Diversity Of Culturable Halotolerant And Halophilic Bacteria Isolated From Saline-Alkaline Soils In Songnen Plain And Polyphasic Taxonomy Of Two Moderately Halophilic Bacteria

Because of the unique metabolic mechanism, physiological structure and hereditary property ofhalotolerant and halophilic microorganisms, they have attracted widespread attention of scholars athome and abroad. They are two kinds of microbiotic resource with value of academic research andutilization. Exploitation of new halotolerant and halophilic microorganisms is beneficial for getting thekey new salt tolerance related genes, improving saline lands, increasing grain production and so on.In this paper, we considered the bodiversity and polyphasic taxonomy of culturable halotolerantand halophilic bacteria from saline-alkaline soils in Songnen Plain. Halotolerant and halophilic bacteriawere isolated from the enriched cultures of the saline-alkline soil samples through the traditional culturemethod, and16S rDNA sequences were amplified and analyzed for the determination of phylogeneticrelationships. Finally,40representative strains were selected from125isolates. Phylogenetic analysisbased on16S rDNA gene sequences revealed that the culturable halotolerant and halophilic bacteria arevery rich in the saline-alkaline soils in Songnen Plain,40representative strains were classified into34species,16genera,8families and three phyla （Firmicutes, γ-Proteobacteria and Actinobacteria） in theDomain Bacteria. The genus Staphylococcus is the predominant genus, followed by Halomonas,Oceanbacillus, Bacillus, Kocuria and Pseudomonas. The16S rDNA sequences of9strains showed97.0to99.0%similarities with their closest type strains, suggesting that they may be the potential novelspecies. The salt tolerance of the strains is mainly concentrated on the5%-10%NaCl while alkaline pHresistance of the strains is between pH9and12. Based on the NaCl dependence,62.5%of the strainswere grouped into the halotolerant bacteria, and the rest into the moderate halophiles.Two moderately halophilic bacteria NEAU-ST10-25and NEAU-ST10-39were potential novelspecies of the genus Halomonas. In this paper, we describe the taxonomic status of the two strains bypolyphasic taxonomy. The results showed that the strain NEAU-ST10-25^Tis a Gram-negative, aerobicmotile rod. It produces beige-yellow-coloured colonies. Growth occurs at NaCl concentrations （w/v） of0-15%（optimum3%）, at temperatures of4-60℃（optimum35℃） and at pH6-12（optimum pH9）. ItsG+C content is53.8mol%. Phylogenetic analyses based on the separate16S rDNA gene and concatenation of the16S rDNA, gyrB and rpoD genes indicate that it belongs to the genus Halomonasin the class Gammaproteobacteria. The most phylogenetically related species is Halomonas alkaliphilaDSM16354^T, with which strain NEAU-ST10-25^Tshow16S rDNA, gyrB and rpoD gene sequencesimilarity values of99.2%,82.3%and88.2%, respectively. The results of DNA-DNA hybridizationassays showed60.47±0.69%relatedness between strain NEAU-ST10-25^Tand Halomonas alkaliphilaDSM16354^T. The major fatty acids are C_18:1ω7c （58.19%）, un18.846/C19:1ω6c （11.48%）, C_16:1ω7c/C_16:1ω6c （8.99%）, C_16:0（8.97%） and C12:03-OH （4.99%） and the predominant respiratory quinone isubiquinone9（Q-9）. The proposed name is Halomonas zhaodongensis, NEAU-ST10-25T（=CGMCC1.12286^T=DSM25869^T） being the type strain.The strain NEAU-ST10-39Tis aerobic, Gram-negative, rod-shaped and motile by peritrichousflagella. Its colonies are yellow. It grow at NaCl concentrations of0-15%w/v （optimum4%w/v）, attemperatures of4-40℃（optimum35℃） and at pH5-10（optimum pH7）. It do not produce acids fromsugars and alcohols. Its G+C content is57.4mol%. Phylogenetic analyses based on16S rDNA genesequences and concatenated16S rDNA, gyrB and rpoD gene sequences indicate that it belongs to thegenus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species isHalomonas axialensis DSM15723T, with which strain NEAU-ST10-39Tshowe16S rDNA, gyrB andrpoD gene sequence similarities of98.3%,82.7%,84.5%, respectively. The results of DNA-DNAhybridization assays showed50.33±1.48%relatedness between strain NEAU-ST10-39TandHalomonas axialensis DSM15723^T,46.27±1.54%between strain NEAU-ST10-39^Tand Halomonasmeridiana DSM5425^Tand42.70±1.35%between strain NEAU-ST10-39^Tand Halomonasaquamarina DSM30161^T. The major fatty acids are C_18:1ω7c （41.07%）, C_16:0（19.05%）, C_16:1ω7c/C_16:1ω6c （15.97%） and C_18:0（12.66%） and the respiratory quinone detected was ubiquinone9（Q-9） only.The proposed name is Halomonas songnenensis, NEAU-ST10-39^T（=CGMCC1.12152T=DSM25870^T）being the type strain.