Sulfate-dependent acetate oxidation under extremely natron-alkaline conditions by syntrophic associations from hypersaline soda lakes.

So far, anaerobic sulfate-dependent acetate oxidation at high pH has only been demonstrated for a low-salt-tolerant syntrophic association of a clostridium 'Candidatus Contubernalis alkalaceticum' and its hydrogenotrophic sulfate-reducing partner Desulfonatronum cooperativum. Anaerobic enrichments at pH 10 inoculated with sediments from hypersaline soda lakes of the Kulunda Steppe (Altai, Russia) demonstrated the possibility of sulfate-dependent acetate oxidation at much higher salt concentrations (up to 3.5 M total Na(+)). The most salt-tolerant purified cultures contained two major components apparently working in syntrophy. The primary acetate-fermenting component was identified as a member of the order Clostridiales forming, together with 'Ca. Contubernalis alkalaceticum', an independent branch within the family Syntrophomonadaceae. A provisional name, 'Ca. Syntrophonatronum acetioxidans', is suggested for the novel haloalkaliphilic clostridium. Two phylotypes of extremely haloalkaliphilic sulfate-reducing bacteria of the genus Desulfonatronospira were identified as sulfate-reducing partners in the acetate-oxidizing cultures under extreme salinity. The dominant phylotype differed from the two species of Desulfonatronospira described so far, whilst a minor component belonged to Desulfonatronum thiodismutans. The results proved that, contrary to previous beliefs, sulfate-dependent acetate oxidation is possible, albeit very slowly, in nearly saturated soda brines.

Analysis of community composition of sulfur-oxidizing bacteria in hypersaline and soda lakes using soxB as a functional molecular marker.

The diversity of soxB gene encoding a key enzyme of the Sox pathway sulfate thiohydrolase has been investigated in pure cultures of various halophilic and haloalkaliphilic sulfur-oxidizing bacteria (SOB) and in salt and soda lakes in southwestern Siberia and Egypt. The gene was detected in the majority of strains belonging to eleven SOB genera excluding members of genera Thiohalospira and Thioalkalimicrobium. The uncultured diversity of soxB in salt and soda lakes was low with a majority of detected sequences belonging to autotrophic SOB from the Gammaproteobacteria. In addition, the soxB analysis allowed detection of putative heterotrophic Gamma- and Alphaproteobacterial SOB yet unknown in culture. All clone libraries obtained from soda lakes contained soxB belonging to the genus Thioalkalivibrio in agreement with the cultivation results. Besides, representatives of the genera Halothiobacillus, Marinobacter, and Halochromatium and of the family Rhodobacteraceae have been detected in both type of saline lakes.

Sulfidogenesis in hypersaline chloride-sulfate lakes of Kulunda Steppe (Altai, Russia).

The activity and culturable diversity of sulfidogens were investigated in anoxic sediments of four hypersaline lakes with pH 7.6-8.2 in the Kulunda Steppe (Altai, Russia). Sulfate reduction rates were low, varying from 0.1 to 6.0 nmol HS(-) /(cm(3) h) with a maximum in the top 10 cm layer. Potential sulfidogenic rates with thiosulfate and sulfur as the e-acceptors were higher than with sulfate and were stimulated by formate, lactate, and acetate. Sulfidogenesis was optimal at salt concentrations below 2 M NaCl. Cultivation at 2 M NaCl resulted in the isolation of several strains of moderately halophilic SRB, but no growth of SRB was observed at 4 M NaCl. At lithotrophic conditions (i.e., with formate or H(2) as e-donors), several closely related alkalitolerant strains belonging to the genus Desulfonatronovibrio were isolated. Enrichments at heterotrophic conditions with lactate, propionate, acetate, or butyrate using sulfate or thiosulfate as e-acceptors yielded isolates related to Desulfosalsimonas propionicica, Desulfohalobium utahense, and Desulfocella halophila. Sulfur-reducing enrichments at 2 M NaCl with ethanol produced a member of the genus Halanaerobium, while enrichments at 4 M NaCl with acetate were dominated by archaea, demonstrating for the first time such type of catabolism in haloarchaea.

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)).

Diversity of RuBisCO and ATP citrate lyase genes in soda lake sediments.

Sediments from six soda lakes of the Kulunda Steppe (Altai, Russia) and from hypersaline alkaline lakes of Wadi Natrun (Egypt) were analyzed for the presence of cbb and aclB genes encoding key enzymes Ci assimilation (RuBisCO in Calvin-Benson and ATP citrate lyase in rTCA cycles, respectively). The cbbL gene (RuBisCO form I) was found in all samples and was most diverse, while the cbbM (RuBisCO form II) and aclB were detected only in few samples and with a much lower diversity. The cbbL libraries from hypersaline lakes were dominated by members of the extremely haloalkaliphilic sulfur-oxidizing Ectothiorhodospiraceae, i.e. the chemolithotrophic Thioalkalivibrio and the phototrophic Halorhodospira. In the less saline soda lakes from the Kulunda Steppe, the cbbL gene comprised up to ten phylotypes with a domination of members of a novel phototrophic Chromatiales lineage. The cbbM clone libraries consisted of two major unidentified lineages probably belonging to chemotrophic sulfur-oxidizing Gammaproteobacteria. One of them, dominating in the haloalkaline lakes from Wadi Natrun, was related to a cbbM phylotype detected previously in a hypersaline lake with a neutral pH, and another, dominating in lakes from the Kulunda Steppe, was only distantly related to the Thiomicrospira cluster. The aclB sequences detected in two samples from the Kulunda Steppe formed a single, deep branch in the Epsilonproteobacteria, distantly related to Arcobacter sulfidicus.

Sulfidogenesis under extremely haloalkaline conditions in soda lakes of Kulunda Steppe (Altai, Russia).

Sulfidogenic activity (SA) in anoxic sediments of several soda lakes with variable salinity in south Kulunda Steppe (Altai, Russia) has been investigated. The study included in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries in which sulfate, thiosulfate or elemental sulfur were used as electron acceptors. Despite the extreme conditions (high salt concentrations and high pH), the SA values were relatively high (ranging from 0.02 to 1.20 micromol HS(-) cm(-3) h(-1)), and only hampered under salt-saturated conditions. The highest SA was observed with elemental sulfur, followed by thiosulfate, while the lowest SA was determined in the presence of sulfate. Of all the electron donors tested, the addition of formate resulted in the highest SA with all three sulfur electron acceptors. Surprisingly, hydrogen as an electron donor had very little effect. Acetate was utilized as an electron donor only under sulfur-reducing conditions. Indigenous populations of sulfidogens in soda lake sediments showed an obligately alkaliphilic pH response of SA, showing a pattern that corresponded well to the in situ pH conditions. Sulfate reduction was much more susceptible to salt inhibition than thiosulfate and sulfur reduction. Microbiological investigations indicated that sulfate-reducing bacteria belonging to the orders Desulfovibrionales and Desulfobacterales could very likely be responsible for the SA with sulfate and thiosulfate as electron acceptors at moderate salt concentrations. Sulfur reduction at moderate salinity was carried out by a specialized group of haloalkaliphilic sulfur-reducing bacteria that utilize volatile fatty acids. In saturated soda brine, extremely natronophilic representatives of the order Halanaerobiales were responsible for the sulfur-dependent respiration.

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)).

Microbial sulfide oxidation in the oxic-anoxic transition zone of freshwater sediment: involvement of lithoautotrophic Magnetospirillum strain J10.

The oxic-anoxic transition zone (OATZ) of freshwater sediments, where opposing gradients exist of reduced iron and sulfide with oxygen, creates a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide. Gradient microcosms incubated with freshwater sediment showed rapid microbial turnover of sulfide and oxygen compared with sterile systems. Microcosms with FeS as a substrate also showed growth at the OATZ and subsequent dilution series resulted in the isolation of three novel strains, of which strain J10 grows chemolithoautotrophically with reduced sulfur compounds under microaerobic conditions. All three strains are motile spirilla with bipolar flagella, related to the genera Magnetospirillum and Dechlorospirillum within the Alphaproteobacteria. Strain J10 is closely related to Magnetospirillum gryphiswaldense and is the first strain in this genus found to be capable of autotrophic growth. Thiosulfate was oxidized completely to sulfate, with a yield of 4 g protein mol(-1) thiosulfate, and autotrophic growth was evidenced by incorporation of (13)C derived from bicarbonate into biomass. A putative gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase type II was identified in strain J10, suggesting that the Calvin-Benson-Bassham cycle is used for autotrophic growth. Analogous genes are also present in other magnetospirilla, and in the autotrophically growing alphaproteobacterium magnetic vibrio MV-1.

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).

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)).

Utilization of aliphatic nitriles under haloalkaline conditions by Bacillus alkalinitrilicus sp. nov. isolated from soda solonchak soil.

Enrichment with isobutyronitrile as the sole carbon, energy and nitrogen source at pH 10, using soda solonchak soils as an inoculum, resulted in the selection of a binary culture consisting of two different spore-forming phenotypes. One of them, strain ANL-iso4, was capable of growth with isobutyronitrile as a single substrate, while the other phenotype only utilized products of isobutyronitrile hydrolysis, such as isobutyroamide and isobutyrate. Strain ANL-iso4 is an obligate alkaliphile and a moderately salt-tolerant bacterium. Apart from isobutyronitrile, it grew on other (C3-C6) aliphatic nitriles at pH 10. Resting cells of ANL-iso4 actively hydrolyzed a number of aliphatic and arylaliphatic nitriles and their corresponding amides. The latter, together with the intermediate formation of amides during nitrile hydrolysis, indicated the presence of a nitrile hydratase/amidase system in the novel bacterium. Although present in an alkaliphilic bacterium, both nitrile- and amide-hydrolyzing activities had a pH optimum within the neutral range, probably due to their intracellular localization. On the basis of phenotypic and phylogenetic analyses, strain ANL-iso4 is proposed as a new species Bacillus alkalinitrilicus sp. nov.

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.

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)).

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.