Use of Purple Non-Sulfur Photosynthetic Bacteria (Rhodobacter sphaeroides) in Promoting Ciliated Protozoa Growth.

Photosynthetic bacterium (PSB) was isolated from sediment samples of Yamagawa Bay, Kagoshima, Japan. Phylogenetic analysis results of PSB isolate were closely related to Rhodobacter sphaeroides, purple non-sulfur photosynthetic bacteria (PNSB). Pink-colored smooth edges of single bacterial colonies were observed after 3-5 days of incubation period on Basic I medium agar plates. Rhodobacter sphaeroides microscopic examination showed a short rod cell (1-2 µm length) with round ends. Sediment and water samples used for ciliates cultivation were collected from Kuwano-ura Bay, Koshiki Island, Japan. Ciliates were cultivated using fish meal with radish leaves medium (MI), with sediment into MI (MII) and algae media (MIII). The use of the algae media (MIII) in cultivation mixture produced the highest total number of ciliates. Big size ciliates were identified as Euplotes minuta and Cyclidium varibonneti, while small size was identified as Micrometopion nutans, based on PCR-DGGE. When ciliates were cultured with the PSB isolate, Rhodobacter sphaeroides as a feed, ciliates grow to 2,081 individual ml-1 72 hrs later. These findings indicate that PNSB can be used to promote ciliates growth.

Shedding light on a Group IV (ECF11) alternative σ factor.

This year marks the 50th anniversary of the discovery of σ70 as a protein factor that was needed for bacterial RNA polymerase to accurately transcribe a promoter in vitro. It was 25 years later that the Group IV alternative σs were described as a distinct family of proteins related to σ70 . In the intervening time, there has been an ever-growing list of Group IV σs, numbers of genes they transcribe, insight into the diverse suite of processes they control, and appreciation for their impact on bacterial lifestyles. This work summarizes knowledge of the Rhodobacter sphaeroides σE -ChrR pair, a member of the ECF11 subfamily of Group IV alternative σs, in protecting cells from the reactive oxygen species, singlet oxygen. It describes lessons learned from analyzing ChrR, a zinc-dependent anti-σ factor, that are generally applicable to Group IV σs and relevant to the response to single oxygen. This MicroReview also illustrates insights into stress responses in this and other bacteria that have been acquired by analyzing or modeling the activity of the σE -ChrR across the bacterial phylogeny.

Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.

Evolutionary origin of the Rhodobacter sphaeroides specialized RpoN sigma factors.

Gene duplication and horizontal gene transfer (HGT) are two events that enable the generation of new genes. Rhodobacter sphaeroides (WS8 and 2.4.1 strains) has four copies of the rpoN gene that are not functionally interchangeable. Until now, this is the only example of specialization of this sigma factor. In this work, we aimed to determine whether the multiple copies of this gene originated from HGT or through gene duplication. Our results suggest a multiplication origin of the different rpoN copies that occurred after the Rhodobacter clade separated. Functional tests indicate that the specialization of the rpoN genes is not restricted to R. sphaeroides. We propose that the rpoN copy involved in nitrogen fixation is the ancestral gene and that the other rpoN genes have acquired new specificities.

Organization and evolution of the biological response to singlet oxygen stress.

The appearance of atmospheric oxygen from photosynthetic activity led to the evolution of aerobic respiration and responses to the resulting reactive oxygen species. In Rhodobacter sphaeroides, a photosynthetic alpha-proteobacterium, a transcriptional response to the reactive oxygen species singlet oxygen ((1)O(2)) is controlled by the group IV sigma factor sigma(E) and the anti-sigma factor ChrR. In this study, we integrated various large datasets to identify genes within the (1)O(2) stress response that contain sigma(E)-dependent promoters both within R. sphaeroides and across the bacterial phylogeny. Transcript pattern clustering and a sigma(E)-binding sequence model were used to predict candidate promoters that respond to (1)O(2) stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides sigma(E)-dependent promoters that control the transcription of 15 (1)O(2)-activated genes. Knowledge of the R. sphaeroides response to (1)O(2) and its regulator sigma(E)-ChrR was combined with large-scale phylogenetic and sequence analyses to predict the existence of a core set of approximately eight conserved sigma(E)-dependent genes in alpha-proteobacteria and gamma-proteobacteria. The bacteria predicted to contain this conserved response to (1)O(2) include photosynthetic species, as well as free-living and symbiotic/pathogenic nonphotosynthetic species. Our analysis also predicts that the response to (1)O(2) evolved within the time frame of the accumulation of atmospheric molecular oxygen on this planet.

Postgenomic adventures with Rhodobacter sphaeroides.

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.

Identification of two gene loci involved in poly-beta-hydroxybutyrate production in Rhodobacter sphaeroides FJ1.

BACKGROUND AND PURPOSE: Polyhydroxyalkanoates (PHAs), biopolyesters of hydroxyl fatty acids, are synthesized and deposited as cytoplasmic inclusions in many bacteria. We isolated a poly-beta-hydroxybutyrate (PHB)-producing bacterium designated Rhodobacter sphaeroides FJ1. To characterize PHB biosynthesis in this organism, we isolated the genes encoding proteins involved in PHB metabolism. METHODS: The genes responsible for the synthesis, accumulation, and degradation of PHB in R. sphaeroides FJ1 were cloned and characterized. RESULTS: Genes involved in the biosynthesis and metabolism of PHB were found to be located in 2 different loci in the genome of R. sphaeroides FJ1. One locus contained genes encoding PHB depolymerase (phbZ), PHB synthase (phbC), phasin (phbP) and the regulator protein (phbR). The other locus contained the beta-ketothiolase gene (phbA) and the acetoactyl-CoA reductase gene (phbB). The phbZ gene was orientated in an opposite direction to that of phbC, phbP and phbR genes that were located in the same cluster. R. sphaeroides FJ1 was able to grow in wastewater released from the human waste treatment plant of Fu-Jen University. Optimal growth and PHB production were achieved when R. sphaeroides FJ1 was grown in tryptic soy broth containing 50% wastewater. PHB production by R. sphaeroides FJ1 varied in media with different carbon to nitrogen ratios, but the level of PHB synthase was constant, suggesting that PHB production depends mainly on substrate supply. CONCLUSIONS: Six genes encoding proteins related to PHB metabolism are clustered in 2 separate loci, phbZCPR and phbAB, in a PHB-producing bacterium R. sphaeroides FJ1 isolated from wastewater. PHB synthase, the key enzyme for PHB synthesis, is constitutively expressed, and its expression level is not affected by different growth conditions.

Isolation and characterization of heterotrophic bacteria able to grow aerobically with quaternary ammonium alcohols as sole source of carbon and nitrogen.

The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon and nitrogen. The strains were compared with a previously isolated TM-degrading bacterium, which was identified as a representative of the species Pseudomonas putida (Syst. Appl. Microbiol. 24 (2001) 252). Two bacteria were isolated with DM, referred to as strains DM 1 and DM 2, respectively. Based on 16S-rDNA analysis, they provided 97% (DM 1) and 98% (DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is typical for representatives of the genus Zoogloea. Using MM we isolated a Gram-negative, non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium revealed 94% identities (best match) to Rhodobacter sphaeroides only. The strains MM 1 and DM 1 exclusively grew with the QAA which was used for their isolation. DM 2 was also utilizing TM as sole source of carbon and nitrogen. However, all of the isolated bacteria were growing with the natural and structurally related compound choline.

Phototrophic utilization of taurine by the purple nonsulfur bacteria Rhodopseudomonas palustris and Rhodobacter sphaeroides.

Taurine metabolism by two phototrophically grown purple nonsulfur bacteria enrichment isolates has been examined. Rhodopseudomonas palustris (strain Tau1) grows with taurine as a sole electron donor, sulfur and nitrogen source during photoautotrophic growth. Rhodobacter sphaeroides (strain Tau3) grows on the compound as sole electron donor, sulfur and nitrogen source, and partial carbon source, in the presence of CO(2) during photoheterotrophic growth. Both organisms utilize an inducible taurine-pyruvate aminotransferase and a sulfoacetaldehyde acetyltransferase. The products of this metabolism are bisulfite and acetyl phosphate. Bisulfite ultimately was oxidized to sulfate, but this was not an adequate source of electrons for photometabolism. Experiments using either [U-(14)C]taurine or (14)CO(2) demonstrated that Rb. sphaeroides Tau3 assimilated the carbon from approximately equimolar amounts of taurine and exogenous CO(2). The taurine-carbon assimilation was not diminished by excess non-radioactive bicarbonate. Malate synthase (but not isocitrate lyase) was induced in these taurine-grown cells. It is concluded that assimilation of taurine carbon occurs through an intermediate other than CO(2). Similar labelling experiments with Rp. palustris Tau1 determined that taurine is utilized only as an electron donor for the reduction of CO(2), which contributes all the cell carbon. Photoautotrophic metabolism was confirmed in this organism by the absence of either malate synthase or isocitrate lyase in taurine+CO(2)-grown cells. Culture collection strains of these two bacteria did not utilize taurine in these fashions.

Farnesyl diphosphate synthase gene of three phototrophic bacteria and its use as a phylogenetic marker.

Farnesyl diphosphate (FPP) synthase is essential not only for phototrophic bacteria in carotenoid biosynthesis, but also for non-phototrophic bacteria in the biosynthesis of physiologically important compounds. The gene encoding FPP synthase was assessed as a molecular marker to investigate the intermingled relationship between the phototropic and non-phototropic bacteria in the alpha-Proteobacteria based on 16S rRNA analysis. The FPP synthase amino acid sequences from three phototropic bacteria, Rhodobacter sphaeroides ATCC 11167(T), Rhodobacter capsulatus ATCC 11166(T) and Rhodovulum sulfidophilum W4(T), were determined and used in conjunction with sequences of other representative members of the alpha-, gamma- and epsilon-Proteobacteria and the low-G+C Gram-positive bacteria for phylogenetic analyses by the neighbour-joining and maximum-likelihood methods. The overall topology of the FPP synthase gene tree is consistent with that of the 16S rRNA tree, producing a distinct cluster of the three phototropic bacteria. A minor discordance between the two trees was observed in the cluster of the non-phototrophic Bradyrhizobiumjaponicum USDA 110 and Mesorhizobium loti MAFF 303099; the FPP synthase genes of these two rhizobial species are highly homologous as compared with their respective 16S rRNA. The results suggest that the FPP synthase and 16S rRNA genes have the same evolutionary pattern, evolving vertically from each common ancestral gene; the FPP synthase gene, therefore, could possibly be used for further study on the molecular systematics of photosynthetic bacteria.

Aeration conditions affecting growth of purple nonsulfur bacteria in an organic wastewater treatment process.

Effects of aeration on purple nonsulfur bacteria (PnSB) were studied in photobioreactors. Bacterial community changes were analyzed by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). DGGE band pattern change was small and only few prominent bands were obtained at non-aeration condition. Sequencing results of the prominent DGGE bands obtained at this condition revealed that they represented mainly the PnSB, Rhodobacter sphaeroides and Rhodopseudomonas palustris. On the other hand, under aerated condition, some prominent bands originated from heterotrophs appeared but no proliferation of PnSB was observed. FISH was applied to detect PnSB and their population was quantified. Maximum PnSB ratio (up to 80%) was obtained both at non-aeration condition and at constant ORPs less than -200 mV. In the presence of DO, Rps. palustris was more competitive to chemoheterotrophs than Rb. sphaeroides.

[Generic interrelations of purple bacteria in the genus Rhodopseudomonas]. / Rodstvennye vzaimootnosheniia pupurnykh bakterii roda Rhodopseudomonas.

The technique of DNA--DNA hybridization was used to study relations offween purple nonsulfur bacteria (the family Rhodospirillaceae). The level of homologies with Rhodopseudomonas sphaeroides 8259 was nearly the same for different species (8-17%) in the genus Rhodopseudomonas under the conditions optimal for hybridization. The same level of homologies was found for the DNA of Rhodospirillum rubrum, a species belonging to another genus of purple nonsulfur bacteria (13%). Rhodomicrobium vannielli was most remote from R. sphaeroides 8259 (3%). Similar results were obtained under other conditions of hybridization. The intraspecial heterogeneity of R. sphaeroides was studied in this work. The thermal stability of hybrid duplexes was analysed. The results are indicative of a considerable divergence of different R. sphaeroides strains (delta T50 = 2.1-11.6).

Dna-Dna hybridization of Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains.

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.

Rhodopseudomonas sphaeroides forma sp. denitrificans, a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides.

From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.