FEMS MicrobiologyLetters 94 (1992) 111-120
© 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00
Published by Elsevier
Ill
FEMSLE 04934
Two forms of ribulose-l,5-bisphosphate carboxylase/oxygenase
from Thiobacillus denitrificans
Robert S. English, Christopher A. Williams ~, Stanley C. Lorbach and Jessup M. Shively
Department of Biological Sciences. Ctemson Unit'ersity, Clemson. SC, USA
Received 7 April 1902
Accepted 8 April Itl92
Key words: Ribulose- 1,5-bisphosphate carboxylase/oxygenase; Thiobacillus denitrifcans;
RuBisCO, form I, form 11
1. SUMMARY
The autotrophic, sulfur-oxidizing bacterium
Thiobaciilus denitrificans possesses two forms of
the Calvin cycle enzyme ribulose-l,5-bisphosphate carboxylase/oxTgenase (RuBisCO). The
form 1 and form !I genes were isolated from a
cosmid library using heterologous DNA probes.
Restriction enzyme analysis indicated that the
genes are within 17 kbp of each other. Other
Calvin cycle enzyme genes are not present. Analysis of T, denitrificans RNA indicated that the
form I genes for the large and small subunits are
co-transcribed with a length of 2800 nucleotides.
The transcript for the form II gene is 1900 nucleotides in length.
Correspondence to: J.M, Shively, Department of Biological
Sciences, Clemson University, Clemson, SC 29634, USA.
t Present address: Sehering Plough Corp., Bloomfield, NJ
07003, USA.
2. INTRODUCTION
Thiobacillus denitrificans is a chemolithotrophic
bacterium which grows anaerobically utilizing
thiosulfate for energy and nitrate as an electron
acceptor. The Calvin cycle is well-established as
the major mechanism of CO2 fixation in this
organism. The key enzyme of this pathway, ribulose- 1,5-bisphosphate earboxylase/oxygenase
(RuBisCO :EC 4.1.1.39), was purified from 7".
denitrificans by McFadden and Denend [1] and
shown to have an approximate molecular mass of
350000. The occurrence of two types of RuBisCO
has since been firmly established. The first, form
I, is a hexadecamer composed of eight large ([.SU)
and eight small subunits (SSU). This form, v.,itc,
an approximate molecular mass of 560000, is
widely distributed among various phototrophic
and chemolithotrophic autotrophs [2]. The second type, form I!, composed of only LSU, has
been found in a number of phototrophic bacteria
with a variable number of subunits [2]. The two
112
forms are dissimilar based upon immunological,
catalytic, and DNA sequence data [3-11],
The presence of both forms of RuBisCO in the
same organism was initially demonstrated in the
phototrophic bacterium Rhodobacter sphaeroides
[12]. Since then, the two forms have also been
shown in the phototrophic bacteria Rhodopseudomonas blastica, and Rhodobacter capsulatus
[13-15].
Herein we report an extension of the enzymoiogicaI evidence of McFadden and Denend to
include the presence of both a form I and form 11
type RuBisCO and the cloning of each of these
genes from T. denitrificans. This is the first
demonstration of the form II RuBisCO in a nonphototrophic organism as well as the first demonstration of a non-phototrophic organism containing both forms.
psi) and the cell debris removed by centrifugation
at 47 8(10× g for 1 h. The supernatant was loaded
onto the surface of linear sucrose gradients (1060%, in TEMB) in VTi50 quick seal tubes (Beckman) which were prepared as described by Shively et al. [15] and centrifuged for 18 h at 76000 × g
(4°C). The tubes were then fractionated from the
bottom in l-ml aliquots and assayed for RuBisCO
activity. Fractions were also subjected to inhibition assays using 6-phosphogluconate.
3.3. RuBisCO assay
Ribulose bisphosphate (RuBP)-dependent assays were carried out at room temperature in
Opticlear shell vials using NaH t4C 0 3 as described by Shively et al. [18]. RuBisCO inhibition
assays were conducted with either 2 mM or 5 mM
6-phosphogtuconate and 25 mM RuBP.
3.4. DNA isolation and Southern blotting
3. MATERIALS AND METHODS
3.1. Cultures
T. denitrificans ATCC 25259, was routinely
grown anaerobically in the medium of Baalsrud
and Baalsrud [16] at 30°C.
Rhodobacter capsulatus PAS 100, from Barry
Marrs, E.I. du Pont de Nemours (Wilmington,
DE) was grown photoheterotrophically with butyrate as the electron donor and NaHCO 3 supplemented to a final concentration of 0.01% as
described by Shively et al. [5].
Escherichia coil DH5a was used as the host
strain for routine plasmid manipulations. E. coil
LE392 was the host strain for the cosmid library.
All E. coli strains were grown in Lmia-Bertani
medium at 37°C [17].
3.2 Enzyme analysis
Twenty liters of T. denitrificans cells (late-log
phase) were harvested in a continuous flow centrifuge (Lahr/Schwarzwald) at 20000 rpm, The
cells were washed twice in TEMB buffer (Tris.
HCI, 10 mM; EDTA, 1 mM; MgCIz.6H20, 15
mM; and NaHCO3, 20 mM; pH 8.0), pelleted,
frozen at -80°C for 15 rain, and resuspended in
50 ml of TEMB buffer. The cells were broken via
two passes through a French pressure cell (20000
Four liters of late-logarithmically grown cells
were centrifuged, washed in TE (Tris. HCI, 10
raM; EDTA, 1 raM; pH 8.0), and frozen at - 80°C
for 15 min to help lysis. DNA purification, restriction endonuclease digestion, agarose gel electrophoresis and Southern blotting were accomplished as previously described [19].
3.5. Library constraction
The vector (pLAFR5) used to construct the
library was kindly provided by N.T. Keen, University of California, Riverside [20]. Double cos site
vectors, as described by Bates and Swift [21], are
more convenient for cosmid cloning because vector DNA need not be treated with phosphatase
and the two sites prevent unwanted concatemerization of vector DNA causing packaging without
inserts. The vector was prepared by sequential
digests with Seal and EcoRI. T. denitrificans
DNA was prepared by partial digestion with
EcoRl and subsequently phosphatased (calf intestinal phosphatase).
Optimal ligation was achieved with a 2:1 insert to vector ratio. Ligation was performed with
1 Weiss unit of T4 DNA ligase in a total volume
of 20 ~1. To ensure no blunt-end ligation of the
Scal sites, ATP was added to give a final concentration of 5 raM; cohesive-end ligation is not
i13
affected by the high ATP level [22]. Packaging
extracts were prepared from E. coli BHB2690
and BHB2688 according to Sambrook et al. [23].
E. coil LE392 infected with the packaged DNA
was spread onto LB-tetracycline plates (50
/~g/ml), Approximately 1200 colonies were transferred by toothpick to new plates for screening.
3.6. Probe preparation and library screening
DNA probes were prepared as described by
Shively et ai. [19]. Colony lifts and lysis of the
cosmid library were accomplished according to
Sambrook et aL [23]. The library was initially
screened with rbcL from Anacystis ni&dans ( EcoRl/Pstl fragment from pANPlI55) then with
rbpL from Rhodospiriilum ntbn~m (Sail fragments from pRR264 and pRR321). Washing, prohybridization, and hybridization were carried out
according to Shively et al. [19].
3. 7. Hybridization conditions
High stringency prehybridization and hybridization of Southern blots were accomplished
in 50% formamide, 5 X Denhardt's, 0.1% SDS,
and 5 X SSPE. Blots were placed into Seal-a-Meal
bags and prehybridized for a minimum of 6 h at
65°C. Hybridizations were performed with approximately 1 x 10r' CPM nick-translated probe
per blot at 42°C for a minimum of 12 h. Two
washes were typically done at room temperature
in 2 X SSC and 0.1% SDS for 20 min each.
When probing with the small subunit (SSU)
gene from Pisum s,tticum, low stringency conditions were used. The prehybridization solution
consisted of 5 X Dcnhardt's and 6 X SSC. The
blot was prehybridized for I h at 60°C and hybridized for 24 h at the same temperature in 1 X
Denhardt's and 6 X SSC. Two washes were accomplished in 6 X SSC and 0.1% SDS which was
followed by two washes in 3 X SSC and 0.1%
SDS. All washes were done at room temperature.
3.8. Mapping
Mapping of the form I and form II genes,
contained within a single pLAFR5 clone
(pTDF12), was accomplished with restriction enzymes which recognized few sites within the insert (e.g. HindlIl). A detailed map of the geue
regions was facilitated by subcloning the EcoR!
fragments into pUC8.
To map and orient the form II gone within the
EcoRl subclone (pTDF2E), another probe from
the R. mbmm gone was used. The initial fragments (pRR264 and pRR321) are locate:l in tandem starting at 260 bp within the R. mbn~m gone
and extending 585 bp [7]. The second probe, a
Pstl/Bglll fragment starts at 1001 bp and extends 396 bp toward the 3' end (isolated from
pRRII6). By mapping the two hybridizing regions relative to each other, the form II gone was
oriented and positioned.
Orientation of the form I gone within the
EcoRl subclone ~pTDFIE) was accomplislled in
a similar manner, using the P. saticum SSU gene
as a secondary probe (isolated from pSSU).
3.9. RNA isolation and blotting
T. denitrificans was harvested in early log phase
to ensure sufficient quantities of RttBisCO
mRNA transcripts. Isolation of RNA was accomplished according to the method of Scherrer [24].
RNA was electrophoresed in a 1.5% agarose/6%
formaldehyde gel and transferred to Zeta Probe
nylon membranes by the use of Trans-Blot Cell
system from BioRad. The resulting blots were
baked at 80°C for 2 h and placed into Seal-a-Meal
bags for storage.
3.10. Hybridization of RNA blots
RNA blots were washed in 0.1% SSC, 0.5%
SDS for I h at 65°C then prehybridized for 12 h
at 42°C in 5 X SSC, 25 mM NaPO4 (pH 6.5), 10 X
Denhardt's, and 50% formamide. Hybridization
was completed in 2 X Denhardt's without BSA
for 24 h (42°C). The blots were then washed in 2
X SSC, 0.1% SDS for 1 h, followed by two 30-rain
washes in 0.1% SSC, 0.1% SDS Cone at room
temperature and one at 55°C).
Total RNA was probed with the form 1 and
for,n 11 .~enc regions from T. denitrificans (1.75
kt'p Ec'. Rl/ttindlil fragment from pTDF1E and
540 bp Sail fragment from pTDF2E). Total RNA
v,:~s also probed with the SSU gone area from T.
.Je,,r Orleans (1.75 kbp Eco Rl/Pst I fragment from
-~DFIEk
114
4. RESULTS
4.1. Enzyme analysis
RuBisCO activity profiles of sucrose gradients
show two distinct peaks of activity (Fig. 1). These
RuBisCO peaks sediment identically to the form
I and form II types from Rb capsulatus [15].
While fractions 19-22 from the T. denitrificans
gradient showed less than 2% inhibition with
either 2 mM or 5 mM 6-phosphogluconate, fractions 24-27 were inhibited greater than 30% and
65% with 2 mM and 5 mM 6-phosphogluconate,
respectively (data not shown).
4.2. DNA analysis
Southern blots of various restriction enzyme
digests of T. denitrificans genomic DNA when
probed with either pANPl155 or pRR264/
pRR321, the genes encoding the form I or the
form II type RuBisCO respectively, revealed different hybridization patterns indicating the probes
did not cross hybridize and are specific for single
copy loci (Fig. 2).
Approximately 1200 library clones were
screened with the form I large subunit gene probe
12
5
80"I'TOM
TOP
10
4
and subsequently with the form I! gene probe.
Miniprep analysis of the positive clones revealed
one which contained only the form I gene, one
which contained both the form I and form II
genes and several which contained only the form
I1 gene. The form I clone, one of the form Ii
clones, and the clone which contained both the
form I and form II genes were selected for further analysis and named pTDF1, pTDF2, arid
pTDF12, respectively (Fig. 3).
4.2.1. Mapping. The three clones were subjected to restriction enzyme digests with various
combinations of enzymes and reprobed with the
form I and form II probes (Fig. 3). This allowed
the mapping of pTDF12 and the orientation of
the two RuBisCO genes (Fig. 4). To confirm that
indeed the T. denitrificans form 1 gene included
the small subunit gene, the cosmid clones were
probed with the SSU gene from P. sativum [25].
The resulting bands show that pTDF1 and
pTDF12 both contain SSU genes and that pTDF2
does not (Fig. 3). The SSU gene is located on the
same EcoRI fragment as the LSU gene.
4.3. mRNA transcripts
Total RNA from T. denitrificans screened with
a form i probe revealed a mRNA transcript of
approximately 2800 nucleotides (Fig. 5). Probing
with the T. denitrificans SSU gene area demonstrated a transcript the same size as the form 1
gene transcript (data not shown). Probing with a
form II probe from T. denitrificans showed a
transcript of approximately 1900 nucleotides (Fig.
5).
4.4. Additional probing
15
_
_'0
~o
Fraction (roll
Fig. 1. Sucrose gradient separation of the two RuBisCO
forms. RuBisCOactivityprofilesof sucrose gradient fractions
from Rb. capsulatusand 7".denitriflcans.Left peak represents
the smaller form II enzyme while the right peak represents
the form I.
When the three clones and genomic DNA
were screened for the presence of other Calvin
cycle genes by using probes for the genes from
the two R. sphaeroides gene clusters (Table 1),
only genomic DNA showed positive hybridization
with the phosphoribulokinase and cfx genes (data
not shown). The three clones, pTDF1, pTDF2,
and pTDF12, (which span an area of 53 kbp)
showed no positive hybridization with any of the
probes.
115
5. DISCUSSION
McFadden and Denend [1] purified an apparent form 11 RuBisCO from T. denitri~cans with
an approximate molecular mass of 350 000. Later,
Tabita and McFadden [10] showed this RuBisCO
to be insensitivite to 6-phosphogluconate inhibition, a characteristic of form I1 enzymes. Our
A
research coupled with these observations establishes the presence of two forms of RuBisCO in
T. denitrificans+ We have demonstrated the separation of the two RuBisCO forms by usiag sucrose density gradients, the inhibition of form l
by and the insensitivity of form I1 to 6-phosphogluconate, the expression of both forms at the
level of transcription and the isolation and map-
B
C
Fig. 2. Hybridization of T. denitrificans genomic DNA with RuBisCO probes. A. Agarose gel of genomic DNA digested with
EcoRl, Hindill, Pstl and BamHl, respectively.Last lane is lambda DNA digested wi~h HindlII. B. Autoradiographof Southern
blot probed with the form I RuBisCO gene (pANPlI55). C. Autoradiographof Southern blot probed with the form II gene
(pRR264/pRR321).
116
ping of the respective genes. The earlier research
[l,10] showing one form of RuBisCO can be
explained by independent regulation of the two
genes. Kinetic differences between the form 1
and form I1 enzymes became apparent when Jordan and Ogren [6] compared the substrate specificity factors for RuBisCO isolated from various
organisms. The specificity factor altows the comparison of the relative rates of carboxylation and
oxygenation at any given CO 2 and O 2 concentr.~:tion. It was found that the form 1 enzyme favors
the carboxylation reaction much more than does
the form II type, i.e. this means that under the
A
B
C
same conditions, the form ! enzyme will more
efficiently fix CO 2. It has been demonstrated that
increasing levels of CO 2 significantly repress the
form I enzyme in Rb. capsulatus and Rb.
sphaeroides [12,15,28]. in the course of this study
on T. denitrificans, the same effect was seen with
different levels of NaHCO:~ added to the medium.
The relative magnitude of the form 1 RuBisCO
peak in the sucrose gradients increased with decreasing NaHCO 3 levels (data not shown).
We also show the genes for both forms of
RuBisCO are separated by only about 17 kbp.
This is the smallest distance reported between
D
E
-- ]
--/__
Q
u
w
W
Fig, 3. Hybridization of 7", denitrificans cosmid clones with rbeL, rbcS and rbpL. A. Lambda DNA digested with Hindlll. B.
Agarose gel of EcoR! digested pTDFI, pTDFI2 and pTDF2 respectively. C. Autoradiograph of Southern blot probed v,ith the
form I RuBisCO gene probe (pANP1155). D. Autoradiograph of Southern blot probed with the SSU gene. E. Autoradiograph of
Southern blot probed with the form II gene probe (pRR264/pRR321). Upper bands in panels D and E are a result of vector
contamination in the probe preparations.
tl7
Table !
Plasmids used
Plasmid
Relevantcharacteristic
Source o r /
reference
pLAFR5
cosmidcloning vehicle;
20
double cos sites
pANPl155 rbcL and rbcS. A. nidulans
8
pRR116 rbpL R. ncbnmJ
26
pRR264 subcloneof internal Sail
This study
fragment from rbpL, 264 bp
pRR321 subcloneof internal Sail
This study
fragment from rbpL, 321 bp
pSSU
rbcS, P. sativum
25
pBSEK80 ]bpA, Rb. sphaeroides
27
pBS420BB prkA, Rb. sphaeroides
27
pBS550P cfxA, Rb. sphaeroides
27
pG
gapB, Rb. sphaeroides
27
pTDFI
7". denitrificans pLAFR5 clone
This study
containing rbcL and rbcS
pTDF2
T. denitrificans pLAFR5 clone
This study
containing rbpL
pTDF|2
T. denitri]icans pLAFR5 clone
This study
containing both RuBisCO genes
pTDFIE EcoR! subclone of r&'L and rbcS This study
in pUCS. T. clenitrificans
pTDF2E &'oR1 subclone e,f rbpL in pUC8. This study
T. denitriJicans
EZ
I
MCS
I
i
II
-
I
I
\
I
I
I
I
the two RuBisCO genes. The clustering of Calvin
cycle genes has been observed in several organisms, e.g. Rb. sphaeroides, AIcaligenes e, trophus,
Xanthobacter flat'us, Nitrobacter ruigaris and Rb.
rpsulatus [29-32]. The proximity of the RuBisCO genes in T. denitri]~cans led to the speculation that other Calvin cycle genes might be
clustered near the RuBisCO genes. However,
probing of the three cosmid clones (spanning 53
kbp) with the Calvin cycle genes from Rb.
sphaeroides did not reveal the clustering of the
phosphoribuiokinase or tyx genes. This does not
preclude the possibility that these and other
Calvin cycle genes are clustered elsewhere. Since
the genes are transcribed in opposite directions,
the region between the two appears to be a
logical regulatory region which warrants further
investigation.
In T. denitrifica,s, like many other organisms,
the mRNA transcript for the form I gene suggests
that the LSU and SSU genes are cotranscribed
[2,9,27,30,32,33]. Assuming the molecular masses
of the subunits to be similar to other reported
molecular masses (i.e. 560110 for the LSU and
i L
•
LII
i kbp
\
*,it,,. I
/
\ \
/ /
",
-
" \
/ /
Lu
\Lu
I
rtJc S
rbc L
~
I
I
I
I
~-
Lu
500 bp
rbp L
Fig. 4. Physical and genetic map of T. denitri~cans RuBisCO genes. Map indicates the EcoRI insert of pTDFI2. MCS refers to the
multicloning site of pLAFR5. The thick lines represent areas which have been mapped completely. The shaded boxes represent
genes. The direction of transcfip'ien is indicated b~, lines with arrows.
tl8
A
C
m
message length observed for both the LSU and
SSU probes, 2800, is certainly reasonable. Following a similar line of reasoning, the form !! transcript size (19110)seems appropriate.
This report demonstrates that the presence of
two forms of RuBisCO is not limited to the
phototrophic bacteria. As more organisms are
carefully examined for the presence of the form
II type, the occurrence of this phenomenon may
allow the evolutionary relationships of the RuBisCO forms to be elucidated.
:J',/i.:'/!
ACKNOWLEDGEMENTS
We would like to thank Dr. E.S. Maxwell,
North Carolina State University, Raleigh, NC, for
his assistance and hospitality during the RNA
isolation procedures and Sally Brock, Department of Biological Sciences, Clemson University,
Clemson, SC for her assistance with figure preparation. This work was supported in part by subcontract C88-102022 from EG&G Idaho, to JMS.
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Fig. 5. Expression of RuBisCO genes in Z denhrificans. A,
Agarose gel of total RNA isolated from T. denitrificans, B,
Autoradiograph of Northern blot probed with the form l
RuBisCO gen¢ probe from T. denitrificans. C. Autoradiograph of Northern blot probed with the form II RuBisCO
gene probe from T. denitrificans,
14000 for the SSU), a message length of about
2000 nucleotides would be required. Because the
lengths of the leader, the intergenic space, and
the transcript tail sequences are not known, the
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