E. M. Spiridonova

An Oligonucleotide Primer System for Amplification of the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes of Bacteria of Various Taxonomic Groups

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 fragments of these genes about 800 bp long to be PCR-amplified for 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.

[Phylogeny of anoxygenic filamentous phototrophic bacteria of the family Oscillochloridaceae as inferred from comparative analyses of the rrs, cbbL, and nifH genes].

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed a lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the “red-like” form I RuBisCO were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated a lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their allocation as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.

Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum-Alkalilimnicola group

Four new isolates were obtained from denitrifying enrichments with various electron donors using sediment samples from hypersaline soda lakes. Based on 16S rRNA gene analysis and DNA-DNA hybridization results, they were all identified as members of the Gammaproteobacteria closely associated with the Alkalispirillum–Alkalilimnicola group. Two isolates were obtained from samples enriched with nitrate as electron acceptor and H2 or polysulfide as electron donors, and another two strains were obtained with N2O as the electron acceptor and sulfide or acetate as electron donors. All four new isolates, together with the type strains of the genera Alkalispirillum and Alkalilimnicola originally described as obligate aerobes, were capable of anaerobic growth with acetate using either nitrate or N2O as electron acceptors. Their denitrification pathway, however, was disrupted at the level of nitrite. RuBisCO form I gene was detected and sequenced in the new isolates and in Alkalilimnicola halodurans but not in Alkalispirillum mobile. These data, together with the evidence of Oremland et al. (Appl Environ Microbiol 68:4795–4802, 2002) on the potential of Alkalilimnicola sp. MLHE-1 for autotrophic growth with arsenite as electron donor and nitrate as electron acceptor, demonstrate much higher metabolic diversity of this specific group of haloalkaliphilic Gammaproteobacteria than was originally anticipated.

Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, cbbL, and pmoA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine bivalve Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of thiotrophic mytilid’s symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.

Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khito-Gol hydrogen-sulfide springs, Buryatia

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8–9.5, with an optimum at pH 9.3 and in a temperature range of 5–39°C, with an optimum at 28–32°C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3–64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505T (= ATCC 8158T). Despite the phenotypic similarity, the hybridization level was as low as 21–29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO2 assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HGT (= VKM B-2365T).

Isolation and characterization of nitrogen-fixing bacteria of the genus Azospirillum from the soil of a Sphagnum peat bog

Abstracthe presence of nitrogen-fixing bacteria of the genus Azospirillum in the soils of acidic raised Sphagnum bogs is revealed for the first time. Three Azospirillum strains, B2, B21, and B22, were isolated as a component of methane-oxidizing enrichment cultures, whereas attempts to isolate them directly from peat samples have failed. The results of comparative analysis of the nucleotide sequences of 16S rRNA genes, DNA-DNA hybridization, and the analysis of the sequences of the functional genes encoding nitrogenase and ribulose-1, 5-bisphosphate carboxylase reveal that all the newly obtained strains can be classified as Azospirillum lipoferum. Yet, unlike A. lipoferum, the isolates do not require biotin and utilize sucrose, inositol, and glycerol for growth. The cell morphology of strain B2 differs from that of the type strain and strains B21 and B22. The results obtained indicate the variability of morphological, physiological, and biochemical properties in closely related Azospirillum strains and suggest the existence of metabolic relationships between methanotrophic bacteria and the representatives of the genus Azospirillum under peat bog conditions.

[Phylogeny of ribulose-1,5-bisphosphate carboxylase/oxygenase genes in haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio].

Fragments of genes of the “green-like” form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of eight species of haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio have been revealed and sequenced using previously developed oligonucleotide primers. The data obtained are used for the construction of phylogenetic trees on the basis of nucleotide sequences of RuBisCO genes and their conceptual translations into amino acid sequences. Comparative analysis of the 16S rRNA and RuBisCO gene trees reveals discrepancies between their topologies. According to a RuBisCO gene analysis, the genus Thioalkalivibrio is not monophyletic, and its inner divergence conforms to the significant morphological differences observed between the species. Presumably, horizontal (interspecies) gene transfer was involved in the evolution of the genus Thioalkalivibrio.