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
2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês
10.1074/jbc.m211267200
ISSN1083-351X
AutoresJames M. Dubbs, F. Robert Tabita,
Tópico(s)Microbial Community Ecology and Physiology
ResumoIn 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, threecbbFII::lacZtranslational fusion plasmids were constructed containing various lengths of sequence 5′ to the cbbII operon ofR. sphaeroides CAC. Expression of β-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 thecbbII promoter from +38 to −227 bp contained a CbbR binding site and conferred low level regulatedcbbII expression. The region from −227 to −1025 bp contained six RegA binding sites and conferred enhancedcbbII 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 thecbbI promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of thecbbI operon is RegA independent, indicated that the mechanisms controlling cbbI andcbbII operon expression during chemoautotrophic growth are quite different. 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, threecbbFII::lacZtranslational fusion plasmids were constructed containing various lengths of sequence 5′ to the cbbII operon ofR. sphaeroides CAC. Expression of β-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 thecbbII promoter from +38 to −227 bp contained a CbbR binding site and conferred low level regulatedcbbII expression. The region from −227 to −1025 bp contained six RegA binding sites and conferred enhancedcbbII 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 thecbbI promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of thecbbI operon is RegA independent, indicated that the mechanisms controlling cbbI andcbbII operon expression during chemoautotrophic growth are quite different. Calvin-Benson-Bassham ribulose bisphosphate carboxylase/oxygenase The nonsulphur purple bacterium Rhodobacter sphaeroidesutilizes 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 CO2serves primarily as an electron sink, with excess reducing equivalents generated by the oxidation of fixed carbon compounds funneled to CO2 (1Tabita F.R. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 885-914Google Scholar). 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 andcbbII (2Gibson J.L. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1107-1124Google Scholar, 3Gibson J.L. Tabita F.R. Arch. Microbiol. 1996; 166: 141-150Crossref PubMed Scopus (52) Google Scholar). 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 ) (4Gibson J.L. Falcone D.L. Tabita F.R. J. Biol. Chem. 1991; 266: 14646-14653Abstract Full Text PDF PubMed Google Scholar) whereas the cbbII operon encodes the large subunit of a form II type Rubisco (cbbMII ) (5Chen J.-H. Gibson J.L. Macue L.A. Tabita F.R. J. Biol. Chem. 1991; 266: 20447-20452Abstract Full Text PDF PubMed Google Scholar). The regulation of cbb gene expression in R. sphaeroides is quite complex (2Gibson J.L. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1107-1124Google Scholar). 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 (6Paoli G.C. Tabita F.R. Arch. Microbiol. 1998; 170: 8-17Crossref PubMed Scopus (26) Google Scholar). 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 (7Falcone D.A. Tabita F.R. J. Bacteriol. 1991; 173: 2099-2108Crossref PubMed Google Scholar, 8Gibson J.L. Tabita F.R. J. Bacteriol. 1977; 132: 818-823Crossref PubMed Google Scholar, 9Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1736-1748Crossref PubMed Google Scholar, 10Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1749-1761Crossref PubMed Google Scholar, 11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). 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 (11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). In addition to the apparent independent regulation of cbbI andcbbII 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 (4Gibson J.L. Falcone D.L. Tabita F.R. J. Biol. Chem. 1991; 266: 14646-14653Abstract Full Text PDF PubMed Google Scholar, 7Falcone D.A. Tabita F.R. J. Bacteriol. 1991; 173: 2099-2108Crossref PubMed Google Scholar, 9Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1736-1748Crossref PubMed Google Scholar, 10Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1749-1761Crossref PubMed Google Scholar). The cbbR gene, which encodes a LysR-type transcriptional regulator, is located immediately upstream and divergently transcribed from cbbFI (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar) and mediates this compensatory effect. CbbR is a positive regulator of the expression of both the cbbI andcbbII operons (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar, 13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar). 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 (14Inoue K. Kouadio J.L. Mosley C.S. Bauer C.E. Biochemistry. 1995; 34: 391-396Crossref PubMed Scopus (60) Google Scholar, 15Mosley C.S. Suzuki J.Y. Bauer C.E. J. Bacteriol. 1994; 176: 7566-7573Crossref PubMed Google Scholar) and R. sphaeroides (16Eraso J.M. Kaplan S. J. Bacteriol. 1995; 177: 2695-2706Crossref PubMed Google Scholar), theregA-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 andcbbII expression during photoautotrophic growth in a 1.5% CO2/98.5% H2 atmosphere (17Qian Y. Tabita F.R. J. Bacteriol. 1996; 178: 12-18Crossref PubMed Google Scholar). It was subsequently shown that regA is required forcbbI and cbbII expression during incubation under photoautotrophic growth conditions (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). It has also been demonstrated that RegA binds directly to cbboperon promoters in both R. capsulatus and R. sphaeroides (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 19Vichivanives P. Bird T.H. Bauer C.E. Tabita F.R. J. Mol. Biol. 2000; 300: 1079-1099Crossref PubMed Scopus (45) Google Scholar). 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 (20Joshi H.M. Tabita F.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14515-14520Crossref PubMed Scopus (164) Google Scholar, 21Qian Y. Tabita F.R. J. Bacteriol. 1998; 180: 4644-4649Crossref PubMed Google Scholar, 22Elsen S. Dischert W. Colbeau A. Bauer C.E. J. Bacteriol. 2000; 182: 2831-2837Crossref PubMed Scopus (72) Google Scholar), hydrogen utilization and evolution (20Joshi H.M. Tabita F.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14515-14520Crossref PubMed Scopus (164) Google Scholar, 22Elsen S. Dischert W. Colbeau A. Bauer C.E. J. Bacteriol. 2000; 182: 2831-2837Crossref PubMed Scopus (72) Google Scholar), electron transport (23Swem L.R. Elsen S. Bird T.H. Swem D.L. Koch H.-G. Myllkallio H. Daldal F. Bauer C.E. J. Mol. Biol. 2001; 309: 121-138Crossref PubMed Scopus (89) Google Scholar), and the oxidation of formaldehyde (24Barber R.D. Donohue T.J. J. Mol. Biol. 1998; 280: 775-784Crossref PubMed Scopus (33) Google Scholar).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::lacZpromoter 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 (13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar). 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) (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). 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) (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Although earlier work determined that the cbbII promoter occurred within 1000 bp of the cbbII transcription start (25Xu H.H. Tabita F.R. J. Bacteriol. 1994; 176: 7299-7308Crossref PubMed Google Scholar), details of the structure of the R. sphaeroides cbbII promoter have not been investigated previously. In this study,cbbII ::lacZtranslational 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 (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), with consequent effects on differential regulation of the cbb operons.DISCUSSIONThis investigation and previous studies (13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar, 18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) indicate that theR. sphaeroides cbbI andcbbII 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 incbbII ) 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 thecbbI 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 asreg (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar, 13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar, 18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Thus far, the involvement of upstream activating sequences appears to be unique to R. sphaeroidesas such sequences are not involved with the regulation of thecbbI and cbbII operons of the related organism R. capsulatus (19Vichivanives P. Bird T.H. Bauer C.E. Tabita F.R. J. Mol. Biol. 2000; 300: 1079-1099Crossref PubMed Scopus (45) Google Scholar).The discovery that maximal aerobic chemoautotrophic expression ofcbbII required regA was unexpected, given that chemoautotrophic expression of cbbI is regA-independent (34Gibson J.L. Dubbs J.M. Tabita F.R. J. Bacteriol. 2002; 184: 6654-6664Crossref PubMed Scopus (22) Google Scholar). 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 O2serves 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 (35O, Gara J.P. Eraso J.M. Kaplan S. J. Bacteriol. 1998; 180: 4044-4050Crossref PubMed Google Scholar). 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 thecbbI operon, because it encodes the form I (L8S8) Rubisco, used as the major autotrophic enzyme in the CBB pathway (1Tabita F.R. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 885-914Google Scholar). 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 (11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). 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 regAbackground, the level of chemoautotrophic cbbII expression from the promoter proximal regulatory region (pVKCIIXcm) was significantly higher than that in parental strain CAC. This increase incbbII expression in the regA mutant could be because of either activation by CbbR or additionalcbbII -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 ofR. sphaeroides. Moreover, it is apparent that the global two-component Reg/Prr system is only selectively involved withcbb control, with Reg/Prr required to activate transcription of only the cbbII operon, and not thecbbI 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. The nonsulphur purple bacterium Rhodobacter sphaeroidesutilizes 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 CO2serves primarily as an electron sink, with excess reducing equivalents generated by the oxidation of fixed carbon compounds funneled to CO2 (1Tabita F.R. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 885-914Google Scholar). 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 andcbbII (2Gibson J.L. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1107-1124Google Scholar, 3Gibson J.L. Tabita F.R. Arch. Microbiol. 1996; 166: 141-150Crossref PubMed Scopus (52) Google Scholar). 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 ) (4Gibson J.L. Falcone D.L. Tabita F.R. J. Biol. Chem. 1991; 266: 14646-14653Abstract Full Text PDF PubMed Google Scholar) whereas the cbbII operon encodes the large subunit of a form II type Rubisco (cbbMII ) (5Chen J.-H. Gibson J.L. Macue L.A. Tabita F.R. J. Biol. Chem. 1991; 266: 20447-20452Abstract Full Text PDF PubMed Google Scholar). The regulation of cbb gene expression in R. sphaeroides is quite complex (2Gibson J.L. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1107-1124Google Scholar). 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 (6Paoli G.C. Tabita F.R. Arch. Microbiol. 1998; 170: 8-17Crossref PubMed Scopus (26) Google Scholar). 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 (7Falcone D.A. Tabita F.R. J. Bacteriol. 1991; 173: 2099-2108Crossref PubMed Google Scholar, 8Gibson J.L. Tabita F.R. J. Bacteriol. 1977; 132: 818-823Crossref PubMed Google Scholar, 9Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1736-1748Crossref PubMed Google Scholar, 10Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1749-1761Crossref PubMed Google Scholar, 11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). 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 (11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). In addition to the apparent independent regulation of cbbI andcbbII 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 (4Gibson J.L. Falcone D.L. Tabita F.R. J. Biol. Chem. 1991; 266: 14646-14653Abstract Full Text PDF PubMed Google Scholar, 7Falcone D.A. Tabita F.R. J. Bacteriol. 1991; 173: 2099-2108Crossref PubMed Google Scholar, 9Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1736-1748Crossref PubMed Google Scholar, 10Hallenbeck P.L. Lerchen R. Hessler P. Kaplan S. J. Bacteriol. 1990; 172: 1749-1761Crossref PubMed Google Scholar). The cbbR gene, which encodes a LysR-type transcriptional regulator, is located immediately upstream and divergently transcribed from cbbFI (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar) and mediates this compensatory effect. CbbR is a positive regulator of the expression of both the cbbI andcbbII operons (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar, 13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar). 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 (14Inoue K. Kouadio J.L. Mosley C.S. Bauer C.E. Biochemistry. 1995; 34: 391-396Crossref PubMed Scopus (60) Google Scholar, 15Mosley C.S. Suzuki J.Y. Bauer C.E. J. Bacteriol. 1994; 176: 7566-7573Crossref PubMed Google Scholar) and R. sphaeroides (16Eraso J.M. Kaplan S. J. Bacteriol. 1995; 177: 2695-2706Crossref PubMed Google Scholar), theregA-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 andcbbII expression during photoautotrophic growth in a 1.5% CO2/98.5% H2 atmosphere (17Qian Y. Tabita F.R. J. Bacteriol. 1996; 178: 12-18Crossref PubMed Google Scholar). It was subsequently shown that regA is required forcbbI and cbbII expression during incubation under photoautotrophic growth conditions (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). It has also been demonstrated that RegA binds directly to cbboperon promoters in both R. capsulatus and R. sphaeroides (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 19Vichivanives P. Bird T.H. Bauer C.E. Tabita F.R. J. Mol. Biol. 2000; 300: 1079-1099Crossref PubMed Scopus (45) Google Scholar). 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 (20Joshi H.M. Tabita F.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14515-14520Crossref PubMed Scopus (164) Google Scholar, 21Qian Y. Tabita F.R. J. Bacteriol. 1998; 180: 4644-4649Crossref PubMed Google Scholar, 22Elsen S. Dischert W. Colbeau A. Bauer C.E. J. Bacteriol. 2000; 182: 2831-2837Crossref PubMed Scopus (72) Google Scholar), hydrogen utilization and evolution (20Joshi H.M. Tabita F.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14515-14520Crossref PubMed Scopus (164) Google Scholar, 22Elsen S. Dischert W. Colbeau A. Bauer C.E. J. Bacteriol. 2000; 182: 2831-2837Crossref PubMed Scopus (72) Google Scholar), electron transport (23Swem L.R. Elsen S. Bird T.H. Swem D.L. Koch H.-G. Myllkallio H. Daldal F. Bauer C.E. J. Mol. Biol. 2001; 309: 121-138Crossref PubMed Scopus (89) Google Scholar), and the oxidation of formaldehyde (24Barber R.D. Donohue T.J. J. Mol. Biol. 1998; 280: 775-784Crossref PubMed Scopus (33) Google Scholar). 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::lacZpromoter 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 (13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar). 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) (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). 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) (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Although earlier work determined that the cbbII promoter occurred within 1000 bp of the cbbII transcription start (25Xu H.H. Tabita F.R. J. Bacteriol. 1994; 176: 7299-7308Crossref PubMed Google Scholar), details of the structure of the R. sphaeroides cbbII promoter have not been investigated previously. In this study,cbbII ::lacZtranslational 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 (18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), with consequent effects on differential regulation of the cbb operons. DISCUSSIONThis investigation and previous studies (13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar, 18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) indicate that theR. sphaeroides cbbI andcbbII 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 incbbII ) 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 thecbbI 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 asreg (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar, 13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar, 18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Thus far, the involvement of upstream activating sequences appears to be unique to R. sphaeroidesas such sequences are not involved with the regulation of thecbbI and cbbII operons of the related organism R. capsulatus (19Vichivanives P. Bird T.H. Bauer C.E. Tabita F.R. J. Mol. Biol. 2000; 300: 1079-1099Crossref PubMed Scopus (45) Google Scholar).The discovery that maximal aerobic chemoautotrophic expression ofcbbII required regA was unexpected, given that chemoautotrophic expression of cbbI is regA-independent (34Gibson J.L. Dubbs J.M. Tabita F.R. J. Bacteriol. 2002; 184: 6654-6664Crossref PubMed Scopus (22) Google Scholar). 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 O2serves 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 (35O, Gara J.P. Eraso J.M. Kaplan S. J. Bacteriol. 1998; 180: 4044-4050Crossref PubMed Google Scholar). 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 thecbbI operon, because it encodes the form I (L8S8) Rubisco, used as the major autotrophic enzyme in the CBB pathway (1Tabita F.R. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 885-914Google Scholar). 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 (11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). 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 regAbackground, the level of chemoautotrophic cbbII expression from the promoter proximal regulatory region (pVKCIIXcm) was significantly higher than that in parental strain CAC. This increase incbbII expression in the regA mutant could be because of either activation by CbbR or additionalcbbII -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 ofR. sphaeroides. Moreover, it is apparent that the global two-component Reg/Prr system is only selectively involved withcbb control, with Reg/Prr required to activate transcription of only the cbbII operon, and not thecbbI 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. This investigation and previous studies (13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar, 18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) indicate that theR. sphaeroides cbbI andcbbII 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 incbbII ) 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 thecbbI 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 asreg (12Gibson J.L. Tabita F.R. J. Bacteriol. 1993; 175: 5778-5784Crossref PubMed Google Scholar, 13Dubbs J.M. Tabita F.R. J. Bacteriol. 1998; 180: 4903-4911Crossref PubMed Google Scholar, 18Dubbs J.M. Bird T.H. Bauer C.E. Tabita F.R. J. Biol. Chem. 2000; 275: 19224-19230Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Thus far, the involvement of upstream activating sequences appears to be unique to R. sphaeroidesas such sequences are not involved with the regulation of thecbbI and cbbII operons of the related organism R. capsulatus (19Vichivanives P. Bird T.H. Bauer C.E. Tabita F.R. J. Mol. Biol. 2000; 300: 1079-1099Crossref PubMed Scopus (45) Google Scholar). The discovery that maximal aerobic chemoautotrophic expression ofcbbII required regA was unexpected, given that chemoautotrophic expression of cbbI is regA-independent (34Gibson J.L. Dubbs J.M. Tabita F.R. J. Bacteriol. 2002; 184: 6654-6664Crossref PubMed Scopus (22) Google Scholar). 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 O2serves 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 (35O, Gara J.P. Eraso J.M. Kaplan S. J. Bacteriol. 1998; 180: 4044-4050Crossref PubMed Google Scholar). 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 thecbbI operon, because it encodes the form I (L8S8) Rubisco, used as the major autotrophic enzyme in the CBB pathway (1Tabita F.R. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 885-914Google Scholar). 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 (11Jouanneau Y. Tabita F.R. J. Bacteriol. 1986; 165: 620-624Crossref PubMed Google Scholar). 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 regAbackground, the level of chemoautotrophic cbbII expression from the promoter proximal regulatory region (pVKCIIXcm) was significantly higher than that in parental strain CAC. This increase incbbII expression in the regA mutant could be because of either activation by CbbR or additionalcbbII -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 ofR. sphaeroides. Moreover, it is apparent that the global two-component Reg/Prr system is only selectively involved withcbb control, with Reg/Prr required to activate transcription of only the cbbII operon, and not thecbbI 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. We thank Dr. Janet L. Gibson for comments and suggestions on the manuscript.
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