy in the form of adenosine triphosphate
(ATP) and be used to drive energy-consuming anabolic reactions.
To generate ATP, different approaches have evolved, including
aerobic respiration, anaerobic respiration and fermentation. In the
electron transport chain, electrons donated by an electron donor, are
passed along a series of membrane-bound enzyme complexes to a
terminal electron acceptor. The transfer of electrons is coupled to the
translocation of protons across the inner membrane, producing a
proton gradient which is used to drive the synthesis of ATP.
Microorganisms can utilize a wide range of terminal electron
acceptors for their respiration. In aerobic respiration the terminal
electron acceptor is oxygen. In the absence of oxygen this pathway is
not possible. The ability to use alternative electron acceptors, such as
fumarate, nitrate and chlorate, allows electron transport when the
access to oxygen is limited. The use of respiration using electron
acceptors other than oxygen is called anaerobic respiration. These
alternative electron acceptors have lower reduction potentials than
oxygen and, hence, less energy is produced during anaerobic
compared to aerobic respiration. Therefore oxygen is often the
preferred electron acceptor in respiration.
1.3.1. Aerobic and anaerobic bacteria
The need for, or tolerance or sensitivity to molecular oxygen (O2)
varies widely among microorganisms. Bacteria that use oxygen as
terminal electron acceptor for energy metabolism are called aerobes.
If the requirement for oxygen is absolute, they are called obligate
aerobes. The opposite, obligate anaerobes, are unable to use oxygen
11
and cannot grow in the presence of oxygen. Facultative anaerobes, on
the other hand, use oxygen as the primary electron acceptor when it is
present but alternative electron acceptors when the supply of oxygen
is low. The switch from aerobic to anaerobic respiration can require
synthesis of alternative terminal reductases. There are also bacteria
referred to as microaerobes, that require oxygen but only at low levels.
1.4.
Chlorate- and perchlorate-respiring bacteria
Early investigations of the chlorate reduction microbiology suggested
that this action was performed by nitrate-respiring organisms and
chlorate was assumed to be an alternative substrate for the nitrate
reductase (de Groot and Stouthamer, 1969). This, however, could not
explain the existence of chlorate reductase C in Proteus mirabilis, an
enzyme only able to use chlorate as a substrate (Oltmann et al.,
1976a). The nitrate and chlorate reduction pathways in different
prokaryotes has been found to have many similarities (Oosterkamp et
al., 2011) and membrane-bound respiratory nitrate reductases have
been shown to utilize chlorate as an alternative electron acceptor (Bell
et al., 1990).
Today it is known that specialized microorganisms have evolved that
are able to couple growth to (per)chlorate metabolism. These bacteria
are phylogenetically diverse, with representatives in the α-, β-,
γ- and ε-Proteobacteria (Bardiya and Bae, 2011; Coates et al., 1999;
Nilsson et al., 2013). Common to all strains are that they are
facultative anaerobes or microaerobes.
The model organism chosen for this study is Ideonella dechloratans
(Malmqvist et al., 1994) which is a gram-negative rod and belongs to
the β-subclass of Proteobacteria. I. dechloratans is reported to be
cytochrome c oxidase positive (Malmqvist et al., 1994) and can utilize
both oxygen and chlorate as terminal electron acceptors.
1.4.1. The biochemical pathway of (per)chlorate reduction
During the biodegradation of perchlorate and/or chlorate, chlorite
and oxygen are sequentially produced by the oxidation of acetate or
12
other electron donor illustrated in figure 1. Perchlorate reducing
bacteria (PRB) can reduce both perchlorate and chlorate to chlorite
(step 1 and 2) with the enzyme perchlorate reductase (Pcr) (Kengen et
al., 1999), while chlorate respiring bacteria (CRB) can only reduce
chlorate (step 2) and use the enzyme chlorate reductase (Clr)
(Danielsson Thorell et al., 2003; Wolterink et al., 2003). Both PRB
and CRB possess a chlorite dismutase (Cld) that can decompose the
chlorite to chloride ions and molecular oxygen (step 3) (van Ginkel et
al., 1996).
For the reduction of chlorate six electrons are required, whereas the
reduction of perchlorate requires eight electrons. The free energy
from the redox reactions in the electron transport chain creates a
proton gradient over the cell membrane which provides the driving
force for ATP production by an ATP synthase. The molecular oxygen
produced during this process is assumed to further be used as
electron acceptor due to the facultative anaerobic nature of bacteria
able to perform dissimilatory (per)chlorate reduction (Rikken et al.,
1996).
Figure 1 The (per)chlorate is reduced via a multi-step pathway. Perchlorate
reducing bacteria can reduce both perchlorate and chlorate (step 1 and 2)
with a perchlorate reductase enzyme, while chlorate reducing bacteria,
which use the enzyme chlorate reductase, only can reduce chlorate
(step 2). In both perchlorate and chlorate reducing bacteria a chlorite
dismutase can be found that enables the last step (step 3) in the
(per)chlorate metabolism.
13
The chlorate metabolism in the CRB I. dechloratans is catalyzed by
the enzymes chlorate reductase, Clr (Danielsson Thorell et al., 2003),
and chlorite dismutase, Cld (Stenklo et al., 2001) and takes place in
the periplasm as illustrated in figure 2. The transport of donated
electrons includes both a membrane-bound terminal cytochrome cbb3
oxidase and a periplasmic cytochrome c able to function as electron
carrier (Smedja Bäcklund et al., 2009; Smedja Bäcklund and Nilsson,
2011).
Figure 2 The proposed electron transport in I. dechloratans. The reduction of one
chlorate molecule requires six electrons passed from the quinone pool; two
are used by the chlorate reductase and the remaining four is passed on to
the membrane-bound terminal oxidase and is used for the reduction of O2
to H2O. X illustrates the suggested presence of additional cytochromes
able to donate electrons to the terminal oxidase, but not to chlorate
reductase. The accumulation of protons in the periplasmic space is used as
driving force for the ATP synthase in the production of ATP.
14
During anaerobic conditions the bacteria must be capable of using the
oxygen produced in the chlorite dismutase reaction. Oxygen at higher
concentrations, though, has been shown to inhibit (per)chlorate
reduction (Chaudhuri et al., 2002; Song and Logan, 2004).
1.4.2. Genes involved in (per)chlorate reduction
The organization and transcriptional direction of the genes involved
in (per)chlorate reduction differs between the bacterial strains
possessing this trait. In PRB the operon pcrABCD can be found,
encoding perchlorate reductase, and in CRB the operon clrABDC,
encoding chlorate reductase, is found instead. Both PRB and CRB
possess a gene encoding the chlorite dismutase (cld) which can be
located either upstream or downstream the pcr or clr operon.
The capability of (per)chlorate reduction has been shown to be subject
of horizontal gene transfer over phylogenetic boundaries (Clark et al.,
2013; Melnyk et al., 2011). This can explain that the key enzymes for
(per)chlorate respiration have a wide dispersal over such a diverse
group of prokaryotes. The genes involved in the reduction of
(per)chlorate in PRB and reduction of chlorate in CRB are flanked by
insertion sequences and have shown to be part of genomic islands.
The concept of genomic islands and the relation to horizontal gene
transfer will be discussed in more detail in section 2.2. The variability
of gene content and orientation in the cluster together with the
diversity of insertion sequences between organisms indicate that
these mobile genetic element have evolved multiple times (Clark et
al., 2013; Melnyk et al., 2011). However, except for the key enzymes
Pcr/Clr and Cld not much is known of the functionality of other genes
included in these clusters.
1.4.2.1.
Perchlorate reductase and chlorate reductase
Several perchlorate and chlorate reductases have been isolated and
studied from different bacterial strains (Danielsson Thorell et al.,
2003; Giblin and Frankenberger Jr., 2001; Okeke and Frankenberger,
2003; Steinberg et al., 2005; Wolterink et al., 2003) and have been
found to originate from different classes of enzymes regarding
15
structure and biochemical aspects (Bardiya and Bae, 2011; Nilsson et
al., 2013). The perchlorate reductases known today consist of two
different subunits (pcrAB) compared to the identified chlorate
reductases which are built up of three different subunits (clrABC).
Phylogenetic analysis of PcrA (Bender et al., 2005) and ClrA
(Danielsson Thorell et al., 2003) have shown that these proteins are
members of the dimethylsulfoxide (DMSO) reductase family, which in
turn belongs to the family of molybdopterin-containing enzymes
(Kisker et al., 1999). DMSO reductases can degrade oxyanions and the
reaction can be summarized as follows: XO + 2H + + 2e− ⇄ X + H2 O
(McEwan et al., 2002). The transfer of one oxygen atom is coupled to
the relocation of two electrons between substrates and other
cofactors, such as iron-sulfur clusters and heme (Hille, 1999; Kisker et
al., 1999).
The pcrD and clrD genes included in each of the clusters likely encode
chaperone-like proteins thought to be involved in the insertion of the
molybdopterin cofactor (Bender et al., 2005; Danielsson Thorell et al.,
2003) and the pcrC gene, found in PRB, is suggested to encode a
cytochrome c involved in the electron transport (Bender et al., 2005).
Chlorate reductase has previously been isolated from I. dechloratans
(Danielsson Thorell et al., 2003). The purified protein was found to
contain molybdopterin and iron. An optical spectrum of the protein
indicated presence of heme b, which later could be confirmed as part
of the cytochrome b subunit, encoded by clrC (Karlsson and Nilsson,
2005).
1.4.2.2.
Chlorite dismutase
Chlorite dismutase is a heme b-containing protein that has the ability
to catalyze the decomposition of chlorite into molecular oxygen and
chloride ions (Schaffner et al., 2015; van Ginkel et al., 1996) and can
be found in bacteria capable of (per)chlorate respiration (Mehboob et
al., 2009; Stenklo et al., 2001; van Ginkel et al., 1996). The enzyme is
vital for the organism due to the high toxicity of chlorite to bacterial
16
cells (de Groot and Stouthamer, 1969; Oltmann et al., 1976b) and for
the production of oxygen that can be utilized by a terminal oxidase.
The reaction usually associated with the formation of a novel
oxygen-oxygen bond is photosynthetic water oxidation and the
production of molecular oxygen catalyzed by the Cld enzyme
(Goblirsch et al., 2010; Streit and DuBois, 2008).
The I. dechloratans Cld enzyme, located in the periplasm, has
previously been isolated and purified (Stenklo et al., 2001) followed
by the cloning and characterization of the cld gene (Danielsson
Thorell et al., 2002).
1.4.2.3.
Additional genes found in (per)chlorate respiring clusters
In the gene clusters for (per)chlorate reduction different families of
c-type cytochromes have been identified (Bohlin et al., 2010; Melnyk
et al., 2011). These are predicted to be involved in the electron
transport of (per)chlorate reduction, but none of them have as yet a
validated function in vivo. The putative cytochrome c gene (cyc) in
the chlorate respiring cluster of I. dechloratans has previously been
heterologusly expressed in E. coli, but it could not be verified if the
reconstituted protein could donate electrons to purified Clr (Bohlin et
al., 2010). Instead another chromosomally encoded cytochrome c,
denoted cyt c-Id1, was shown to deliver electrons to chlorate
reductase (Smedja Bäcklund and Nilsson, 2011), as illustrated in
figure 2.
Along with the cyc gene also a mobB gene and an arsR gene is found
in the chlorate respiring cluster in I. dechloratans (Bohlin et al., 2010;
Clark et al., 2013). The mobB gene is thought to encode a gene
product that is involved in the molybdenum co-factor biosynthesis
(Iobbi-Nivol et al., 1995) and arsR is a putative transcriptional
regulator usually associated with sensing metals, but has been shown
to be involved in other non-metal signals as well (Gao et al., 2012).
17
1.5.
Aim of present study
The main focus of the research presented in this thesis was to
investigate the oxygen dependent regulation of proteins involved in
the anaerobic respiration of chlorate in I. dechloratans. It is
important with deeper understanding regarding the regulation of
these corresponding genes in response to different growth conditions
for an efficient use of chlorate-reducing bacteria in bioremediation
applications.
The aims with the subprojects in this thesis have been to:
• Investigate the effect of oxygen on the expression of enzymes
involved in chlorate respiration (paper I).
• Investigate the expression and regulation of additional genes
(cyc and mobB) present in the chlorate respiring genomic
island (paper II).
• Investigate potential regulatory sequences using a reporter
system to obtain more detailed understanding of the
mechanisms involved in the anaerobic induction of enzymes
responsible for chlorate respiration (paper III and IV).
18
2. Theory
The adaption to different growth conditions requires that the cell can
regulate its content of metabolic enzymes to suit current needs. The
specifications for all cellular proteins are encoded in the
corresponding genes, which together constitute the genome.
2.1. From DNA to protein
When a cell requires a specific protein, the DNA sequence encoding
the corresponding gene is copied into a messenger RNA (mRNA) in a
process called transcription. Following transcription, translation, i.e.
the reading of the mRNA to make a protein, begins.
Bacterial transcription is carried out by the multi-subunit RNA
polymerase (RNAP). The subunits α2ββ'ω form the RNAP core
enzyme which is capable of DNA-dependent RNA synthesis. However,
the core enzyme cannot locate genes by itself and needs to interact
with a sigma (σ) factor to form the RNAP holoenzyme (α2ββ'ωσ)
capable of initiation of transcription. Once RNA synthesis has been
initiated, however, the σ factor dissociates and the core enzyme
proceeds with RNA transcription.
The σ factor recognizes a sequence upstream of the gene known as
promoter. Promoters are located close to the transcription start site
(TSS) of genes. The TSS is the first nucleotide of the RNA transcript
and is numbered +1, nucleotides upstream of the TSS are given
negative numbers. Bacteria often have a set of alternative σ factors
which can recognize different promoter elements. The association of
appropriate alternative σ factor with the RNAP core enzyme enables
the redirection of transcription initiation from different promoters in
response to environmental changes. The majority of known σ factors
today belong to the σ70-family. Four different DNA sequence elements
have been found which has been shown to be important for promoter
recognition by the σ70 factor.
These are the -10 element,
the -35 element, the upstream promoter (UP) element and the
extended -10 element (Browning and Busby, 2004; Paget and
Helmann, 2003). The consensus sequences, i.e. the calculated order
19
of most frequent nucleotides, of these elements have been determined
by the analysis of numerous prokaryotic promoters.
The -10 and -35 element are located about 10 and 35 base pairs,
respectively, upstream of the TSS (figure 3). The consensus sequence
for the -10 element is TATAAT, where the last T and the first A is the
most conserved bases and seems to be most important for promoter
melting (Heyduk and Heyduk, 2014). The most conserved nucleotides
in the -35 element seem to be the first three bases (TTG) of the
consensus TTGACA. For the spacer between the -35 and -10 element
there is no known consensus sequence, but a consensus length of
usually 17 bp. The -10 extended motif (TGn) can be found in a number
of prokaryotic promoters and is located direct upstream of the -10
element. Presence of this DNA motif can be particular important for
promoters that contain a -35 element that not match the consensus
sequence that well (Dombroski, 1997; Keilty and Rosenberg, 1987;
Kumar et al., 1993) or has a longer spacer between the -10 and -35
element (Mitchell et al., 2003). In some promoters an UP element can
be found which comprises an AT-rich region located around -40
to -60 bp relative the TSS.
Figure 3 A typical structure of a σ70 bacterial promoter with -10 and -35 regions.
Promoters can also contain an extended -10 element and an upstream
promoter (UP) element. The consensus sequences for the -10 (TATAAT),
extended -10 (TGn) and -35 (TTGACA) elements are shown. The
transcription start site (TSS) are marked as +1.
The RNA polymerase is designed to transcribe RNA, but not all RNA
molecules need to be synthesized in the equal amounts. For a fully
function promoter it is not necessary with presence of all four
upstream elements, but the combination of these sequences and
20
similarity to the known consensus determines the rate at which the
RNAP holoenzyme forms a stable initiation complex with the
promoter. A variation in the consensus sequence of just one
nucleotide can decrease the rate of binding dramatically. The result is
that RNA polymerase will recognize some promoters well (referred to
as a strong promoter) and others less well (referred to as a weak
promoter).
In bacteria, the mRNA can contain more than one cistron, i.e. a region
of DNA that encodes a polypeptide. Such mRNAs are called
polycistronic mRNAs (Snyder and Champness, 2003) and can be
regulated in such fashion that each gene in the mRNA is translated
into an individual polypeptide independently. The regions between
the cistrons can be very short and the open reading frames may even
overlap. If the polycistronic mRNA contains more than one ribosome
binding site, it is possible with simultaneous translation of more than
one region of the mRNA.
2.2. Regulation of gene expression
Bacteria use their genes very efficiently and do not transcribe mRNA
for proteins that are not needed at a certain moment. The regulation
of gene expression can occur by a variety of mechanisms and can take
place at any step in the production of a functional protein, e.g. at the
transcriptional, translational or post-translational level.
In addition of transcriptional control by different σ factors, bacteria
can also regulate transcription by proteins which act either as
up-regulators or down-regulators of genes (Barnard et al., 2004;
Browning and Busby, 2004; Lee et al., 2012). Some activator proteins
bind to specific sites on DNA and then recruits the RNA polymerase
to the target promoter by protein-protein interactions (Ptashne and
Gann, 1997). Alternative mechanisms instead involve activator
proteins that change the conformation of the target promoter and
induce binding of the RNA polymerase (Brown et al., 2003). Another
possibility is by pre-recruitment which involves the formation of an
activator-RNA polymerase complex before binding to the target DNA
promoter (Griffith et al., 2002; Martin et al., 2002).
21
The fumarate and nitrate reduction (FNR) protein of E. coli is a
transcriptional regulator, encoded by the fnr gene and is required,
directly or indirectly, for the expression of numerous genes involved
in the anaerobic metabolism (Crack et al., 2004; Fleischhacker and
Kiley, 2011). The active FNR protein acts as a homodimer and each
subunit has a DNA-recognition helix in the C-terminal that interacts
with the sequence TTGAT (Eiglmeier et al., 1989) and binds to the
DNA consensus sequence TTGATNNNNATCAA, referred to as the
FNR-box. Exposure to oxygen converts the protein-bound [4Fe-4S]2+
cluster to its [2Fe-2S]2+ form which decreases dimerization and
causes loss of the specific DNA binding ability (Khoroshilova et al.,
1997). FNR homologues have been found in a variety of taxonomically
diverse bacteria, not all using the same signaling mechanism as the
E. coli FNR protein (Spiro, 1994).
Another transcriptional regulator is the ArcA-protein which is part of
the two-component anoxic redox control system, ArcBA (Bekker et
al., 2010). ArcB is a sensory kinase that phosphorylates and, hence,
activates the cytoplasmic response regulator ArcA during
oxygen-limited conditions. The active form of ArcA (ArcA~P) then
binds to DNA and can either repress the genes of aerobic metabolism
during anaerobic conditions (Unden and Bongaerts, 1997) or activate
the expression of operons encoding enzymes important for the
adaption to microaerobic or anaerobic conditions (Lynch and Lin,
1996; Nesbit et al., 2012).
The transcription of a gene into an mRNA does not automatically
mean production of the corresponding protein. Degradation of mRNA
occurs all the time by ribonucleases and the lifetime of individual
RNAs in the bacteria have shown to be dependent on the
RNA-ribosome association (Deana and Belasco, 2005). The
production of a functional protein can also be regulated at the
translational or post-translational level by for example translational
blocking by antisense regulation, in which a short complementary
RNA binds to the mRNA and prevent binding to the ribosome or
create a target for mRNA cleavage by ribonucleases (Thomason and
Storz, 2010).
22
2.3. Mobile genetic elements
Mobile genetic elements are DNA segments that can move within or
between genomes. Horizontal gene transfer refers to the movement of
genes between different species of bacteria and has played a major
role for bacterial evolution (Gogarten and Townsend, 2005; Ochman
et al., 2000) and can be mediated by several mechanisms (Frost et al.,
2005; Holmes and Jobling, 1996; Thomas and Nielsen, 2005). The
acquisition of certain genes can enhance the survival chances for a
bacterium, as for example with antibiotic resistance or virulence
genes (Juhas, 2015; Rice, 2002).
A transposable element, or transposon, is a mobile element that
encodes enzymes which enable it to translocate within the same or to
another DNA molecule (Mahillon and Chandler, 1998) and can be
found in both eukaryotes and prokaryotes. The simplest transposable
elements are the so-called insertion sequences or IS elements
(Mahillon and Chandler, 1998). If two IS elements with the ability to
transpose are located close to each other, the intervening DNA
sequence becomes mobile. These entities are termed composite
transposons (Wagner, 2006) and contain one or more gene(s) in
addition to those needed for transposition.
The term genomic island is used for a region in the genome distinct
from its surroundings, often with an origin in horizontal gene transfer
(Bellanger et al., 2014). Examples of features used to recognize
genomic islands are different GC-content and unusual codon usage
compared to the chromosome of the host (Bellanger et al., 2014;
Juhas et al., 2009). They are often relatively large (10-200 kb)
(Hacker and Kaper, 2000) and can contain genes which enhance the
fitness of the host such as novel metabolic pathways or resistance to
antibiotics (Dobrindt et al., 2004). Some genomic islands may have
been translocated by transposase or recombinase genes encoded by
the island itself, but other mechanisms are known as well (Bellanger
et al., 2014; Juhas et al., 2009).
23
3. Methods used in present study
3.1. Polymerase chain reaction
DNA with a known sequence can be conveniently and rapidly
multiplied in vitro using the polymerase chain reaction (PCR). This
method can be used both to detect and to amplify specific sequences
for the construction of recombinant DNA molecules with starting
from as little as a single copy.
To be able to perform a PCR one needs template DNA, primers,
heat-stable DNA polymerase (usually Taq polymerase) and free
nucleotides. The amplification then is carried out with repeated cycles
of heat denaturation of the DNA, primer hybridization and extension
of the template DNA. The amount of the specific DNA sequence
increases exponentially since amplified products from the previous
cycle serves as template for the next cycle of amplification. Typically
20 to 30 cycles is enough to generate sufficient amounts of DNA to be
detected in a subsequent agarose gel electrophoresis or used for
recombinant cloning. The applications of the PCR are numerous and
have led to a large number of variants of this method.
3.2. Expression analysis
The expression of genes is carefully controlled by bacteria and can be
increased or decreased in response to environmental changes. To
provide insights into gene function it is important to understand
where and when specific genes are used. Today several methods are
known that can be used to quantitatively measure the mRNA level
from a certain gene. Analysis of expression can also be determined by
directly measure protein levels.
3.2.1. Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction (RT-PCR) is a
method performed with RNA as template which is reversed
transcribed during the process into an equivalent amount of
complementary DNA (cDNA) by the enzyme reverse transcriptase
(RT). RNA is extremely susceptible to degradation and can be difficult
24
to analyze. The ability to synthetize cDNA from an RNA template
opens the possibility to study RNA with the same methods used for
the more thermostable DNA molecule.
RT-PCR can be performed with sequence-specific primers, oligo(dT)
primers or random hexamer primers, alternatively with combination
of these. The use of sequence-specific primers offer the greatest
specificity but a new cDNA synthesis must be performed for each gene
to be studied. If the purpose is to synthesize large pools of cDNA,
other primer options are more suitable. Oligo(dT) primers can be
used for eukaryotic mRNA that have a 3´- poly(A) tail. For
non-polyadenylated RNA, such as bacterial RNA, random hexamer
primers are the ideal choice to produce a large pool of cDNA
molecules. The mixture of random primers consists of
oligonucleotides representing all possible hexamer sequences. These
primers can bind to all types of RNA molecules and gives random
coverage to all regions of the RNA. This gives rise to a cDNA pool with
various lengths of cDNA.
In present study both random hexamer primers and sequence-specific
primers have been used to synthesize cDNA from bacterial total RNA
preparations.
3.2.2. Quantitative polymerase chain reaction
The PCR reaction can be used for the quantitation of the amount of
template present in a sample. In this case, it is not sufficient to
analyze the end product after temperature cycling. Instead, the
reaction is followed and quantified in real time by the use of a
fluorescent reporter that interacts with double stranded DNA
molecules. It is possible to use either DNA or RNA as template. When
using RNA as template the quantitative PCR (qPCR) must be
preceded by a RT-PCR step. Combining these two methods yields
quantitative reverse-transcription PCR (qRT-PCR) which can be
performed in one or two steps (Bustin, 2000).
To validate that the signal obtained from the target are from the
specific amplicon to be observed, it is important to include certain
25
controls in the PCR. Such controls are the no-template control (NTC)
and the no-reverse transcriptase control (no-RT) which is required for
qRT-PCR reactions and is used to verify successful genomic DNA
(gDNA) removal. Carryover of gDNA in RNA samples is a concern
when determining expression levels. If gDNA is present in the
samples, it can be co-amplified with the target transcript of interest
and give rise to invalid data. Therefore, it is of utmost importance to
include the no-RT control in qRT-PCR reactions.
When the reaction is followed in real time, progress curves such as
those seen in figure 3 are obtained. Initially, the amount of product
and the fluorescence increase exponentially, and then level off as DNA
polymerase activity and the supply of primers becomes limiting.
Figure 4 The amplification curve for target and reference DNA in quantitative
PCR. The fluorescence signal from each sample is plotted against cycle
number. To obtain the relative expression level (∆CT), the target CT value
is normalized to the reference CT value.
The threshold cycle (CT ) is defined as the point at which the
fluorescence of the reporter dye increases over an arbitrary placed
threshold. It is a relative measure of the amount of target nucleic acid
26
in a sample provided that the threshold is chosen so that it is crossed
during the exponential phase of amplification.
Depending on the goal with the experiment there are different ways to
determine the appropriate quantification method. We have studied
the relative expression of genes at different growth conditions. The
relative expression level (∆CT ) is obtained when normalizing the CT
value from the target to the CT value from a reference gene. Genes
with constant expression levels, so called house-keeping genes, are
often good choices as reference genes. In our studies we have used
a gene for 16S ribosomal RNA as normalization gene. The variability
of the reference gene value represents the cumulative errors that can
occur during the different stages of the procedure including variations
in extraction yield, variations in reverse-transcription yield and
efficiency of amplification (Bustin et al., 2009). Hence, the
normalization of the target gene to the reference gene enables direct
comparisons of the same target in different samples.
In our gene expression studies we have used comparative
quantification to calculate the fold difference by the 2−∆∆CT method
(Livak and Schmittgen, 2001) (Eq. 1 and 2). This method can be used
when comparing a target gene of interest in one sample (growth
condition A) with the same target gene in another sample (growth
condition B). The results are presented as fold- up or down-regulation
of the growth condition A compared to growth condition B. Each
target gene is normalized to the reference gene.
∆∆CT = �CT target A − CT reference A � − �CT target B − CT reference B � (Eq. 1)
Fold difference of relative expression levels = 2−∆∆C T
(Eq. 2)
Equation 2 can be used provided that the reaction efficiency for all
target and reference genes is as similar as possible. The efficiency of a
PCR reaction depends on several factors including choice of primers,
master mix and sample quality and can affect the CT value. If the
27
reaction is 100 % efficient, the amount of target sequence is doubled
in each cycle. In practice the efficiency may be lower and it is then
important for quantitation that the efficiency for amplification of
target and reference sequences are known. If there is a variation in
efficiency between targets it is possible to use alternative quantitative
models that correct for amplification differences (Pfaffl, 2001).
3.3. Enzyme activity measurements
Enzyme activity measurements have been performed to compare the
activity of the two enzymes Clr and Cld during different growth
conditions.
The Clr activity was determined colorimetrically by following the
chlorate dependent oxidation of reduced methyl viologen (MV) under
anaerobic conditions. In our measurements the assay mixture
consisted of a bis-tris solution, MV and periplasmic extracts
containing Clr enzyme. The MV is reduced by the addition of
dithionite and when a stable absorbance is reached, the reaction is
started by the addition of chlorate. In its reduced form, the otherwise
colorless MV is blue with an absorption maximum at A600 nm. The
oxidation of MV is monitored spectrophotometrically at 600 nm and
the linear decrease in absorbance can be used to calculate the Clr
activity. Values were normalized to the total protein content, and one
unit of activity is defined as the amount of enzyme required to oxidize
1 µmol MV min-1 mg protein-1.
The Cld activity was measured by following chlorite-dependent
oxygen evolution using a Clark-type electrode. Periplasmic extracts
(including Cld enzyme) and phosphate buffer with the addition of
EDTA was added to the reaction chamber and the reaction was
started by the addition of chlorite. The time course of oxygen
evolution in this assay is non-linear, so it is important that the initial
reaction rate is used to calculate the enzyme activity. Also in this assay
the samples was normalized to the total protein content. The enzyme
activity is expressed as mmol O2 produced min-1 mg protein-1.
28
3.4. Creation and cloning of recombinant DNA
The definition of recombinant DNA is an artificially created DNA
molecule in which DNA sequences have been brought together
forming a new molecule not normally found in nature. Recombinant
DNA technology enables individual fragments of DNA to be inserted
into a vector, such as bacterial plasmids or bacteriophages, and
amplified in bacteria. The reproduction of the recombinant DNA
inside the host cell contributing to the production of many copies of
the specific sequence is known as cloning. This technique has
numerous practical applications and enables the expression of genes,
and hence produces proteins, not originally found in the organism.
The basic procedure is to obtain many copies of the DNA to be
inserted in the vector and this can be achieved with PCR. The DNA is
then cut with two different restriction enzymes followed by ligation
with a vector cut with the same set of restriction enzymes. By the use
of two different restriction enzymes, it is possible to control the
direction of the fragment when inserted into the vector. A thus
constructed recombinant molecule can be introduced into bacterial
cells in different ways. A common method is by transformation of
chemically competent cells. Typically these cells are exposed to
divalent cations (often calcium chloride) under cold conditions and
thereafter heat shocked, this makes the bacteria’s cell membrane
more permeable and increases the likelihood that the bacteria take up
the new DNA molecule. Another method is electroporation, in which
an electric field is applied to cells, creating holes in the bacterial
membrane and enables the bacteria to take up the vector (Weaver and
Chizmadzhev, 1996).
Transformation is a highly inefficient process. Vectors must therefore
contain selectable markers to allow the selection of transformed
organisms. Common selective markers are genes that confer
resistance to antibiotics.
It is possible to co-transform a bacterium with more than one
plasmid. However, if two plasmids use the same mechanism for
replication, they belong to the same incompability group and cannot
co-exist in the same bacterium (Bergquist, 1987). If the two plasmids
29
belong to different incompability groups, the regulation of plasmid
replication, and hence copy numbers of the plasmids, are independent
and both can be stably maintained in the population.
3.4.1. Reporter gene technology
There are numerous different vectors available. Expression vectors
are designed in such way that cloned fragment is inserted
downstream of a promoter in the vector. The cloned insert can then,
hopefully, often be transcribed and translated into a protein. In our
studies we have instead used reporter vectors, and these are
constructed for studying regulating elements upstream of genes such
as promoters. The promoter of interest is cloned and ligated upstream
of a reporter gene as illustrated in figure 5.
Figure 5 Reporter vectors are constructed for the insertion of regulating
elements, such as promoters, upstream of a reporter gene. Followed
digestion by restriction enzymes (1) and ligation with the promoter(2), the
vector is transferred into a host bacterium (3).The produced reporter gene
can then be measured at the mRNA- or protein-level (4 and 5) in the host.
30
Reporter gene technology can be used to study gene regulation, the
structure of regulatory elements or signaling pathways (Naylor, 1999).
The main considerations when selecting a reporter gene are (i) that
the activity of the reporter gene should be easily distinguished from
the endogenous proteins produced in the host cell; (ii) ensure that the
reporter activity do not interfere with enzyme activities in the host
cell; (iii) the chosen assay for detection of the gene product should be
sensitive and reproducible.
In this work reporter gene technology has been used to study clr and
cld promoter functionality. This has been performed by the use of a
vector containing a promoterless lacZ gene, encoding the enzyme
β-galactosidase. By promoter-lacZ fusion constructs transcriptional
expression encoded by promoter regulating elements can be studied.
The amount of active β-galactosidase, transcribed from the upstream
promoter, can be monitored a by using a colorimetric β-galactosidase
assay (Miller, 1972). The measured enzyme activity is thereafter
calculated with equation 3.
Units = 1000 ×
OD420 −1.75 ×OD550
𝑡𝑡×𝑣𝑣×OD600
(Eq. 3)
The OD600 denotes the cell density before the assay, OD420 the
absorbance of the o-nitrophenol and 1.75 × OD550 the light scattering
of cell debris. Included in the formula are also the time of the reaction
specified in minutes (t) and milliliters of the culture used in the
assay (v).
31
4. Results and discussion
Paper I
In this paper we wanted to investigate if the presence of oxygen
effected the expression of the I. dechloratans enzymes Cld and Clr.
Both the enzyme activity and the mRNA levels were investigated
under three different growth conditions; aerobic without chlorate,
aerobic with chlorate and anaerobic with chlorate. Whole cell extract
preparations of the cultures were used for enzyme activity
measurements. The Cld activity was measured as chlorite-dependent
oxygen evolution and the Clr activity was measured as
chlorate-dependent oxidation of reduced methyl viologen. The
enzyme activities were normalized to total protein content and the
comparisons between different growth regimes were performed on
cell cultures at different OD600-values. The highest activity was found
for cells in mid log phase (OD 0.4-0.6), and therefore subsequent
measurements were performed with bacterial cultures harvested
within this span.
Figure 6 The measured enzyme activities of Cld (A) and Clr (B), normalized to
total protein content. Black circles represent cells grown under anaerobic
growth conditions and white circles aerobic growth conditions.
Enzyme activities for both Cld and Clr could be measured at basal
levels at aerobic growth conditions with oxygen as sole electron
acceptor (figure 6). An induction in enzyme activity could be observed
after transfer to an anaerobic chlorate reducing environment, for Cld
32
about five times and for Cld 200 times. Absence of oxygen was
required for enzyme activity induction since no significant difference
could be observed when adding chlorate to the aerobic cultures.
To study the genetic regulation of the cld and clrA genes, the
corresponding mRNA’s were analyzed using qRT-PCR. RNA
preparations were performed followed by cDNA synthesis with RT
using random hexamer primers. All three growth conditions were
then analyzed in the same qPCR with primers specific for 16S rRNA,
cld and clrA. The mRNA levels of the two target genes, cld and clrA,
were normalized to the reference gene 16S rRNA.
Figure 7 The relative expression ratio of the cld (white) and clrA (grey) genes for
comparison of different growth conditions. Anaerobic compared to
aerobic without chlorate and aerobic with chlorate compared with
aerobic without chlorate. The error bars indicate standard errors of the
mean (SEM; n=9).
The results show that both genes are transcribed at all three growth
conditions, which is consistent with the results from the enzyme
activity measurements. The expression of both the genes cld and clrA
was shown to be induced under anaerobic conditions with basal
expression levels at aerobic growth. The presence of chlorate during
33
aerobic conditions did not result in an induction of either the cld or
the clrA gene (figure 7).
Based on the results in this study both the genes cld and clrA seems to
be regulated at the transcriptional level. At anaerobic growth an
eight- to nine-fold increase in mRNA levels can be observed for both
genes. For the cld gene this can account for the observed increase in
corresponding enzyme activity of about five times. For the clrA gene,
on the other hand, the difference in corresponding enzyme activity is
much greater, suggesting that other regulating mechanisms are also
involved.
34
Paper II
In addition to the enzymes chlorate reductase and chlorite dismutase,
chlorate respiration requires a carrier for the transport of electrons
from the membrane-bound components of the respiratory chain to
the periplasmic chlorate reductase. A c cytochrome, cyt c-Id1 encoded
by the I. dechloratans chromosome, has been shown to be able to
donate electrons to chlorate reductase and constitutes a major part of
the periplasmic c cytochromes (Smedja Bäcklund and Nilsson, 2011).
A candidate cytochrome c gene (cyc) is present in the gene cluster for
chlorate respiration, but the corresponding protein has hitherto not
been detected in the periplasm.
This raises the question of the expression of the cyc gene. The gene
has previously been cloned and heterologously expressed in
E. coli, but the refolded gene product could not be shown to serve as
an immediate electron transfer to Clr (Bohlin et al., 2010). The work
presented in this chapter was initiated to investigate if the cyc gene is
transcriptionally active in I. dechloratans, and if so, whether there is
any difference in expression under different growth regimes.
We were able to verify the presence of cyc mRNA in total RNA
preparations from I. dechloratans by qRT-PCR. It was found that the
mRNA levels increased about nine-fold when transferring cells from
aerobic to anaerobic conditions. Moreover, the calculated ∆CT -values,
that is the amount of cyc mRNA normalized to the reference gene,
were very similar to those found for clrA under aerobic and anoxic
conditions in paper I, indicating the same absolute copy numbers for
cyc and clrA under both conditions. This suggested the possibility
that both sequences are present in the same mRNA molecule, which is
possible since the gene for cyc follows directly downstream the gene
for clrC (figure 8).
To investigate a co-transcription scenario, PCR reactions were set up
with total cDNA as template. The primer pairs were constructed to
span two open reading frames (ORF) with the forward primer specific
for the 3´end in one ORF and the downstream primer specific for the
5´end in the nearest downstream ORF (figure 8). Precautions were
taken to ensure elimination of gDNA before cDNA synthesis. A PCR
35
product could only be formed if the template polynucleotide, in this
case the mRNA, contains both genes in the same fragment.
PCR products with expected sizes for all reactions could be verified on
an agarose gel. The PCR products were then ligated into the T-tailed
vector pGEM-T Easy and transformed into E. coli XL-1 Blue cells
followed by sequencing of the insert. The correct sequence targeted by
the primers with cDNA as template could be confirmed. This shows
that the genes clrABDC, cyc, mobB, arsR are co-transcribed in a
polycistronic mRNA.
Figure
8 Gene arrangement in the chlorate metabolism cluster of
I. dechloratans. clrA, clrB, clrC encode the α-, β- and γ-subunits of
chlorate reductase respectively, clrD is assumed to encode a specific
chaperone, cyc encodes a c-type cytochrome, mobB is assumed to encode a
protein involved in molybdenum cofactor biosynthesis, arsR is a putative
member of the ArsR transcriptional regulator family and h denotes a
hypothetical protein. ISIde1 is an insertion sequence positioned between
the clrABDC and cld genes and ISAav1 is one of the two insertion
sequences flanking the composite transposon (Clark et al., 2013). Positions
of primer pairs used in the PCR reactions are indicated with arrow heads.
This figure is reproduced and reprinted from paper Hellberg Lindqvist,
M., Nilsson, T., Sundin, P., Rova, M. 2015. Chlorate reductase is
co-transcribed with cytochrome c and other downstream genes in the
gene cluster for chlorate respiration of Ideonella dechloratans. FEMS
Microbiology Letters. 362:fnv019, with permission from Oxford
University Press.
An additional cDNA synthesis was performed to confirm this. This
time a specific primer was used instead of random hexamer primers
for cDNA synthesis. The reverse primer in the hypothetic protein was
the only primer added to this reaction with total RNA as template.
36
PCR reactions were set up with cDNA as template, primer pairs used
for the different reactions were: clrA-clrB, clrD-clrC and
arsR-hypothetic protein. The products could be verified on an agarose
gel as single bands, whereas no products could be detected in the
no-RT controls from the cDNA synthesis containing all components
without addition of RT. This verifies complete elimination of gDNA
before cDNA synthesis.
In paper II the expression of the cyc gene could be verified at the
mRNA level. The transcript is quite long (over 5.1 kbp) and includes
genes that are quite dissimilar regarding characteristics and function.
If all genes included in the mRNA are translated into functional
proteins still needs to be further investigated. It is not certain that the
bacteria needs equal amounts of all corresponding proteins and the
most logical scenario would then be an additional regulation at the
translational level. Despite several attempts, the cyc gene product has
not been found in cell extracts and it is possible that a role as electron
donor to Clr has been taken over by the chromosomally encoded
cyt cId-1 in I. dechloratans.
37
Paper III
In order to investigate the presence of regulating elements in the cld
and clr promoters the sequences upstream of these genes were cloned
into a broad host range reporter plasmid upstream of a lacZ gene and
transformed into E. coli. The pBBR1MCS vector series (Kovach et al.,
1995) was known to be possible to electroporate into I. dechloratans
from earlier work and was chosen with the intention of future
expression studies in I. dechloratans, after initial characterization in
E. coli.
The sequence upstream of the clr gene was ligated correctly to the
broad-host range plasmid, as verified by sequencing, but did not
function as a promoter in the reporter system. The sequence
upstream of cld, on the other hand, was found to function as a
promoter in the reporter system, with expression sensitive to the
oxygen level and the presence of a functional FNR protein in the host.
These results are presented in this paper.
In a search for recognition sequences for known bacterial
transcription factors (using the Virtual footprint server,
http://www.prodoric.de/vfp), a putative binding site for FNR and a
possible ArcA binding region have been noted (figure 9).
To assess the role of FNR and/or ArcA as possible anaerobic
transcriptional regulators, four reporter constructs were made; one
with the wild type cld promoter (pBBR1MCS-4-lacZ_cld(200)), one
with a shortened version of the cld promoter (pBBR1MCS-2lacZ_cld(82)), one with a four-nucleotide substitution in the putative
FNR-box (pBBR1MCS-4-lacZ_cld(mut1)), and one construct with a
four-nucleotide substitution in the possible ArcA-binding region
(pBBR1MCS-4-lacZ_cld(mut2)) (figure 9). All four promoter-lacZ
fusion plasmids were transformed into E. coli XL-1 Blue cells. The
functionality of the cloned regions as promoters was analyzed in the
reporter system with a β-galactosidase assay as described in section
3.3.1. and the results are presented in figure 10.
38
39
Figure 9 Illustration of the four constructs in plasmids pBRR1MCS-4-lacZ(200, mut1 and mut2) or plasmid
pBBR1MCS-2-lacZ(82) containing the wild type (200) or modified versions of the region upstream of the cld gene. The
putative FNR- and ArcA-binding regions are underlined. In construct (mut1) the FNR binding site is changed to
ATTAATTAAAACAA and in construct (mut2) the ArcA binding site is changed to TCCGAAGTTTG, mutated nucleotides
are marked with asterisks. The construct (82) contains the shortened version of the cld promoter which includes the
putative FNR binding site and the same downstream sequence as (200). Two suggested alternative promoters have been
noted in the wild type sequence, here marked as shaded areas. The FNR-dependent promoter consists of -10(1) and -35(1)
and the FNR-independent promoter consists of -10(2) and -35(2). The previously determined transcription start site
(TSS) is marked with +1.
The β-galactosidase activity from cells containing the plasmid
pBBR1MCS-4-lacZ_cld(200) grown under aerobic conditions was
clearly detectable, and at a level about eight times above the
background level obtained with the reporter plasmid without insert
(pBBR1MCS-4-lacZ). After anaerobic growth, significantly higher
activity was obtained. At anaerobic conditions an about four-fold
induction of the reporter enzyme could be observed compared to the
activity from aerobically grown cells and this is in the same order of
magnitude as the induction of Cld activity measured in
I. dechloratans (paper I). This result show that the regulatory
sequences that account for the anaerobic induction of cld from
I. dechloratans is present in the cloned fragment and are functional in
the used E. coli reporter system.
Figure 10 Results from the β-galactosidase assays with E. coli XL-1 Blue cells or
RM101 harboring plasmids pBBR1MCS-4-lacZ (200, mut1 or mut2) or
pBBR1MCS-2-lacZ (82). Neg K1 and Neg K2 show reported values
obtained with plasmids pBBR1MCS-4-lacZ and pBBR1MCS-2-lacZ,
respectively, without insert which serves as negative controls. The white
bars represent values from aerobically grown cells and the grey bars
anaerobically grown cells. The error bars indicate standard error of the
mean (SEM; n=6-12).
40
The site-directed mutagenesis of four nucleotides in the putative
FNR-box in plasmid pBBR1MCS-4-lacZ_cld(mut1) was found to
knock out the transcription the downstream lacZ gene. No induction
of β-galactosidase activity could be observed in the absence of oxygen
and both the aerobic and anaerobic values were in the same order of
magnitude, reduced to about two times the background level. This
indicates that this region is critical for the transcription of the
downstream gene, irrespective of cultivation conditions.
The I. dechloratans genome was found to contain a gene that might
encode a FNR homologue. It appeared therefore possible that the
anaerobic induction of the cld gene is dependent of an FNR-type
transcription factor. To further investigate the role of FNR as
transcriptional regulator of the cld gene the plasmid pBBR1MCS-4lacZ_cld(200) was transferred to the fnr-negative E. coli strain
RM101 (Bekker et al., 2010). Under aerobic conditions the observed
β-galactosidase activity was about nine times higher compared to the
background level. This is comparable to the basal aerobic activity
observed in E. coli XL-1 Blue with the same plasmid. No induction
could be seen in RM101 cells grown under anaerobic fermentative
conditions compared to the aerobically grown cultures (figure 10).
This result demonstrates that the E. coli FNR protein is involved in
the anaerobic induction from the cld promoter in the reporter
constructs.
FNR-dependent class II promoters usually have a single FNR binding
site centered at-41.5 bp relative the TSS (Browning and Busby, 2004).
Based on the position of the FNR binding site the most likely -35
and -10 promoter elements found is AACACAN14TGCGAAGAT and
includes a putative -10 extended motif, as shown in figure 9.
The plasmids pBBR1MCS-4-lacZ_cld(mut2) and pBBR1MCS-2lacZ_cld(82) was shown to contain functional promoters. The relative
β-galactosidase enzyme activity from both plasmids was about 40 %
at aerobic conditions and >60 % at anaerobic conditions compared to
measured values for the plasmid pBBR1MCS-4-lacZ_cld(200)
containing the full-length wild type cld promoter. It thus appears that
the mutated nucleotides in plasmid pBBR1MCS-4-lacZ_cld(mut2)
41
and the region missing in plasmid pBBR1MCS-2-lacZ_cld(82) affect
the transcription pattern in the same way. The region upstream of the
putative FNR-box seems to be needed for maximal expression at both
aerobic and anaerobic conditions, but has greater impact on the
aerobic expression.
To be able to draw any conclusions regarding possible involvement of
an ArcA protein in the reporter-system used, additional work is
needed. However, no gene encoding an arcA homologue has been
found in the I. dechloratans genome, and it is therefore questionable
if the cld gene is regulated by an active ArcA~P protein in vivo in the
host bacterium.
Previous work revealed only one possible TSS in the cld promoter
from I. dechloratans (Danielsson Thorell et al., 2002). The findings in
this study, however, raise the question of the presence of alternative
TSS, one for the basal aerobic expression and one for FNR-dependent
anaerobic transcription (figure 9).
Similar examples with overlapping promoters are known for other
bacteria. As example the E. coli focApfl operon can be mentioned,
which is under the control of two alternative TSS, separated by 10
nucleotides (Kaiser and Sawers, 1997). The transcription under
aerobic growth is regulated by a factor independent promoter. In
contrast, the anaerobic transcription is dependent on the presence of
a functional FNR protein and transcribes from the alternative TSS.
An analogous scenario for cld expression would be that aerobic
transcription occurs from the promoter upstream of the previously
determined TSS (figure 11). This promoter would then account for the
basal aerobic expression of the cld gene. The anaerobic expression, on
the other hand, takes place from the promoter downstream from the
FNR-box and is dependent on the presence of a functional FNR
protein.
42
Figure 11 Illustration of a possible scenario with two alternative overlapping
promoters in the region upstream of the cld gene. a) The anaerobic
promoter consists of TSS (1), -10 (1) and -35(1) and the transcription from
this promoter is dependent on the binding of a functional FNR protein.
b) The aerobic promoter consists of TSS (2), -10 (2) and -35 (2) and
account for the basal Cld activity observed at aerobic growth.
43
Paper IV
In this paper the candidate FNR homologue in the I. dechloratans
genome is further investigated. The expression of the fnr gene in
I. dechloratans was confirmed by the detection of the corresponding
mRNA using qRT-PCR. These findings thereby support the
conclusion in paper III suggesting an FNR-dependent induction from
the cld promoter in the absence of oxygen. The expression levels of
the I. dechloratans fnr gene was shown to be equal independent of
cultivation conditions (aerobic vs. anaerobic).
Previously studies of the expression from the cld promoter in plasmid
pBBR1MCS-4-lacZ_cld(200) in the fnr-negative E. coli strain RM101
showed that only basal expression of the downstream gene could be
observed at both aerobic and anaerobic growth (paper III). Therefore
we wanted to investigate if the I. dechloratans FNR homologue could
complement the fnr mutation in RM101.
The I. dechloratans fnr gene, including promoter region, was
amplified by PCR and cloned into the plasmid pBR322, creating
pBR322_Id(fnr).
RM101
was
then
co-transformed
with
pBR322_Id(fnr) or plasmid pBR322 together with plasmid
pBBR1MCS-2-lacZ_cld(prom) or pBBR1MCS-2-lacZ. This resulted in
RM101 clones with four different plasmid combinations presented in
table 1.
Table 1 Designations of RM101 cells containing four different plasmid
combinations.
Clone
Plasmids
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 + 𝑐𝑐𝑐𝑐𝑐𝑐+ )
pBR322_Id(fnr)
pBBR1MCS-2-lacZ_cld(prom)
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 + 𝑐𝑐𝑐𝑐𝑐𝑐− )
pBR322_Id(fnr)
pBBR1MCS-2-lacZ
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 − 𝑐𝑐𝑐𝑐𝑐𝑐+ )
pBR322
pBBR1MCS-2- lacZ_cld(prom)
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 − 𝑐𝑐𝑐𝑐𝑐𝑐− )
pBR322
pBBR1MCS-2-lacZ
44
The
four
clones
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 + 𝑐𝑐𝑐𝑐𝑐𝑐 + ),
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 + 𝑐𝑐𝑐𝑐𝑐𝑐 − ),
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 − 𝑐𝑐𝑐𝑐𝑐𝑐 + ) and RM101(𝑓𝑓𝑓𝑓𝑓𝑓 − 𝑐𝑐𝑐𝑐𝑐𝑐 − ), were grown in a defined
medium under aerobic or batch fermentative conditions followed by a
β-galactosidase assay.
The clones RM101(𝑓𝑓𝑓𝑓𝑓𝑓 − 𝑐𝑐𝑐𝑐𝑐𝑐 − ) and
RM101(𝑓𝑓𝑓𝑓𝑓𝑓 + 𝑐𝑐𝑐𝑐𝑐𝑐 − ) lacking the cld promoter served as negative
controls. The measured activity from clone RM101(𝑓𝑓𝑓𝑓𝑓𝑓 − 𝑐𝑐𝑐𝑐𝑐𝑐 + ) was
about eight times higher than the negative controls, independent of
culture conditions. This result shows that the cld promoter is
compatible with the transcription machinery in RM101. The basal
transcription from the cld promoter is in the same order of magnitude
independent of growth conditions and presence of a functional FNR
protein. These data confirms previous results presented in paper III,
but with another plasmid.
Figure 12 The result from the β-galactosidase assay from RM101cells with four
different plasmid combinations. The white bars represent aerobically
grown cells and the grey bars anaerobically grown cells. The error bars
indicate standard error of the mean (SEM; n=9).
The presence of I. dechloratans fnr gene in clone RM101(𝑓𝑓𝑓𝑓𝑓𝑓 + 𝑐𝑐𝑐𝑐𝑐𝑐 + )
resulted in an induction of β-galactosidase of about five times during
anaerobic fermentative conditions compared to aerobic conditions.
45
This shows that the I. dechloratans fnr gene is able to complement
the ∆fnr mutation in RM101 and that the gene product can induce
expression from the cld promoter to the same extent as seen with the
E. coli XL-1 FNR protein in paper III. These findings thereby support
the result in paper III indicating that the anaerobic transcriptional
regulation of the cld gene is mediated by a functional FNR protein
in vivo.
In the E. coli FNR an N-terminal cluster of four cysteine residues can
be found. Three of these together with one internal cysteine residue
have been found to serve as ligands for the iron-sulfur cluster (Green
et al., 2001). Corresponding regions in the I. dechloratans putative
FNR homologue was also found to contain four cysteine residues;
three in the N-terminal and one internal. This argues in favor for that
the I. dechloratans gene encodes a true FNR homologue. Hitherto no
attempts have been made to try to find this protein in the host
bacterium and this remains to be done to confirm that the detected
fnr mRNA actually is translated into a functional protein in vivo.
The finding that a gene encoded outside the genomic island for
chlorate respiration serves an important function is an interesting
parallel to the role of cyt c-Id1 discussed in paper II, and may reflect
integration of a function acquired by horizontal gene transfer into the
regulatory machinery of the host.
46
5. Concluding remarks
The aim with this study was to clarify how the genes involved in the
chlorate respiration in I. dechloratans are regulated in response to
different growth conditions. We have demonstrated that both the
genes cld and clrA are transcribed both at aerobic and anaerobic
conditions with an upregulation of about eight to nine times in the
absence of oxygen. This can correspond for the upregulation seen for
Cld enzyme activity of about five times at anaerobic growth compared
to cells grown under aerobic conditions. For Clr enzyme activity, on
the other hand, which is upregulated about 200 times in the same
comparison it is possible that other regulating mechanisms are
involved as well. The presence of chlorate itself during oxygen rich
conditions did neither affect the transcription nor the enzyme activity
of either of the two proteins.
Interesting, the work presented in paper II demonstrates that
clrABDC, cyc, arsR and mobB are co-trancribed in a polycistronic
mRNA. It still needs to be clarified, though, if the genes downstream
of the clr operon are translated.
Due to difficulties in the transformation process of the bacterium
I. dechloratans, we decided to work with an E. coli reporter system in
paper III and IV. Unfortunately, we were not able to clone a
functional clr promoter, but the anaerobic induction from the cld
promoter, on the other hand, could be demonstrated to be dependent
on the presence of a functional FNR protein in the host. Presence of
fnr mRNA was demonstrated in I. dechloratans and the gene product
was able to complement the ∆fnr mutation in E. coli strain RM101. In
conclusion, these findings thereby support that the reported
anaerobic induction of the Cld enzyme is mediated by a functional
FNR homologue in vivo.
47
6. Future perspective
To obtain a deeper understanding of the mechanisms involved in the
anaerobic induction of enzymes responsible for chlorate respiration,
also functional regions in the clr promoter have to be studied in more
detail. It would be very interesting if one could be able to create
functional clr promoter:lacZ fusion construct to be analyzed into a
reporter system as for the cld gene.
When using a reporter system there is always a risk that regulatory
sequences present in the promoter region are not recognized by the
transcription machinery in the reporter. Therefore, a complementary
approach would be to study both cld and clr promoter:lacZ fusion
construct into the host bacterium I. dechloratans. The
pBBR1MCS-series of vectors were chosen for this study as they can be
electroporated into the bacterium, although with very low efficiency.
Several attempts have been made to electroporate I. dechloratans
with the cld promoter:lacZ construct, unfortunately with as yet
limited success. To pursue this line of work, improvement of
transformation, as well as the establishment of conditions for reporter
enzyme assay in I. dechloratans is needed.
The cyc and arsR genes downstream of the clr operon were shown to
be transcribed in paper II. The arsR might encode a transcriptional
regulator and the cyc gene may encode a cytochrome c involved in the
electron transport to Clr. Further investigations are needed to find out
if these genes are translated.
48
7. Populärvetenskaplig sammanfattning
Miljögifter i naturen kan finnas kvar länge och påverka oss människor
i generationer. Med användingen av potentiellt skadliga kemikalier så
ingår också ett ansvar som innefattar lämplig hantering av dessa.
Blekning av pappersmassa med klordioxid inom pappers- och
massaindustrin ger upphov till biprodukten klorat, en kemikalie som
kan ha negativ inverkan på den omgivande miljön om den släpps ut i
närliggande vattendrag.
Kloraten som bildas kan renas bort från avloppsvattnet genom
biorening, det vill säga rening med bakteriers hjälp. Nedbrytningen av
klorat sker i två steg och resulterar i slutprodukterna syre och
kloridjoner med hjälp av de två enzymerna kloratreduktas (Clr) och
kloritdismutas (Cld). Syftet med denna avhandling har varit att
studera om, och i så fall hur, dessa två enzymer påverkas av tillgången
på syre i bakterien Ideonella dechloratans.
Biorening
Själva bioreningstekniken har varit känd sedan början av 1900-talet
och används idag världen över för att rena avloppsvatten från
industrier och hushåll. Cirka 30 000 av de ämnen som återfinns i vår
omgivande miljö har sitt ursprung i kemikalier som vi använder varje
dag. Konceptet med biorening är relativt enkel och man använder sig
av naturligt förekommande bakterier som bryter ner komplexa
molekyler till mindre komplexa föreningar. Då det inte är möjligt för
en enskild organism att bryta ner alla ämnen krävs en uppsättning
med många olika sorters bakterier för att kunna ta hand om alla
kemikalier som förekommer i avloppsvattnet. Hur effektiv
nedbrytningen av vissa ämnen är bestäms även delvis av yttre faktorer
såsom tillgång på syre, temperatur och pH.
Det klorat som förekommer i naturen har generellt hamnat där på
grund av olika utsläpp från industriella processer. Kemikalien har
också historiskt använts som ogräsmedel och produceras som en
biprodukt vid disinfektion av dricksvatten med hypoklorit. Klorat har
49
visat sig vara giftigt för alger i Östersjön och kan orsaka hemolytisk
anemi (vilket innebär att de röda blodkropparna går sönder) hos
däggdjur om det intas via dricksvatten i höga doser under lång tid.
Det är idag känt att kloratrespirerande bakterier kan användas för
biologisk nedbrytning av klorat, I. dechloratans som vi studerar är en
av dem. Som tidigare nämnt så sker nedbrytningen av klorat i
I. dechloratans med hjälp av de två enzymerna Clr och Cld. Clr
katalyserar nedbrytningen av klorat till klorit och Cld katalyserar
nedbrytningen av klorit till kloridjoner och syre (figur 1). Det
molekylära syre (O2) som bildas används av bakterien själv i en
reaktion med vatten som sluprodukt. Hela denna kedja av reaktioner
sker i bakterien för att i slutänden kunna producera kemisk energi i
form av ATP (adenosintrifosfat). Denna energi kan sedan användas av
bakterien för att exempelvis tillverka proteiner och att dela sig.
Figur 1 Nebrytningen av klorat till klorit katalyseras av enzymet kloratreduktas
(Clr). Enzymet kloritdismutas (Cld) katalyserar i sin tur nedbrytningen
av klorit till kloridjoner och syre. Syret som produceras används vidare i
en ytterligare reaktion och ger upphov till slutprodukten vatten.
Om det finns syre närvarande i det omgivande avloppsvattnet kan
detta ställa till med problem för nedbrytningen av klorat. Då syre
finns att tillgå är det mer förmånligt för bakterien att använda detta
än att bryta ner klorat och bakterien stänger av förmågan att bryta ner
klorat temporärt. Denna förmåga kan dock återupptas igen när allt
syre är konsumerat men fördröjningen kan vara lång. På grund av att
den biorening som sker av andra ämnen i avloppsvattnet från ett
pappersbruk ofta kräver syre, kan det här uppstå en konflikt mellan
att effektivt bryta ner andra ämnen och nedbrytningen av klorat. Det
är viktigt att öka förståelsen för hur och varför biorening av klorat
50
fungerar för att på så vis kunna utveckla denna reningsmetod till att
bli så effektivt som möjligt.
Genreglering
Även om en bakterie väljer att stänga av produktionen av ett visst
protein, till exempel tillverkningen av enzymerna Clr och Cld, så finns
ändå generna som kodar för detta protein kvar. Genom att styra vilka
gener som är aktiva och vilka som inte är det kan bakterien enbart
producera de proteiner som behövs just för tillfället. Produktionen av
proteiner är en mycket kostsam process och därför tillverkar bakterier
enbart de proteiner som de har användning för just för tillfället.
Figur 2 Tillverkningen av proteiner i cellen kan delas in i två steg; i det första
steget (a), transkriptionen, så utgör genen på DNA-molekylen en mall för
hur mRNA-molekylen ska se ut, i det andra steget (b), translationen, så
översätts mRNA-molekylen till ett protein.
Koden för hur varje enskilt protein (inklusive enzymer) i en cell ska se
ut finns lagrat i cellens DNA. En DNA-molekyl innehåller gener vilka
utgör en mall för tillverkningen av mRNA-molekyler (efter engelskans
messenger RNA) i en process som kallas transkription. I nästa steg,
51
translationen, så används mRNA-molekylen som en ritning för
tillverkningen av själva proteinet (figur 2). Alla celler, inklusive
bakterier, kan själva styra vilka proteiner som bildas vid en given tid
genom att reglera transkriptionen av en gen eller translationen av ett
mRNA. Regleringen kan även ske av det färdiga proteinet.
Om regleringen av en gen sker på transkriptionsnivå så påverkar det
hur många mRNA-molekyler som bildas. En nedreglering innebär att
färre mRNA-molekyler bildas (och därmed färre proteinmolekyler)
medan en uppreglering är det motsatta och medför att fler
mRNA-molekyler bildas (och även fler proteinmolekyler).
Vad har denna avhandling bidragit till?
Arbetet i denna avhandling har handlat om att försöka förstå hur de
gener som är involverade i nedbrytningen av klorat i I. dechloratans
regleras i närvaro respektive frånvaro av syre och även tillgången på
klorat. Vi har därför låtit bakterier få växa under tre olika
förhållanden; i medium rikt på syre men inget klorat, i medium med
både syre och klorat, i medium utan syre men med klorat. Efter några
timmars tillväxt har bakteriecellerna löst upp (lyserats) och
enzymaktiviteten av Clr och Cld har mätts i cellextrakten med lite
olika metoder.
När bakterierna får växa i medium som är rikt på syre (aerobt) så kan
man bara uppmäta mycket låga aktiviteter för enzymerna Clr och Cld i
cellextrakten. Det innebär att bakterien inte kan bryta ner klorat i
någon större utsträckning när syre finns tillgängligt i odlingsmediet.
Den aktivitet som har uppmätts i cellextrakt från bakterier som
istället har fått växa utan syre (anaerobt) men med klorat är betydligt
högre för båda enzymerna. Närvaron av klorat i sig hade ingen effekt
så slutsaten blev att det är frånavon av syre som leder till en ökning av
enzymaktiviteten för både Clr och Cld.
Varken Clr eller Cld fyller någon funktion när syre finns att tillgå och
det är onödigt kostsamt för bakterien att tillverka en stor mängd
aktivt enzym som inte ska användas. Det var inte känt innan detta
52
arbete om regleringen av Clr och Cld i bakterien I. dechloratans
skedde på transkriptions- eller translationsnivå. Förutom att mäta
mängden aktivt enzym vid de tre olika odlingsförhållandena valde vi
därför att även mäta mängden mRNA som bildades av de båda
enzymerna Clr och Cld. Intressant nog så visade dessa mätningar att
ett större antal mRNA-molekyler producerades av både
kloratreduktas och kloritdismutas vid anaeroba förhållanden vilket
innebär att dessa båda enzymer regleras på transkriptionsnivån. Vi
kan dock inte utesluta att även andra reglermekanismer kan vara
involverade i regleringen av Clr.
Aktiveringen av Cld har visat sig vara beroende av ett protein, kallat
FNR. Denna typ av protein finns hos vissa bakterier och är endast
aktivt i frånvaro av syre. Under anaeroba förhållanden hjälper
FNR-proteiner till att slå på gener som behövs och stänga av gener
som inte behövs när syre inte finns att tillgå. Hos bakterien
I. dechloratans så binder troligtvis ett FNR-liknande protein till en
sekvens på DNA-molekylen precis innan genen för kloritdismutas
under anaeroba förhållanden. Detta leder till att produktionen av
kloritdismutas mRNA-molekyler uppregleras i frånvaro av syre och
det i sin tur innebär att många fler Cld enzymer bildas. Det är därför
vi kan uppmäta en högre enzymaktivitet av Cld i cellextrakt anaerobt
än aerobt.
Förutom att vi kan uppmäta ett större antal mRNA-molekyler av
kloratreduktas i frånvaro av syre, så råder det fortfarande en viss
osäkerhet kring hur induktionen av Clr sker under anaeroba
förhållanden. Detta spännande arbete hoppas jag att framtida
doktorander i biokemi får möjlighet att undersöka djupare.
53
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64
Oxygen-dependent regulation of key
components in microbial chlorate respiration
Contamination of perchlorate and chlorate in nature is primarily the result of
various industrial processes. The microbial respiration of these oxyanions of
chlorine plays a major role in reducing the society’s impact on the environment.
The focus with this thesis is to investigate the oxygen-dependent regulation
of key components involved in the chlorate respiration in the gram-negative
bacterium Ideonella dechloratans. Chlorate metabolism is based on the action of
the enzymes chlorate reductase and chlorite dismutase and results in the end
products molecular oxygen and chloride ion. Up-regulation of chlorite dismutase
activity in the absence of oxygen is demonstrated to occur at the transcriptional
level, with the participation of the transcriptional fumarate and nitrate reduction
regulator (FNR). Also, the chlorate reductase enzyme was shown to be regulated
at the transcriptional level with the possible involvement of additional regulating
mechanisms as well. Interestingly, the corresponding chlorate reductase operon
was found to be part of a polycistronic mRNA which also comprises the gene for
a cytochrome c and a putative transcriptional regulator protein.
ISBN 978-91-7063-692-9
ISSN 1403-8099
DOCTORIAL THESIS | Karlstad University Studies | 2016:13