The genetic basis of anoxygenic photosynthetic arsenite oxidation.

'Photoarsenotrophy', the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H2 S, H2 and NO2-. Photoarsenotrophy was recently identified from Paoha Island's (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases.

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

Description of Ectothiorhodospira salini sp. nov.

Strain JA430(T) is a Gram-negative, vibrioid to spiral shaped phototrophic purple sulfur bacterium isolated from anoxic sediment of a saltern at Kanyakumari in a mineral salts medium that contained 2% NaCl (w/v). Strain JA430(T) grows optimally at 5-6% NaCl and tolerates up to 12% NaCl. Intracellular photosynthetic membranes were of the lamellar type. Bacteriochlorophyll a and carotenoids of the spirilloxanthin series are present as photosynthetic pigments. Major cellular fatty acids are C(18:1)ω7c, C(16:0), C(19:0)cycloω8c and C(16:1)ω7c/C(16:1)ω6c. Strain JA430(T) exhibits photoorganoheterotrophy and chemoorganoheterotrophy and requires para-aminobenzoic acid, pantothenate and pyridoxal phosphate for growth. Phylogenetic analysis on the basis of 16S rRNA gene sequence analysis showed that strain JA430(T) forms monophyletic group in the genus Ectothiorhodospira. The highest sequence similarity for strain JA430(T) was found with the type strains of Ectothiorhodospira variabilis DSM 21381(T) (96.1%) and Ectothiorhodospira haloalkaliphila ATCC 51935(T) (96.2%). Morphological and physiological characteristics discriminate strain JA430(T) from other species of the genus Ectothiorhodospira, for which we describe this as a novel species, Ectothiorhodospira salini sp. nov. ( = NBRC 105915(T) = KCTC 5805(T)).

Ectothiorhodospira variabilis sp. nov., an alkaliphilic and halophilic purple sulfur bacterium from soda lakes.

During studies of moderately halophilic strains of Ectothiorhodospira from steppe soda lakes, we found a novel group of bacteria related to Ectothiorhodospira haloalkaliphila with salt optima at 50-80 g NaCl l(-1). Phylogenetic analysis using 16S rRNA gene sequences of strains from soda lakes in Mongolia, Egypt and Siberia revealed separation of the group of new isolates from other Ectothiorhodospira species, including the closely related Ect. haloalkaliphila. DNA-DNA hybridization studies demonstrated that the new isolates form a homogeneous group at the species level, but at the same time are distinct from related species such as Ect. haloalkaliphila, Ect. vacuolata, Ect. shaposhnikovii and Ect. marina. The new isolates are considered to be strains of a novel species, for which the name Ectothiorhodospira variabilis sp. nov. is proposed, with the type strain WN22(T) (=VKM B-2479(T) =DSM 21381(T)). Photosynthetic pigments of the novel species are bacteriochlorophyll a and carotenoids of the spirilloxanthin series with spirilloxanthin and derivatives thereof, together with small amounts of lycopene and rhodopin. Gas vesicles are formed by most of the strains, particularly in media containing yeast extract (0.5 g l(-1)) and acetate (0.5-2.0 g l(-1)). Sequence analysis of nifH (nitrogenase) and cbbL (RuBisCO) confirmed the assignment of the strains to the genus Ectothiorhodospira and in particular the close relationship to Ect. haloalkaliphila. The novel species Ect. variabilis is found in soda lakes separated by great geographical distances and is an alkaliphilic and halophilic bacterium that tolerates salt concentrations up to 150-200 g NaCl l(-1).

[An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups]. / Sistema oligonukleotidnykh praimerov dlia amplifikatsii genov ribulozo-1,5-bisfosfatkarboksilazy/oksigenazy u bakterii razlichnykh taksonomicheskikh grupp.

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.

Cloning a gene encoding a light-harvesting I polypeptide from Ectothiorhodospira sp.

Trying to detect the genes coding for light harvesting II polypeptides of the purple bacteria Ectothiorhodospira sp. a gene corresponding to a light harvesting I polypeptide was cloned. This paper discusses the probable reasons of this result. The sequence of this polypeptide underlines the strong similarity with light harvesting complexes from bacteria such as Rhodospirillum molischianum and Chromatium vinosum.

A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter.

Flavocytochrome c-sulfide dehydrogenase (FCSD), an enzyme that catalyzes the reversible conversion of sulfide to elemental sulfur in vitro, is common to bacteria that utilize reduced sulfur compounds as electron donors in the process of carbon dioxide fixation. FCSD is a heterodimer containing two different cofactors, a flavin (FAD) and one or two heme c groups, located on the separate protein subunits. Efforts to produce the holoproteins of the soluble Allochromatium vinosum FCSD and the membrane-bound Ectothiorhodospira vacuolata protein in Escherichia coli using several expression systems were unsuccessful. Although all systems used were able to export the recombinant FCSDs to the periplasm, the proteins did not incorporate heme. In order to develop a new expression system involving photosynthetic hosts (Rhodobacter capsulatus, Rhodobacter sphaeroides and Ect. vacuolata), plasmid mobilisation from E. coli donors was studied. In the search for efficient promoters for such hosts, a system was developed combining the broad-host-range plasmid pGV910 and the promoter of the A. vinosum RuBisCo gene, rbcA. Conjugation was used to enable transfer from the expression plasmid of E. coli into Rba. capsulatus, Rba. sphaeroides strains and into Ect. vacuolata. Both Rhodobacter hosts were able to transcribe the genes coding for FCSD from the rbcA promoter and to produce detectable amounts of recombinant FCSD holoprotein. Western blots showed that the best production was obtained from cells grown photosynthetically on malate or acetate with sulfide. This system may prove to be of general use for the production of recombinant c-type cytochromes in homologous or related host systems.

Improved osmotolerance of recombinant Escherichia coli by de novo glycine betaine biosynthesis.

The genes from the extreme halophile Ecto-thiorhodospira halochloris encoding the biosynthesis of glycine betaine from glycine were cloned into Escherichia coli. The accumulation of glycine betaine and its effect on osmotolerance of the cells were studied. In mineral medium with NaCl concentrations from 0.15 to 0.5 M, the accumulation of both endogenously synthesized and exogenously provided glycine betaine stimulated the growth of E. coli. The intracellular levels of glycine betaine and the cellular yields were clearly higher for cells receiving glycine betaine exogenously than for cells synthesizing it. The lower level of glycine betaine accumulation in cells synthesizing it is most likely a consequence of the limited availability of precursors (e.g. S-adenosylmethionine) rather than the result of a low expression level of the genes. Glycine betaine also stimulated the growth of E. coli and decreased acetate formation in mineral medium with high sucrose concentrations (up to 200 g.l(-1)).

Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase.

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.

A membrane-bound flavocytochrome c-sulfide dehydrogenase from the purple phototrophic sulfur bacterium Ectothiorhodospira vacuolata.

The amino acid sequence of Ectothiorhodospira vacuolata cytochrome c-552, isolated from membranes with n-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of Chlorobium limicola f. sp. thiosulfatophilum flavocytochrome c-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against Chromatium vinosum FCSD. When aligned according to the three-dimensional structure of Chromatium FCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (C. vinosum numbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of E. vacuolata FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.

Revision of species delineation in the genus Ectothiorhodospira.

When the type strains and other strains of the six currently defined species of the genus Ectothiorhodospira were examined by DNA-DNA reassociation and RFLP of 16S/23S rDNA (ribotype), only four genospecies could be found. The possibility of defining taxonomically meaningful species corresponding to these four genospecies was investigated by combining DNA relatedness and ribotype data with other genotypic and phenotypic characters already described in the literature, an approach known as polyphasic taxonomy. Following this comparison, the type strain and another strain of Ectothiorhodospira vacuolata were found to be very similar to the type strain of Ectothiorhodospira shaposhnikovii and have been transferred to this latter species. Also, the type strain of Ectothiorhodospira marismortui and another previously unidentified strain were found to be very similar to the type strain of Ectothiorhodospira mobilis and have been transferred to this latter species. Due to the limited degree of reciprocal DNA relatedness, strains belonging either to Ectothiorhodospira marina or to Ectothiorhodospira haloalkaliphila are still considered as belonging to separate species, even though they show a remarkable phenotypic similarity. This revision has led to the delineation of only four species in the genus Ectothiorhodospira, namely E. mobilis, E. shaposhnikovii, E. marina and E. haloalkaliphila. E. vacuolata is recognized as a junior synonym of E. shaposhnikovii and E. marismortui as a junior synonym of E. mobilis.