From the Department of Microbiology and the Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, Ohio 43210-1292
Received for publication, November 4, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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In a previous study (Dubbs, J. M.,
Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000)
J. Biol. Chem. 275, 19224-19230), it was demonstrated
that the regulators CbbR and RegA (PrrA) interacted with both promoter
proximal and promoter distal regions of the form I
(cbbI) promoter operon specifying genes of the
Calvin-Benson-Bassham cycle of Rhodobacter
sphaeroides. To determine how these regulators interact with the
form II (cbbII) promoter, three
cbbFII::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbbII operon of
R. sphaeroides CAC. Expression of The nonsulphur purple bacterium Rhodobacter sphaeroides
utilizes the Calvin-Benson-Bassham
(CBB)1 reductive pentose
cycle as its primary pathway for CO2 fixation. In this
metabolically diverse organism the CBB cycle plays two very different
roles. Under autotrophic growth conditions, CO2 serves as
the sole carbon source, and the CBB cycle is the primary source for
nearly all of the fixed carbon utilized by the cell. This may entail
aerobic chemoautotrophic growth in the dark (i.e. in a
minimal medium lacking organic carbon under an atmosphere of 5%
CO2/45% H2/50% air) or anaerobic
photoautotrophic growth in the light (i.e. in a minimal
medium bubbled with 1.5% CO2/98.5% H2).
Photoheterotrophic growth in the presence of a fixed carbon source
causes the role of the CBB cycle to shift, such that CO2 serves primarily as an electron sink, with excess reducing equivalents generated by the oxidation of fixed carbon compounds funneled to
CO2 (1). When grown under conditions where the CBB cycle is
required, R. sphaeroides maintains the appropriate level of CBB cycle activity through the coordinate expression of two CBB cycle
operons, denoted cbbI and
cbbII (2, 3). In addition to structural genes
that encode CBB cycle enzymes, each operon encodes one of two distinct
forms of ribulose bisphosphate carboxylase/oxygenase (Rubisco).
The cbbI operon contains the genes for a
form I (L8S8) Rubisco
(cbbLIcbbSI) (4)
whereas the cbbII operon encodes the large
subunit of a form II type Rubisco (cbbMII) (5).
The regulation of cbb gene expression in R. sphaeroides is quite complex (2). Expression of the genes in both
the cbbI and cbbII
operons is highly induced during anaerobic phototrophic growth and
moderately induced during aerobic chemoautotrophic growth (6). During growth under CO2 fixing conditions, expression of each
operon is modulated independently in response to a number of
environmental parameters such as the level of CO2 and the
reduction state of organic carbon compounds supplied for growth
(7-11). This independent regulation results in shifts in the relative
abundance of proteins encoded within each operon. In general, growth
under photoheterotrophic conditions, with a fixed (organic) carbon
source, results in an excess of cbbII expression
over cbbI. Maximal expression from both operons
is observed under photoautotrophic and chemoautotrophic conditions;
i.e. when CO2 is used as the sole carbon source,
with cbbI operon expression exceeding that for
the cbbII operon (11). In addition to the
apparent independent regulation of cbbI and cbbII gene expression, a mechanism for
interdependent regulation also exists that results in a compensatory
increase in the expression of one operon when the other is inactivated
(4, 7, 9, 10). The cbbR gene, which encodes a LysR-type
transcriptional regulator, is located immediately upstream and
divergently transcribed from cbbFI (12) and
mediates this compensatory effect. CbbR is a positive regulator of the
expression of both the cbbI and cbbII operons (12, 13). The regA-regB
(prrA-prrB) two component regulatory system, encoding sensor
kinase RegB (PrrB) and response regulator RegA (PrrA) also plays a role
in cbb regulation. Although originally identified as a
regulator of photosystem biosynthesis genes in both Rhodobacter
capsulatus (14, 15) and R. sphaeroides (16), the
regA-regB (prrA-prrB) two-component regulatory
system was implicated in cbb regulation by genetic studies
that demonstrated that a R. sphaeroides regB insertion
mutant exhibited reduced cbbI and
cbbII expression during photoautotrophic growth
in a 1.5% CO2/98.5% H2 atmosphere (17). It
was subsequently shown that regA is required for
cbbI and cbbII expression
during incubation under photoautotrophic growth conditions (18). It has
also been demonstrated that RegA binds directly to cbb
operon promoters in both R. capsulatus and R. sphaeroides (18, 19). A growing number of studies have shown that
the regA-regB (prrA-prrB) two-component system
and its homologs regulate the expression of genes involved in a wide
variety of metabolic processes such as nitrogen fixation and nitrogen
metabolism (20-22), hydrogen utilization and evolution (20, 22),
electron transport (23), and the oxidation of formaldehyde (24).
The overall goal of our ongoing investigation is to understand the
mechanism(s) involved in the regulation of cbb gene
expression in R. sphaeroides. Prior to this work, the
primary model system for our cbb gene regulation studies was
the R. sphaeroides cbbI operon. Previous
studies, using cbbI::lacZ
promoter fusions showed that the cbbI promoter
contains a promoter proximal region ( Bacterial Strains, Plasmids, and Culture
Conditions--
R. sphaeroides strains CAC (6), and
CAC::regA Purification of RegA*, RegB", and CbbR--
RegA* was purified
from E. coli strain BL21(DE3) carrying the plasmid
pET29CBD::regA* using a method described
previously (29). RegB", a truncated form of RegB lacking a membrane
spanning region, was purified from E. coli strain BL21(DE3)
carrying plasmid pET28alt::regB" as described
previously (30). Preparations of recombinant R. sphaeroides
CbbR were obtained from E. coli strain BL21(DE3) carrying
plasmid pET11R-11 as described previously (13).
DNaseI Footprint Analysis--
Probes for DNase I footprint
analyses were prepared by PCR amplification of selected regions of the
cbbII operon promoter of R. sphaeroides, using pJG3 (31) as a template. Selective labeling of
DNA strands was performed by 5' end labeling of one of the
oligonucleotide primers with T4 polynucleotide kinase (New England
Biolabs, Beverly, MA) and [
DNase I footprint assays were performed as described previously (18).
RegA* was phosphorylated in a reaction containing RegA* (128 µM), RegB" (128 µM), ATP (1 mM)
in a buffer containing 50 mM HEPES, pH 7.8, 5 mM MgCl2, 100 mM KCl, 2 mM CaCl2, 1.5 mM dithiothreitol, 25 µg/ml bovine serum albumin, and 25% glycerol. Standard G+A DNA
sequence ladders were generated by the chemical cleavage method as
described by Ausubel et al. (32). The sequences of the
oligonucleotides used to generate the probes used for the CbbR
footprinting experiments are as follows: cbbII-1,
5'-CATGGCTCCTCCTGCCTCTG-3'; cbbII-2, 5'-TCCTCGGAGCGGGGTGAGCG-3'. The
sequences of the oligonucleotides used to generate the probes used
in the RegA* footprinting experiments are as follows: cbbII-5,
5'-CAGGGCCGGTGGGGGGCCTC-3'; cbbII-11, 5'-GCCCTGCCCAAGCCTGACGC-3';
cbbII-13, 5'-GACGCCTGCGGACACGGACC-3'; cbbII-14,
GCCCGGCGAGACGCTCGCTC-3'; cbbII-15B, 5'-CCCTGCCTTGGGGTCGCTGC-3'; cbbII-16, 5'-GCAAGGCAGAACTCGAGGAG-3'; cbbII-19,
5'-CGCCGCCTGTGCCACCATGC-3'; cbbII-20, 5'-GCATGGTGGCACAGCCGCCG-3'.
Preparation of Extracts for Column Chromatography and Gel
Mobility Shift Assays--
Extracts of R. sphaeroides
strains grown to late exponential phase (i.e.
A660 nm = ~1.0) were sonicated in a buffer containing Buffer A (10 mM KCl, 20 mM HEPES, pH
8.5, 1 mM EDTA, and 5 mM Construction of cbbII::lacZ Promoter Fusion
Plasmids--
A 1056-bp fragment containing the
cbbII promoter was amplified from pJG3 (31) via
PCR. The primers used for the amplification were designed to create an
EcoRI site 1020 bp upstream of the cbbII transcription start and a BglII
site 47 bp downstream of the cbbII transcription
start, within cbbFII. This fragment was blunt-end cloned into the HincII site of pK18 to generate
plasmid pKcbbII and sequenced. The 1056-bp
EcoRI/BglII fragment of pKcbbII was
ligated into EcoRI/BamHI-digested pMC1403 to
yield plasmid pMCCII. The BamHI/BglII ligation in
pMCCII resulted in an in-frame fusion of cbbFII
to lacZ that contains the first three codons of
cbbFII. EcoRI-digested pMCCII was
then ligated into the EcoRI site of the conjugative plasmid
pVK101 (33) to yield plasmid pVKCII.
To construct plasmid pVKCIIXho, the sequence upstream of the
XhoI site at Regulation of the cbbII Operon in R. sphaeroidesCAC--
To identify the DNA sequences required for transcriptional
regulation of the cbbII operon a number of
lacZ translational fusion plasmids containing increasing
amounts of DNA upstream of the cbbFII
translation start were constructed. Previous studies (25) using
transcriptional fusions to xylE determined that the
cbbII promoter was located within 991 bp of the
cbbFII translation start. It was with this in
mind that we constructed
cbbFII::lacZ translational fusion plasmids containing 1025 bp (pVKCII), 545 bp (pVKCIIXho), and
227 bp (pVKCIIXcm) of sequence upstream of the
cbbFII transcription start (Fig.
1). These plasmids were transferred into
R. sphaeroides CAC, a spontaneous gain of function mutant of
R. sphaeroides HR that has the ability to grow
chemoautotrophically in the presence of 10% O2
concentrations (i.e. in minimal medium lacking organic carbon bubbled with 5% CO2/45% H2/50% air)
(6). The plasmid-containing strains were then assayed for
It is known that form II Rubisco synthesis and cbbM
transcription is enhanced under photoautotrophic growth conditions (4, 11). The enhanced level of LacZ activity during photoautotrophic growth
using plasmid pVKCIIXcm is consistent with these prior studies and
suggested that the cbbII promoter contained a
proximal regulatory region within 227 bp upstream of the
cbbFII transcription start (pVKCIIXcm) that
conferred the proper regulatory pattern.
Two regions upstream of Regulation of cbbII Promoter Activity in R. sphaeroides
cbbR Insertion Strain 1312--
To investigate the role that the
trans-acting transcriptional activator CbbR plays in
cbbII operon regulation, fusion plasmids pVKCIIXcm, pVKCIIXho, and pVKCII were introduced into cbbR
insertion strain 1312, and lacZ expression was monitored
under chemoheterotrophic and photoheterotrophic growth conditions
(Table II). Strain 1312 harbors an inactivated CbbR because of the
insertion of a trimethoprim resistance cassette within the
cbbR gene (12). Consequently, strain 1312 does not
synthesize form I Rubisco and accumulates only low levels of form II
Rubisco compared with wild-type R. sphaeroides. As a result,
strain 1312 is capable of growth under chemoheterotrophic and
photoheterotrophic conditions but not under photoautotrophic or
chemoautotrophic conditions (12). It was apparent that normal regulated
expression exhibited by each of the
cbbFII-lacZ fusion plasmids upon
switching from chemoheterotrophic to photoheterotrophic growth was
abolished in this strain (Table II) indicating that a functional
cbbR gene was necessary for the upstream activating sequence
contained in plasmids pVKCIIXho and pVKCII to influence lacZ expression.
Regulation of cbbII Promoter Activity in R. sphaeroides
regA Insertion Mutant Strain CAC::regA Binding of R. sphaeroides CbbR to the cbbII
Promoter--
Previous gel mobility shift studies had indicated that
CbbR binds to the cbbII promoter within 200 bp
of the cbbII transcription start (data not
shown). To define the site of CbbR binding, DNase I protection assays,
using [32P]-labeled probes spanning the region from +41
to Binding of R. capsulatus RegA* to the R. sphaeroides
cbbII Promoter--
Previous studies (17, 18) had shown
that the RegA-RegB (PrrA-PrrB) system is involved in cbb
regulation in R. sphaeroides, and a constitutively
active RegA protein (RegA*) from the related nonsulphur purple
bacterium R. capsulatus was shown to bind specific regions
of the cbbI operon promoter-operator of R. sphaeroides. To define the RegA binding sites, we used R. capsulatus RegA* in DNase I footprinting experiments of the
R. sphaeroides cbbII promoter-operator region.
[32P]-Labeled probes that covered a region from +41 to
Detection of cbbII Promoter Binding Activities in
Extracts of R. sphaeroides Strain CAC--
The results of the
cbbII::lacZ fusion studies
suggested that other proteins or other factors might be involved in
cbbII regulation in addition to CbbR and RegA
under aerobic chemoautotrophic growth conditions. This was particularly
evident, because a
cbbII::lacZ fusion plasmid
containing 227 bp of sequence upstream of the
cbbII transcription start (pVKCIIXcm) showed a
higher level of lacZ expression under chemoautotrophic
growth conditions in a regA mutant background, compared with
the parental strain. In addition, the involvement of potential
alternative regulators was consistent with the observation that
cbbII expression during aerobic chemoautotrophic growth was significantly lower than during anaerobic photoautotrophic growth. To determine whether additional cbbII
regulatory proteins were present in R. sphaeroides, a
crude extract of chemoautotrophically grown R. sphaeroides
CAC::regA This investigation and previous studies (13, 18) indicate that the
R. sphaeroides cbbI and
cbbII promoters have similar structural
features. Both promoters are composed of a promoter proximal regulatory
region, containing a CbbR binding site sufficient to confer low level
regulated cbb expression; in addition, a more distal
upstream activating region, containing RegA binding sites, enhances
expression. Although each upstream activating region contains multiple
RegA binding sites, a single site in each operon (located at -galactosidase was
monitored under a variety of growth conditions in both the parental
strain and knock-out strains that contain mutations that affect
synthesis of CbbR and RegA. The binding sites for both CbbR and RegA
were determined by DNase I footprinting. A region of the
cbbII promoter from +38 to
227 bp contained a
CbbR binding site and conferred low level regulated cbbII expression. The region from
227 to
1025 bp contained six RegA binding sites and conferred enhanced
cbbII expression under all growth conditions.
Unlike the cbbI operon, the region between
227 and
545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were
required for maximal cbbII expression. Two
potentially novel and specific cbbII
promoter-binding proteins that did not interact with the
cbbI promoter region were detected in crude
extracts of R. sphaeroides. These results, combined with
the observation that chemoautotrophic expression of the
cbbI operon is RegA independent, indicated that
the mechanisms controlling cbbI and
cbbII operon expression during
chemoautotrophic growth are quite different.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 to +1 bp) that confers low
level regulated expression of cbbI that is
CbbR-dependent (13). DNaseI footprinting studies showed that this region contained a binding site for CbbR (
10 to
70 bp),
along with two RegA binding sites (
61 to
110) (18). A promoter
distal upstream activating region was also identified, between
280
and
636 bp, that significantly enhanced cbbI
expression under all growth conditions tested. This region was found to
contain two RegA binding sites (
301 to
415 bp) (18). Although
earlier work determined that the cbbII promoter
occurred within 1000 bp of the cbbII
transcription start (25), details of the structure of the R. sphaeroides cbbII promoter have not been investigated previously. In this study,
cbbII::lacZ
translational fusions with different amounts of upstream sequence were
constructed to facilitate monitoring of gene expression under a variety
of growth conditions. Evidence for upstream activating sequences was
obtained within the cbbII promoter region, and
DNaseI footprint analyses enabled binding sites for both CbbR and RegA
to be identified within the cbbII promoter
region. An important byproduct of these studies was the demonstration
that two potentially novel and specific cbbII
promoter-binding proteins were present in cell extracts of R. sphaeroides. The results of this investigation indicated that the
structure of the R. sphaeroides cbbII promoter
exhibited both similarities and differences to the R. sphaeroides
cbbI promoter (18), with consequent effects on
differential regulation of the cbb operons.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(17) (Table I) were grown
photoautotrophically under a gas atmosphere of 1.5%
CO2/98.5% H2 and chemoautotrophically under a
gas atmosphere of 5% CO2/45% H2/50% air as
described previously (11, 13). R. sphaeroides strains CAC
(6), CAC::regA
(26), and 1312 (12)
were also grown photoheterotrophically under a 100% argon atmosphere
in Ormerod's medium supplemented with 0.4% malate (27). Aerobic
chemoheterotrophic growth was performed using Ormerod's medium
supplemented with 0.4% malate while shaking vigorously in the dark at
30 °C. Growth on solid medium was performed with the addition of
1.5% agar, and anaerobiosis was achieved using a BBL Gas Pak anaerobic
system (BD Biosciences). Triparental matings were performed
using helper plasmid pRK2013 according to methods described previously
(28). Antibiotics were added to the medium, as required, at the
following concentrations (in µg/ml): for Escherichia coli,
ampicillin (100-200), tetracycline (12.5), spectinomycin (20), and
kanamycin (50); for R. sphaeroides, trimethoprim (50), kanamycin (25), tetracycline (3.5), and spectinomycin (20).
-Galactosidase Assays--
Cultures of R. sphaeroides strains CAC,
CAC::regA
, and 1312 were sampled (10 ml) at an A660 nm of between 0.8 and 1.1. Sonicated extracts were generated in a buffer containing 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, and 5 mM
-mercaptoethanol. Total protein was determined using
the Bio-Rad protein assay dye-binding reagent (Bio-Rad).
-Galactosidase assays were performed as described previously
(13).
-32P] ATP (7000 Ci/nmol;
ICN Biochemicals, Costa Mesa, CA) prior to amplification. The PCR
reactions consisted of 1 µmol of each labeled and unlabeled
oligonucleotide primer, 28 ng of template DNA, and 2.5 units of
Taq polymerase (Invitrogen). Amplification was performed as
follows: denaturation at 95 °C for 5 min, then 30 cycles of 1 min at
94 °C, 1 min at 55 °C, and 1 min at 72 °C. A final elongation
step was performed for 7 min at 72 °C. PCR fragments were purified
on non-denaturing polyacrylamide gels followed by electroelution. The
isolated probe DNA was then ethanol-precipitated and resuspended in 100 µl of buffer containing 50 mM HEPES, pH 8.0, and 100 mM sodium acetate.
-mercaptoethanol).
The cleared supernatant was precipitated with ammonium sulfate at 70%
saturation, and the precipitated proteins were resuspended and dialyzed
against Buffer A. The sample was then bound to a 3-ml Uno-Q (Bio-Rad)
column equilibrated with Buffer A and eluted with a 10 mM
to 1.5 M KCl gradient in Buffer A. Column chromatography
was performed using a Biologic Duo-Flow FPLC (Bio-Rad). Eluted
fractions were assayed for cbbII promoter binding activity by the gel mobility shift assay. Gel mobility shift
DNA binding reactions were performed in a 50-µl total volume containing 50% eluted fraction, 36% Buffer A, 1.8 µg poly(dI-dC), 10% glycerol, and 20,000 cpm of 32P-radiolabeled probe
DNA. Gel electrophoresis was performed as described previously (13).
Gel mobility shift probes were generated by either filling in
restriction fragment overhanging ends with DNA polymerase I Klenow
fragment in the presence of [
-32P]dCTP and dNTPs or
PCR amplification using [
-32P] end-labeled
oligonucleotides as described earlier for DNaseI footprint analysis.
Gel mobility shift probes 1, 2, and 3 in Fig. 5 were generated using
the cbbII promoter restriction fragments XhoI/BamHI, XhoI/XcmI, and
XcmI/BamHI, respectively. The oligonucleotide pairs used to generate probes 4, 5, and 6 in Fig. 5 were
cbbII-16:cbbII-3, cbbII-15B:cbbII-3, and cbbII-1:cbbII-2, respectively.
The sequence of oligonucleotide cbbII-3 is 5'-CGCGTCGAACAGTGGGGCGCC-3'.
The sequences of the other oligonucleotides used are listed earlier under "Experimental Procedures" under "DNaseI Footprint Analysis."
638 bp was deleted by digestion of
pKcbbII with SmaI/XhoI. The
overhanging ends were filled using Klenow polymerase and dNTPs followed
by religation. Ligation products that had not deleted the
SmaI/XhoI fragment were selected against by
digestion with KpnI before transformation into E. coli JM109. The plasmid (pKII
Xho) selected was found to have
deleted an additional 93 bp downstream of the XhoI site to
545 bp. A 606-bp EcoRI/BglII fragment of
pKII
Xho was ligated into EcoRI/BamHI-digested
pMC1403 to generate pMCCIIXho. EcoRI-digested pMCCIIXho was
then ligated into the EcoRI site of the conjugative plasmid
pVK101 to yield pVKCIIXho. The method used for the construction of
pVKCIIXcm was the same as that for pVKCIIXho except that
XcmI was used in place of XhoI to generate
plasmids pKII
Xcm and pMCCIIXcm. This resulted in a lacZ
fusion containing 227 bp upstream of the cbbII
transcription start.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity under chemoheterotrophic, photoheterotrophic, photoautotrophic, and chemoautotrophic growth conditions (Table II). As expected,
aerobic chemoheterotrophic growth in malate medium yielded the
lowest LacZ activity for each plasmid-containing strain. There was a
dramatic increase in LacZ activity in cells grown under
photoautotrophic conditions (i.e. CO2 as the
sole carbon source), whereas photoheterotrophic growth with malate as
the carbon source resulted in significantly lower activity. During
aerobic chemoautotrophic growth, fusion-plasmids pVKCIIXcm, pVKCIIXho,
and pVKCII mediated levels of LacZ activity that were 77, 44, and
74% lower, respectively, than levels obtained under
photoautotrophic growth conditions.
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Fig. 1.
Map of cbbII::lacZ
fusion plasmids. The restriction fragments corresponding to each
fusion construct are shown, and the amount of sequence upstream of the
cbbII transcription start site is indicated. The
positions of three cbbII upstream open reading
frames are indicated. Sections of the cbbII
promoter designated as upstream regulatory regions 1 and 2 are also
shown. The function of open reading frames U1, U2, and V have not been
determined. It should be noted that the distal end of the insert in
plasmid (pVKCIIXho) occurs at 545 bp, 93 bp downstream of the
XhoI site (see "Experimental Procedures").
Strains and plasmids
227 bp were found to affect
cbbII transcription. The first upstream
regulatory region (region 1) strongly activated
cbbII expression under all growth conditions and
is situated between
227 and
545 bp (Fig. 1). This region activated
cbbII expression 10-, 16-, and 39-fold under
photoheterotrophic, photoautotrophic, and chemoautotrophic growth
conditions, respectively (Table II,
compare pVKCIIXcm to pVKCIIXho). Even during aerobic chemoheterotrophic
growth conditions, region 1 conferred a 2-fold enhancement of
cbbII expression. The second upstream regulatory region (region 2) spans the sequence between
545 and
1025 bp. This
region enhanced cbbII expression only during
chemoheterotrophic and photoautotrophic growth conditions, where it
conferred 6- and 1.5-fold increases in cbbII
expression, respectively (Table II, compare pVKCII to pVKCIIXho).
During chemoautotrophic growth region 2 had a slightly negative effect,
reducing cbbII expression by ~29%.
Expression of cbbII-lacZ translational fusions in R. sphaeroides strains CAC, 1312, and CAC::regA under varying
growth conditions
) indicate conditions under which a particular strain is incapable
of growth.
--
Previous
studies had demonstrated that the RegA-RegB (PrrA-PrrB) two-component
regulatory system is also required, along with cbbR, for
maximum cbb expression in R. sphaeroides (17,
18). The regA insertion mutant strain R. sphaeroides
CAC::regA
is unable to grow under phototrophic growth
conditions; however, it does have the ability to grow under aerobic
chemoheterotrophic and aerobic chemoautotrophic conditions.
Consequently, the cbbFII-lacZ fusions
were introduced into R. sphaeroides CAC::regA
;
lacZ expression was monitored during growth under aerobic
chemoheterotrophic and chemoautotrophic conditions and compared with
strain CAC (Table II). In chemoautotrophically grown R. sphaeroides CAC::regA
, the promoter proximal
regulatory region mediated a dramatic 7-fold increase in
cbbII expression relative to that in the
parental strain (Table II, pVKCIIXcm). These results indicated that the Reg/Prr system may have an indirect negative regulatory affect on this
region under this growth condition. Under aerobic chemoautotrophic growth conditions, the positive regulatory effect of region 1 and
the somewhat lessened affect of region 2 in the wild type were
completely negated in the regA background. In fact, region 2 (Table II, pVKCII) conferred a moderate enhancement of
cbbII expression during aerobic chemoautotrophic
growth suggesting that the negative effect of this region is
regA-dependent in the wild type. Clearly,
regA was necessary for high level regulated expression of
the cbbII operon during aerobic chemoautotrophic
growth. Interestingly, regA appeared also to be necessary
for maximal aerobic chemoheterotrophic expression of the
cbbII operon as well, because
cbbII activation in the wild-type strain via
region 1, and especially region 2, was no longer observed in the
regA strain (Table II, pVKCIIXho and pVKCII).
149 bp relative to the cbbII transcription
start, were performed. CbbR protected two closely spaced regions, as
seen by DNaseI digestion (Fig. 2). The
first site (site A) was located from
1 to
31 bp, and the second
site (site B) stretched from
35 to
61 bp. A DNaseI hypersensitive
site was found between sites A and B at
32 bp.
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Fig. 2.
DNase I footprint analysis of the binding of
CbbR to the R. sphaeroides cbbII
promoter-operator region. The oligonucleotides cbbII-1
(bottom strand) and cbbII-2 (top strand) were
used to PCR amplify the probes used in the experiment. The labeled
oligonucleotide (see "Experimental Procedures") used to generate
each probe is indicated above each panel. The
amount of partially purified CbbR (µg) added to each reaction is
indicated at the top of the panel. Standard
(Std) lanes contain a Maxam and Gilbert A+G
cleavage ladder of the probe used in the experiment. Bars
indicate regions of protection, and asterisks (*) denote
DNase I hypersensitive sites. Sequence reference positions are
indicated.
1038 bp were employed. The results indicated that RegA* bound to the
R. sphaeroides cbbII promoter at six distinct
sites (Fig. 3,
A-D). Protection at the first site (site 1) was
found from
282 to
308 bp with DNase I hypersensitive sites
occurring at
280 and
302 bp (Fig. 3A). The second site
(site 2) was widely separated from site 1 and consisted of protection
from
583 to
601 bp with a hypersensitive site located at
597 bp
(Fig. 3B). The third RegA* binding site (site 3) protected
the region from
733 to
749 bp with a hypersensitive site at
744
bp (Fig. 3C). The fourth site (site 4) was comprised of an
area of protection from
761 to
784 bp (Fig. 3C).
Protection at site 5 spanned the region from
836 to
851 bp with a
DNase I hypersensitive site at
839 bp (Fig. 3D). The final
RegA* binding site (site 6) showed protection from
857 to
883
bp with DNaseI hypersensitive sites at
867 and
882 bp (Fig.
3D).
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Fig. 3.
DNase I footprint analysis of the binding of
phosphorylated RegA* to the R. sphaeroides
cbbII promoter-operator region. The
oligonucleotide pairs (see "Experimental Procedures") used to
generate the probes used in each experiment are as follows: cbbII-15B
(top strand) and cbbII-5 (bottom strand)
(panel A), cbbII-16 (top strand) and cbbII-11
(bottom strand) (panel B), cbbII-20 (top
strand) and cbbII-13 (bottom strand) (panel
C); cbbII-14 (top strand) and cbbII-19
(bottom strand) (panel C). The labeled
oligonucleotide used to generate each probe is indicated at the
top of each panel, along with the concentration
of RegA* (µM) in each reaction mixture. Standard
(Std) lanes contain a Maxam and Gilbert A+G
cleavage ladder of the probe used in the experiment. Bars
indicate regions of protection, and asterisks (*) denote
DNase I hypersensitive sites. Sequence reference positions are
indicated. Arrows ( ) indicate the position of the
cbbII transcription start.
was subjected to ammonium sulfate precipitation to 70% saturation. The 70% ammonium sulfate fraction was separated by anionic exchange chromatography (Uno-Q; Bio-Rad) and
eluted with a 50 mM to 1.5 M KCl gradient. The
resulting fractions were tested for cbbII
promoter binding activity in a gel mobility shift assay. The results of
the gel mobility shift assay indicated that two DNA binding activities
were present that bound to a probe spanning a region from
4 to
637
bp (BamHI/XhoI fragment) upstream of the
cbbII transcription start (Fig.
4). The first
cbbII promoter binding activity (activity X)
eluted in several fractions between 0.23 and 0.44 M KCl.
The second binding activity (activity Y) eluted in a single fraction at
~0.62 M KCl. Both of the cbbII promoter binding activities could also be separated using
heparin-Sepharose affinity chromatography (data not shown). To more
closely define the region of the cbbII promoter
to which activity X binds, additional gel mobility shifts were
performed using a heparin-agarose fraction derived from extracts of
R. sphaeroides grown under photoautotrophic conditions using
DNA probes spanning various regions between
4 and
637 bp upstream
of the cbbII transcription start (Fig.
5, A and B).
Activity X bound to probe 4 (spanning
91 to
650 bp) resulting in
one high intensity shifted band and an additional less abundant more
slowly migrating band. A gel mobility shift assay using the shorter
probe 5, spanning
91 to
357 bp, produced a single high intensity
shifted band. Use of probe 6 (spanning +41 to
149 bp) detected a
single faint shifted signal. The gel mobility shift experiments also
showed that cleavage of probe 1, spanning
4 to
637 bp, by digestion
with XcmI, resulted in no binding to either of the resulting
fragments (probes 2 and 3 in Fig. 5B). The results suggest
that activity X binds to the cbbII promoter
region at a high affinity site between positions
91 and
357 bp,
probably in the vicinity of the XcmI site at
221 bp.
Additional lower affinity binding sites may also occur between +41 and
149 bp and between
357 and
650 bp.
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Fig. 4.
Detection of cbbII
promoter-binding proteins in extracts of a R. sphaeroides
regA mutant grown chemoautotrophically. A phosphorimage of a
gel shift assay performed on fractions eluted from an anionic exchange
column (lanes 1-14 and 15-28) of an
extract of chemoautotrophically grown R. sphaeroides
CAC::regA is shown. Positive control reactions
using a heparin-agarose fraction containing protein X were included
(lanes C). Fractions containing DNA binding activities X and
Y are indicated.
View larger version (25K):
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Fig. 5.
Binding of protein X to the cbbII
promoter region. A, restriction sites used to generate
probes 1, 2, and 3 are shown. The start and end positions of each probe
are indicated in parentheses. Probes 4, 5, and 6 are
described under "Experimental Procedures." B, shown is a
compilation of phosphorimages of several gel mobility shift assays
using probes of varying sizes that span different regions of the
cbbII promoter-operator (A) and 15 µl of a heparin-Sepharose fraction containing protein X. Lane
1, probe 4, no protein added; lane 2, probe 4 plus
protein X; lane 3, probe 5, no protein added; lane
4, probe 5 plus protein X; lane 5, probe 6, no protein
added; lane 6, probe 6 plus protein X; lane 7,
probe 1, no protein added; lane 8, probe 1 plus protein X;
lane 9, probe 2, no protein added; lane 10, probe
2 plus protein X; lane 11, probe 3, no protein added;
lane 12, probe 3 plus protein X. All reactions contained 1.8 µg of poly(dI-dC).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
301 bp
in cbbI and
282 bp in
cbbII) is responsible for the majority of this
activation suggesting that both of these sites function similarly
during cbb activation. The placement of the CbbR and RegA
binding sites and the involvement of upstream sequences in regulated
expression of the cbbII operon is summarized (Fig. 6). Not surprisingly the regulation
of the cbbII operon mirrored that of the
cbbI operon with low expression during aerobic chemoheterotrophic growth and high expression during phototrophic growth. Maximal cbb expression during phototrophic growth
has been shown to be dependent on cbbR, as well as
reg (12, 13, 18). Thus far, the involvement of upstream
activating sequences appears to be unique to R. sphaeroides
as such sequences are not involved with the regulation of the
cbbI and cbbII operons of the related organism R. capsulatus (19).
View larger version (44K):
[in a new window]
Fig. 6.
Summary of DNase I footprinting results of
CbbR and RegA* binding to the R. sphaeroides
cbbII promoter-operator region. The sequence is
numbered relative to the cbbII transcription
start at +1. Brackets indicate regions of protection on the
top (above) and bottom (below) strands with DNase
I hypersensitive sites indicated by asterisks. The
translation start site for cbbFII is indicated,
along with the cbbII transcription start sites
( ). The relative increase in LacZ activity (i.e. 1X, 16X,
and 1.5X) conferred by the three promoter fusions used in this study
during photoautotrophic growth is indicated above the
sequence.
The discovery that maximal aerobic chemoautotrophic expression of cbbII required regA was unexpected, given that chemoautotrophic expression of cbbI is regA-independent (34). This indicates that molecular mechanisms involved with regulating the two operons are quite distinct under this growth condition. Although the nature of the different chemoautotrophic regulatory mechanisms is not known, the need for different control mechanisms may stem from the fact that O2 serves as a terminal electron acceptor during chemoautotrophic growth. Previously, it was shown that the action of the Reg/Prr two-component system of R. sphaeroides may be mediated by electron flow through a cbb3-type terminal cytochrome oxidase, because inactivation of the operon (ccoNOPQ) that encodes this oxidase resulted in aberrant regA-dependent activation of photopigment gene expression under aerobic growth conditions (35). It is possible that during chemoautotrophic growth electron flow to O2 via the cbb3-oxidase may dampen the Reg/Prr-mediated activation of cbb gene expression to the extent that RegA activation alone would produce an insufficient level of CBB cycle enzymes to support optimal growth. The fact that the cbbII promoter is expressed at reduced levels during chemoautotrophic growth relative to photoautotrophic growth is consistent with this idea. An inability to support high level cbb expression might necessitate the recruitment of an additional positive regulatory system(s). The most probable target for chemoautotrophic up-regulation would be the cbbI operon, because it encodes the form I (L8S8) Rubisco, used as the major autotrophic enzyme in the CBB pathway (1). However, form II Rubisco and enzymes encoded by the cbbII operon allow the CBB pathway to play a somewhat more specialized role such that CO2 may be employed as a terminal electron acceptor (11). Thus, retaining Reg/Prr control over cbbII gene expression during chemoautotrophic growth may give R. sphaeroides an enhanced ability to regulate redox poise when growing at the expense of highly reduced electron donors (i.e. molecular H2).
Additional regulators may also affect cbbII expression during aerobic chemoautotrophic growth. In a regA background, the level of chemoautotrophic cbbII expression from the promoter proximal regulatory region (pVKCIIXcm) was significantly higher than that in parental strain CAC. This increase in cbbII expression in the regA mutant could be because of either activation by CbbR or additional cbbII-specific regulatory proteins whose expression may or may not be affected by regA. The detection of two cbbII promoter-binding proteins, X and Y, in extracts of R. sphaeroides grown both chemoautotrophically and photoautotrophically, provided direct evidence for additional cbbII-specific proteins that bind physiologically significant regulatory sequences. Although proteins X and Y have not yet been identified, binding cannot be attributed to RegA because of the fact that these proteins were present in extracts of a R. sphaeroides regA strain. Moreover, protein X was detected in extracts of photoheterotrophically grown R. sphaeroides cbbR mutant strain 1312.
In conclusion, despite the involvement of similar upstream activation
sequences, it is clear that distinct molecular mechanisms serve to
regulate gene expression of the two major cbb operons of
R. sphaeroides. Moreover, it is apparent that the global
two-component Reg/Prr system is only selectively involved with
cbb control, with Reg/Prr required to activate transcription
of only the cbbII operon, and not the
cbbI operon under aerobic chemoautotrophic growth conditions. Future studies will focus on further elucidating regulatory mechanism(s) involved in cbb activation that are
both distinct and common to both operons, as well as identifying and determining the role of recently discovered proteins that bind specifically to the cbbII promoter.
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ACKNOWLEDGEMENTS |
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We thank Dr. Janet L. Gibson for comments and suggestions on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-45404.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology,
The Ohio State University, 484 West 12th Ave., Columbus, OH 43210-1292. Tel.: 614-292-4297; Fax: 614-292-6337; E-mail: tabita.1@osu.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M211267200
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ABBREVIATIONS |
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The abbreviations used are: CBB, Calvin-Benson-Bassham; Rubisco, ribulose bisphosphate carboxylase/oxygenase.
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