[Phylogenetic diversity of bacteria in soda lake stratified sediments].

Various previously developed techniques for DNA extraction from the samples with complex physicochemical structure (soils, silts, and sediments) and modifications of these techniques developed in the present work were tested. Their usability for DNA extraction from the sediments of the Kulunda Steppe hypersaline soda lakes was assessed, and the most efficient procedure for indirect (two-stage) DNA extraction was proposed. Almost complete separation of the cell fraction was shown, as well as the inefficiency of nested PCR for analysis of the clone libraries obtained from washed sediments by amplification of the 16S rRNA gene fragments. Analysis of the clone library obtained from the cell fractions of stratified sediments (upper, medium, and lower layers) revealed that in the sediments of Lake Gorchina-3 most eubacterial phylotypes belonged to the class Clostridia, phylum Firmicutes. They were probably specific for this habitatand formed a new, presently unknown high-rank taxon. The data obtained revealed no pronounced stratification of the spe- cies diversity of the eubacterial component of the microbial community inhabiting the sediments (0-20 cm) in the inshore zone of Lake Gorchina-3.

Utilization of arylaliphatic nitriles by haloalkaliphilic Halomonas nitrilicus sp. nov. isolated from soda soils.

An enrichment culture from saline soda soils, using acetate as carbon and energy source and 2-phenylpropionitrile as nitrogen source (PPN) at pH 10, resulted in the isolation of strain ANL-alpha CH3. The strain was identified as a representative of the genus Halomonas in the Gammaproteobacteria. The bacterium was capable of PPN utilization as a nitrogen source only, while phenylacetonitrile (PAN) served both as carbon, energy and nitrogen source. This capacity was not described previously for any other haloalkaliphilic bacteria. Apart from the nitriles mentioned above, resting cells of ANL-alpha CH3 also hydrolyzed mandelonitrile, benzonitrile, acrylonitrile, and phenylglycinonitrile, presumably using nitrilase pathway. Neither nitrile hydratase nor amidase activity was detected. The isolate showed a capacity to grow with benzoate and salicylate as carbon and energy source and demonstrated the ability to completely mineralize PAN. These clearly indicated a potential to catabolize aromatic compounds. On the basis of unique phenotype and distinct phylogeny, strain ANL-alpha CH3 is proposed as a novel species of the genus Halomonas--Halomonas nitrilicus sp. nov.

Dethiobacter alkaliphilus gen. nov. sp. nov., and Desulfurivibrio alkaliphilus gen. nov. sp. nov.: two novel representatives of reductive sulfur cycle from soda lakes.

Anaerobic enrichments with H2 as electron donor and thiosulfate/polysulfide as electron acceptor at pH 10 and 0.6 M total Na+ yielded two non sulfate-reducing representatives of reductive sulfur cycle from soda lake sediments. Strain AHT 1 was isolated with thiosulfate as the electron acceptor from north-eastern Mongolian soda lakes and strain AHT 2-with polysulfide as the electron acceptor from Wadi al Natrun lakes in Egypt. Both isolates represented new phylogenetic lineages: AHT 1-within Clostridiales and AHT 2-within the Deltaproteobacteria. Both bacteria are obligate anaerobes with respiratory metabolism. Both grew chemolithoautotrophically with H2 as the electron donor and can use thiosulfate, elemental sulfur and polysulfide as the electron acceptors. AHT 2 also used nitrate as acceptor, reducing it to ammonia. During thiosulfate reduction, AHT 1 excreted sulfite. dsrAB gene was not found in either strain. Both strains were moderate salt-tolerant (grow up to 2 M total Na+) true alkaliphiles (grow between pH 8.5 and 10.3). On the basis of the phenotypic and phylogenetic data, strains AHT 1 and AHT 2 are proposed as new genera and species Dethiobacter alkaliphilus and Desulfurivibrio alkaliphilus, respectively.

Desulfurispirillum alkaliphilum gen. nov. sp. nov., a novel obligately anaerobic sulfur- and dissimilatory nitrate-reducing bacterium from a full-scale sulfide-removing bioreactor.

Strain SR 1(T)was isolated under anaerobic conditions using elemental sulfur as electron acceptor and acetate as carbon and energy source from the Thiopaq bioreactor in Eerbeek (The Netherlands), which is removing H(2)S from biogas by oxidation to elemental sulfur under oxygen-limiting and moderately haloalkaline conditions. The bacterium is obligately anaerobic, using elemental sulfur, nitrate and fumarate as electron acceptors. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while nitrate is dissimilatory reduced to ammonium. Furthermore, in the presence of nitrate, strain SR 1(T) was able to oxidize limited amounts of sulfide to elemental sulfur during anaerobic growth with acetate. The new isolate is mesophilic and belongs to moderate haloalkaliphiles, with a pH range for growth (on acetate and nitrate) from 7.5 to 10.25 (optimum 9.0), and a salt range from 0.1 to 2.5 M Na(+) (optimum 0.4 M). According to phylogenetic analysis, SR 1(T) is a member of a deep bacterial lineage, distantly related to Chrysiogenes arsenatis (Macy et al. 1996). On the basis of the phenotypic and genetic data, the novel isolate is placed into a new genus and species, Desulfurispirillum alkaliphilum (type strain SR(T)= DSM 18275 = UNIQEM U250).

Extremely halophilic denitrifying bacteria from hypersaline inland lakes, Halovibrio denitrificans sp. nov. and Halospina denitrificans gen. nov., sp. nov., and evidence that the genus name Halovibrio Fendrich 1989 with the type species Halovibrio variabilis should be associated with DSM 3050.

Anaerobic enrichments with acetate as electron donor and nitrate as electron acceptor at 4 M NaCl from inland, hypersaline lake sediments from Central Asia resulted in the isolation of several extremely halophilic bacteria that comprised two subgroups, most with vibrio-shaped cells and a single strain with rod-shaped cells. Members of both subgroups were extremely halophilic, with growth occurring in 2-5 M NaCl with an optimum at 2-3 M. 16S rRNA gene sequence analysis showed a close affiliation of the new isolates with Pseudomonas halophila DSM 3050 in the Gammaproteobacteria. However, phenotypic comparison of the denitrifying halophiles with the original description of P. halophila demonstrated that they were more similar to another bacterium isolated from the same source at the same time, the extremely halophilic Halovibrio variabilis, which has since been reclassified as Halomonas variabilis (DSM 3051). Direct cross-comparison showed that the characteristics of these two halophilic bacteria do not correspond with the original descriptions associated with these names and DSM numbers. While it is desirable that this problem be solved, in connection with the present investigations, this is a matter that can only be solved by a Request for an Opinion. On the basis of the phenotypic and genetic comparison of these isolates, it is proposed that the new denitrifying vibrio-shaped isolates represent a novel species, Halovibrio denitrificans sp. nov. (type strain HGD 3T=DSM 15503T=UNIQEM U232T) and that the rod-shaped isolate represents a novel genus and species, Halospina denitrificans gen. nov., sp. nov. (type strain HGD 1-3T=DSM 15505T=UNIQEM U233T).

The status of the genus name Halovibrio Fendrich 1989 and the identity of the strains Pseudomonas halophila DSM 3050 and Halomonas variabilis DSM 3051. Request for an opinion.

During the course of studies on halophilic, Gram-negative bacteria, a comparison of two strains of halophilic bacteria, Pseudomonas halophila DSM 3050 and Halomonas variabilis DSM 3051 (formerly Halovibrio variabilis) demonstrated that the characteristics of strain DSM 3050 corresponded to the original description of Halovibrio variabilis and those of DSM 3051 to P. halophila, both of which had been isolated from the Great Salt Lake in Utah [Fendrich, C. (1988). Syst Appl Microbiol 11, 36-43]. It was concluded that these two strains did not correspond with their original descriptions, e.g. the original description of Halovibrio variabilis matched that of Pseudomonas halophila DSM 3050 and the original description of Pseudomonas halophila matched that of Halovibrio variabilis DSM 3051, which was subsequently transferred to the genus Halomonas as Halomonas variabilis DSM 3051. These findings raise the question of whether the genus name Halovibrio, with the type species Halovibrio variabilis, should be associated with type strain DSM 3050 and recognition of strain DSM 3051 as the type strain of Pseudomonas halophila.

Citreicella thiooxidans gen. nov., sp. nov., a novel lithoheterotrophic sulfur-oxidizing bacterium from the Black Sea.

Four strains of obligately heterotrophic bacteria isolated from the oxygen-sulfide interface of the Black Sea are characterized. The bacteria are aerobic, Gram-negative, with lemon-like, nonmotile cells. Bacteriochlorophyll a is not detected. They are mesophilic and neutrophilic with a temperature range of 8-35 degrees C (optimum 25) and pH range of 6.5-8.5 (optimum 7.8). Their growth is NaCl dependent within a range of 5 and 60 (optimum 20) g l(-1). They are able to oxidize thiosulfate, sulfide and elemental sulfur to sulfate and to use metabolic energy from these reactions (lithoheterotrophy). According to the level of DNA reassociation of more than 40%, all isolates represent a single generic group. The G+C content of the DNA was in the range of 67.5-69.2mol%. According to phylogenetic analysis, the new isolates form a separate branch in the alpha-3 subdivision of the Proteobacteria together with two undescribed marine bacterial strains. On the basis of phenotypical and genomic properties, the new isolates are described as a new genus and species Citreicella thiooxidans gen. nov., sp. nov. The type strain is CHLG 1T ( = DSM 10146, UNIQEM U 228).

Characterization of molybdenum-free nitrate reductase from haloalkalophilic bacterium Halomonas sp. strain AGJ 1-3.

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.

Ion transport coupled to terminal oxidase functioning in the extremely alkaliphilic halotolerant bacterium Thioalkalivibrio.

Proton transport in the terminal part of the respiratory chain in the extremely alkaliphilic halotolerant bacterial strain Thioalkalivibrio versutus was studied under near-optimum growth conditions (pH 9.0-9.5). Under these conditions, bacterial cells generated electric potential with the negative charge being inside the cells. When only the terminal part of the respiratory chain functioned, it was found that: 1) unlike other bacteria known, this bacterium did not acidify the medium in the presence of K(+) and valinomycin; 2) in the presence of an uncoupler, CCCP, but in the absence of valinomycin, reversible alkalinization of the medium occurred as a result of proton influx into the cells. Cyanide prevented this alkalinization. The difference spectra indicate that cell membranes contained cytochromes c and (b+o), some of which reacted with CO. The respiratory activity of membranes in the terminal part of the respiratory chain was optimal at pH 9.5 and specifically depended on sodium ions (C(1/2) = 10 mM). The data suggest the presence of a Na(+)-pump in the terminal part of the respiratory chain of the studied strain which can pump Na(+) out of the cells.