Genetic and physical mapping of the Rhodobacter sphaeroides photosynthetic gene cluster from R-prime pWS2

Plasmid pWS2 is an R68.45 chimera originally isolated as an R-prime which complemented the Rhodobacter sphaeroides bch-420 allele. Our experiments have shown that pWS2 is also able to complement a wide range of R. sphaeroides pigment and photosynthetic mutants employing nitrosoquanidine, transposon or insertion-generated mutations effecting puhA, puc, puf, cycA, bch, and crt genes. A combination of orthogonal-field-alternation gel electrophoresis, transverse alternating field gel electrophoresis, and conventional electrophoresis have been used to estimate the size of pWS2 at congruent to 168.3 +/- 3.5 kb. A restriction map of the congruent to 109 kb of R. sphaeroides insert DNA was generated by partial and complete restriction endonuclease digestion coupled with Southern hybridization analysis using either gene-specific or junction fragment probes. Genes encoding bacteriochlorophyll (Bchl)-binding proteins (pufBALMX, pucBA, and puhA), cytochrome c2 (cycA), and enzymes involved in Bchl (bch) and carotenoid (crt) biosynthesis have been shown to reside within a contiguous 53-kb region of the R. sphaeroides DNA present on pWS2. The puf operon lies at one end of the 53-kb segment, while the genes puhA, cycA, and pucBA, the latter two of which are located within congruent to 12.0 kb of each other, define the other end of this 53-kb region. The genetic and physical mapping data provided in this paper are discussed in terms of the similarities and differences in the organization of the photosynthetic gene cluster between R. sphaeroides and other photosynthetic bacteria as well as highlighting the use of pWS2 in studies of photosynthetic gene structure and function.     

Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity

In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100?W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24?h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24?h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48?h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24?h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.     

Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity

In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100?W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24?h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24?h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48?h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24?h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.     

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.