Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.     

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.     

A single flavoprotein,AppA, integrates both redox and light signals in Rhodobacter sphaeroides

Anoxygenic photosynthetic proteobacteria exhibit various light responses, including changing levels of expression of photosynthesis genes. However, the underlying mechanisms are largely unknown. We show that expression of the puf and puc operons encoding structural proteins of the photosynthetic complexes is strongly repressed by blue light under semi-aerobic growth in Rhodobacter sphaeroides but not in the related species Rhodobacter capsulatus. At very low oxygen tension, puf and puc expression is independent of blue light in both species. Photosynthetic electron transport does not mediate the blue light repression, implying the existence of specific photoreceptors. Here, we show that the flavoprotein AppA is likely to act as the photoreceptor for blue light-dependent repression during continuous illumination. The FAD cofactor of AppA is essential for the blue light-dependent sensory transduction of this response. AppA, which is present in R. sphaeroides but not in R. capsulatus, is known to participate in the redox-dependent control of photosynthesis gene expression. Thus, AppA is the first example of a protein with dual sensing capabilities that integrates both redox and light signals.     

Gene transfer system for Rhodopseudomonas viridis.

A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis.     

The rate of decay of Rhodobacter capsulatus-specific puf mRNA segments is differentially affected by RNase E activity in Escherichia coli.

In Rhodobacter capsulatus the puf operon encodes proteins of the photosynthetic apparatus. The polycistronic puf mRNA is comprised of segments that show differential stability. Here, we show that the rate of decay of the 2.7-kb pufBALMX mRNA species in Escherichia coli depends on the activity of ribonuclease E (RNase E), whereas the degradation of the 0.5-kb pufBA mRNA segment is not affected by a mutation in the rne gene. The RNase E-promoted decay of the pufLMX mRNA depends on the presence of a 1.4-kb pufLM mRNA segment, in which rate-limiting endonucleolytic cleavage was postulated to occur in R. capsulatus. The insertion of 185 bp of this 1.4-kb segment into pufB results in an RNase E-dependent decay of the modified pufBA mRNA segment in E. coli. Our findings suggest that in R. capsulatus an RNase E-like activity is responsible for the rate-limiting endonucleolytic cleavage occurring within the pufLM mRNA segment, whereas the 0.5-kb pufBA mRNA segment is degraded by a different RNase E-independent decay mechanism.     

The cytochrome bc1 complex of Rhodobacter sphaeroides can restore cytochrome c2-independent photosynthetic growth to a Rhodobacter capsulatus mutant lacking cytochrome bc1.

Plasmids encoding the structural genes for the Rhodobacter capsulatus and Rhodobacter sphaeroides cytochrome (cyt) bc1 complexes were introduced into strains of R. capsulatus lacking the cyt bc1 complex, with and without cyt c2. The R. capsulatus merodiploids contained higher than wild-type levels of cyt bc1 complex, as evidenced by immunological and spectroscopic analyses. On the other hand, the R. sphaeroides-R. capsulatus hybrid merodiploids produced only barely detectable amounts of R. sphaeroides cyt bc1 complex in R. capsulatus. Nonetheless, when they contained cyt c2, they were capable of photosynthetic growth, as judged by the sensitivity of this growth to specific inhibitors of the photochemical reaction center and the cyt bc1 complex, such as atrazine, myxothiazol, and stigmatellin. Interestingly, in the absence of cyt c2, although the R. sphaeroides cyt bc1 complex was able to support the photosynthetic growth of a cyt bc1-less mutant of R. capsulatus in rich medium, it was unable to do so when C4 dicarboxylic acids, such as malate and succinate, were used as the sole carbon source. Even this conditional ability of R. sphaeroides cyt bc1 complex to replace that of R. capsulatus for photosynthetic growth suggests that in the latter species the cyt c2-independent rereduction of the reaction center is not due to a structural property unique to the R. capsulatus cyt bc1 complex. Similarly, the inability of R. sphaeroides to exhibit a similar pathway is not due to some inherent property of its cyt bc1 complex.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

Acetate metabolism in purple non-sulfur bacteria

Abstract The photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus E1F1 can grow on acetate or dl -malate photoheterotrophically under anerobic conditions or chemoheterotrophically in the dark in the presence of dioxygen. Bacterial cells grown under both anaerobic and aerobic conditions exhibited high amounts of the tricarboxylic acid cycle enzymes especially in dark-aerobic cultures. A high activity of isocitrate lyase was found in cells of R. capsulatus E1F1 and, to a lesser extent, in those of R. capsulatus IP2, Rhodobacter sphaeroides and Rhodospirillum rubrum grown photoheterotrophically on acetate under anaerobic conditions. The second enzyme of the glyoxylate shunt, malate synthase, appears to be constitutive. Itaconate, a powerful inhibitor of isocitrate lyase, severely inhibited growth of R. capsulatus, R. rubrum and R. sphaeroides on acetate, thus corroborating a physiological role of the enzyme in acetate metabolism by Rhodospirillaceae.     

Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases.

The nucleotide sequence of the 1794-bp fragment containing the crtD gene from Rhodobacter sphaeroides 2.4.1 encoding for methoxyneurosporene dehydrogenase has been determined. A 63% sequence identity was found when compared with the nucleotide sequence of the crtD gene from Rhodobacter capsulatus. A putative regulatory palindromic motif present in the crtD gene from R. capsulatus also exists in this gene from R. sphaeroides. The translated open reading frame of the crtD gene of R. sphaeroides has identified a polypeptide of 495 amino acids which shares a 56% sequence identity with the same CrtD protein of R. capsulatus. The N- and C-termini of these CrtD proteins present a high degree of similarity with the N- and C-termini of other carotenoid dehydrogenases including those encoded by crtI genes. This is in good agreement with the previously hypothesized homology between CrtI and CrtD proteins.