Cloning and expression of the Rhodobacter sphaeroides reaction center H gene

Donohue, Timothy J.; Hoger, J H; Kaplan, Samuel

doi:10.1128/jb.168.2.953-961.1986

Cited by 67 publications

(49 citation statements)

References 40 publications

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“…These transcript levels are normally observed in cells growing photosynthetically at moderate to low light intensity (13,14,24,26). Thus, these measurements are compatible with observations made in T 1a (28) with respect to light-harvesting complex formation, or RC assembly (50), as well as those described above.…”

Section: Vol 177 1995 Oxygen Regulation Of Gene Expression In R Spmentioning

confidence: 97%

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

Eraso¹,

Kaplan²

1995

J Bacteriol

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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: Vol 177 1995 Oxygen Regulation Of Gene Expression In R Spmentioning

confidence: 97%

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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“…Therefore, we examined the effects of the mutations in PUC705-BA and PUC-Pv on the expression of both puf and puhA operons. Specific RNA probes corresponding to the pufRA and puhA structural genes (StyI fragment of puJBA genes [9] and SphI-XhoI internal fragment of puhA gene [11]) were used for Northern blot hybridization analysis. RNA derived from PUC-Pv showed about the same level of pufBA-specific transcript as in wild type (Fig.…”

Section: B800-850 Complexmentioning

confidence: 99%

“…The LH complexes capture and funnel photons as excitation energy for the photo-induced reversible oxidation-reduction of the RC, ultimately giving rise to chemical energy (32). The fixed photosynthetic unit is composed of B875 and RC complexes (1,5) encoded by the puf (42) and puh (11) operons. The ratio of B875 to RC complexes is fixed at approximately 12:1 to 15:1 irrespective of the incident light intensity.…”

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Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides

1989

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The puc operon of Rhodobacter sphaeroides comprises the pucBA structural genes which encode B800-850 light-harvesting ,I and a polypeptides, respectively. Northern (RNA) blot hybridization analysis of puc operon expression has identified two pucBA-specific transcripts. The small (0.5-kilobase [kb]) transcript encodes the 0i and a polypeptides and, under photoheterotrophic growth conditions, was approximately 200-fold more abundant than the large (2.3-kb) transcript. The 5' end of the 0.5-kb transcript was mapped at 117 nucleotides upstream from the start ofpucB. The 3' ends of the 0.5-kb transcript were mapped to two adjacent nucleotides, which follow a stem-loop structure immediately 3' to the pucA stop codon. Two mutant strains, PUC705-BA and PUC-Pv, were constructed by replacement of the pucBA genes and adjacent DNA in the former case or by insertional interruption of the DNA downstream of the pucBA genes in the latter case. The two mutant strains were devoid of B800-850 complexes during photosynthetic growth but were otherwise apparently normal. The B800-850-phenotype of both PUC705-BA and PUC-Pv was not complemented in trans with a 2.5-kb PstI restriction endonuclease fragment extending from 0.75 kb upstream of pucBA to 1.3 kb downstream of pucBA, despite the presence of the 0.5-kb pucBA-specific transcript. Both of the mutant strains, however, showed restoration of B800-850 expression with a 10.5-kb EcoRI restriction endonuclease fragment in trans encompassing the 2.5-kb PstI fragment. Western immunoblot analysis revealed no B800-850-I polypeptide as well as no polypeptide designated 15A in either mutant. Nonetheless, under photoheterotrophic growth conditions, the 0.5-kb pucBA-specific transcript was present in PUC-Pv, although no 2.3-kb transcript was detectable. We suggest that the DNA region immediately downstream of pucBA encodes a gene product(s) essential for translational or posttranslational expression of the B800-850 0i and a polypeptides.The purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides is an ideal system for the study of bacterial photosynthesis as well as membrane development (22). In response to lower partial pressures of oxygen, this bacterium can form the unique photosynthetic membrane system referred to as the intracytoplasmic membrane (ICM), in addition to the normal gram-negative membrane system found during aerobic growth (4, 17). The three major bacteriochlorophyll (Bchl)-protein complexes found in the ICM are the light-harvesting complexes B800-850 (LHII) and B875 (LHI) and the reaction center (RC) complex. The LH complexes capture and funnel photons as excitation energy for the photo-induced reversible oxidation-reduction of the RC, ultimately giving rise to chemical energy (32). The fixed photosynthetic unit is composed of B875 and RC complexes (1, 5) encoded by the puf(42) and puh (11) operons. The ratio of B875 to RC complexes is fixed at approximately 12:1 to 15:1 irrespective of the incident light intensity. On the other hand, B800-850 complex formation is va...

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“…Through the isolation and analysis of R. sphaeroides mutants altered in these spectral complexes, we hope to determine what factors and genetic regions are necessary for synthesis of functional spectral complexes in vivo. Since the structural genes for the B875, B800-850, and RC polypeptides from R. sphaeroides have been cloned and sequenced, and their operon structure has been determined (16,26,27,44,45,47,48), such an approach provides the foundation for further studies to understand the genetic and molecular mechanisms regulating the synthesis and stoichiometry of individual LH complexes in vivo.…”

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Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides

1988

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Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-oa polypeptide (and presumably the ,( polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.

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Cloning and expression of the Rhodobacter sphaeroides reaction center H gene

Cited by 67 publications

References 40 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

Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides

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