Adaptation Mechanisms Of A Novel Moderately Halophilic Species Halomonas Huangheensis Response To Salinity Stress

Moderately halophilic bacteria is a kind of bacteria which have optimum growth performance in the moderate and high salt concentrations range from 3-15%. The accumulation of intracellular compatible solutes play an important role in responsing to the salinity stress. It is theoretically and practically significant for creating new seed resources, improving saline-alkaline land and developing new bio-products to use these functional genes which isolated and identified from extreme environment, by analysing the accumulation rules of their compatible solutes, exploring relative functional genes and revealing its molecular mechanism of adapting salt stress. A new Halomonas strain is isolated and identified in the study, we test the accumulation of its major compatible solutes, conduct identification and evolutionary analysing of its genes related to these compatible solutes synthesis, metabolism and transport, finally predict the molecular regulation mechanism of its major compatible solutes. The results are as follows.1. A moderately halophilic bacterium, designated as BJGMM-B45T, was isolated from a saline-alkali soil collected from Shandong province. It is indicated that strain BJGMM-B45 is a novel species of the genus Halomonas through the analysis of its physiological and biochemical characters, phylogenetic analysis based on 16S rRNA gene sequence, and DNA-DNA hybridization homology assay. It was named as Halomonas huangheensis sp. nov..2. The results, from its growth performance under 7 LB media with different salt concentration and test of Na+, K+, and eight kinds of compatible solutes, showed that Halomonas huangheensis BJGMM-B45T grow best in the media with 2-15% NaCl, its major compatible solutes are glutamate, glycine-betaine, ectoine and proline with different accumulation under different salt concentration environment. Glutamate accumulation is far beyond the other three compatible solutes in all 7 media with different NaCl content. Ectoine is the second highest compatible solute in the media with 5-18% NaCl, its accumulation level decrease with NaCl increase. Glycine-betaine content exceeds that of ectoine when NaCl concentration reach 20%. The accumulation level of proline is very few under all 7 salt concentration environment, with a little change.3. The complete genome sequencing of Halomonas huangheensis BJGMM-B45T is conducted by integrating Illumina Hiseq 2000 and the next-next-generation sequencing. The result indicates that the genome size of Halomonas huangheensis BJGMM-B45T is 4.7 Mb, G+C content is 58.5%, including 3993 coding sequences,12 rRNA and 63 tRNA. The comparison of all ORF with COG is completed. The possible synthesis pathways of glutamate, ectoine and glycine-betaine are mapped by comparison with KEGG, meanwhile,29 genes or regulators are identified including 6 newly predicted genes. The evolutionary analysis is completed for the genes coding synthases of the above three compatible solutes, these genes are ectA, ectB, ectC, lysA, betAl, betA2, betA3, betA4, glnAl, glnA2, glnA3 and glnA4.4. Transcriptome RNA-Seq sequencing result of Halomonas huangheensis BJGMM-B45T grown on 1%,10% and 18% NaCl concentration indicate that there are 10 high-expression genes in all three conditions, most of these 10 genes encode molecular chaperones. Differential expression of gene among this three salt concentration environments show that 82 genes were up-regulated and 109 genes down-regulated in the group of 10% NaCl,224 genes were up-regulated and 180 genes down-regulated, compared with 1% NaCl as control group.5. The metabolic pathways for glutamate, glycine-betaine, ectoine and proline are predicted through transcriptome analysis of these compatible solutes under three salt concentration environment. For glutamate, putA is up-regulated in moderately salt concentration, then more glutamane are produced. gltA is significantly up-regulated when the NaCl concentration increase to 18%, glutamine will be catalyzed to glutamate, meanwhile much more intercellular glutamine are absorbed through activation of glutamine ABC transport system. For ectoine, ectB, ectC, lysC were up-regulated with the NaCl concentration increasing, ectoine synthesis increase gradually not only from aspartate semialdehyde but also from L-aspartic acid. For glycine-betaine, its accumulation also is regulated by betAl and betBl, at the same time, transport BetT and BCCT transport system are involved in the regulation of glycine-betaine to different extent. Proline is accumulated through regulating of proline:sodium symporter SSS2.In summary, the study identify a novel strain of genus halomonas as Halomonas huangheensis BJGMM-B45T, deeply and systematically analyses the accumulation mechanism of its major compatible solutes through physiological characters, completes genome sequencing and transcriptome sequencing among lower, moderate and higher salt circumstances. The metabolic pathways of glutamate, glycine-betaine and ectoine are mapped on the basis of this analysis. These studies lay the foundation for further exploration of functional genes from this moderately halophilic bacterium and its future development and application.