Interactions of the cbbII Promoter-Operator Region with CbbR and RegA (PrrA) Regulators Indicate Distinct Mechanisms to Control Expression of the Two cbb Operons of Rhodobacter sphaeroides*

James M. Dubbs and F. Robert TabitaDagger

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (-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

Bacterial Strains, Plasmids, and Culture Conditions-- R. sphaeroides strains CAC (6), and CAC::regAOmega (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::regAOmega (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).

beta -Galactosidase Assays-- Cultures of R. sphaeroides strains CAC, CAC::regAOmega , 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 beta -mercaptoethanol. Total protein was determined using the Bio-Rad protein assay dye-binding reagent (Bio-Rad). beta -Galactosidase assays were performed as described previously (13).

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 [gamma -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.

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 beta -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 [alpha -32P]dCTP and dNTPs or PCR amplification using [gamma -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."

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 -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 (pKIIDelta 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 pKIIDelta 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 pKIIDelta 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

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 beta -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").


                              
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Table I
Strains and plasmids

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 -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%.


                              
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Table II
Expression of cbbII-lacZ translational fusions in R. sphaeroides strains CAC, 1312, and CAC::regAOmega under varying growth conditions
All values are the average of values determined in three independent experiments. The S.D. for each value appears in parentheses. Dashes (---) indicate conditions under which a particular strain is incapable of growth.

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::regAOmega -- 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::regAOmega 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::regAOmega ; 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::regAOmega , 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).

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 -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.

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 -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 (right-arrow) indicate the position of the cbbII transcription start.

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::regAOmega 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::regAOmega 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.


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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

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 -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).


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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 (right-arrow). 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.

    ACKNOWLEDGEMENTS

We thank Dr. Janet L. Gibson for comments and suggestions on the manuscript.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: CBB, Calvin-Benson-Bassham; Rubisco, ribulose bisphosphate carboxylase/oxygenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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