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Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5-10, and within a salt range from 0.2 to 4 M Na(+), with an optimum at 0.5-1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles.

A new plasmid was isolated from the haloalkaliphilic archaeon, Natronobacterium sp. strain AS7091 and named pNB101. Sequence analysis revealed that pNB101 consists of 2,538 bp, in which three major open reading frames (ORF1, ORF2, and ORF3) were identified in the same strand. The ORF1 encodes a putative replication (Rep) protein with three typical motifs (I, II, and III) found in rolling-circle (RC) replicating plasmids. The putative double-stranded origin (DSO) and single-stranded origin (SSO) were detected within ORF3 and downstream of ORF1, respectively. S1 nuclease digestion and Southern blot analysis demonstrated the existence of the single-stranded DNA (ssDNA) intermediate from pNB101, which corresponds to the leading strand in RC replication and was further confirmed by strand-specific RNA probes. A single transcript for ORF1 ( rep) was detected by Northern blotting, and the 5' end of this transcript was determined by primer extension. Both results indicate that the three motifs (I-III) are located at the very end of the N-terminal of this Rep protein. Northern blot analysis also revealed that the ORF3 was transcribed at a very high level, which may play an important role in plasmid replication because the putative DSO is located in this gene. Together, our results indicate that pNB101, the first plasmid isolated from haloalkaliphilic Archaea, represents a novel RC-replicating plasmid.

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23,000) and of the oligomer determined by sedimentation equilibrium experiments (125,000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [gamma-32P]ATP, and Mg2+ was required for the incorporation of phosphate. The kinase preserved the ability to transfer gamma-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.