Salinity-induced succession of phototrophic communities in a southwestern Siberian soda lake during the solar activity cycle.

A variety of lakes located in the dry steppe area of southwestern Siberia are exposed to rapid climatic changes, including intra-century cycles with alternating dry and wet phases driven by solar activity. As a result, the salt lakes of that region experience significant fluctuations in water level and salinity, which have an essential impact on the indigenous microbial communities. But there are few microbiological studies that have analyzed this impact, despite its importance for understanding the functioning of regional water ecosystems. This work is a retrospective study aimed at analyzing how solar activity-related changes in hydrological regime affect phototrophic microbial communities using the example of the shallow soda lake Tanatar VI, located in the Kulunda steppe (Altai Region, Russia, southwestern Siberia). The main approach used in this study was the comparison of hydrochemical and microscopic data obtained during annual field work with satellite and solar activity data for the 12-year observation period (2011-2022). The occurrence of 33 morphotypes of cyanobacteria, two key morphotypes of chlorophytes, and four morphotypes of anoxygenic phototrophic bacteria was analyzed due to their easily recognizable morphology. During the study period, the lake surface changed threefold and the salinity changed by more than an order of magnitude, which strongly correlated with the phases of the solar activity cycles. The periods of high (2011-2014; 100-250 g/L), medium (2015-2016; 60 g/L), extremely low (2017-2020; 13-16 g/L), and low (2021-2022; 23-34 g/L) salinity with unique biodiversity of phototrophic communities were distinguished. This study shows that solar activity cycles determine the dynamics of the total salinity of a southwestern Siberian soda lake, which in turn determines the communities and microorganisms that will occur in the lake, ultimately leading to cyclical changes in alternative states of the ecosystem (dynamic stability).

Desulfonatronobacter acidivorans gen. nov., sp. nov. and Desulfobulbus alkaliphilus sp. nov., haloalkaliphilic heterotrophic sulfate-reducing bacteria from soda lakes.

Two types of heterotrophic sulfate-reducing bacteria (SRB) were isolated from anoxic sediments of hypersaline soda lakes in Kulunda Steppe (Altai, Russia). The isolates used propionate as an energy and carbon source. Strain APT2(T) was enriched and isolated with thiosulfate as the electron acceptor. Strains APS1(T) and ASS1 were isolated with sulfate. Strain APT2(T) was a short rod and motile with a single subpolar flagellum, while strains APS1(T) and ASS1 were lemon-shaped oval rods and motile with a single polar flagellum and thin flagella-like filaments. Strain APT2(T) grew by complete oxidation of C(3)-C(8) fatty acids with thiosulfate or sulfate as the electron acceptor, while strains APS1(T) and ASS1 were much less versatile and utilized only propionate and pyruvate as the electron donor and carbon source with sulfate or sulfite as the electron acceptor. Furthermore, strains APS1(T) and ASS1 oxidized propionate incompletely to form acetate. All of the isolates were moderately halophilic and obligately alkaliphilic. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolates in the order Desulfobacterales of the class Deltaproteobacteria. Strain APT2(T) belonged to the family Desulfobacteraceae and clustered with a halophilic SRB, Desulfosalsimonas propionicica PropA(T). Strains APS1(T) and ASS1 were closely related to each other and clustered with the genus Desulfobulbus of the family Desulfobulbaceae. On the basis of phenotypic and phylogenetic analysis, the isolates are proposed to represent two novel taxa, Desulfonatronobacter acidivorans gen. nov., sp. nov. (type strain of the type species APT2(T) = DSM 24257(T) = UNIQEM U853(T)) and Desulfobulbus alkaliphilus sp. nov. (type strain APS1(T) = DSM 24258(T) = UNIQEM U900(T)).

Complete genome sequence of "Thioalkalivibrio sulfidophilus" HL-EbGr7.

"Thioalkalivibrio sulfidophilus" HL-EbGr7 is an obligately chemolithoautotrophic, haloalkaliphilic sulfur-oxidizing bacterium (SOB) belonging to the Gammaproteobacteria. The strain was found to predominate a full-scale bioreactor, removing sulfide from biogas. Here we report the complete genome sequence of strain HL-EbGr7 and its annotation. The genome was sequenced within the Joint Genome Institute Community Sequencing Program, because of its relevance to the sustainable removal of sulfide from bio- and industrial waste gases.

Plasticicumulans acidivorans gen. nov., sp. nov., a polyhydroxyalkanoate-accumulating gammaproteobacterium from a sequencing-batch bioreactor.

Here, we describe a novel bacterium, strain TUD-YJ37(T), which can accumulate polyhydroxybutyrate (PHB) to more than 85 % (w/w) dry cell weight. The bacterium was isolated from a mixed-culture bioreactor by using a feast-famine regime and its properties were characterized. Phylogenetic analysis based on full 16S rRNA gene sequences revealed that the isolate is a member of the Gammaproteobacteria, forming an independent, deep phylogenetic lineage. It is most closely related to members of the genera Methylocaldum, Methylococcus and Natronocella, with sequence similarities below 91 %. Strain TUD-YJ37(T) was an obligately aerobic, ovoid, Gram-negative bacterium, motile by means of a polar flagellum. It utilized C2-C10 fatty acids as carbon and energy sources. The temperature range for growth was 20-35 °C, with an optimum of 30 °C; the pH range was 6.0-8.0, without a clear optimum. The major respiratory quinone was Q-8. Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids, an unidentified aminolipid and another unidentified lipid. The predominant fatty acids in the membrane polar lipids were C16:1ω7c, C16:0 and C18:1ω7c. The G+C content of the genomic DNA was 67.4 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, the isolate is proposed to represent a novel genus and species, for which the name Plasticicumulans acidivorans gen. nov., sp. nov. is proposed. The type strain of Plasticicumulans acidivorans is TUD-YJ37(T) ( = DSM 23606(T)  = CBS 122990(T)).

Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats.

The presence and diversity of the cbb genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (a key enzyme of the Calvin-Benson cycle of autotrophic CO(2) assimilation) were investigated in pure cultures of seven genera of halophilic chemolithoautotrophic sulfur-oxidizing bacteria (SOB) and in sediments from a hypersaline lake in which such bacteria have been recently discovered. All of the halophilic SOB strains (with the exception of Thiohalomonas nitratireducens) possessed the cbbL gene encoding RuBisCO form I, while the cbbM gene encoding RuBisCO form II was detected only in some of the pure cultures. The general topologies of the CbbL/CbbM trees and the 16S rRNA gene tree were different, but both markers showed that the halophilic SOB genera formed independent lineages in the Gammaproteobacteria. In some cases, such as with several strains of the genus Thiohalospira and with Thioalkalibacter halophilus, the cbbL clustering was incongruent with the positions of these strains on the ribosomal tree. In the cbbM tree, the clustering of Thiohalospira and Thiohalorhabdus strains was incongruent with their branching in both cbbL and 16S rRNA gene trees. cbbL and cbbM genes related to those found in the analysed halophilic SOB were also detected in a sediment from a hypersaline lake in Kulunda Steppe (Russia). Most of the cbbL and cbbM genes belonged to members of the genus Thiohalorhabdus. In the cbbL clone library, sequences related to those of Halothiobacillus and Thiohalospira were detected as minor components. Some of the environmental cbbM sequences belonged to as yet unknown phylotypes, representing deep lineages of halophilic autotrophs.

Aerobic carboxydotrophy under extremely haloalkaline conditions in Alkalispirillum/Alkalilimnicola strains isolated from soda lakes.

Aerobic enrichments from soda lake sediments with CO as the only substrate resulted in the isolation of five bacterial strains capable of autotrophic growth with CO at extremely high pH and salinity. The strains belonged to the Alkalispirillum/Alkalilimnicola cluster in the Gammaproteobacteria, where the ability to oxidize CO, but not growth with CO, has been demonstrated previously. The growth with CO was possible only at an oxygen concentration below 5 % and CO concentration below 20 % in the gas phase. The isolates were also capable of growth with formate but not with H(2). The carboxydotrophic growth occurred within a narrow pH range from 8 to 10.5 (optimum at 9.5) and a broad salt concentration from 0.3 to 3.5 M total Na(+) (optimum at 1.0 M). Cells grown on CO had high respiration activity with CO and formate, while the cells grown on formate actively oxidized formate alone. In CO-grown cells, CO-dehydrogenase (CODH) activity was detectable both in soluble and membrane fractions, while the NAD-independent formate dehydrogenase (FDH) resided solely in membranes. The results of total protein profiling and the failure to detect CODH with conventional primers for the coxL gene indicated that the CO-oxidizing enzyme in haloalkaliphilic isolates might differ from the classical aerobic CODH complex. A single cbbL gene encoding the RuBisCO large subunit was detected in all strains, suggesting the presence of the Calvin cycle of inorganic carbon fixation. Overall, these results demonstrated the possibility of aerobic carboxydotrophy under extremely haloalkaline conditions.

Thiohalobacter thiocyanaticus gen. nov., sp. nov., a moderately halophilic, sulfur-oxidizing gammaproteobacterium from hypersaline lakes, that utilizes thiocyanate.

A moderately halophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain HRh1(T), was obtained from mixed sediment samples from hypersaline chloride-sulfate lakes in the Kulunda Steppe, in south-western Siberia (Russia), using aerobic enrichment culture at 1 M NaCl with thiocyanate as substrate. Cells of the isolate were short, non-motile rods with a Gram-negative type of cell wall. The bacterium was an obligate aerobe capable of chemolithoautotrophic growth with thiocyanate and thiosulfate. With thiosulfate, it grew at NaCl concentrations of 0.2-3.0 M (optimum 0.5 M) and at pH 6.3-8.0 (optimum pH 7.3-7.5). During growth on thiocyanate, cyanate was identified as an intermediate. The dominant cellular fatty acids were C(16 : 0) and C(18 : 1)omega7. Phylogenetic analysis based on 16S rRNA gene sequencing placed the isolate in the class Gammaproteobacteria as an independent lineage, with an unclassified marine sulfur-oxidizing bacterium as the closest culturable relative (93 % sequence similarity). A single cbbL gene (coding for the key enzyme of the Calvin-Benson cycle of autotrophic CO(2) assimilation) with relatively low similarity to any homologous genes found in chemolithoautotrophs was detected in strain HRh1(T). On the basis of our phenotypic and phylogenetic analysis, the halophilic isolate is proposed to represent a new genus and novel species, Thiohalobacter thiocyanaticus gen. nov., sp. nov. The type strain of Thiohalobacter thiocyanaticus is HRh1(T) (=DSM 21152(T) =UNIQEM U249(T)).

Improved method for direct screening of true lipase-producing microorganisms with particular emphasis on alkaline conditions.

A rapid and effective method for direct detection, selection and testing of microorganisms able to produce both cell-bound and extracellular true lipases is described. The method is based on formation of clearance zones on turbid solid media with emulsified olive oil around or under the colonies, cell fractions or culture supernatant of lipase-producing organisms. The method was successfully applied for the screening and isolation of microorganisms producing alkaline lipases.

Nitriliruptor alkaliphilus gen. nov., sp. nov., a deep-lineage haloalkaliphilic actinobacterium from soda lakes capable of growth on aliphatic nitriles, and proposal of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov.

A novel bacterial strain, designated ANL-iso2(T), was obtained from an enrichment culture inoculated with a mixture of soda lake sediments by using isobutyronitrile (iBN) as the carbon, energy and nitrogen source at pH 10. The enrichment resulted in a stable binary culture containing iBN-degrading Gram-positive rods and a satellite Gram-negative gammaproteobacterium Marinospirillum sp. strain (ANL-isoa) scavenging the products of nitrile hydrolysis. Cells of the iBN-degrading strain, ANL-iso2(T), were short, non-motile, non-spore-forming rods. Strain ANL-iso2(T) was capable of utilizing propionitrile (C(3)), butyronitrile (C(4)), isobutyronitrile (C(4)), valeronitrile (C(5)) and capronitrile (C(6)) as the only growth substrate. Growth on nitriles was biphasic with fast initial hydrolysis of nitriles to the corresponding amides, carboxylic acids and ammonia and slow further utilization of these products resulting in biomass growth. Cells of strain ANL-iso2(T) grown with iBN were capable of extremely active hydration of a wide range of nitriles into the corresponding amides and much slower hydrolysis of these amides to the corresponding carboxylic acids. This indicated the presence of the nitrile hydratase/amidase pathway of nitrile degradation in the novel bacterium. Strain ANL-iso2(T) showed obligately alkaliphilic growth on iBN within the pH range 8.4-10.6, with optimum growth at 9.0-9.5. It was moderately salt-tolerant, with a salt range for growth of 0.1-2.0 M Na(+) and an optimum salt concentration for growth of 0.2-0.3 M. The dominant fatty acids in the polar lipids were C(16 : 0), iso-C(14), C(14 : 0), iso-C(16) and C(16 : 1)omega7. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic analysis placed strain ANL-iso2(T) within the class Actinobacteria as an independent lineage with only uncultured bacteria from soda lakes as its nearest relatives. On the basis of its unique phenotype and distinct phylogeny, strain ANL-iso2(T) is considered to represent a novel species of a new genus, for which the name Nitriliruptor alkaliphilus gen. nov., sp. nov. is proposed. The type strain of the type species, Nitriliruptor alkaliphilus, is ANL-iso2(T) (=DSM 45188(T)=NCCB 100119(T)=UNIQEM U239(T)). Phylogenetic data suggest that the novel bacterium forms the basis of a new family Nitriliruptoraceae fam. nov. and a novel order Nitriliruptorales ord. nov. within the class Actinobacteria.

Thiohalorhabdus denitrificans gen. nov., sp. nov., an extremely halophilic, sulfur-oxidizing, deep-lineage gammaproteobacterium from hypersaline habitats.

Seven strains of extremely halophilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) were enriched and isolated at 4 M NaCl from sediments of hypersaline inland lakes in south-eastern Siberia and a Mediterranean sea solar saltern. Cells of the novel isolates were spindle-like, long and non-motile rods with a Gram-negative type of cell wall. They were obligately chemolithoautotrophic SOB using thiosulfate and tetrathionate as electron donors and represent the first example of extremely halophilic chemolithoautotrophs that are able to grow anaerobically with nitrate as electron acceptor. The characteristic feature of the group was the production of large amounts of tetrathionate as an intermediate during the oxidation of thiosulfate to sulfate. With thiosulfate, the novel strains grew within the pH range from 6.5 to 8.2 (optimum at pH 7.5-7.8) and at NaCl concentrations from 1.5 to 4.0 M (optimum at 3.0 M). Cells grown at 4 M NaCl accumulated extremely high concentrations of glycine betaine as a compatible solute. The dominant cellular fatty acids were 10MeC(16 : 0) and C(16 : 0). Based on the DNA-DNA relatedness values, the isolates consisted of a single genomic species and had a similar phenotype. Phylogenetic analysis placed the novel bacteria in the class Gammaproteobacteria as an independent lineage with no significant relationship to any other genera in this class. On the basis of phenotypic and genotypic analysis, the group is proposed to represent a new genus, Thiohalorhabdus gen. nov., with Thiohalorhabdus denitrificans gen. nov., sp. nov. as the type species (type strain HL 19(T)=DSM 15699(T)=UNIQEM U223(T)).

Bacillus alkalidiazotrophicus sp. nov., a diazotrophic, low salt-tolerant alkaliphile isolated from Mongolian soda soil.

Strain MS 6(T) was obtained from a microoxic enrichment with a soda soil sample from north-eastern Mongolia in nitrogen-free alkaline medium at pH 10. The isolate had clostridia-like motile cells and formed ellipsoid endospores. It was able to fix dinitrogen gas growing on nitrogen-free alkaline medium. Strain MS 6(T) was a strictly fermentative bacterium without a respiratory chain, although it had a high catalase activity and tolerated aerobic conditions. It was an obligate alkaliphile with a pH range for growth between 7.5 and 10.6 (optimum at 9.0-9.5). Growth and nitrogen fixation at pH 10 were possible at a total salt content of up to 1.2 M Na(+) (optimum at 0.2-0.3 M). The dominant cellular fatty acids included C(16 : 0), C(16 : 1)omega7, anteiso-C(15 : 0) and C(14 : 0). The dominant isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. 16S rRNA gene sequencing identified strain MS 6(T) as a member of the genus Bacillus. Its closest relative was Bacillus arseniciselenatis E1H(T). The key functional nitrogenase gene nifH was detected in both strain MS 6(T) and its close relative and these strains formed a novel lineage in the nifH gene family. On the basis of these phenotypic and genetic comparisons, strain MS 6(T) is proposed to represent a novel species of the genus Bacillus, Bacillus alkalidiazotrophicus sp. nov. with the type strain MS 6(T) (=NCCB 100213(T)=UNIQEM U377(T)).

Haloalkaliphilic diazotrophs in soda solonchak soils.

Nitrogen fixation (NF) potential was measured in more than 40 samples of soda solonchak soils with the pH of water extract between 9.5 and 11.0 collected in several locations of Central Asia and in Egypt, using the acetylene reduction method. NF was detected in most of the samples. Maximal rates were observed under microaerophilic-anaerobic conditions with glucose as a substrate. In most cases, the NF negatively correlated with salt content and alkalinity. Five enrichments at pH 10 under micro-oxic conditions with glucose resulted in stable haloalkaliphilic mixed cultures, with diazotrophic component(s) active up to 2.0-3.0 M total Na(+). The cultures were dominated by Gram-positive spore-forming bacteria. Molecular cloning of nifH genes demonstrated the presence of two phylogenetic lineages of diazotrophs in the enrichments affiliated with the low-GC Gram-positive bacteria (in rRNA groups 1 and 6 of bacilli and in Clostridiales). Isolation of pure cultures of haloalkaliphilic diazotrophs from micro-oxic enrichments yielded nine strains, comprising two phylogenetic lineages. Most of the isolates (eight) were affiliated with the aerotolerant fermentative haloalkaliphilic bacterium Amphibacillus tropicus and a single strain clustered with the obligately anaerobic haloalkaliphile Bacillus arseniciselenatis. Diazotrophy has never been recognized previously in these groups of Gram-positive bacteria. Overall, the results demonstrated the existence, in soda solonchak soils, of a novel group of free-living fermentative diazotrophic bacteria active at extremely haloalkaline conditions.

Thiohalospira halophila gen. nov., sp. nov. and Thiohalospira alkaliphila sp. nov., novel obligately chemolithoautotrophic, halophilic, sulfur-oxidizing gammaproteobacteria from hypersaline habitats.

A previously unknown ecotype of obligately chemolithoautotrophic, sulfur-oxidizing bacteria was discovered in sediments of various inland hypersaline lakes and a solar saltern. The salt requirements for these bacteria were similar to those of haloarchaea, representing the first example of extreme halophiles occurring among the chemolithoautotrophs. They were enriched and isolated at 4 M NaCl under aerobic conditions with thiosulfate or tetrathionate as the electron donor or under micro-oxic conditions with sulfide. In total, 20 strains were obtained from hypersaline inland lakes in central Asia, central Russia and Crimea and a sea saltern of the Adriatic Sea. The isolates were thin, motile spirilla, some of which possessed a yellow, membrane-bound pigment. They were obligately aerobic, chemolithoautotrophic, sulfur-oxidizing bacteria that used thiosulfate, sulfide, sulfur and tetrathionate as electron donors. The characteristic feature of the group was the production of large amounts of tetrathionate as an intermediate during the oxidation of thiosulfate to sulfate. All but one of the strains grew within the pH range 6.5-8.2 (optimally at pH 7.3-7.8) and at NaCl concentrations from 2.0 to 5 M (optimally at 3.0 M). A single strain, designated ALgr 6sp(T), obtained (by enrichment) from the hypersaline alkaline lakes of the Wadi Natrun valley, was found to be moderately halophilic and facultatively alkaliphilic (capable of growth at pH 10). The predominant cellular fatty acids were quite unusual, with 10-methyl C(16 : 0) and C(16 : 0) predominating. Cells grown at 4 M NaCl accumulated extremely high concentrations of glycine betaine as a compatible solute. The 20 neutrophilic isolates contained three genospecies (on the basis of DNA-DNA relatedness data) but could not be discriminated phenotypically. On the basis of the phenotypic and genotypic analyses, the isolates constitute a novel genus and species, for which the name Thiohalospira halophila gen. nov., sp. nov. is proposed. The type strain of Thiohalospira halophila is HL 3(T) (=DSM 15071(T)=UNIQEM U219(T)). The haloalkaliphilic strain ALgr 6sp(T) represents a second species of the new genus, for which the name Thiohalospira alkaliphila sp. nov. is proposed. The type strain of Thiohalospira alkaliphila is ALgr 6sp(T) (=DSM 17116(T)=UNIQEM U372(T)).

Sulfidogenesis under extremely haloalkaline conditions by Desulfonatronospira thiodismutans gen. nov., sp. nov., and Desulfonatronospira delicata sp. nov. - a novel lineage of Deltaproteobacteria from hypersaline soda lakes.

High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na(+) and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H(2) and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5-10, and grew in soda brines containing 1-4 M total Na(+) (optimum at 1.0-2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1(T) and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6(T) from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. nov.

Bacterial diversity and activity along a salinity gradient in soda lakes of the Kulunda Steppe (Altai, Russia).

Here we describe the diversity and activity of sulfate reducing bacteria along a salinity gradient in four different soda lakes from the Kulunda Steppe (South East Siberia, Russia). For this purpose, a combination of culture-dependent and independent techniques was applied. The general bacterial and SRB diversity were analyzed by denaturing gradient gel electrophoresis (DGGE) targeting the 16S rDNA gene. DNA was used to detect the microbial populations that were present in the soda lake sediments, whereas ribosomal RNA was used as a template to obtain information on those that were active. Individual DGGE bands were sequenced and a phylogenetic analysis was performed. In addition, the overall activity of SRB was obtained by measuring the sulfate reduction rates (SRR) and their abundance was estimated by serial dilution. Our results showed the presence of minor, but highly active microbial populations, mostly represented by members of the Proteobacteria. Remarkably high SRR were measured at hypersaline conditions (200 g L(-1)). A relatively high viable count indicated that sulfate reducing bacteria could be highly active in hypersaline soda lakes. Furthermore, the increase of sodium carbonate/bicarbonate seemed to affect the composition of the microbial community in soda lakes, but not the rate of sulfate reduction.

Methylohalomonas lacus gen. nov., sp. nov. and Methylonatrum kenyense gen. nov., sp. nov., methylotrophic gammaproteobacteria from hypersaline lakes.

Aerobic enrichment at 4 M NaCl, pH 7.5, with methanol as carbon and energy source from sediments of hypersaline chloride-sulfate lakes in Kulunda Steppe (Altai, Russia) resulted in the isolation of a moderately halophilic and obligately methylotrophic bacterium, strain HMT 1(T). The bacterium grew with methanol and methylamine within a pH range of 6.8-8.2 with an optimum at pH 7.5 and at NaCl concentrations of 0.5-4 M with an optimum at 2 M. In addition to methanol and methylamine, it can oxidize ethanol, formate, formaldehyde and dimethylamine. Carbon is assimilated via the serine pathway. The main compatible solute is glycine betaine. 16S rRNA gene sequence analysis placed the isolate as a new lineage in the family Ectothiorhodospiraceae (Gammaproteobacteria). It is proposed, therefore, to accommodate this bacterium within a novel genus and species, Methylohalomonas lacus gen. nov., sp. nov., with HMT 1(T) (=DSM 15733(T) =NCCB 100208(T) =UNIQEM U237(T)) as the type strain. Two strains were obtained in pure culture from sediments of soda lake Magadi in Kenya and the Kulunda Steppe (Russia) on a mineral medium at pH 10 containing 0.6 M total Na(+) using methanol as a substrate. Strain AMT 1(T) was enriched with methanol, while strain AMT 3 originated from an enrichment culture with CO. The isolates are restricted facultative methylotrophs, capable of growth with methanol, formate and acetate as carbon and energy sources. With methanol, the strains grew within a broad salinity range from 0.3 to 3.5-4 M total Na(+), with an optimum at 0.5-1 M. The pH range for growth was between 8.3 and 10.5, with an optimum at pH 9.5, which characterized the soda lake isolates as obligate haloalkaliphiles. Carbon is assimilated autotrophically via the Calvin-Benson cycle. Sequence analysis of the gene coding for the key enzyme RuBisCO demonstrated that strain AMT 1(T) possessed a single cbbL gene of the 'green' form I, clustering with members of the family Ectothiorhodospiraceae. Analysis of the 16S rRNA gene sequence showed that strains AMT 1(T) and AMT 3 belong to a single species that forms a separate lineage within the family Ectothiorhodospiraceae. On the basis of phenotypic and genetic data, the novel haloalkaliphilic methylotrophs are described as representing a novel genus and species, Methylonatrum kenyense gen. nov., sp. nov. (type strain AMT 1(T) =DSM 15732(T) =NCCB 100209(T) =UNIQEM U238(T)).

Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans.

Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the <>, high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed.

Phylogeny and evolution of the family Ectothiorhodospiraceae based on comparison of 16S rRNA, cbbL and nifH gene sequences.

The occurrence of genes encoding nitrogenase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was investigated in the members of the family Ectothiorhodospiraceae. This family forms a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and mostly includes photo- and chemoautotrophic halophilic and haloalkaliphilic bacteria. The cbbL gene encoding the large subunit of 'green-like' form I RubisCO was found in all strains, except the type strains of Alkalispirillum mobile and Arhodomonas aquaeolei. The nifH gene encoding nitrogenase reductase was present in all investigated species of the phototrophic genera Ectothiorhodospira, Halorhodospira and Thiorhodospira, but not of the genus Ectothiorhodosinus. Unexpectedly, nifH fragments were also obtained for the chemotrophic species Thioalkalispira microaerophila and Alkalilimnicola halodurans, for which diazotrophic potential has not previously been assumed. The cbbL-, nifH- and 16S rRNA gene-based trees were not highly congruent in their branching patterns since, in the 'RubisCO' and 'nitrogenase' trees, representatives of the Ectothiorhodospiraceae are divided in a number of broadly distributed clusters and branches. However, the data obtained may be regarded as evidence of the monophyletic origin of the cbbL and nifH genes in most species within the family Ectothiorhodospiraceae and mainly corresponded to the current taxonomic structure of this family. The cbbL phylogeny of the chemolithoautotrophic sulfur-oxidizers Thioalkalivibrio nitratireducens and Thioalkalivibrio paradoxus and the nitrifier Nitrococcus mobilis deviated significantly from the 16S-rRNA gene-based phylogeny. These species clustered with one of the duplicated cbbL genes of the purple sulfur bacterium Allochromatium vinosum, a member of the family Chromatiaceae.

Thiohalomonas denitrificans gen. nov., sp. nov. and Thiohalomonas nitratireducens sp. nov., novel obligately chemolithoautotrophic, moderately halophilic, thiodenitrifying Gammaproteobacteria from hypersaline habitats.

A novel group of moderately halophilic, obligately chemolithoautotrophic, sulfur-oxidizing Gammaproteobacteria was found in sediments of various inland hypersaline lakes and a solar saltern. These bacteria were enriched and isolated with thiosulfate as electron donor and nitrate as electron acceptor at 2 M NaCl. Ten isolates (HLD strains) were long non-motile rods. They grew anaerobically as complete denitrifiers, and aerobically under micro-oxic conditions. Sulfate was the final product of thiosulfate and sulfide oxidation, and nitrite and N(2)O were intermediates of nitrate reduction to N(2). The HLD strains grew optimally at pH 7.3-7.8, and at NaCl concentrations of 1.5-2.0 M. On the basis of phenotypic and genetic analysis, the moderately halophilic, thiodenitrifying isolates are proposed to be assigned to a new genus and species, Thiohalomonas denitrificans gen. nov., sp. nov. The type strain is HLD 2(T) (=DSM 15841(T)=UNIQEM U222(T) ). A single strain, HRhD 3sp(T), with vibrio-shaped cells, was obtained from a co-culture capable of complete denitrification of nitrate in the presence of either thiocyanate or thiosulfate as electron donor. It grew anaerobically with thiosulfate, reducing nitrate to nitrite, or under micro-oxic conditions at 1.0-2.5 M NaCl with an optimum at 1.0 M. Strain HRhD 3sp(T) was genetically related to the HLD strains at the level of a separate species and is described as Thiohalomonas nitratireducens sp. nov. The type strain is HRhD 3sp(T) (=DSM 16925(T)=UNIQEM U248(T)).

Acetonitrile degradation under haloalkaline conditions by Natronocella acetinitrilica gen. nov., sp. nov.

Nitriles are important environmental compounds, both as natural products and industrial pollutants. Until now, there have been no data on the possibility of microbial nitrile degradation at high pH/salt conditions. Acetonitrile (CH(3)C(triple bond)N) is the simplest organic nitrile. Here, evidence is provided of microbial utilization of acetonitrile as a carbon, energy and nitrogen source at extremely high pH and moderate salinity. Positive enrichment cultures with acetonitrile at pH 10 and salt content equivalent to 0.6 M total Na(+) were obtained from mixed sediment samples from soda lakes, but not from soda soils. Purification of these cultures resulted in the isolation of two bacterial strains capable of growth with acetonitrile as sole carbon, energy and nitrogen source under haloalkaline conditions. Apart from acetonitrile, the bacteria also grew with propionitrile. Nitrile hydrolysis to acetamide was identified as the rate-limiting step of acetonitrile degradation via the nitrile hydratase/amidase pathway. The new bacteria belonged to moderately salt-tolerant obligate alkaliphiles with optimum growth at pH 10 and 0.5 M total Na(+). The cells were yellow-coloured due to a high concentration of carotenoids dominated by zeaxanthin. Phylogenetic analysis placed the isolates into a new lineage within the family Ectothiorhodospiraceae in the Gammaproteobacteria. On the basis of unique phenotypic properties and their separate phylogenetic position, the new bacteria are placed into a new genus and species for which the name Natronocella acetinitrilica gen. nov., sp. nov is proposed.