Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides

Davis, John Gorton; Donohue, Timothy J.; Kaplan, Samuel

doi:10.1128/jb.170.1.320-329.1988

Cited by 182 publications

(188 citation statements)

References 39 publications

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“…Ampicillin was used at 150 g/ml and chloramphenicol was used at 25 g/ml for E. coli cultures. Valinomycin was used at 2 M. R. sphaeroides cultures were grown as described previously (12,14). E. coli GM2163 was used to isolate unmethylated DNA for subcloning.…”

Section: Methodsmentioning

confidence: 99%

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Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase

Eraso¹,

Kaplan²

1995

J Bacteriol

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121

204

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Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides. prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA. Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities. Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis. Previously, J. K. Lee and S. Kaplan (J. Bacteriol. 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation. This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78). Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression. Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes. Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA. The genetic organization of the photosynthesis response regulatory (PRR) region is discussed.Rhodobacter sphaeroides is a purple, nonsulfur photoheterotrophic bacterium whose mode of growth depends on the concentration of oxygen in the surrounding environment (49). At high oxygen concentrations, R. sphaeroides grows chemoheterotrophically, and morphologically it resembles a typical gram-negative bacterium (3). Under low oxygen or anaerobiosis, the photosynthetic membrane system, designated the intracytoplasmic membrane (ICM), is induced; the ICM fills the interior of the cell, and its abundance is inversely related to the incident light intensity (25). The ICM contains all of the structural and functional components of the photosynthetic apparatus. Included are three distinct bacteriochlorophyll (Bchl) a pigment-protein complexes: the photochemical reaction center (RC) (36) and the two light-harvesting complexes, designated the B800-850 and B-875 spectral complexes (7, 9). When grown anaerobically in the dark, in the presence of alternative electron acceptors, the gratuitous synthesis of the ICM occurs (53).Lee and Kaplan (28) provided the first demonstration that a relatively simple switching mechanism must be involved in the repression of photosynthesis gene expression by oxygen in the genus Rhodobacter when they isolated the spontaneous mutant T 1a , which accumulates photosynthetic membranes in the presence of high oxygen. Subsequen...

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Section: Methodsmentioning

confidence: 99%

“…Genetic techniques. Plasmid DNA was mobilized into R. sphaeroides by conjugation, using techniques previously described (12). The need to supplement Sistrom's minimal medium with K 2 TeO 2 could be bypassed by using the auxotroph E. coli DH5␣phe as the donor in conjugations (17).…”

Section: Methodsmentioning

confidence: 99%

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase

Eraso¹,

Kaplan²

1995

J Bacteriol

Self Cite

121

204

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“…The resulting plasmid was mobilized from Escherichia coli S17-1 into R. sphaeroides by conjugation as described previously (25), and the ␤-galactosidase activity (Miller units) was determined as described previously (26).…”

Section: Construction Of Bchc::lacz Transcriptional Fusion Plasmids Amentioning

confidence: 99%

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Kim

Lee

et al. 2008

Journal of Biological Chemistry

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Chlorophyllide a reductase of Rhodobacter sphaeroides, which were reconstituted with the purified subunits of BchX, BchY, and BchZ, reduced ring B of chlorophyllide a using NADH under anaerobic conditions. Interestingly, suppressor mutations rescuing the inability of R. sphaeroides Fe-SOD mutant to grow in succinate-based minimal medium were predominantly mapped to BchZ subunit of chlorophyllide a reductase. The enzyme is labile in the presence of O 2 . However, it generates superoxide at low O 2 . The enzymes reconstituted with BchX, BchY, and the mutein subunit of BchZ from suppressor mutants showed less activity not only for chlorophyllide a reduction but also for superoxide generation compared with the enzyme reconstituted with the wild-type subunits. BchX, which contains FMN, and BchY are iron-sulfur proteins, whereas BchZ is a hemoprotein containing b-type heme. Neither chlorophyllide a reduction nor superoxide generation was observed with the enzyme reconstituted with the wild-type subunits of BchX and BchY, and the apo-subunit of BchZ that had been refolded without heme, in which FMN of BchX was fully reduced. Thus, superoxide is generated not from FMN of BchX but from heme of BchZ. Consistently, the heme of BchZ muteins was half-reduced in its redox state compared with that of wild-type BchZ.Rhodobacter sphaeroides, a facultative photosynthetic bacterium, contains two SODs 3 ; CuZn-SOD is detected only under the conditions where photosynthetic complexes are formed (1), whereas Fe-SOD is constitutively expressed, although its activity of the aerobically grown cell nearly doubled as compared with that of the anaerobically grown cell. The role of CuZn-SOD in protecting the photoheterotrophic cells from periplasmic superoxide upon exposure to O 2 was proposed (1). A cambialistic SOD using manganese or iron is the only SOD found in Rhodobacter capsulatus and is essential for cell viability (2, 3). Oxidative stress defense in R. sphaeroides and R. capsulatus is also mediated by catalases as well as by glutathione-glutaredoxin and thioredoxin systems that act as thiol-disulfide redox buffer to reduce the protein thiols that were oxidized (4, 5). Mutations in glutathione-glutaredoxin and thioredoxin systems lowered the formation of the photosynthetic complex (4, 5).The formation of photosynthetic complexes of R. sphaeroides is redox-dependent. Lowering oxygen tension induces the formation of intracytoplasmic membrane housing the photosynthetic complexes of R. sphaeroides. Light captured by B800 -850 and B875 LH complexes is transferred to reaction center complex, where the redox reactions are initiated to convert light energy into ATP and reducing power. The oxygenregulated expression of apoproteins of LH and reaction center complexes, which are encoded by puc, puf, and puh operons, was elucidated (for reviews see Refs. 6 and 7).The expression of several enzymes for Bchl a synthesis is also subject to anaerobic induction (8 -10). Protoporphyrin IX, which is synthesized from 5-aminolevulinic acid, is a common in...

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“…The copy number of RK2-derived plasmids has been estimated to be -5 to 10 per R. sphaeroides genome (5,9). From the immunoblot analysis with cyt c2 antiserum, it is evident that the fusion proteins are being accumulated to higher levels than is the wild-type cyt C2 encoded by the 2.4.1 chromosome.…”

Section: Methodsmentioning

confidence: 99%

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

et al. 1991

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Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.

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Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides

Cited by 182 publications

References 39 publications

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

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