Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na(+)/H(+) antiporter of Halobacillus dabanensis.

NhaH is a novel Na(+)/H(+) antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na(+)(Li(+))/H(+) antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na(+) and Li(+) without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na(+) and Li(+) but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H(+) binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na(+) and Li(+), indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li(+), without affecting that for Na(+), suggesting that E229 may be mainly responsible for Li(+) binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na(+) and Li(+), indicating that H87 and H88 are involved in H(+) binding or pH regulation.

Oceanobacillus manasiensis sp. nov., a moderately halophilic bacterium isolated from the salt lakes of Xinjiang, China.

Three Gram reaction positive, rod-shaped, moderately motile halophilic bacterial strains, designated YD3-56(T), YD16, and YH29, were isolated from the sediments of Manasi and Aiding salt lakes in the Xinjiang region of China, respectively. The strains grew optimally at 30-37 degrees C, pH 8-11, in the presence of 5-10% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to members of the genus Oceanobacillus, exhibiting 99.1-99.2% similarity to O. kapialis KCTC 13177(T), 99.2-99.3% to O. picturae KCTC 3821(T), and 94.2-96% sequence similarity to other described Oceanobacillus species. SDS-PAGE of whole cell proteins preparations demonstrated that the strains exhibited high similarity to each other, but distinguished from O. kapialis KCTC 13177(T) and O. picturae KCTC 3821(T) (75%). DNA-DNA hybridization revealed that the similarity between the representative strain YD3-56(T) and O. kapialis KCTC 13177(T) was 35.3%, and the similarity between YD3-56(T) and O. picturae KCTC 3821(T) was 22.3%. Chemotaxonomic analysis of the strains showed menaquinone-7 was the predominant respiratory quinine. Major cellular fatty acids were anteiso-C(15:0) and anteiso-C(17:0). The polar lipid pattern for strain YD3-56(T) predominantly contained phosphatidylcholine, and trace to moderate amounts of phosphatidyl ethanolamine and hydroxy-phosphatidyl ethanolamine. The diamino acid in murein was meso-diaminopimelic acid. The DNA G+C content of the strains was 39.7-40.1 mol%. On the basis of these results, the three strains should be classified as a novel species of the genus Oceanobacillus, for which the name Oceanobacillus manasiensis sp. nov. has been proposed, with the type strain as YD3-56(T) (=CGMCC 1.9105(T) =NBRC 105903(T)).

[Current status on proteomics of extremophilic microorganisms--a review].

We summarized the key handicap and troubleshooting when proteomic techniques were used to investigate extremophilic microorganisms, and the actual state of their proteomes research in recent years. Up to now, proteomics techniques keep developing and improving rapidly, but they has not been widely used to explore proteome of extremophilic microorganisms including halophiles, thermophiles, psychophiles, acidophiles and alkaliphiles due to specific problems including incomplete dissociation of protein-protein complexes of extremophiles, and a lot of proteins synthesized by extremophiles are resistant to the conditions which dissociated and denatured proteins synthesized by mesophilic organisms. However, the foreground of potential application of the techniques draws people on attempting zealously multifarious methods. At the present time, several technical problems for separating halophilic proteins, integral membrane proteins and predicting the function of new proteins have been solved availably. Proteomics data have validated some conclusions of genome predictions, and revealed many novel proteins and a few properties of extremophiles can not be resolved fully by genome data. The investigation of extremophiles proteomes indicated that a comprehensive view of protein expression profiles should rely on more than one proteomic method. In addition, the mutual verification of conclusions on the basis of genome and proteome and combination of these two techniques must accelerate the study of extremophilic microorganisms, and redound to uncover deeply and wholly the unique mechanisms of microorganisms adaptation to extreme environments. Moreover, it would clarify the mechanisms of their survival, and point out new direction of survey for improving damage result from stresses, finally contribute to human survival and healthy.

[Study progress on compatible solutes in moderately halophilic bacteria].

Moderately halophilic bacteria which grow best in media with 3% to 15% salt constitute a heterogenous group of microorganisms which belong to different genera. These bacteria can inhabit the salt or soda lakes, coastal lagoons or man-made salterns. Moderately halophilc bacteria living in higher saline environments can not only cope with high osmotic stress but also adapt osmotic shock in short time. To adapt to these environments, all the species make a osmoprotection by the accumulation a restricted range of low molecular mass molecules, small, organic compatible solutes, such as sugars, amino acids, betaines and ectoines. Therefore, the osmoadaptation of moderately halophilc bacteria is regulated by the so-called "compatible solute" strategy. Compatible solutes are operationally defined as organic osmolytes that can be amassed by the cell in exceedingly high concentrations without disturbing vital cellular functions and the correct folding of proteins. As a result, compatible solutes can make important contributions to the restoration of the turgor under conditions of low water activity by counteracting the efflux of water from the cell. In addition, they have a stabilizing, both in vivo and vitro, on the native structure of proteins and cell components. This mechanism has a minimal requirement for genetic change and a high degree of flexibility in allowing moderate halophiles to adapt to saline environment. In this review, the adaptation to saline environments, the variety and characteristic of compatible solutes, and the functional mechanism of moderately halophilic bacteria are reviewed and discussed.

[Biosynthesis of polyhydroxyalkanoates and its metabolic pathway in Comamonas sp. strain CNB-1].

Comamonas sp. strain CNB-1 degrades chloronitrbenzene and nitrobenzene for carbon and nitrogen sources. In this study, accumulation of polyhydroxyalkanoic acids (PHAs) within strain CNB-1 cells was investigated under various conditions. Results indicated that strain CNB-1 was able to synthesize PHA from various short-chain fatty acid and alcohols, and 57 w% of the dry cell weight (DCW) PHA was obtained when valerate and 1,4-butanediol were co-fed. Supplements of short-chain alcohols stimulated the accumulation of PHAs, and this stimulatory effect was attributed to the more amount of reductant generated from alcohol dehydrogenation. The genes encoding for PHA polymerase (phaC), for acetoacetyl-CoA thiolase (phaA), and acetoacetyl-CoA reductase (phaB) were cloned in Escherichia coli, and the recombinant E. coli synthesized PHA and showed enzymatic activities of PHA polymerase, acetoacetyl-CoA thiolase, and acetoacetyl-CoA reductase. The three genes occurred as a cluster of pha(C-A-B). To optimize their expression, the three genes were cloned to the pET vector and expressed respectively. Mass of expressed protein was detected and the enzyme activities increased greatly in contrast to wild CNB-1 strain, which is about 4.1, 71, and 2882 folds of activities of CNB-1.

Wenxinia marina gen. nov., sp. nov., a novel member of the Roseobacter clade isolated from oilfield sediments of the South China Sea.

An aerobic and heterotrophic, Gram-negative bacterial isolate, strain HY34(T), was isolated from sediment of an oilfield in the South China Sea, China. The taxonomy of strain HY34(T) was studied by phenotypic and phylogenetic methods. Strain HY34(T) formed faint-pink colonies on marine agar 2216. Cells of strain HY34(T) were non-motile, ovoid or short rods. Strain HY34(T) was positive for catalase and oxidase, and nitrate was reduced to nitrite. The nearly complete 16S rRNA gene sequence of strain HY34(T) was obtained and sequence analysis showed that it, together with the genus Rubellimicrobium, formed a distinct clade close to some members of the Roseobacter clade in the family Rhodobacteraceae, and it showed highest sequence similarities to Oceanicola granulosus HTCC2516(T) (93.8 %), Silicibacter lacuscaerulensis ITI-1157(T) (93.3 %), Dinoroseobacter shibae DFL 12(T) (93.3 %) and Rubellimicrobium thermophilum C-lvk-R2A-2(T) (92.2 %). Bacteriochlorophyll a was not detected. The ubiquinone system was Q-10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid. The major fatty acids (>10 %) were C(18 : 1) omega 7c and C(16 : 0). The DNA G+C content of this strain was 69.4 mol%. A polyphasic analysis supported the conclusion that this strain represents a novel genus and species, which we designated Wenxinia marina gen. nov., sp. nov. The type strain of Wenxinia marina is HY34(T) (=CGMCC 1.6105(T) =JCM 14017(T)).

Salegentibacter catena sp. nov., isolated from sediment of the South China Sea, and emended description of the genus Salegentibacter.

A novel marine bacterial strain, HY1T, was isolated from sediment of the South China Sea. The strain was aerobic and heterotrophic and formed saffron yellow-pigmented colonies on marine agar 2216. Cells were non-motile, Gram-negative rods, frequently occurring in chains. BLASTN searches revealed that the 16S rRNA gene sequence of strain HY1T showed high similarity with those of members of the genera Gillisia (91.7-93.8 %) and Salegentibacter (92.6-93.5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain clustered with members of both Salegentibacter and Gillisia and phylogenetic trees constructed using three different methods (neighbour-joining, maximum-parsimony and minimum-evolution) indicated that strain HY1T clustered more frequently with members of the genus Salegentibacter. The DNA G+C content of strain HY1T was 44.4 mol% and its major cellular fatty acids (>or=5 % of the total fatty acids) were iso-15 : 1 (5.0 %), iso-15 : 0 (6.8 %), anteiso-15 : 0 (6.4 %), 15 : 0 (10.4 %), iso-16 : 0 (13.5 %), summed feature 3 (comprising iso-15 : 0 2-OH and/or 16 : 1omega7c; 6.3 %), iso-17 : 0 3-OH (5.2 %) and 17 : 0 2-OH (5.0 %). Cells contained menaquinone 6. Based on the phylogenetic and phenotypic analyses, strain HY1T should be classified as representing a novel species within the genus Salegentibacter, for which the name Salegentibacter catena sp. nov. is proposed. The type strain is HY1T (=CGMCC 1.6101T=JCM 14015T). Based on this study and on previously described Salegentibacter species, an emended description of the genus Salegentibacter is given.

Cloning and characterization of the genes encoding a glycine betaine ABC-type transporter in Halobacillus trueperi DSM10404T.

By using Southern blot hybridization and inverse polymerase chain reaction, a 5.5-kb DNA fragment was obtained from the genomic DNA of Halobacillus trueperi DSM10404(T). Sequence analysis revealed that it contained a potential operon with high levels of sequence similarity to the opuA operon encoding glycine betaine transporter from Bacillus subtilis, which is a member of the ATP-binding cassette (ABC) substrate binding the protein-dependent transporter superfamily. The potential operon, designated as qatA (quaternary amine transporter), consists of three structural genes, which are predicted to encode an ATP-binding protein (QatAA), a membrane-associated protein (QatAB), and an extracellular substrate-binding protein (QatAC). Moreover, the putative promoter region of the operon was found with close homology to the sigma(A)-dependent promoter of B. subtilis. Reverse transcription (RT)-PCR analysis revealed that qatAA, qatAB, and qatAC genes were transcribed in cells of H. trueperi. Cells of Escherichia coli mutant MKH13 harboring qatA on pAY41 were able to grow on selective M9 salt medium containing glycine betaine and accumulated glycine betaine in the cytoplasm, showing that qatAA, qatAB, and qatAC genes together encode a functional glycine betaine transporter.

[Sodium ion transportation system and its possible mechanisms in bacteria].

Sodium ion with high concentration is toxic to living cells, and microorganisms adapt to the environment containing high concentration of salt by the strategies of salt-in-cytoplasm and compatible solutes. The Na+ extrusion system plays important roles in maintaining cytoplasmic Na+ homeostasis and pH level in microbial cells. Two possible mechanisms of Na+ circulation across the cytoplasmic membrane have been proposed, namely primary Na+ pump and secondary Na+/H+ antiporter. Primary sodium pumps coupled the extrusion of Na+ to respiration, and the activity of which was insensitive to uncoupler CCCP ( carbonyl-cyanide m-chlorophenylhydrazone). There were two types of secondary Na+/H+ antiporters-encoding genes designated single gene and multiple subunits, respectively. The types of transportation systems for Na+, possible mechanisms of Na+ extrusion, and projects for further study in bacteria are reviewed.

[Two-dimensional gel electrophoresis analysis of moderately halophilic bacterium Halobacillus dabanensis D-8T under hypoosmotic shock condition].

Halobacillus dabanensis D-8T was isolated from the saline deposits of Daban lake in Xinjiang of China, and is able to grow in complex medium containing 0.5% to 25% salt. To figure out the survival mechanisms of Gram-positive moderately halophilic bacteria under hypoosmotic shock conditions, two-dimensional gel electrophoresis (2-DE) was carried out to investigate differential protein expression profiles of H. dabanensis D-8T in response to low osmotic challenge. The 2-D gels were stored as dry gels and their images were taken by ImageScanner and analyzed by Imagemaster 2D Platinum software. About 650 protein spots were detected in 2-D gel. Most of proteins were distributed in molecular mass of 17.5 - 66kDa and the range of isoelectric point 4.0 - 5.9. A total of 34 protein spots were found to alter their expression after strain D-8T was subjected to hypoosmotic shock from 20% to 0% salinity for 5 min and 50 min. Among them, the expression of 20 protein spots is up-regulated including 6 new protein spots, while that of 14 protein spots is down-regulated in answer to sudden osmotic down-shift. Protein spots of interest were excised from the gels and digested by trypsin. By means of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) and MASCOT search engine, 4 up-regulated protein spots were identified with peptide mass fingerprint, and are similar to heat shock protein DanK, rod shape-determining protein, penicillin-binding protein (PBP-1A) and 5-enolpyruvoylshikimate-3-phosphate synthase, respectively. Noticeably, PBP-1A firstly was up-regulated after shock of 5 min but disappeared after shock of 50 min. This indicated that the strain activate a minor mechanism of peptidoglycan synthesis to compensate the major synthesis mechanism for cells survival through a down-shift challenge. In addition, this paper was the first report that heat shock proteins were up-regulated in response to sudden osmotic down-shift.

Cyclobacterium lianum sp. nov., a marine bacterium isolated from sediment of an oilfield in the South China Sea, and emended description of the genus Cyclobacterium.

The marine bacterial strain HY9(T) was isolated from sediment from the South China Sea. Strain HY9(T) is aerobic, heterotrophic and rose-pigmented. The cells are non-motile and curved, i.e. ring-like or horseshoe-shaped. The 16S rRNA gene sequence of strain HY9(T) was determined and blast searches revealed that it possessed significant sequence similarities with respect to Cyclobacterium species (92.8-93.6 %). Phylogenetic analysis confirmed that strain HY9(T) was tightly clustered with members of the genus Cyclobacterium. The cellular morphology and chemotaxonomic and phenotypic properties of strain HY9(T) showed that it should be classified as a member of the genus Cyclobacterium. Significant evolutionary distances and a range of phenotypic features distinguished strain HY9(T) from previously described Cyclobacterium species. Hence, strain HY9(T) represents a novel species in the genus Cyclobacterium, for which the name Cyclobacterium lianum sp. nov. is proposed. The type strain is HY9(T) (=CGMCC 1.6102(T)=JCM 14011(T)). On the basis of this study and previously described properties of Cyclobacterium species, an emended description of the genus Cyclobacterium is proposed.

The pha2 gene cluster involved in Na+ resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter.

Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.

[Transcriptional regulation of noeAB from Sinorhizobium meliloti 042BM].

The expression regulation of S. meliloti 042BM noeAB was studied. The results showed that trigonelline could not elevate the level of noeAB expression, which indicated that these genes are not regulated by nodD2. Since association of nodD3 and syrM could not change the level of the genes expression, they aren't also controlled by nodD3-syrM system. However, induction of luteolin resulted in 16 times increase of noeAB expression, which indicated that noeAB was regulated by nodD1. Most interestingly, more than 30 times increase in its expression was observed on TY medium without any flavonoid. Thus, it was suggested that noeAB may be controlled by other unknown factors.

[Construction of the genomic library of Halobacillus sp. D8 and isolation of the glycine betaine transporter betH gene].

Halobacillus sp. D8 is a sporing-forming, gram-positive moderately halophilic bacterium which could tolerate up to 25% (W/V) NaCl. A genomic library of Halobacillus sp. D8 was constructed using pUC18 as vector, and 9000 recombinant plasmids were obtained. By dot blot hybridization, colony PCR and DNA sequencing, the entire glycine betaine transporter betH gene was isolated from the constructed library. Inspection of the sequenced 4.3 kb DNA region revealed the presence of three ORFs. The putative ORF of betH is 1515bp long, encoding a 505-residue protein (BetH) with a calculated molecular mass of 56.1kD. Hydrophobicity plot analysis of BetH indicated a transmembrane protein containing 12 transmembrane regions. Homology searches for BetH of strain D8 in the GenBank using the BLAST program revealed significant sequence identities to other glycine betaine transporters: the putative glycine betaine transporter of O. iheyensis (64% identity), OpuD of B. subtilis (51% identity), BetH of H. trueperi (49% identity), BetL of L. monocytogenes (48% identity), BetM of M. halophilus (43% identity) and the putative glycine betaine transporter of B. halodurans (44% identity).

[The expression of gene related to salt tolerance from Sinorhizobium meliloti 042BM in Escherichia coli and purification of its fusion protein].

A 1.9kb DNA fragment related to salt tolerance of S. meliloti strain 042BM containing two open reading frames were obtained by PCR amplification and ligated into shuttle vector pBBR1-MCS2. The complementation experiment showed that ORF2 is related to salt tolerance and named as rstA gene. Then the gene was cloned into the expression vector pThio-HisA, B and C, respectively, and recombinant expression vectors pGSA, pGB and pGC were constructed, and transformed into E. coli Top10. Inducing by IPTG and analyzing with SDS-PAGE, the fusion protein encoded by pGSA was obtained,and it is 36% content of whole cell protein. It was isolated and purified by affinity chromography on ProBond, and the inclusion body precipitated by saturated sulfate ammonium, and 95% purity of fusion protein was obtained. The final product displayed a single band with a corresponding molecular weight 43kD in SDS-PAGE, and was verified by the Western blot.

[Cloning, deletion and functional analysis of noeA from Sinorhizobium meliloti 042BM].

042BM noeA was obtained by PCR. It is identical to that of S. meliloti 1021 at 99% level, and similarity of their NoeA is 97%. In addition, it was found that this protein shares significant homology with the SAM-dependent methyltransferase of Mesorhizobium sp. BNC1 (32% similarity), and the similarity of its 303-362 region to the 160- 220 region of Ll11 methyltransferases of E. coli (PrmA) is 41%. Compared to 042BM, the noeA deletion mutant 042BMA-Km showed different degrees of increase in number of nodule, fresh weight of nodule and plant top dry weight on alfalfa cultivars of Putong Zihua, Baoding, Ningxia, Baifa and Aohan, but decrease on Milu. However, this mutant has no significant change in ability to nodulate cultivars of Huanghou and Zahua. Hence, noeA is involved in alfalfa cultivar-specific nodulation.

[Positional analysis of a gene related to salt tolerance in Sinorhizobium meliloti by transposon rescue].

Salt sensitive mutant 042BML-2 was obtained by mutating Sinorhizobium meliloti 042BM with transposon Tn5 carried on the plasmid pRL1063a. By transposon rescue, a 1.179 kb of DNA sequence of S. meliloti flanking the Tn5 insertion site was obtained. The sequence similarity analysis through BLAST analysis in GenBank revealed the transposon was inserted into a functionally unknown gene, which is 6 270 bp in length, of S. meliloti, and the gene was named rtsC. This study indicated that rtsC was associated with salt tolerance in S. meliloti 042BM. Hydrophobicity profile analysis of the putative amino acid sequence showed that two predicted transmembrane domains existed in N-terminal portion of RtsC. The significance of RtsC protein in the salt-tolerance in S. meliloti was discussed.

Cloning and characterization of the Halobacillus trueperi betH gene, encoding the transport system for the compatible solute glycine betaine.

Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A fragment of the glycine betaine transporter betH gene was obtained from the genome of H. trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions. BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family. Its putative promoter region was highly homologous to sigmaB-dependent promoter of B. subtilis. A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13. The accumulation of glycine betaine in transformed E. coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy.

[A Sinorhizoboium meliloti strain that can nodulate soybean plants].

Sinorhizobium meliloti XJ96077 was isolated from root nodules of alfalfa (Medicago sativa) in Xinjiang Region of China. Nodulation experiments showed that both soybean and alfalfa were effectively nodulated by XJ96077. The DNA (G+ C) mol% of strain XJ96077 was 61.9%. The DNA homologies of strain XJ96077 were 93% and 80% with S. meliloti USDA1002T and 042BM, respectively. These results showed that XJ96077 belongs to Sinorhizobium meliloti. To prove the capability of XJ96077 to nodulate both soybean and alfalfa, constitutively expressed green fluorescence protein gene gfp was introduced to XJ96077, and the recombinant strain XJ96077(G) was obtained. Root nodules of the soybean and alfalfa inoculated with XJ96077(G) and the expression of gfp were observed using the confocal laser scanning microscope. XJ96077 showed various nodulation capacities with different soybean cultivars.

[Analysis of 16S rDNA sequences and DNA-DNA hybridization of moderately halophilic bacteria from Xinjiang region].

Based on the previous studies on numerical taxonomy and 16S rDNA PCR-RFLP analysis, the moderately halophilic bacteria isolated from Xinjiang Region constituted a new cluster, and the phylogenetic tree was constructed by comparing with the 16S rDNA sequences of the other moderately halophilic bacteria species. In the phylogenetic tree, most of the reference strains were clustered in a group, and the similarity values of 16S rDNA sequence were above 96%. However, AI-3, Alcanivorax borkumensis and Halobacillus litoralis were clustered in another group, and the similarity value of 16S rDNA sequences between AI-3 and Alcanivorax borkumensis was 96%, and that of 16S rDNA sequences between AI-3 and Halobacillus litoralis was 99%. The results indicated that AI-3 was different from the reference strains in phylogeny. The values of DNA homology in the new cluster were more than 70%, but the value between AI-3 and Halomonas elongata was less than 50%. Thus, the strain AI-3 possibly represent a new moderately halophilic bacteria species.