Olga L. Kovaleva

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.

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.

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)).

Ectothiorhodospira magna sp. nov., a New Large Alkaliphilic Purple Sulfur Bacterium

Two strains of purple sulfur bacteria of the family Ectothiorhodospiraceae were isolated from moderately saline steppe lakes (with pH above 9.0) of the Transbaikal region (strain B7-7) and Mongolia (strain M10). The cells of the novel strains were spiral-shaped, 2.0–3.2 × 9.6–20.0 μm, motile due to a polar tuft of flagella. Photosynthetic pigments were represented by bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Photosynthetic membranes were represented by long strands of lamellae distributed throughout the whole cell; unlike most Ectothiorhodospiraceae species, the membranes were not packed into regular stacks. Bacteria were capable of weak growth on sulfide and slow grow on hydrogen under photoautotrophic conditions. The best growth was noted on sulfide in the presence of acetate and bicarbonate. Thiosulfate did not stimulate phototrophic growth, even in the presence of organic substrates. The new isolates were alkaliphiles growing at a pH optimum of 9–10. Growth was possible within a salinity range of 0–80 g/l NaCl, with an optimum at 5–15 g/l NaCl. The morphology, the structure of the photosynthetic apparatus (strands of lamellae), and the physiology of the new strains were similar to those of Thiorhodospira sibirica. However, analysis of the 16S rRNA gene sequences demonstrated that the studied isolates were closely related to the type strain Ectothiorhodospira shaposhnikovii (99% similarity) of the family Ectothiorhodospiraceae, whereas the level of similarity between the new strains and Thiorhodospira sibirica was only 94–95%. According to the results of DNA-DNA hybridization, the DNA-DNA homology level between the tested strains was almost 100%; the similarity between the new isolates and the type strain Ectothiorhodospira shaposhnikovii was only 58%. The isolates differed from other representatives of the genus Ectothiorhodospira in the structure of the gene encoding the key enzyme of autotrophic CO2 fixation, ribulose-1,5-bisphosphate carboxylase (RuBisCo), which was similar to the RuBisCo genes of members of another family of sulfur bacteria, Chromatiaceae. The new isolates of purple bacteria were described as a new species of the genus Ectothiorhodospira, Ect. magna sp. nov. with the type strain B7-7T (= VKM B-2537 = DSM 22250).

Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov., thermophilic anaerobic representatives of the phylum Planctomycetes

Two novel strains of thermophilic planctomycetes were recovered from terrestrial and subterranean habitats. Strain R1(T) was isolated from a hot spring (Kunashir Island, Russia) and strain SBP2(T) was isolated from a deep gold mine (South Africa). Both isolates grew in the temperature range 30-60 °C and pH range 5.0-8.0. Strain R1(T) grew optimally at 60 °C and pH 6.0-6.5; for SBP2(T) optimal conditions were at 52 °C and pH 7.5-8.0. Both strains were capable of anaerobic respiration with nitrate and nitrite as electron acceptors as well as of microaerobic growth. They also could grow by fermentation of mono-, di- and polysaccharides. Based on their phylogenetic position and phenotypic features we suggest that the new isolates represent two novel species belonging to a new genus in the order Planctomycetales, for which the names Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov. are proposed. The type strain of Thermogutta terrifontis, the type species of the genus, is R1(T) ( = DSM 26237(T) = VKM B-2805(T)), and the type strain of Thermogutta hypogea is SBP2(T) ( = JCM 19991(T) = VKM B-2782(T)).

Tepidisphaera mucosa gen. nov., sp. nov., a moderately thermophilic member of the class Phycisphaerae in the phylum Planctomycetes, and proposal of a new family, Tepidisphaeraceae fam. nov., and a new order, Tepidisphaerales ord. nov.

Three strains of facultatively aerobic, moderately thermophilic bacteria were isolated from terrestrial hot springs in Baikal Lake region and Kamchatka (Russia). Cells of the new isolates were cocci reproducing by binary fission. The temperature range for growth was between 20 and 56 °C and the pH range for growth from pH 4.5 to 8.5, with optimal growth at 47-50 °C and pH 7.0-7.5. The organisms were chemoheterotrophs preferring sugars and polysaccharides as growth substrates. 16S rRNA gene sequences of strains 2842, 2813 and 2918Kr were nearly identical (99.7-100 % similarity) and indicated that the strains belonged to the phylum Planctomycetes. The phylogenetically closest cultivated relatives were Algisphaera agarilytica 06SJR6-2(T) and Phycisphaera mikurensis FYK2301M01(T) with 16S rRNA gene sequence similarity values of 82.4 and 80.3 %, respectively. The novel strains differed from them by higher growth temperature, sensitivity to NaCl concentration above 3.0 % and by their cellular fatty acids profile. On the basis of phylogenetic and physiological data, strains 2842(T), 2813 and 2918Kr represent a novel genus and species for which we propose the name Tepidisphaera mucosa sp. nov. The type strain is 2842(T) ( = VKM B-2832(T) = JCM 19875(T)). We also propose that Tepidisphaera gen. nov. is the type genus of a novel family, Tepidisphaeraceae fam. nov. and a novel order, Tepidisphaerales ord. nov.

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.

Application of ribulose-1,5-bisphosphate carboxylase/oxygenase genes as molecular markers for assessment of the diversity of autotrophic microbial communities inhabiting the upper sediment horizons of the saline and soda lakes of the Kulunda Steppe

The genes encoding the key metabolic reactions are often used as functional markers for phylogenetic analysis and microbial ecology studies. The composition and structure of the genes encoding ribulose-1,5-bisphosphate carboxylase (RuBisCO) of various photoautotrophic bacteria, representatives of the order Chromatiales, including collection strains and the strains isolated from saline and soda lakes, were studied in detail. The green-like form I RuBisCO was detected in the majority of the studied strains. In some strains, the genes encoding both form I and form II RuBisCO were present, which has not been previously known for the representatives of this group of bacteria. Moreover, RuBisCO genes were used as functional markers to investigate the autotrophic microbial community inhabiting the upper horizons of bottom sediments of two saline soda lakes and two hypersaline neutral lakes of the Kulunda Steppe. In general, the diversity of autotrophic bacteria in the studied sediment horizons was low. In soda lakes, haloalkaliphilic cyanobacteria and sulfuroxidizing bacteria (SOB) of the genus Halorhodospira were predominant. In saline lakes, halophilic chemoautotrophic SOB Halothiobacillus and Thioalkalivibrio were found, as well as photoautotrophic bacteria of the genus Ectothiorhodosinus and cyanobacteria. Many phylotypes remained unidentified, which indicates the presence of groups of microorganisms with an unknown type of metabolism.

Genomic analysis of Caldithrix abyssi, the thermophilic anaerobic bacterium of the novel bacterial phylum Calditrichaeota

The genome of Caldithrix abyssi, the first cultivated representative of a phylum-level bacterial lineage, was sequenced within the framework of Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. The genomic analysis revealed mechanisms allowing this anaerobic bacterium to ferment peptides or to implement nitrate reduction with acetate or molecular hydrogen as electron donors. The genome encoded five different [NiFe]- and [FeFe]-hydrogenases, one of which, group 1 [NiFe]-hydrogenase, is presumably involved in lithoheterotrophic growth, three other produce H2 during fermentation, and one is apparently bidirectional. The ability to reduce nitrate is determined by a nitrate reductase of the Nap family, while nitrite reduction to ammonia is presumably catalyzed by an octaheme cytochrome c nitrite reductase εHao. The genome contained genes of respiratory polysulfide/thiosulfate reductase, however, elemental sulfur and thiosulfate were not used as the electron acceptors for anaerobic respiration with acetate or H2, probably due to the lack of the gene of the maturation protein. Nevertheless, elemental sulfur and thiosulfate stimulated growth on fermentable substrates (peptides), being reduced to sulfide, most probably through the action of the cytoplasmic sulfide dehydrogenase and/or NAD(P)-dependent [NiFe]-hydrogenase (sulfhydrogenase) encoded by the genome. Surprisingly, the genome of this anaerobic microorganism encoded all genes for cytochrome c oxidase, however, its maturation machinery seems to be non-operational due to genomic rearrangements of supplementary genes. Despite the fact that sugars were not among the substrates reported when C. abyssi was first described, our genomic analysis revealed multiple genes of glycoside hydrolases, and some of them were predicted to be secreted. This finding aided in bringing out four carbohydrates that supported the growth of C. abyssi: starch, cellobiose, glucomannan and xyloglucan. The genomic analysis demonstrated the ability of C. abyssi to synthesize nucleotides and most amino acids and vitamins. Finally, the genomic sequence allowed us to perform a phylogenomic analysis, based on 38 protein sequences, which confirmed the deep branching of this lineage and justified the proposal of a novel phylum Calditrichaeota.

Use of genes of carbon metabolism enzymes as molecular markers of Chlorobi phylum representatives

This work examined the feasibility of using certain genes of carbon metabolism enzymes as molecular markers adequate for studying phylogeny and ecology of green sulfur bacteria (GSB) of the Chlorobi phylum. Primers designed to amplify the genes of ATP citrate lyase (aclB) and citrate synthase (gltA) revealed the respective genes in the genomes of all of the newly studied GSB strains. The phylogenetic trees constructed based on nucleotide sequences of these genes and amino acid sequences of the conceptually translated proteins were on the whole congruent with the 16S rRNA gene tree, with the single exception of GltA of Chloroherpeton thalassium, which formed a separate branch beyond the cluster comprised by other representatives of the Chlorobi phylum. Thus, the aclB genes but not gltA genes proved to be suitable for the design of primers specific to all Chlorobi representatives. Therefore, it was the aclB gene that was further used as a molecular marker to detect GSB in enrichment cultures and environmental samples. AclB phylotypes of GSB were revealed in all of the samples studied, with the exception of environmental samples from soda lakes. The identification of the revealed phylotypes was in agreement with the identification based on the FMO protein gene (fmo), which is a well-known Chlorobi-specific molecular marker.