Characterization and complementation of a mutant of Rhodobacter sphaeroides with a chromosomal deletion in the light-harvesting (LH2) genes

An LH2- strain of Rhodobacter sphaeroides, DBC1, has been constructed by deleting the puc operon, which encodes the LH2 alpha and beta polypeptides, from the chromosome and replacing it with a kanamycin resistance gene. Southern blot analysis indicates that the 950 bp BamHI restriction fragment which contains the puc operon has been lost and has been replaced by the 1.25 kb Km(R) cassette derived from Tn903. Strain DBC1 lacked the LH2 complex, as shown by loss of the characteristic absorbance bands at 800 and 850 nm. The LH2 polypeptides were also found to be absent after SDS-PAGE. The wild-type phenotype was restored to DBC1 by the transfer of a 3.8 kb BscI fragment containing the puc operon in plasmid pMA81. Transconjugants possessed a wild-type absorbance spectrum and LH2 polypeptides.     

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.     

Phosphatidylcholine is required for the efficient formation of photosynthetic membrane and B800-850 light-harvesting complex in Rhodobacter sphaeroides

No phosphatidylcholine (PC) was detected in the membrane of Rhodobacter sphaeroides pmtA mutant (PmtA1) lacking phosphatidylethanolamine (PE) N-methyltransferase, whereas PE in the mutant was increased up to the mole % comparable to the combined level of PE and PC of wild type. Neither the fatty acid composition nor the fluidity of membrane was altered by pmtA mutation. Consistently, aerobic and photoheterotrophic growth of PmtA1 were not different from wild type. However, PmtA1 showed an extended lag phase (15 h) after the growth transition from aerobic to photoheterotrophic conditions, indicating the PC requirement for the efficient formation of intracytoplasmic membrane (ICM). Interestingly, the B800-850 complex of PmtA1 was decreased more than twofold in comparison with wild type, whereas the level of the B875 complex comprising the fixed photosynthetic unit was not changed. Since puc expression is not affected by pmtA mutation, PC appears to be required for the proper formation of the B800-850 complex in the ICM of R. sphaeroides.     

Characterization of light-harvesting mutants of Rhodopseudomonas sphaeroides. I. Measurement of the efficiency of energy transfer from light-harvesting complexes to the reaction center

Light-harvesting mutants of Rhodopseudomonas sphaeroides lacking either the B800-850 complex or the B875 complex have been characterized by their absorption spectra in the visible and near-infrared region, and by their ability to transfer energy from the light-harvesting complexes to the reaction center. A new method of measuring the relative efficiency of energy transfer from the light-harvesting complexes to the reaction center is described. The B875- mutant had absorption maxima in the near-infrared at 800 and 849 nm with no evidence of an 875-nm shoulder. The efficiency of energy transfer from the light-harvesting complexes to the reaction center in the B875- mutant was 24% of the value measured for the wild-type strain and the B800-850- mutant. Yet, despite the fact that the efficiency of energy transfer for the B800-850- mutant and the wild-type strain were the same, there was a large difference in their photosynthetic unit size. These results are discussed in the context of a model in which light energy captured by the B800-850 complexes is transferred through the B875 complexes to the reaction center.     

Differential carotenoid composition of the B875 and B800-850 photosynthetic antenna complexes in Rhodobacter sphaeroides 2.4.1: involvement of spheroidene and spheroidenone in adaptation to changes in light intensity and oxygen availability.

Rhodobacter sphaeroides 2.4.1 is a member of the nonsulfur purple facultative photosynthetic proteobacteria, capable of growth under a variety of cultivation conditions. In addition to the structural polypeptides and bacteriochlorophyll, the two major antenna complexes, B875 and B800-850, contain a variety of carotenoids which are an important structural and functional component of the membrane-bound photosynthetic complexes of this bacterium. Two major carotenoids, spheroidene and its keto derivative, spheroidenone, are differentially synthesized by R. sphaeroides, depending on the growth conditions. Spheroidene prevails during growth under anaerobic conditions and low light intensities, whereas spheroidenone is predominant in semiaerobically grown cells or during anaerobic growth at high light intensities. In this study, we demonstrate that in wild-type cells, spheroidene is predominantly associated with the B800-850 photosynthetic antenna complex and spheroidenone is more abundant in the B875 complex. Exploiting mutants defective in the biosynthesis of either the B875 or B800-850 light-harvesting complex, we demonstrate an association between the formation of either the B875 or B800-850 complex, on the one hand, and the accumulation of spheroidenone or spheroidene, on the other. The possible involvement of the conversion of spheroidene to spheroidenone as a significant control mechanism involved in the adaptation of R. sphaeroides to changes in light intensity and oxygen tension is discussed.     

Quantum efficiency of the minor channel for excitation migration from B800 to B875 bacteriochlorophyll fractions in purple bacteria

Excitation migration in the light-harvesting bacteriochlorophyll complexes LH1 and LH2 of purple bacteria has been studied in many experimental and theoretical works. According to the widely accepted notions, it proceeds along the descending energy stairway, B800* → 850* → 875* → P870*, where symbol * stands for excitations in the corresponding BChl fraction. In this paper we demonstrate the existence of one more way of direct excitation delivery from B800 to B875, bypassing the main route via B850. The comparative modeling enables the estimation of the mean portion of excitation that uses this minor migration way. In some real cases it may reach 9–9.5%. The values of the critical distances for excitation migration from B800 to B850 and from B800 to B875, as well as their values for arbitrary spectral shifts in BChl molecules, are determined.     

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

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-alpha polypeptide (and presumably the beta 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.     

A Rhodobacter sphaeroides puf L, M and X deletion mutant and its complementation in trans with a 5.3 kb puf operon shuttle fragment

A Rhodobacter sphaeroides puf L, M and X deletion mutant was constructed using interposon mutagenesis. The puf L, M and X genes were replaced with a kanamycin resistance cartridge isolated from the transposon Tn5. The deletion strain PUFLMX 21 did not grow photoheterotrophically and was resistant to kanamycin. Southern blot analysis of genomic DNA from the deletion strain confirmed that the kanamycin resistance was inserted specifically into the puf operon and that the L, M and X genes were deleted. A spontaneous carotenoid mutant of PUFLMX was selected and was found to accumulate primarily neurosporene. Spectroscopic analysis of chromatophores isolated from the deletion strain showed normal B875 and B800-850 expression providing further evidence that the photosynthetic minus phenotype was not the result of insertional inactivation of the promoter region of the puf operon, or the puf Q region. The deletion strain could be returned to the photosynthetic plus phenotype by complementation in trans with a 5.3 kb puf operon shuttle fragment, although the generation time of the complemented strain was 30% longer than wild type.     

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

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.     

Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism.

A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.     

The cbb3 terminal oxidase of Rhodobacter sphaeroides 2.4.1: structural and functional implications for the regulation of spectral complex formation

We have previously shown that the flow of reductant through the cbb3 terminal cytochrome c oxidase of Rhodobacter sphaeroides is essential to the repression of photosynthesis (PS) gene expression in the presence of oxygen by inhibiting the functional activity of the Prr two-component activation system. To gain further insight into the role of the cbb3 oxidase and the cognate ccoNOQP operon in the oxygen regulation of PS gene expression, we constructed nonpolar, in-frame deletions within the ccoN and ccoQ genes. Whereas mutations in ccoN, ccoQ, and ccoP resulted in PS gene expression in the presence of oxygen, only the ccoQ mutation showed both the normal flow of reductant through the cbb3 oxidase and the absence of any alteration in the relative levels of spheroidene and spheroidenone, as is observed for those mutations in the cco operon that result in the loss of terminal oxidase activity. Consistent with these findings is the observation that extra copies of the ccoNOQP operon in trans resulted in the decreased formation of both the B800-850 and B875 spectral complexes under anaerobic growth conditions. These results in conjunction with our earlier findings indicate that (1) the flow of reductant through the cbb3 terminal oxidase is a prerequisite to the regulation of PS gene expression by the Prr two-component regulatory system, (2) the CcoQ protein is involved in conveying the signal derived from reductant flow through the cbb3 terminal oxidase to the Prr regulatory pathway, (3) there is reductant flow through this terminal oxidase under anaerobic conditions, and as a result, the activity of the Prr system is still subject to cbb3 regulation, and (4) the acceptor for reductant flow through cbb3 under anaerobic conditions is in whole or in part involved in the conversion of spheroidene to spheroidenone.