Phylogenetic Analysis And Exoenzme Charateristics Of Halophilic And Halotolerant Bacteria Isolated From Mangrove Soil From The Moon Bay In Wenchang City,Hainan Province,China

To explore the biodiversity of halotolerant and halophilic bacteria isolated from mangrove soil collected from the Moon Bay?19.40.39.49 N?19.40.47.33 N,111.00.30.14 E?111.00.36.49 E?in Wenchang City in Hainan Province,PR China,bacteria strains were isolated from the soil samples using culture-dependent method;the biodiversity including taxonomic diversity,species diversity and genetic diversity,were studied by using 16S r RNA gene sequence analysis;the extracellular enzymatic characteristics were observed using hydrolysis method to understand the diversity of physiological types.Some strains that represent potential new taxa/species were studied using polyphasic taxonomic approach to investigated their taxonomic position.A total of 399 halophilic/halotolerant bacteria were isolated from the mangrove soil samples on 4 halophilic media?JMC611,MA,MH and NA?with 15%or 20%?w/v?Na Cl.After morphological observation and salt tolerance tests,we selected 217representative strains for further phylogenetic analysis and extracellular exoenzyme tests.The results of salt tolerance experiment showed that,70 strains?32.3%?,125?57.6%?,16?7.4%?and 6?2.8%?could tolerate 30%,25%,20%and 15%Na Cl,respectively,and that most of them were halophilic strains?211,97.2%?,and 6 were halotolerant?JSM 1782015,JSM 1782048,JSM 1782057,JSM 1782114,JSM 1782358,JSM 1782361?.The results of phylogenetic analysis based on 16S r RNA gene sequence comparison showed that 217 representative strains belonged to 39 species,being members of 16 genera?Allobacillus,Bacillus,Citricoccus,Gracilibacillus,Halobacillus,Halomonas,Marinococcus,Marinobacter,Nocardiopsis,Oceanobacillus,Pontibacillus,Salinicola,Staphylococcus,Thalassobacillus,Virgibacillus,Zhihengliuella?of 6 families?Alteromonadaceae,Bacillaceae,Halomonadaceae,Micrococcaceae,Nocardiopsaceae,Staphylococcaceae?in 3 phyla?Firmicutes,Actinobacteria,Proteobacteria?.The phylum Firmicutes had the most abundant strains?198 strains,91.2%?,and the family Bacillaceae had the most dominant strains?196,90.3%?,and the genus Halobacillus was the most dominant genus?104,47.9%?.Only16 strains were members of the phylum Proteobacteria,and 3 strains were members of the phylum Actinobacteria.At least 7 strains?JSM 1782048,JSM 1782050,JSM1782141,JSM 1782161,JSM 1782259,JSM 1782309 and JSM 1782372?should represent7 potential novel species.The results of enzymatic tests showed that,out of the 217 strains tested,a total of173?79.7%?strains produced at least one kind of exoenzyme,and 62 strains?28.6%?,52?24.0%?and 59?27.2%?showed respectively 1,2 and 3 exoenzyme-activities;and that 79 strains?36.4%?showed amylase-positive,131?60.4%?esterase-positive and133?61.3%?gelatinase-positive.The results of extracellular enzymatic tests also showed that at least 6 strains?JSM 1782036,JSM 1782161,JSM 1782248,JSM 1782293,JSM1782296,JSM 1782399?exhibited relatively strong amylase-activity,in which JSM1782248 and JSM 1782399 belonged to the genus Halobacillus,JSM 1782293 and JSM1782296 belonged to the genus Pontibacillus,JSM 1782036 belonged to the Gracilibacillus,and JSM 1782161 belonged to the Marinobacter;that 9 strains?JSM1782040,JSM 1782041,JSM 1782225,JSM 1782231,JSM 1782239,JSM 1782265,JSM 1782270,JSM 1782277,JSM 1782321?showed relatively high gelatinase-activity,in which 8 strains belonged to the genus Halobacillus,and JSM 1782277 belonged to the genus Pontibacillus;that 10 strains?JSM 1782009,JSM 1782066,JSM 1782091,JSM 1782109,JSM 1782120,JSM 1782121,JSM 1782145,JSM 1782152,JSM1782172,JSM 1782338?exhibited relatively strong esterase-activity,in which 9 strains belonged to the family Bacillaceae,8 strains belonged to the Halobacillus,and JSM1782152 belonged to the genus Oceanobacillus,and JSM 1782009 belonged to the genus Nocardiopsis.Exploying the polyphasic taxonomic approach,strain JSM 1782161 was studied in order to investigate the taxonomic position.The results showed that strain JSM 1782161was aerobic,gram-stain-negative,motile,slightly halophilic,rod-shaped.Growth occured at 15-40°C?optimum 30-35°C?,p H 5.5-10.5?optimum p H 7.5-8?with 1-20%?w/v?Na Cl?optimum 4-8%?.The DNA G+C content was 61.8mol%.The predominant ubiquinone is Q-9,and the major polar lipids were phosphatidylethanolamine,diphosphatidylglycerol and phosphatidylglycerol.The major fatty acids were C_16:1?9c,C_16:0,C_18:1?7c and summed feature 3(iso-C_15:0 2-OH and/or C_16:1?7c).16S r RNA gene sequence analysis showed that,JSM 1782161 belonged the genus Marinobacter,being most closely related to M.nanhaiticus D15-8W^T?96.72%16S r RNA gene sequence similarity?.On the phylogenetic tree,they clustered together.The combination of results from phenotypic,chemotaxonomic characterization and results of the phylogenetic analysis supported that strain JSM 1782161 represents a novel Marinobacter species.We propose the name Marinobacter mangrove for the novel species.Strain JSM 1782161^T?=CGMCC1.13826^T=KCTC 62890^T?is the the type strain of Marinobacter mangrove.