Studies On The Production Of Ectoine Of Two Borderline Extreme Halophilic Bacteria And Their Genomic Analysis

The salt tolerance gradient test of 28 halophilic actinomycetes isolated from Jilantai salt lake soil was carried out in this paper.According to the taxonomic criteria of halophilic microorganisms,two strains of borderline extreme halophilic bacteria(JG5 and JG9),17 strains of moderate halophilic bacteria,6 strains of weak halophilic bacteria and 3 strains of non-halophilic bacteria were obtained.We observed the culture characteristics and biological characteristics of JG5 and JG9,and analyzed their phylogeny based on 16 s rRNA gene sequence.The results showed that both JG5 and JG9 belonged to Streptomyces,and their homology was similar.Using the most conserved ectB gene in the ectoine gene cluster,degenerate primers were synthesized to screen the strains containing the ectoine gene cluster.In order to explore the expression of ectoine gene cluster in different strains,we carried out the extraction of intracellular ectoine with 80 % ethanol,and qualitatively detected the presence of ectoine by TLC.The PCR results showed that 17 of 28 halophilic strains contained ectoine gene cluster.TLC showed that 11 of 17 strains could produce ectoine.The intracellular ectoine accumulation in different NaCl concentrations(0%,5%,10%,15%,m/v)was quantitatively determined by HPLC.The results of HPLC showed that both two strains could produce intracellular ectoine at different concentrations of NaCl.With the NaCl concentration of 10%,the intracellular ectoine of JG5 reached a maximum value of 80.35mg/L.JG9 reached a maximum concentration of 97.89mg/L when the NaCl concentration was 5%.Using the Illumina PE150 sequencing platform,we determined and assembled the whole genome of two borderline extreme halophilic bacteria.Whole-genome sequencing and bioinformation analysis showed that the genome size of strain JG5 was 8373014 bp,GC content of it was 72.44%.It contained 7143 coding genes and 89 ncRNA.The genome size of strain JG9 was 8273778 bp,GC content of it was 72.54%.It contained 7073 coding genes and93 ncRNA.The results of antiSMASH analysis of two strains showed that both of two strains contained large number of secondary metabolic gene clusters including ectoine gene clusters,46 gene clusters have been predicted of JG5 and 43 in JG9.It showed that two strains contained abundant secondary metabolites resources,which had great value of development and utilization.