Department of Microbiology and Molecular
Genetics, The University of Texas Health Science Center Medical School,
Houston, Texas 77030
Under anaerobic-dark growth conditions, in the presence of the
alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), Rhodobacter
sphaeroides 2.4.1T respires anaerobically using the
molybdoenzyme DMSO reductase (DMSOR). Genes encoding DMSOR and
associated proteins are encoded by genes of the dor locus.
Previously, we demonstrated that the expression of DMSOR is regulated
by both the oxygen status of the cell via the FnrL protein and by the
presence of DMSO or TMAO, presumably through the DorS-DorR
two-component sensor-regulator system. Here we further investigate
expression of the dor genes through the use of
transcriptional lacZ fusions to the dorS,
dorR, and dorC promoters. The expression of
dorC::lacZ was strongly induced by
the absence of oxygen and presence of DMSO. In accordance with our
previous findings of DMSOR activity,
dorC::lacZ expression was reduced by
up to one-third when cells were grown photosynthetically in the
presence of DMSO with medium or high light, compared to the expression
observed after anaerobic-dark growth. The induction of
dorC::lacZ expression in the presence
of DMSO was dependent on the DorS and DorR proteins. Expression of the
dorS and dorR genes was also induced in the
absence of oxygen. In an FnrL mutant, dorS::lacZ expression was not induced
when oxygen tensions in the media were lowered, in contrast to what
occurred in the wild-type strain. The expression of
dorS::lacZ and
dorR::lacZ was dependent on the DorS
and DorR proteins themselves, suggesting the importance of
autoregulation. These results demonstrate a cascade regulation of
dor gene expression, where the expression of the regulatory proteins DorS and DorR governs the downstream regulation of the dorCBA operon encoding the structural proteins of DMSOR.
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INTRODUCTION |
Rhodobacter sphaeroides
2.4.1T is a facultative phototrophic bacterium which is
capable of a wide range of metabolic lifestyles including aerobic,
anaerobic, photosynthetic, and diazotrophic growth modes. Under
anoxygenic growth conditions, in the absence of light, R. sphaeroides 2.4.1T respires anaerobically using
dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO)
as the terminal electron acceptor (for a review see reference
14). Reduction of both compounds is achieved by a
single enzyme, DMSO reductase (DMSOR), which is a monomeric periplasmic
protein containing a molybdopterin cofactor and whose structure has
recently been determined (12, 20).
As part of our low-redundancy sequencing strategy for chromosome II of
R. sphaeroides 2.4.1T, we sequenced a 13-kb
region containing genes homologous to the previously sequenced
dmsCBA genes of R. sphaeroides f. sp.
denitrificans and to the tor genes of
Escherichia coli, which encode components of TMAO reductase
(2, 9, 15, 16, 22, 25, 28). The dorC,
dorB, and dorA genes form a single operon, and,
respectively, encode a soluble c-type cytochrome, a membrane
protein of unknown function, and DMSOR (16). Upstream of the
dorCBA operon are two adjacent genes, dorS and
dorR, that are transcribed divergently inward toward each
other and which, respectively, encode putative sensor kinase and
response regulator proteins of the two-component signal transduction
family of proteins (16, 23). Strains with mutations in any
of the dorS, dorR, or dorCBA genes are
unable to grow anaerobically in the dark when DMSO or TMAO is supplied as the terminal electron acceptor and show negligible amounts of
DMSOR-specific activity (16). These results indicate that the dor genes encode the sole system responsible for the
reduction of both DMSO and TMAO in this bacterium.
We showed that the expression of the DorA protein was regulated by both
the oxygen status of the cell and by the presence of DMSO or TMAO in
the growth medium (16). We also demonstrated that the FnrL
protein has a positive role in the regulation of DorA expression,
suggesting that the expression of the dorCBA operon is
responsive to redox control (31). Further, DorS and DorR
mutants failed to accumulate DorA under any growth condition, demonstrating a positive role for these proteins in dorCBA
expression (16). In a separate study, it was shown that the
DmsR protein of R. sphaeroides f. sp.
denitrificans induced the dmsCBA operon in
response to DMSO, by binding to specific sites in the dmsC promoter (26). The DmsR protein of R. sphaeroides
f. sp. denitrificans and the DorR protein of R. sphaeroides 2.4.1T are almost identical at the amino
acid level, and thus it seems likely that the DorR protein plays a
similar role in R. sphaeroides 2.4.1T. However,
the authors did not report a corresponding DorS homolog and the cognate
sensor protein for DmsR has not been identified.
We wished to further investigate how the dor genes of
R. sphaeroides 2.4.1T are regulated by both
oxygen and DMSO and at what level these two signals interact. Here we
examine transcriptional regulation of the dorS,
dorR, and dorC promoters and present data which
demonstrate the requirement for DMSO and anaerobiosis for the
regulation of these promoters. We also show that dorC
expression is governed either directly or indirectly by light
intensity. We further demonstrate that this system is under
autoregulation and propose a cascade model for the regulation of DMSOR
expression in R. sphaeroides 2.4.1T.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The bacterial strains and plasmids used in this work are listed in
Table 1. E. coli strains were
grown at 37°C in Luria-Bertani medium, and R. sphaeroides
strains were grown at 30°C in Sistrom's minimal medium A containing
succinate as the carbon source (3, 19). Where appropriate,
DMSO was added at a final concentration of 60 mM and TMAO was added at
a final concentration of 30 mM. Cells were grown anaerobically in
sealed glass tubes, which were first sparged with nitrogen gas, and
were incubated in the dark for chemoheterotrophic growth or in front of
a 10-W m
2 light source for photoheterotrophic growth,
except in the experiments involving different light intensities.
Aerobic cultures were grown by continuous sparging with a mixture of
30% O2-69% N2-1% CO2. For
oxygen shift assays, cultures were grown aerobically for five to six
culture doublings and then shifted to 2% O2-97%
N2-1% CO2. One culture sample was removed
prior to the oxygen shift, and further samples were assayed at
appropriate intervals after the shift.
The media were supplemented with antibiotics, where appropriate, to
maintain selection for plasmids or to select for recombinant strains.
The final concentrations were as follows: ampicillin, 100 µg
ml1 (E. coli); kanamycin, 25 µg
ml1 (R. sphaeroides); spectinomycin, 25 µg
ml1 (R. sphaeroides); streptomycin, 25 µg
ml1 (R. sphaeroides); and tetracycline, 1 µg
ml1 (R. sphaeroides) and 10 µg
ml1 (E. coli).
Materials and reagents.
All reagents and materials used were
of analytical grade and, except where noted, were purchased from Sigma
Chemical Co. (St. Louis, Mo.).
Construction of lacZ reporter fusion plasmids.
Standard recombinant DNA techniques were used throughout
(19). Enzymes were purchased from New England Biolabs, Inc.
(Beverly, Mass.), Stratagene (La Jolla, Calif.), Promega Corp.
(Madison, Wis.), and Boehringer Mannheim Biochemicals, Bethesda
Research Laboratories Life Technologies Inc. (Gaithersburg, Md.).
The upstream regulatory sequences (URS) of the dorS,
dorR, and dorC genes were amplified by the PCR
using Vent DNA polymerase (New England Biolabs) and oligonucleotides
purchased from Bethesda Research Laboratories Life Technologies. The
reaction conditions for each of the PCR amplifications were identical,
consisting of 25 cycles of denaturation at 95°C for 1 min, annealing
at 50°C for 1 min, and extension at 72°C for 1 min. PCR
amplification of the dorS promoter region was performed
using the primers DORSP1 (5'-CGCCTAGGACCTCGCGGATCGG-3') and
DORSP2 (5'-GCGTTCGAACCCGCGCCTCGGCG-3'), generating a 662-bp
product. The PCR product was purified by using the Wizard PCR
purification kit (Promega Corp.), and the ends were filled in by using
Pfu polymerase (Stratagene). The blunt-ended PCR product was
cloned into SmaI-digested pBS II, resulting in plasmid
pNMT69. The dorR promoter region was amplified by using the
primers DORRPBAM (5'-CGGCGCGGATCCCGCATCGAGTGGC-3') and
DORRPHIND (5'-CGCGGCAAGCTTGCGCAGATACATCG-3') to generate a
525-bp product. The PCR product was cloned into pBS II, as above, to
give plasmid pNMT92. The dorC promoter region was amplified
by using the primers DORCP1 (5'-GCGCGACGTCGCGCGCTTCGCTGACTTCG-3')
and DORCP2 (5'-GCGCGACGTCCCGCATCGAGTGG-3') to generate
a 659-bp product. The PCR product was cloned into pBS II, as above, to
give plasmid pNMT68. The sequence and orientation of the cloned
products were confirmed before proceeding with additional cloning
steps. All of the cloned products were subcloned into the promoterless
lacZ vector pML5, using
BamHI-HindIII double digestions. This
resulted in the following plasmids:
dorS::lacZ, pNMT77;
dorR::lacZ, pNMT94; and
dorC::lacZ, pNMT78 (Fig.
1). Each of the lacZ fusion
plasmids was conjugated into R. sphaeroides 2.4.1T by triparental matings with pRK2013, as described
previously (4).
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FIG. 1.
Physical map of plasmids containing transcriptional
lacZ fusions to the dorC (A), dorS
(B), and dorR (C) URS of R. sphaeroides
2.4.1T. Putative DorR binding sites in the dorC
and dorR URS are boxed (boxes 1 to 4). The putative FnrL
binding motif in the dorS URS is shown in bold type.
Putative Shine-Dalgarno sequences are underlined. The arrows indicate
the direction of transcription. See the text for further details.
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DNA sequencing.
Automated DNA sequencing was performed using
an ABI 373A automatic DNA sequencer (Applied Biosystems Inc., Foster
City, Calif.) at the DNA Core Facility of the Department of
Microbiology and Molecular Genetics, The University of Texas Health
Science Center, Houston. Oligonucleotides used for priming the
sequencing reactions were purchased from Bethesda Research Laboratories
Life Technologies. Sequences were analyzed by using the Genetics
Computer Group programs and the BLAST server at the National Center for
Biotechnology Information (5).
Cell extract preparation and assays of 
-galactosidase
activity.
Preparation of crude cell extracts and determination of
-galactosidase activities were performed as described previously (18, 24). Cell extract protein concentrations were
determined by using the Pierce BCA Protein Assay Reagent (Pierce,
Rockford, Ill.) with bovine serum albumin as a reference standard.
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RESULTS |
Regulation of dorC::lacZ
expression.
Previous experiments revealed that the DorA protein
was produced only in the absence of oxygen and in the presence of DMSO or TMAO (16). Since the dorA gene is in an operon
downstream of the dorB and dorC genes, with a
putative promoter region upstream of dorC, we constructed a
dorC::lacZ fusion in order to measure expression from the dorC URS. After introduction of this
fusion plasmid (pNMT78) (Fig. 1) into wild-type or various mutant
strains of R. sphaeroides 2.4.1T, we measured
-galactosidase activities after growth under a number of different
conditions.
dorC::lacZ expression was maximal after
anaerobic-dark growth in the presence of DMSO (Fig.
2). In contrast, very little or no
-galactosidase activity was evident after aerobic growth or photosynthetic growth in the absence of DMSO. Intriguingly, the expression of dorC::lacZ was
approximately ninefold lower after anaerobic growth in the dark with
TMAO than after growth with DMSO under similar conditions. Most
interesting is the lower activity observed during photosynthetic growth
in the presence of DMSO when compared to the activity after growth
under anaerobic-dark conditions (Fig. 2). We previously observed that
the specific activity of DMSOR after photosynthetic growth in the
presence of DMSO was approximately 50% of that for anaerobic-dark
conditions (16). Therefore, it appears that there is a good
correlation between dorC transcription and DMSOR activity.
We grew wild-type cells containing the various
dor::lacZ fusions photosynthetically in
the presence of DMSO at different light intensities and assayed -galactosidase activities of extracts from these cultures in order
to determine if there was an effect of light intensity on dor gene expression.
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FIG. 2.
-Galactosidase activities from cell extracts of
R. sphaeroides strains containing the
dorC::lacZ transcriptional fusion
plasmid pNMT78. Growth conditions are as follows: AER ( ),
aerobically with 60 mM DMSO; ANA+DMSO ( ), anaerobically in the dark
with 60 mM DMSO; ANA+TMAO (), anaerobically in the dark with 30 mM
TMAO; PS ( ), photosynthetically; PS+DMSO ( ), photosynthetically
with 60 mM DMSO. Photosynthetic cultures were grown with a light
intensity of 10 W m2. Results are the mean values from
triplicate assays of at least three independent cultures and are
corrected for activity from the vector alone under the same conditions
(pML5, <35 µmol of o-nitrophenol formed
min1 mg of protein1). Vertical bars
represent the standard deviation from the mean.
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For the dorC::lacZ fusion, decreased
-galactosidase activities were observed after cells were grown under
low (3 W m2), medium (10 W m2), or high
(100 W m2) light intensity compared to the activity after
anaerobic-dark growth (Table 2). Growth
under high light intensity resulted in the maximal decrease in
activity, resulting in a level approximately threefold lower than that
observed under anaerobic-dark conditions (Table 2). In contrast,
expression of dorS::lacZ did not vary significantly with differing light intensity, although a small decrease
in -galactosidase activity was observed when cells were grown under
low or medium light intensity (Table 2). Only when cells were grown
under high light, did dorR::lacZ
expression decrease, to a level approximately 50% of that observed in
the dark or under low or medium light intensity (Table 2).
It is clear that maximum expression of
dorC::lacZ is observed after growth in
the dark, in the absence of oxygen and in the presence of DMSO. We
previously observed that the DorA protein was absent in DorR and DorS
mutants, and it is believed that DorS and DorR form a two-component
sensor-regulator system (16). To determine whether the DorSR
system acts at the dorC URS, we examined the expression of
dorC::lacZ in DorR and DorS mutant backgrounds after photosynthetic growth in the presence of DMSO, since
the mutants are unable to grow anaerobically in the dark with DMSO or
TMAO. The -galactosidase activities in both mutants were very low
when compared to activities in the wild-type background (Fig. 2).
Similar low levels of activity were observed after photosynthetic growth in the absence of DMSO (data not shown). These results demonstrate a positive role for the DorSR system in the control of the
dorCBA operon.
dorS::lacZ and
dorR::lacZ expression.
To
further establish the roles of the DorS and DorR proteins in the
regulation of dor expression, we were interested to learn how the genes encoding these proteins are themselves regulated. We
constructed dorS::lacZ and
dorR::lacZ fusions and measured the
resulting -galactosidase activities from these fusions in wild-type
and mutant backgrounds after growth under different conditions. For
both fusions, only very low levels of activity were observed after
aerobic growth (Fig. 3 and
4). An approximately 20-fold induction of
dorS::lacZ expression was observed
after anaerobic-dark growth in the presence of DMSO (Fig. 3). The
activity after anaerobic-dark growth with TMAO was approximately 45%
of that when cells were grown with DMSO, in contrast to the approximate 90% decrease in dorC::lacZ expression
observed between TMAO- and DMSO-grown cultures. In further contrast to
dorC::lacZ expression, dorS::lacZ expression decreased only
slightly after photosynthetic growth in the presence of DMSO, compared
to the expression observed after anaerobic-dark growth. The expression
of dorS::lacZ also appeared to be under
DMSO-dependent control, as an approximately twofold increase in
activity was observed after photosynthetic growth in the presence of
DMSO compared to that in the absence of DMSO.
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FIG. 3.
-Galactosidase activities from cell extracts of
R. sphaeroides strains containing the
dorS::lacZ transcriptional fusion
plasmid pNMT77. Growth conditions are as follows: AER (),
aerobically with 60 mM DMSO; ANA+DMSO (), anaerobically in the dark
with 60 mM DMSO; ANA+TMAO (), anaerobically in the dark with 30 mM
TMAO; PS (), photosynthetically; PS+DMSO (), photosynthetically
with 60 mM DMSO. Photosynthetic cultures were grown with a light
intensity of 10 W m2. Results are the mean values from
triplicate assays of at least three independent cultures and are
corrected for activity from the vector alone under the same conditions
(pML5, <35 µmol of o-nitrophenol formed
min1 mg of protein1). Vertical bars
represent the standard deviation from the mean.
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FIG. 4.
-Galactosidase activities from cell extracts of
R. sphaeroides strains containing the
dorR::lacZ transcriptional fusion
plasmid pNMT94. Growth conditions are as follows: AER (),
aerobically with 60 mM DMSO; ANA+DMSO (), anaerobically in the dark
with 60 mM DMSO; ANA+TMAO (), anaerobically in the dark with 30 mM
TMAO; PS (), photosynthetically; PS+DMSO (), photosynthetically
with 60 mM DMSO. Photosynthetic cultures were grown with a light
intensity of 10 W m2. Results are the mean values from
triplicate assays of at least three independent cultures and are
corrected for activity from the vector alone under the same conditions
(pML5, <35 µmol of o-nitrophenol formed
min1 mg of protein1). Vertical bars
represent the standard deviation from the mean.
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The expression of dorR::lacZ was much
lower relative to that of the
dorS::lacZ and
dorC::lacZ fusions, but since the
values presented are the results of three independent growths, each
performed in triplicate, we believe that these represent the true
values. Measurement of -galactosidase activities from the
dorR::lacZ fusion revealed that the
expression of dorR, like that of dorS, is also
induced by anaerobiosis, although only an approximately fivefold
induction is observed in this case (Fig. 4). Similar to the reduction
in dorS::lacZ expression,
anaerobic-dark growth with TMAO resulted in an approximately 40%
reduction in dorR::lacZ expression when
compared to the levels observed when cells were grown with DMSO. When
cells were grown photosynthetically,
dorR::lacZ expression was stimulated
twofold by the presence of DMSO. No significant differences were
observed in the levels of dorR::lacZ expression between anaerobic-dark- and photosynthetically grown cultures in the presence of DMSO, in contrast to levels of both dorC::lacZ and
dorS::lacZ expression (Fig. 4). When
the light intensity was increased to 100 W m2,
dorR::lacZ expression was reduced by
approximately 50% relative to that seen under low or medium light
intensity (Table 2). This demonstrates that dorR expression
is also under light-responsive regulation, but not as stringently as
dorC expression.
Since the DorSR system positively regulated
dorC::lacZ expression, we wondered if
the expression of the dorS and dorR genes themselves was under autoregulation by the DorS and DorR proteins. Both
the dorS::lacZ and
dorR::lacZ fusions were introduced into the respective DorS and DorR mutant strains, NM15 and NM16, and the
-galactosidase activities were measured after photosynthetic growth
in the presence of DMSO. The activities from both fusions were much
lower than those for the wild-type strain under similar growth
conditions and resembled the levels observed under aerobic conditions
for each fusion, indicating a positive role for the DorS and DorR
proteins in the autoregulation of dorS and dorR gene expression (Fig. 3 and 4). Further, it should also be noted that
we assayed dorS::lacZ and
dorR::lacZ expression in the mutant strains after photosynthetic growth in the absence of DMSO and found
the levels of expression to be very similar irrespective of whether
DMSO was present or not (data not shown).
FnrL positively regulates
dorS::lacZ.
It was previously
demonstrated than an FnrL mutant was unable to synthesize the DorC
c-type cytochrome or the DorA protein, even when grown in
low oxygen in the presence of DMSO (31). This suggested that
at least one component of the dor cluster was under
FnrL-mediated regulation. Upon inspection of the dor sequences, it was observed that within the dorS URS, there
is a putative FnrL binding site, TTGAC-N4-ATCAA, differing
from the consensus Fnr motif by only one nucleotide change (Fig. 1)
(32). To determine whether the expression of dorS
is regulated by FnrL, the dorS::lacZ
fusion plasmid pNMT77 was introduced into the FnrL mutant strain JZ1678
and an oxygen shift experiment was performed where high-oxygen (30%)
cultures were shifted to low-oxygen (2%) conditions in the presence of
DMSO.
Again, an extremely low level of -galactosidase activity was
observed for both the wild-type and FnrL mutant strains under high-oxygen (30%) conditions (Fig. 5).
After the cultures were shifted to 2% oxygen, a rapid increase in
dorS::lacZ expression was observed in
the wild-type strain which attained a plateau level 2 to 4 h
postshift. After this time, dorS::lacZ
expression decreased toward the steady-state level previously observed
for anaerobic-dark cultures (Fig. 3). In contrast, no such increase was
observed for dorS::lacZ expression in
the FnrL mutant strain. In fact,
dorS::lacZ expression actually
decreased after the oxygen shift.
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FIG. 5.
Kinetics of induction of
dorS::lacZ transcriptional fusion in
the wild-type strain 2.4.1T ( ) and FnrL mutant strain
JZ1678 ( ) following a shift from 30 to 2% oxygen, indicated by the
vertical arrow. Cultures were sampled at the times indicated, and
extracts from 15-ml samples were assayed for -galactosidase
activity. The values represent the means of triplicate assays from two
independent growth experiments. The standard error in each case did not
exceed 20% of the mean value.
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DISCUSSION |
Our previous results indicated that the expression of the
dorCBA operon, encoding the structural components of DMSO
reductase, is dually regulated by oxygen and DMSO (16). In
order to further investigate the regulation of the dor genes
in R. sphaeroides 2.4.1T, we constructed
transcriptional lacZ fusions to the upstream regulatory
sequences of the dorS, dorR, and dorC
genes for use as reporters of promoter activity.
As predicted, expression of dorC::lacZ
was induced by both the presence of DMSO and the absence of oxygen.
After aerobic growth or photosynthetic growth in the absence of DMSO,
negligible levels of -galactosidase activity are observed. Further,
it would appear that TMAO is less efficient as an inducer of
dorC::lacZ expression than DMSO. We
previously demonstrated that the dorCBA-encoded DMSOR is
also responsible for TMAO reductase activity and that Dor mutants are
unable to utilize TMAO as the terminal electron acceptor
(16). The weaker induction by TMAO may be related to the
possibility that the DorSR system is responsible for dorCBA induction in response to the presence of either DMSO or TMAO
(17). A twofold decrease in
dorS::lacZ and
dorR::lacZ expression was observed
after growth with TMAO compared with expression after growth with DMSO.
Further, in DorS and DorR mutants
dorC::lacZ was expressed at a similar
low level when the cells were grown photosynthetically with either DMSO
or TMAO (data not shown). These data suggest that the DorSR system is
responsible for both DMSO- and TMAO-dependent sensing and regulation.
We also showed that DorS and DorR mutants are unable to synthesize the
DorA protein (16). The expression of
dorC::lacZ in the DorS and DorR mutant
backgrounds was negligible after cells were grown photosynthetically in
the presence of DMSO or TMAO. Since the DorS and DorR proteins are
required for the induction of
dorS::lacZ,
dorR::lacZ, and
dorC::lacZ expression in response to
DMSO (or TMAO), we propose that the DorS and DorR proteins form a DMSO
(and TMAO)-responsive regulatory system, homologous to the TorS and
TorR proteins of E. coli (9, 22). However, since
dorS::lacZ and
dorR::lacZ expression in the absence of
either DMSO or TMAO is still dependent on the DorSR system, we believe that this system is responsive to an additional signal. The fact that
expression of dorC::lacZ is not induced
in the absence of DMSO, in contrast to that of
dorS::lacZ or
dorR::lacZ, even when DorS and DorR are
present, is explained by our recent finding that an additional
regulatory system, which we term DorXY, is positively required for
dorC::lacZ expression but not for
dorS::lacZ or
dorR::lacZ expression (17).
We are currently investigating whether this system is an additional
DMSO-dependent regulatory system.
Presumably, DorR is able to activate transcription by binding to
conserved motifs in the dorC URS (Dor box, consensus
A/CG/TGTTA/CACANC), which were previously identified as DmsR binding
sites in R. sphaeroides f. sp. denitrificans
(Fig. 1) (26). These motifs are very similar to the TorR
binding sites identified in the torCDA URS in E. coli (21). This suggests that DorR is able to activate
transcription in a manner similar to that by DmsR and TorR. In this
respect, it is of importance to note the absence of a homolog of the
TorT protein in R. sphaeroides 2.4.1T. In
E. coli the TorT protein is required for torCDA
expression, acting upstream of the TorS sensor protein (9,
10). The authors conclude that TorT is probably not a
TMAO-binding protein, and its exact role remains unclear. Presumably,
the absence of a TorT homolog in R. sphaeroides
2.4.1T reflects a functional difference in the signal
transduction pathway between the Tor and Dor systems.
The reduction in dorC::lacZ expression
when cells were grown photosynthetically with DMSO compared to
anaerobic-dark growth was in accordance with the 50% decrease in
DMSOR specific activity that we previously observed
(16). Further, we found a correlation between the decrease
in expression of dorC::lacZ and the
increase in light intensity (Table 2). The observation that
dorCBA expression is possibly light responsive is exciting
since mechanisms for light-responsive gene regulation in R. sphaeroides 2.4.1T are poorly characterized. It has
previously been demonstrated that several genes required for
photosynthetic growth, namely crtA, crtI,
puc, and bchF, are under light-dependent
transcriptional regulation (30). In addition, increases in
light intensity have been shown to affect the accumulation of
bacteriochlorophyll in the cell, levels of puc (which
encodes the B800-850 components of the light-harvesting complex) mRNA,
and carotenoid accumulation (11, 29). It has been
demonstrated that the PpsR protein functions in the light-responsive
regulation of B800-850 abundance (8). Recently, it was shown
that the activity of PpsR depends on the AppA protein, which may serve
as a redox-dependent modulator of PpsR activity (8). Since
dorS::lacZ and
dorR::lacZ expression are much less
affected than dorC::lacZ expression, we
believe that the major target for light-dependent regulation is at the dorC URS. We are currently investigating whether the
recently identified DorXY regulatory system is affected by light
intensity or redox and whether
dorC::lacZ expression is dependent on
any of the previously identified redox-dependent regulatory systems of
R. sphaeroides 2.4.1T (17).
In addition to examining the regulation of dorCBA
expression, we were interested in how the genes encoding the DorS and
DorR regulatory proteins themselves were regulated.
dorS::lacZ expression was shown to be
dependent on the absence of oxygen and the presence of DMSO. By
performing an oxygen shift experiment using an FnrL mutant, we
demonstrated the positive role for this protein in the induction of
dorS::lacZ expression in response to
lowering the oxygen concentration (Fig. 5). The presence of an Fnr
binding motif in the dorS URS suggests that this
FnrL-dependent regulation occurs directly at the dorS
promoter. dorS::lacZ expression was also shown to be increased by the presence of DMSO after photosynthetic growth (Fig. 3). As for dorC::lacZ
expression, we believe that this induction is due to the activities of
the DorS and DorR proteins, since in the DorS and DorR mutant strains
dorS::lacZ expression was extremely
low. At present it is unclear as to how the DorS and DorR proteins
affect dorS expression, since there are no putative DorR
binding motifs present in the dorS URS. Experiments are
currently under way to examine this further.
The expression of dorR::lacZ was also
shown to be dependent on the absence of oxygen and the presence of
DMSO. Further, dorR::lacZ expression
was also dependent on the presence of the DorSR system. Since there are
putative DorR binding motifs present in the dorR URS, it is
believed that the positive regulation by DMSO occurs via DorR binding
and activation through these sites. Since
dorR::lacZ expression returned to
aerobic levels in the DorS mutant, even when cells were grown
photosynthetically in the presence of DMSO, we believe that the
anaerobic induction of dorR::lacZ
expression observed is dependent on the induction and activity of the
DorS protein. It was therefore surprising to us to find that the DorS- and DorR-mediated effects on dorS and dorR
expression were manifested even in the absence of DMSO. This suggests
that the DorSR system may be responsive to an additional signal other
than DMSO (or TMAO). It was previously demonstrated that the DmsR
protein of R. sphaeroides f. sp. denitrificans
was able to bind to and retard DNA on a gel of the dmsCBA
URS in the absence of DMSO (26). This would suggest
that DMSO is not the sole signal for the DorSR system.
Taking these results together, we would like to propose the following
model for the regulation of DMSO reductase (dor) gene expression in R. sphaeroides 2.4.1T. Under
oxygen-limited conditions the FnrL protein is able to induce the
transcription of the dorS gene, encoding the DorS
sensor-kinase protein (Fig. 6). DorS is
able to phosphorylate its cognate regulator, DorR, in response to the
presence of DMSO (or TMAO) and to an additional uncharacterized signal.
Phosphorylated DorR is then able to activate transcription from both
the dorR and dorCBA promoters. This leads to an
increase in the synthesis of the DorR protein itself and production of
functional DMSOR. DorR also appears to affect transcription of the
dorS gene, by an as yet uncharacterized mechanism. An
additional regulatory system, encoded by the dorX and
dorY genes, is also required for dorCBA
expression. The dorCBA and dorR promoters are
also subject to light-responsive control. Therefore, it appears that a
regulatory cascade is involved, whereby the regulation of the
dorS and dorR genes, encoding regulatory proteins, controls the downstream expression of the dorCBA
operon, encoding the structural components of the DMSOR enzyme.
Further, we have demonstrated that this regulation is complex,
requiring multiple signals and multiple regulatory proteins.
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FIG. 6.
Model for the regulation of DMSO reductase
(dor) gene expression in R. sphaeroides
2.4.1T. See the text for further details.
|
|
We thank Tony Shaw (University of Queensland, Brisbane,
Australia) and Silke Leimkühler (University of Bielefeld,
Bielefeld, Germany) for generously providing plasmid pML5. We also
thank Jill Zeilstra-Ryalls, Jesus Eraso, and Tracy Palmer for helpful comments and suggestions.
This work was supported by U.S. Public Health Service grant GM15590 to
S.K.
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Abstract
Introduction
