Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River

Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acsF gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-A-L-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP beta-proteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments.     

Horizontal Transfer of the Photosynthesis Gene Cluster and Operon Rearrangement in Purple Bacteria

A 37-kb photosynthesis gene cluster was sequenced in a photosynthetic bacterium belonging to the beta subclass of purple bacteria (Proteobacteria), Rubrivivax gelatinosus. The cluster contained 12 bacteriochlorophyll biosynthesis genes (bch), 7 carotenoid biosynthesis genes (crt), structural genes for photosynthetic apparatuses (puf and puh), and some other related genes. The gene arrangement was markedly different from those of other purple photosynthetic bacteria, while two superoperonal structures, crtEF-bchCXYZ-puf and bchFNBHLM-lhaA-puhA, were conserved. Molecular phylogenetic analyses of these photosynthesis genes showed that the photosynthesis gene cluster of Rvi. gelatinosus was originated from those of the species belonging to the alpha subclass of purple bacteria. It was concluded that a horizontal transfer of the photosynthesis gene cluster from an ancestral species belonging to the alpha subclass to that of the beta subclass of purple bacteria had occurred and was followed by rearrangements of the operons in this cluster.     

Proposal of Pseudorhodobacter ferrugineus gen nov,comb nov,for a non-photosynthetic marine bacterium,Agrobacterium ferrugineum,related to the genus Rhodobacter

The marine gram-negative non-photosynthetic bacterium, Agrobacterium ferrugineum IAM 12616(T) forms one cluster with the species of the photosynthetic genus Rhodobacter in phylogenetic trees based on molecules of 16S rRNA, 23S rRNA and DNA gyrases. Agrobacterium ferrugineum and Rhodobacter species are similar in that growth occurs without NaCl in the culture medium (optimal NaCl concentration for growth of P. ferrugineus is 1%) and their major hydroxy fatty acid compositions are 3-hydroxy decanoic acids (3-OH 10:0) and 3-hydroxy tetradecanoic acids (3-OH 14:1). However, A. ferrugineum differs from Rhodobacter species in G+C content (58 mol% in A. ferrugineum versus 64-73 mol% in Rhodobacter species), in having an insertion in its 16S rRNA gene sequence, and in lacking photosynthetic abilities, bacteriochlorophyll a and intracytoplasmic membrane systems. Furthermore, experiments using PCR and Southern hybridization show that A. ferrugineum does not have puhA gene and puf genes localized near the opposite ends of the photosynthesis gene cluster of Rhodobacter capsulatus. It suggests that A. ferrugineum may not have any genes for photosynthesis. We propose the transfer of A. ferrugineum IAM 12616(T) to the genus Pseudorhodobacter gen. nov. as Pseudorhodobacter ferrugineus comb. nov. Although Pseudorhodobacter ferrugineus disturbs the phylogenetic monophyly of the genus Rhodobacter, this taxonomic proposal seems adequate until it has been clarified whether P. ferrugineus possesses an incomplete photosynthetic apparatus.     

"Green-like" and "red-like" RubisCO cbbL genes in Rhodobacter azotoformans

We found that Rhodobacter azotoformans IFO 16436T contains two different cbbL genes coding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunits. One gene is located within a "green-like" group of the RubisCO phylogenetic tree, and the other is located within a "red-like" group. This is the first report that one organism contains both green-like and red-like RubisCO genes. Moreover, by PCR using primers which amplify two green-like and red-like cbbL genes alternatively and dot blot hybridization, we demonstrated that Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter veldkampii possess only green-like cbbL genes, and Rhodobacter sphaeroides possesses only a red-like cbbL gene. In the cbbL phylogenic analysis, R. spaeroides and R. azotoformans 1 (red-like) formed a cluster within the red-like group, and R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed a cluster within the green-like group. This suggests that red-like cbbL genes of Rhodobacter species were derived from one ancestor, and green-like cbbL genes were derived from another ancestor. On the other hand, molecular phylogeny of the bacteria indicates that R. veldkampii, which has only a green-like cbbL gene, is the earliest evolved Rhodobacter species and that R. azotoformans and R. sphaeroides, which have red-like cbbL genes, are the latest evolved. Consequently, the following hypothesis is proposed: the common ancestor of Rhodobacter had a green-like cbbL gene, the common ancestor of R. azotoformans and R. sphaeroides subsequently obtained a red-like cbbL gene by a horizontal gene transfer, and the ancestor of R. sphaeroides later lost the green-like cbbL gene.     

DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1

This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1^T. The photosynthesis gene cluster is located within a ~73 kb AseI genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R.sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC=cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.     

Redox and light regulation of gene expression in photosynthetic prokaryotes

All photosynthetic organisms control expression of photosynthesis genes in response to alterations in light intensity as well as to changes in cellular redox potential. Light regulation in plants involves a well-defined set of red- and blue-light absorbing photoreceptors called phytochrome and cryptochrome. Less understood are the factors that control synthesis of the plant photosystem in response to changes in cellular redox. Among a diverse set of photosynthetic bacteria the best understood regulatory systems are those synthesized by the photosynthetic bacterium Rhodobacter capsulatus. This species uses the global two-component signal transduction cascade, RegB and RegA, to anaerobically de-repress anaerobic gene expression. Under reducing conditions, the phosphate on RegB is transferred to RegA, which then activates genes involved in photosynthesis, nitrogen fixation, carbon fixation, respiration and electron transport. In the presence of oxygen, there is a second regulator known as CrtJ, which is responsible for repressing photosynthesis gene expression. CrtJ responds to redox by forming an intramolecular disulphide bond under oxidizing, but not reducing, growth conditions. The presence of the disulphide bond stimulates DNA binding activity of the repressor. There is also a flavoprotein that functions as a blue-light absorbing anti-repressor of CrtJ in the related bacterial species Rhodobacter sphaeroides called AppA. AppA exhibits a novel long-lived photocycle that is initiated by blue-light absorption by the flavin. Once excited, AppA binds to CrtJ thereby inhibiting the repressor activity of CrtJ. Various mechanistic aspects of this photocycle will be discussed.     

Putative novel photosynthetic reaction centre organizations in marine aerobic anoxygenic photosynthetic bacteria: insights from metagenomics and environmental genomics

Photosynthetic core complexes of anoxygenic bacteria consist of reaction centres (RCs) surrounded by light-harvesting complexes (LHC). The structural proteins of the RC-LHC1 complex are encoded by the puf-operon. We find diverse operon organizations of puf-operons that reflect structural differences of the core complex in marine aerobic anoxygenic photosynthetic bacteria (AAnP). By analysis of environmental DNA records coming from AAnP bacteria we find several unknown proteins downstream to the pufM, which were assigned as novel PufX proteins. As all known pufX genes belong to Rhodobacter strains which carry out anaerobic photosynthesis, this may be the first observation of a PufX-containing RCs in aerobic anoxygenic photosynthetic bacteria. Phylogenetic analyses of PufM proteins from cultured as well as from uncultured bacteria show that PufM from operons containing putative novel pufX genes are grouped with Rhodobacter and not with Roseobacter strains.     

Oxygen-regulated expression of genes for pigment binding proteins in Rhodobacter capsulatus

Oxygen is the major external factor affecting the expression of photosynthesis genes in facultatively photosynthetic bacteria. Many investigations over the last years mainly carried out on the closely related species Rhodobacter capsulatus and Rhodobacter sphaeroides have identified a number of proteins involved in the oxygen-regulated signal pathway, in which the RegB/RegA two component system plays a central role. While the RegB/RegA system activates photosynthesis genes under low oxygen tension other proteins like CrtJ and PPBP have a repressing function under high oxygen tension. Additional DNA binding proteins like the integration host factor can modulate the expression of photosynthesis genes. The role of alternative sigma factors in this signal pathway is still unclear.     

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.     

Genome organization and localization of the pufLM genes of the photosynthesis reaction center in phylogenetically diverse marine Alphaproteobacteria

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996(T) to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.     

Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3' of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sulfolipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.