INTRODUCTION

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Purple nonsulfur photosynthetic bacteria display exceptional metabolic versatility (20, 31) and assimilate CO₂ via the highly regulated Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway (12, 17, 48). During photo- and chemoautotrophic growth, CO₂ is the sole source of cellular carbon, and maximal levels of the key CBB pathway enzymes, ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase (PRK), are observed (48). Photoheterotrophic growth results in much lower yet substantial levels of RubisCO and PRK; however, under these conditions the CBB pathway functions primarily to help maintain the redox balance of the cell by allowing CO₂ to serve as an electron sink. Alternate electron acceptors such as dimethyl sulfoxide (DMSO) can function in place of CO₂ (7, 43, 56).

The organization and regulation of structural genes encoding enzymes of the CBB pathway have been extensively studied in Rhodobacter sphaeroides, and there are at least three major operons which comprise the cbb regulon of this organism. Two major operons, the cbb_I, or form I, operon and the cbb_II, or form II, operon, are comprised of structural genes of the CBB pathway, some of which are duplicated (13-15). The third operon consists of the cbbXYZ genes, encoding two proteins of unknown function and phosphoglycolate phosphatase, respectively, and is downstream of the cbb_I operon (18). Transcription of both the cbb_I and cbb_II operons is positively regulated by the product of the cbbR gene, which is upstream and divergently transcribed from the R. sphaeroides cbb_I operon (16). By contrast, the form I RubisCO genes (cbbLS) of R. capsulatus are not associated with any CBB pathway structural genes (38, 39), and an open reading frame (ORF) with sequence similarity to cbbQ, which is also downstream of cbbLS of Pseudomonas hydrogenothermophila and Chromatium vinosum (62), is found downstream of R. capsulatus cbbLS (38). The cbbQ gene product has no known function in R. capsulatus (20a). In addition, there are two cbbR genes in R. capsulatus; cbbR_I is upstream and divergently transcribed from the cbbLS genes (38), while cbbR_II is upstream and divergently transcribed from the cbbFPTGAM genes (39).

The recent description of variant cbb gene organization in R. capsulatus and R. sphaeroides, particularly the presence of two cbbR genes in R. capsulatus, suggests potential differences in cbb gene regulation. For example, unlike R. sphaeroides, R. capsulatus does not synthesize form I RubisCO when the organism is grown photoheterotrophically on malate (39, 46). Furthermore, the R. capsulatus form I enzyme is immunologically distinct from the form I enzyme of R. sphaeroides (15, 39) and appears to have been acquired by horizontal gene transfer (38). Thus, to initiate and provide a framework for cbb gene regulation studies in R. capsulatus, specific cbb gene disruption strains and cbb promoter fusions were constructed and characterized.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, media, and growth conditions. Plasmids and R. capsulatus strains used or constructed are listed in Table 1. Escherichia coli JM107 (60), JM109 pir, and S17-1 pir (40) were grown aerobically on LB medium (2) at 37°C. Aerobic cultures of R. capsulatus were grown in PYE medium (57) at 30°C. Photosynthetic cultures were grown in Ormerod's medium (37) supplemented with thiamine (1 µg/ml), nicotinic acid (1 µg/ml), and biotin (0.1 µg/ml). Photo- and chemoautotrophic growth conditions were previously described (38, 39). Antibiotic concentrations used for R. capsulatus strains were as follows: rifampin, 100 µg/ml; kanamycin, 5 µg/ml; spectinomycin, 10 µg/ml; and tetracycline, 2 µg/ml for plasmid maintenance or 0.1 µg/ml for screening during gene disruption experiments. For E. coli, antibiotic concentrations were 30 µg/ml for kanamycin, 50 µg/ml for spectinomycin, 12.5 µg/ml for tetracycline, and 200 µg/ml for trimethoprim. DMSO was used at 30 mM.

DNA manipulations. Routine DNA manipulations, including plasmid preparation, restriction endonuclease digestion, agarose gel electrophoresis, fragment ligation, and bacterial transformation, were performed by standard methods (2). R. capsulatus chromosomal DNA was prepared as previously described (19). For gene disruption experiments, plasmid pJP5603 derivatives were conjugated into R. capsulatus SB1003 by using E. coli S17-1 pir (40). For complementation of mutant strains, plasmids were conjugated into R. capsulatus by triparental matings on filter pads as previously described (57), using the helper plasmid pRK2013 (10).

Southern blotting and hybridization. Southern transfer experiments were performed by using GeneScreen Plus (NEN, DuPont, Boston, Mass.) or Hybond N+ (Amersham, Arlington Heights, Ill.) membranes. Hybridizations were conducted according to the protocols provided by NEN, DuPont, using formamide under stringent conditions. Probes were labeled with [-³²P]dCTP (NEN, DuPont) by the random prime labeling method (9), using a kit purchased from United States Biochemical Corporation (Cleveland, Ohio).

DNA sequencing and analysis. Nucleotide sequences were determined with an ABI Prism 310 Genetic Analyzer. A thermal cycler and dye terminator cycle sequencing kit were used as described by the manufacturer (Perkin-Elmer, Foster City, Calif.). The M13/pUC forward 23-base primer, M13 reverse (48) primer, and sequence-specific synthetic primers were used to complete the double-stranded sequence. Sequence analysis was carried out with the University of Wisconsin Genetics Computing Group software, the EGCG extension programs (The Sanger Centre, Hinxton, England), and the MacVector sequence analysis software (International Biotechnology, Inc., New Haven, Conn.).

Preparation of cell extracts and enzyme assays. Culture samples (20 to 30 ml) were taken in late log phase (A₆₆₀ = 0.9 to 1.2) and washed twice in cold buffer (100 mM Tris-HCl, 1 mM EDTA [pH 8.0]) before freezing at 70°C. Thawed pellets were resuspended in 1 ml of TEM buffer (50 mM Tris-HCl, 1 mM EDTA, 5 mM -mercaptoethanol [pH 7.5]) and disrupted by sonication in an ice bath. Cell debris was removed by centrifugation for 10 min in a microcentrifuge at 4°C.

Western immunoblot analysis. Antibodies raised against R. sphaeroides form II RubisCO and form I PRK (PRK I) were used to detect R. capsulatus form II RubisCO and PRK, respectively. Although R. capsulatus form I RubisCO reacts poorly with antibody raised against R. sphaeroides form I RubisCO, anti-Synechococcus strain PCC 6301 RubisCO antibody cross-reacts well (38, 39) and was used to detect R. capsulatus form I RubisCO. Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (28). After SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, Mass.), using a Bio-Rad Transblot semidry cell (Bio-Rad, Hercules, Calif.) and established protocols (50). The blots were developed by using the Vistra ECF fluorescent detection system (Amersham Corporation, Buckinghamshire, England) and visualized with a Molecular Dynamics Storm 840 imaging system (Molecular Dynamics, Sunnyvale, Calif.).

Construction of mutant strains. R. capsulatus strains with disruptions in cbbL, cbbM, cbbP, cbbR_I, and cbbR_II were constructed by mobilizing the appropriate pJP5603 derivative into strain SB1003 from E. coli S17-1 pir. Homologous recombination of the plasmid-borne disrupted gene into the wild-type copy in the chromosome was forced because pJP5603 does not replicate in R. capsulatus. Recombinant strains were selected by aerobic growth on PYE plates supplemented with the antibiotic corresponding to the disrupting cassette. Rifampin was used to select against the E. coli donor. Resistant clones were screened for sensitivity to the plasmid-encoded antibiotic resistance marker to identify strains that may had undergone a second recombination event. Double recombination was confirmed by Southern blotting and hybridization analysis of chromosomal DNA from the mutant and wild-type strains (data not shown). Specific plasmid and strain constructions are described below.

Strain SBI (cbbL). A 4.7-kb BamHI fragment, containing the R. capsulatus cbbLS genes, was cloned from pRKFIP into pUC1813. The resulting plasmid, pUC1813::FIB, lacked any EcoRI sites in the multiple cloning region so that the 639-bp EcoRI fragment within cbbL could be removed and replaced by the spectinomycin resistance (Sp^r) gene from pHP45. The 6.5-kb BamHI fragment containing the disrupted gene was moved from pUC1813::FI to pJP5603, resulting in plasmid pJP::FI. Plasmid pJP::FI was mobilized into R. capsulatus SB1003 from E. coli S17-1 pir. Six of the Sp^r exconjugants screened were sensitive to kanamycin (Km^s). Southern blot analysis of chromosomal DNA prepared from the six Sp^r Km^s isolates revealed that five of them resulted from double recombination. One of these strains was used for subsequent experiments.

Strain SBII (cbbM). The 2-kb SalI fragment encoding the R. capsulatus cbbM gene was cloned from plasmid pK18FIIS2-I into plasmid pUC1318. The resulting construct, pUC1318FII, lacked HindIII sites within its multiple cloning region. To generate a Km^r cassette with flanking HindIII sites, a 1.4-kb SalI fragment encoding the Tn5 Km^r gene was cloned from pUC1318K into plasmid pUC1813, generating pUC1813K. The 650-bp HindIII fragment within the cbbM gene in vector pUC1318FII was removed and replaced by the HindIII fragment containing the Tn5 Km^r gene from plasmid pUC1813K. The resulting cbbM deletion fragment was cloned as an XbaI fragment into plasmid pTC5603, yielding pTC::FIIKm. E. coli S17-1 pir was used to mobilize pTC::FIIKm into R. capsulatus SB1003. Three hundred Km^r clones were screened for tetracycline sensitivity (Tc^s). All of the exconjugants were sensitive to 2 µg of tetracyline per ml, but only five clones were sensitive to 0.1 µg/ml. Due to the very low resistance to tetracycline, the 300 clones were examined for loss of pTC5603 by colony hybridization. The five Tc^s clones did not hybridize to the pTC5603 probe, but the 295 Tc^s clones did hybridize to the probe. Three of the five Tc^s clones were screened by Southern blot hybridization analysis of chromosomal DNA and found to be the result of double recombination. One of these recombinants was used for subsequent experiments.

Strain SBI-II (cbbL cbbM). To construct a strain lacking genes for both forms of RubisCO, the cbbM deletion plasmid pTC::FIIKm was mobilized into R. capsulatus cbbL strain SBI. Two hundred Km^r colonies were screened, and two were Tc^s. Both of these Tc^s clones had lost the pTC5603 vector as determined by colony hybridization using pTC5603 as a probe. Southern blot analysis using cbbM and cbbL probes revealed that these strains were the result of double recombination, leaving behind a deletion within the chromosomal copy of cbbM, with the cbbL gene deletion still present.

Strain SBP (cbbP). A 543-bp SmaI-BamHI fragment encoding part of the R. capsulatus cbbP gene was cloned from pK18FIIB2.3 into pK18, resulting in plasmid pK18::BSm. The cbbP gene was disrupted by cloning the Sp^r gene from pHP45 as a SalI fragment into the unique XhoI site within the cbbP gene fragment in plasmid pK18::BSm, yielding plasmid pK18CBBP. The resulting disrupted gene fragment was cloned as a BamHI-SmaI fragment into pJP5603, yielding plasmid pJP::CBBP. E. coli S17-1 pir was used to mobilize plasmid pJP::CBBP into R. capsulatus SB1003. Two hundred fifty Sp^r exconjugants were screened, and seven were Km^s. One of these strains was screened by Southern blot hybridization analysis of the chromosomal DNA and found to be the result of a double recombination. This strain, SBP, was characterized further.

Strain SBRI (cbbR_I). The Sp^r cassette from pHP45 was cloned as a SmaI fragment into the unique SspI site within the cbbR_I gene in plasmid pEULA4 to yield pEULA4. The cbbR_I disruption was cloned from pEULA4 into the EcoRI site in pJP5603. The resulting plasmid, pJPLA4, was mobilized into R. capsulatus SB1003 via E. coli S17-1 pir. Of the 1,500 Sp^r colonies screened, 35 were Km^s. Chromosomal DNA was prepared from eight Km^s clones, and Southern blot and hybridization analysis confirmed that each of the clones was the result of double recombination. One strain, SBRI, was characterized further.

Strain SBRII (cbbR_II). The 3.7-kb SalI-SmaI fragment containing the R. capsulatus cbbR_II and cbbF genes was cloned from pK18FIIS4.4 into plasmid pTZ18R, generating pTZ::FII3.7. Removal of the SalI-SmaI fragment from the multiple cloning region of pTZ18R during the construction of pTZ::FII3.7 deleted the BamHI site. This allowed disruption of the cbbR_II gene in pTZ::FII3.7 by insertion of a BamHI fragment containing the Tn5 Km^r gene from plasmid pRL648 into the unique BamHI site within cbbR_II. The resulting construct, pTZ::CBBRKm, was linearized with XbaI and ligated to XbaI-digested pTC5603, resulting in plasmid pTZTC::CBBRKm. This plasmid was mobilized into R. capsulatus SB1003 from E. coli S17-1 pir. Three hundred Km^r colonies were screened, and 299 were sensitive to Tc. Hybridization of colony blots from the 300 Km^r clones using a probe derived from the Tc^r region of pTC5603 (EcoRI to PvuII fragment of pBR322) revealed that only the single Tc^r clone contained the Tc^r gene. Chromosomal DNA was prepared from three of the Tc^s and the Tc^r clone. The Tc^r clone was the result of a single recombination of pTZTC::CBBRKm into the SB1003 chromosome, and each of the three Tc^s clones was the result of double recombination. One of the Km^r Tc^s double-recombinant clones, strain SBRII, was used in subsequent experiments.

Construction of cbb promoter fusions. The translational fusion vector pXBA601 (1) was used for construction of cbbL (cbb_Ip) and cbbF (cbb_IIp) promoter fusions to lacZ. pXBA601 requires that the fusion end of the promoter fragment be ligated to the unique BamHI site within this vector. For construction of the cbb_Ip fusion, the ends of a 3.2-kb SalI-NcoI fragment from pEULA4 were filled with the Klenow fragment of DNA polymerase I. The blunt-ended fragment was cloned into the SmaI site of pK18, yielding plasmid pK18FISN. This resulted in an in-frame fusion of the cbbL ATG initiation codon that is within the NcoI recognition site to lacZ. After screening for the proper orientation, the fusion was confirmed by sequencing. A PstI-BamHI fragment and a BamHI fragment were cloned from pK18FISN into pXBA601, resulting in constructs with 367 bp and 3.2 kb upstream of the cbbL initiation codon fused to lacZ, pXLBP and pXLB, respectively. Inserts were detected by colony blot hybridization, and the orientation of the insert in pXLB was determined by restriction enzyme digestion. The cbb_IIp fusion was constructed by first filling the ends of the 2.44 kb SalI-NcoI fragment from plasmid pKFIIS4.4 with the Klenow fragment of DNA polymerase I and ligating it to SmaI-cut pK18, yielding pK18FIISN. After screening for the orientation of the insert, the fusion was confirmed by nucleotide sequencing. This resulted in an in-frame lacZ fusion to the cbbF ATG initiation codon that is within the NcoI recognition site. A 722-bp BamHI fragment was subcloned from pK18FIISN into pXBA601. The presence of an insert was determined by colony blot hybridization, and the orientation of the insert was determined by nucleotide sequencing. The resulting construct, pXFB, contained 722 bp upstream of cbbF fused to lacZ at the cbbF start codon.

Nucleotide sequence accession number. The nucleotide sequences reported in this paper have been submitted to the GenBank database under accession no. U87282.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Nucleotide sequence analysis and amino acid sequence comparisons. The DNA upstream of the presumptive R. capsulatus cbb_II operon contains at least two regions of interest with respect to cbb gene regulation: the cbbR_II gene, encoding a putative cbb transcriptional activator, and the cbbR_II-cbbF intergenic region, containing the presumptive cbb_II operon promoter. As a prelude to further studies of R. capsulatus cbb gene regulation, the nucleotide sequence of a 4,537-bp region, from the SalI site 1.4 kb downstream of cbbR_II to the 5' end of cbbT_II,, was determined (Fig. 1). In addition to cbbR_II, cbbF, cbbP, and part of the cbbT gene known to be present in this region (39), database searches revealed one full-length ORF and one partial ORF downstream of cbbR_II. One end of the sequenced region contained a partial ORF (Fig. 1) encoding 83 amino acids that were 56.6% identical (74.7% similar) to the C-terminal portion of Agrobacterium tumefaciens phosphoglucomutase and 49.4% identical (69.9% similar) to the human PGM *1+ isoform of phosphoglucomutase (Fig. 2A). A phosphoglucomutase gene had not been previously identified in nonsulfur purple photosynthetic bacteria. An ORF encoding a 322-amino-acid gene product was directly downstream from cbbR_II (Fig. 1). The deduced amino acid sequence of this ORF showed 47.7 and 43.3% identity to the NAD(P)H quinone oxidoreductase (QOR) from Pseudomonas aeruginosa and E. coli, respectively. QOR from E. coli has been crystallized (49), and every residue known to be involved in substrate binding or catalysis is conserved in the R. capsulatus enzyme (Fig. 2B). The AXXGXXG sequence (Fig. 2B) is an unusual nucleotide binding fingerprint motif found only among the QORs (49).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Map of R. capsulatus cbb genes. Arrows indicate direction and size of potential transcripts. The sites of gene disruptions in the mutant strains are indicated. The dark bar denotes the region that was sequenced for this study. , potential transcriptional terminator hairpin structure; , hairpin preceded by an RNase E recognition sequence. Gene designations are as follows: cbbR, LysR-type transcriptional regulator; cbbL, form I RubisCO large subunit; cbbS, form I RubisCO small subunit; cbbQ, gene of unknown function; pgm, phosphoglucomutase; qor, NAD(P)H quinone oxidoreductase; cbbF, fructose 1,6-sedoheptulose 1,7-bisphosphatase; cbbP, phosphoribulokinase; cbbT, transketolase; cbbG, glyceraldehyde 3-phosphate dehydrogenase; cbbA, fructose 1,6-bisphosphate aldolase; cbbM, form II RubisCO. Restriction sites: B, BamHI, E, EcoRI, H, HindIII; S, SalI; Sm, SmaI, X, XhoI.

View larger version (61K): [in this window] [in a new window]   View larger version (39K): [in this window] [in a new window]   FIG. 2.   Comparison and alignment of deduced amino acid sequences to sequences of known proteins. Invariant amino acids are indicated by asterisks and conservative changes are indicated by dots below the amino acids. (A) Alignment of the deduced amino acid sequence of the R. capsulatus (Rcap) partial ORF with that of A. tumefaciens (Atum) (51) and human phosphoglucomutase (58) amino acid sequences. In each case, the threonine residue is the C-terminal residue. (B) Amino acid sequence alignment of R. capsulatus QOR with QOR from E. coli (29) and P. aeruginosa (Paer) (GenBank accession no. X85015). AXXGXXG is an unusual nucleotide binding motif found in this protein (49). Additional residues in contact with the bound NADPH () and those that are within the catalytic site () are indicated. (C) FBPase amino acid alignment. The R. capsulatus deduced amino acid sequence is aligned with the amino acid sequences of R. sphaeroides FBPase I (14) and FBPase II (13) (Rsph I and Rsph II). (D) PRK amino acid sequence alignment. The R. capsulatus PRK amino acid sequence is aligned with the amino acid sequences of R. sphaeroides PRK I (14) and PRK II (13) (Rsph I and Rsph II). The putative ATP binding domain is indicated by the shaded box, and the pyridine nucleotide binding site is indicated by the bar. Residues implicated in the R. sphaeroides form I enzyme in sugar phosphate binding (#) and catalysis () are noted. (E) Alignment of the R. capsulatus (Rc) cbbT partial deduced amino acid sequence with R. capsulatus TktA (4) and R. sphaeroides (Rs) CbbT (13) sequences.

Construction and phenotypes of R. capsulatus cbb mutant strains. Strains SBI (cbbL), SBII (cbbM), SBI-II (cbbL cbbM), SBP (cbbP), SBRI (cbbR_I), and SBRII (cbbR_II) were constructed as described in Materials and Methods. The ability of the wild-type and mutant strains to grow under photoheterotrophic, photoautotrophic, and chemoautotrophic conditions was determined on solid media, and the results are presented in Table 1.

Characterization and complementation of RubisCO-minus strains. Analysis of R. sphaeroides cbbL and cbbM mutant strains revealed that disruption of the gene(s) encoding one RubisCO led to enhanced levels of RubisCO gene transcription, greater amounts of RubisCO protein, and enhanced enzyme activity of the remaining RubisCO. Indeed, the observed level of activity met or exceeded that present in the wild-type strain (14). To determine if a similar compensatory regulatory effect occurred in R. capsulatus, RubisCO activities and protein levels were assessed in cbbL and cbbM strains. The disruption of the cbbL and cbbM genes in R. capsulatus SBI and SBII, respectively, was confirmed by hybridization analyses of Southern blots (data not shown), and Western immunoblotting confirmed the lack of RubisCO protein corresponding to each mutated gene (Fig. 3). Unlike the wild-type strain, in which form II RubisCO is present in both photoheterotrophically and photoautotrophically grown cells (Fig. 3B, lanes 2 and 5), form II RubisCO was not present in extracts of either photoheterotrophically or photoautotrophically grown SBII (Fig. 3B, lanes 4 and 7). Since wild-type strain SB1003 did not synthesize detectable levels of form I RubisCO under photoheterotrophic conditions (Fig. 3A, lane 2), the lack of form I RubisCO in strain SBI was confirmed by Western blot analysis of extracts from photoautotrophically grown cells (Fig. 3A, lane 6). Despite the fact that form I RubisCO is not detectable in photoheterotrophically grown wild-type cells, either form I (strain SBII) or form II (SBI) RubisCO supported photoheterotrophic, photoautotrophic, and chemoautotrophic growth (Table 1); however, the doubling times for the mutant strains were slightly longer than for the wild-type under photoheterotrophic and photoautotrophic conditions (Table 2).

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Western immunoblot analysis of R. capsulatus wild-type and RubisCO-minus strains. Purified Synechococcus sp. strain PCC 6301 RubisCO and purified R. sphaeroides form II RubisCO were loaded into lanes 1 and 8, respectively. Crude extracts (approximately 10 µg of protein) were loaded as follows: lane 2, photoheterotrophically grown SB1003; lane 3, photoheterotrophically grown SBI; lane 4, photoheterotrophically grown SBII; lane 5, photoautotrophically grown SB1003; lane 6, photoautotrophically grown SBI; lane 7, photoautotrophically grown SBII. Blots were incubated with antibody raised against Synechococcus strain PCC 6301 RubisCO (A) and R. sphaeroides form II RubisCO (B). The figure was generated as follows: the region of interest of each blot was converted to TIFF files by using the software provide with the Molecular Dynamics Storm 840 imaging system (Molecular Dynamics), the TIFF files were imported into CorelDraw 7.0 (Corel Corporation, Ottawa, Ontario, Canada), where frames and numbers were added, and the images were printed on a Kodak ColorEase PS printer.

Characterization and complementation of the PRK-minus strain. Unlike R. sphaeroides, R. capsulatus appears to have only a single copy of cbbP (39). Further evidence that there is only one copy of cbbP in R. capsulatus was provided by low-stringency Southern blot analysis of R. capsulatus genomic DNA, using a probe derived from the R. capsulatus cbbP gene. In each case, the size of the hybridizing fragment corresponded to the size predicted for cbbP-containing fragment for the cbbFPTGAM region (data not shown). Hybridization and wash conditions were used such that a second copy of cbbP would be predicted to be less than 60% identical to the cbbP probe. PRK is the only enzyme, other than RubisCO, that is unique to the CBB pathway; therefore, disruption of the R. capsulatus cbbP gene should abolish the CBB pathway. In addition, if the R. capsulatus cbbFPTGAM genes form an operon, disruption of cbbP would be expected to have a polar effect on the expression of downstream genes, including cbbM. The cbbP deletion strain, SBP, was unable to grow photoautotrophically or chemoautotrophically and grew photoheterotrophically only when DMSO was supplied as an exogenous electron acceptor (Table 1). Strain SBP lacked detectable PRK activity when grown photoheterotrophically with DMSO (Table 2) despite the fact that the presence of DMSO did not significantly reduce the level of PRK activity in the wild-type strain (Table 2). Additionally, Western immunoblot analysis showed low but detectable amounts of PRK in extracts from strain SB1003 grown photoheterotrophically in the presence of DMSO, while strain SBP lacked detectable levels of PRK protein (Fig. 4C, lanes 1 and 2). A concomitant loss of detectable levels of form II RubisCO protein was also observed in SBP (Fig. 4B, lanes 1 and 2). The level of RubisCO activity in strain SBP grown photoheterotrophically in the presence of DMSO was much lower than that in wild-type strain SB1003 (Table 2), and unlike in strain SBII, the compensatory synthesis of form I RubisCO was not observed (Fig. 4A, lane 2). Complementation of R. capsulatus SBP with R. sphaeroides cbbP_I in expression vector pRPS-1 (pRPS::RsP_I) resulted in photoheterotrophic growth without a requirement for DMSO. Complementation was dependent on the proper orientation of the inserted DNA fragment. Despite very high PRK activity and PRK protein synthesis in the complemented strain (Table 2; Fig. 4C, lane 3), plasmid pRPS::RsP_I did not complement strain SBP to photoautotrophic growth. A fourfold increase in RubisCO activity was also noted when strain SBP was complemented with plasmid pRPS::RsP_I, and only form I RubisCO protein was detected (Table 2; Fig. 4A and B, lane 3). The loss of form II RubisCO protein in strain SBP and the synthesis of only form I RubisCO in the complemented strain provide additional evidence that the cbb_II genes are cotranscribed.

View larger version (45K): [in this window] [in a new window]   FIG. 4.   Western blot analysis the R. capsulatus wild-type and PRK-minus strains. Crude extracts were prepared and loaded (approximately 10 µg of protein) as follow: lane 1, SB1003 grown photoheterotrophically with malate in the presence of DMSO; lane 2, SBP grown photoheterotrophically with malate in the presence of DMSO; lane 3, strain SBP with plasmid pRPS::RsPI grown photoheterotrophically with malate; lane 4, purified R. sphaeroides PRK I. Blots were incubated with antibody raised against Synechococcus strain PCC 6301 RubisCO (A), R. sphaeroides form II RubisCO (B), and R. sphaeroides PRK I (C). The figure was generated as described in the legend to Fig. 3.

Characterization and complementation of CbbR I- and CbbR II-minus strains. R. capsulatus strains in which cbbR_I and cbbR_II were insertionally inactivated were constructed to establish a physiological role for the respective cbbR gene products. A strain in which the cbbR_I gene was disrupted, strain SBRI, exhibited no phenotype (Table 1), and under photoheterotrophic conditions, RubisCO activity in this strain was not significantly lower than the level in the wild-type strain (Table 2). Interestingly, about one-half of the wild-type RubisCO activity was detected in strain SBRI grown under photoautotrophic conditions (Table 2). Since form I RubisCO is not synthesized in photoheterotrophically grown SB1003, these results (wild-type RubisCO activity under photoheterotrophic conditions and reduced RubisCO activity under photoautotrophic conditions) would be consistent with the absence of form I RubisCO synthesis in strain SBRI. Western immunoblot analysis confirmed that RubisCO activity in strain SBRI was due solely to form II RubisCO synthesis (Fig. 5). Form II RubisCO synthesis in strain SBRI was qualitatively similar to that in the wild-type strain under both photoheterotrophic and photoautotrophic conditions (Fig. 5B, lanes 2 to 5). As in the wild-type strain, no form I RubisCO was present in extracts prepared from photoheterotrophically grown SBRI (Fig. 5A, lanes 2 and 4), but unlike wild-type strain SB1003, strain SBRI did not synthesize form I RubisCO even under photoautotrophic conditions (Fig. 5A, lanes 3 and 5). Although a slight reaction with the anti-form I RubisCO is visible in Fig. 5A, lane 5, this was not due to the presence form I RubisCO in extracts of photoautotrophically grown SBRI because the cross-reacting protein is of higher apparent molecular weight than the form I RubisCO and it was not detected in subsequent Western immunoblots (data not shown). Introduction of the R. capsulatus cbbR_I gene into strain SBRI on plasmid pVK::CbbRI restored the ability to synthesize high levels of form I RubisCO under photoautotrophic conditions (Fig. 5A, lane 7) without an apparent effect on form I RubisCO synthesis under photoheterotrophic conditions (Fig. 5A, lane 6).

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Western immunoblot analysis of R. capsulatus wild-type and CbbR-minus strains. Purified Synechococcus sp. strain PCC 6301 RubisCO and purified R. sphaeroides form II RubisCO were loaded into lanes 1 and 11, respectively. Crude extracts were loaded (approximately 10 µg of protein) as follows: lane 2, photoheterotrophically grown SB1003; lane 3, photoautotrophically grown SB1003; lane 4, photoheterotrophically grown SBRI; lane 5, photoautotrophically grown SBRI; lane 6, photoheterotrophically grown SBRI with pVK::CbbRI; lane 7, photoautotrophically grown SBRI with pVK::CbbRI; lane 8, photoheterotrophically grown SBRII; lane 9, photoheterotrophically grown SBRII with plasmid pVK::CbbRII; lane 10, photoautotrophically grown SBRII with plasmid pVK::CbbRII. Blots were incubated with antibody raised against Synechococcus strain PCC 6301 RubisCO (A) and R. sphaeroides form II RubisCO (B). The figure was generated as described in the legend to Fig. 3.

Analysis of R. capsulatus cbb promoter fusion constructs. Promoter fusions were constructed to further examine the regulation of the cbb operons under photoheterotrophic and photoautotrophic conditions. -Galactosidase activity was measured in extracts from SB1003 containing plasmid-borne fusions of lacZ to cbb_Ip and cbb_IIp (Table 1; Fig. 6). Under photoheterotrophic conditions, -galactosidase activity was nearly undetectable in strain SB1003 containing the cbb_Ip-lacZ fusion pXLB (Fig. 6), consistent with the finding that form I RubisCO was not detected in R. capsulatus grown photoheterotrophically on malate (Table 2; Fig. 3). In agreement with previous studies (39, 46) and data presented here (Table 2; Fig. 3), which show that form II RubisCO is synthesized under photoheterotrophic conditions, -galactosidase activities in photoheterotrophically grown SB1003 containing a cbb_IIp-lacZ fusion (pXFB) indicated that transcription occurred from cbb_IIp under these conditions (Fig. 6). Increased -galactosidase activity was observed in photoautotrophically grown SB1003 harboring either pXLB or pXFB, suggesting that transcription from cbb_Ip and cbb_IIp is induced under photoautotrophic conditions.

View larger version (16K): [in this window] [in a new window]   FIG. 6.   -Galactosidase activity from R. capsulatus cbbp-lacZ fusions. The cbb promoter fragments that were fused to the lacZ gene in vector pXBA601 are represented by arrows. In plasmids pXLB and pXLBP, cbbIp was fused to lacZ at the ATG start codon of cbbL. cbbIIp was fused to the ATG start codon of cbbF in plasmid pXFB. -Galactosidase activity is expressed in nanomoles/minute/milligram. The growth conditions were photoheterotrophically with malate as a carbon source (MAL) and photoautotrophically (PA). Strain SBRII did not grow photoautotrophically, and so the -galactosidase activity could not be determined. N.D., not detectable; , not determined. The values are averages derived from multiple assays of two independent cultures for each strain.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Previous studies established that cbb gene organization in R. capsulatus is different from the situation for the cbb regulon of the closely related organism R. sphaeroides (38, 39). The present study elaborated additional features of cbb gene organization and control in R. capsulatus as a prelude to further detailed investigations of cbb regulation in this organism. The finding that the cbbP ribosomal binding site was within the cbbF coding region and the polar effect of the cbbP disruption on form II RubisCO synthesis provided strong evidence that the R. capsulatus cbbFPTGAM genes make up an operon (cbb_II operon). Moreover, the presence of a potential RNase E cleavage site within the R. capsulatus cbbP-cbbT intergenic region hints that posttranscriptional processing of the R. capsulatus cbb_II message may occur, reminiscent of the suggested posttranscriptional processing of cbb operon transcripts of R. sphaeroides (14). Despite these similarities, a very significant difference between the R. capsulatus and R. sphaeroides cbb_II operons is the presence of a cbbR gene, cbbR_II, divergently transcribed from the R. capsulatus cbb_II operon. In addition, a second cbbR gene, cbbR_I, is upstream and divergently transcribed from the cbbLS (cbb_I) operon. It has become well established that CbbR is involved in the regulation of cbb gene expression in a number of autotrophic bacteria, including C. vinosum (54), Ralstonia eutropha (formerly Alcaligenes eutrophus) (59), Xanthobacter flavus (34), R. sphaeroides (16), Rhodospirillum rubrum (8), and Thiobacillus ferrooxidans (26). These studies are buttressed by the finding that CbbR binds to the promoter region of the cbb operon (26, 27, 52, 53), with a physiological role for CbbR now well established (16, 52, 59). In R. eutropha (59), R. sphaeroides (16), and X. flavus (52), the product of a single cbbR gene regulates transcription from at least two different promoters. Consequently, transcription from these operons is coordinately activated within a single CbbR regulon. Only Thiobacillus denitrificans (30) and R. capsulatus have two potentially functional cbbR genes. The presence of two CbbR proteins in R. capsulatus raises questions concerning the involvement of each form of CbbR in the expression of the two or more cbb promoters found in this organism. The likelihood that CbbR I controls only the cbb_I operon was previously suggested since the cbbR_I, cbbLS, and cbbQ genes were all apparently acquired by horizontal gene transfer (38). Thus, to probe the role of the two CbbRs in R. capsulatus cbb gene regulation, strains with disruptions in cbbR_I and cbbR_II were constructed and characterized. A strain (SBRII) in which the cbbR_II gene was disrupted was unable to grow autotrophically and grew at a reduced rate photoheterotrophically, showing reduced levels of both PRK and RubisCO activity and form II RubisCO protein. In addition, -galactosidase activity derived from cbb_IIp-lacZ fusion pXFB in strain SBRII was only about 9% of the activity of the wild-type strain under photoheterotrophic conditions. These results clearly implicate CbbR II in activation of transcription at cbb_IIp. The presence of PRK activity in strain SBRII indicates that some transcription from cbb_IIp occurred in the absence of CbbR II, albeit at an apparently reduced rate. Whether this was due to cross-talk activation by CbbR I remains to be established; however, it should be noted that transcription from cbb_IIp is not entirely dependent on CbbR in R. sphaeroides (16). The lack of form I RubisCO in photoautotrophically grown strain SBRI provides evidence that CbbR I is involved in the regulation of form I RubisCO synthesis, probably by activating transcription at cbb_Ip. Furthermore, the absence of form I RubisCO in photoautotrophically grown strain SBRI indicates that CbbR II is unable to activate transcription from cbb_Ip. In addition, the ability of strain SBRI to grow under photoheterotrophic and autotrophic conditions, and the apparent normal level of form II RubisCO synthesis in this strain, demonstrate that CbbR I is not required for expression of the cbb_II operon. The data strongly indicate that the CbbR I and CbbR II proteins are necessary for normal regulation of the cbb_I and cbb_II operons, respectively, and that cross-talk activation of the cbb operons by the opposite CbbR protein does not occur. These studies thus provide the first indication that the cbb operons may belong to independent CbbR regulons.

LysR-type transcriptional activators generally bind to the promoter they activate, even under noninducing conditions, and the binding of a low-molecular-weight coinducer molecule to the LysR-type protein is required, in most cases, to activate transcription (45). It will be interesting to determine if independent regulation of the R. capsulatus cbb operons by the cognate CbbR proteins correlates with activation by unique coinducer molecules. Activation of transcription at cbb_IIp by CbbR II under photoheterotrophic conditions, and lack of transcriptional activation at cbb_Ip by CbbR I under the same conditions, indicate that either a repressor binds to cbb_Ip under photoheterotrophic conditions, different inducer molecules bind to the different CbbR proteins, or the CbbR proteins bind the same inducer with different affinities. In the latter case, it is possible that the intracellular concentration of the inducing metabolite increases under photoautotrophic conditions, resulting in activation of transcription at cbb_Ip by CbbR I. Certainly, the presence of two different CbbR proteins raises additional questions about DNA binding specificity. Since CbbR probably binds to the cbb promoter region in the absence of an inducer molecule, binding must be specific to prevent repressive effects on the opposite promoter (i.e., binding of CbbR II to the cbb_I promoter may not activate transcription but could prevent the binding of CbbR I). Current studies are directed at examining the specificity of CbbR I and CbbR II in vitro.

The product of the qor gene discovered downstream of cbbR_II may also serve to regulate cbb gene expression. This gene encodes a soluble NAD(P)H QOR that catalyzes the reversible transfer of electrons from reduced pyridine nucleotides, with a preference for NADPH, to membrane-bound quinones. On the basis of the reaction catalyzed by this enzyme, QOR could function to sense or maintain the redox state of the membrane quinone pool. Interestingly, NADPH has been implicated as the coinducer of CbbR transcriptional activation in X. flavus (53). Thus, as a redox sensor, QOR could be involved in the regulation of cbb gene expression or perhaps in regulating the CBB pathway enzymes.

Although the evidence discussed above demonstrates that the cbb_I and cbb_II operons are differentially regulated by the two CbbR proteins, additional evidence suggests that regulation of these operons is also coordinated. When either cbbM or cbbL was disrupted, the absence of the missing RubisCO was compensated for, such that levels of RubisCO did not differ significantly from that in the wild-type strain. This compensation is analogous to what was observed in R. sphaeroides (14). However, the compensation effect was most dramatically demonstrated in R. capsulatus by the ability of strain SBII to grow photoheterotrophically, concomitant with the synthesis of form I RubisCO. Since the wild-type strain did not synthesize detectable levels of form I RubisCO under photoheterotrophic conditions, the absence of form II RubisCO synthesis in strain SBII somehow signaled the cell to compensate, by making form I RubisCO. However, compensation of form I RubisCO synthesis (Table 2) was not manifested by the cbbP mutant (strain SBP), in which form II RubisCO is not synthesized due to a polar effect of this mutation on cbbM (Fig. 4). These results thus suggest that the balance of various intermediates of the CBB pathway might regulate gene expression, which is an area that is currently being explored.

In summary, R. capsulatus cbb gene regulation is quite complex and differs markedly from that in R. sphaeroides. Two different CbbR transcriptional activators that allow autonomous regulation of the cbb_I and cbb_II operons, perhaps by binding different inducer molecules, are present in R. capsulatus. Obviously, to allow efficient regulation, the CbbR proteins must bind specifically to their respective cbb promoters. The presence of a potential RNase E recognition site within the cbb_II message suggests that it is posttranscriptionally processed. Further study of R. capsulatus cbb gene regulation will not only provide a better understanding of the control of CO₂ fixation but also address more general questions of gene regulation, such as the specificity of DNA-protein interactions and the significance of mRNA processing in prokaryotes.