The Diversity Of Culturable Bacteria And Identification Of Novel Isolates In Polar Marine Sediments

The polar regions are characterized by cold,dry climate and strong radiation,which make the polar microorganisms have their special physiological and functional characteristics.Also,polar marine sediments could be also a treasure trove of microbial resources with complex active components.In this study,the diversity of culturable bacteria in polar marine sediments were analyzed through the isolation and molecular identification from the samples of 26 stations during the 9th Chinese National Atctic Expedition and samples of 11 stations during the 34th Chinese National Antactic Expedition.Colwellia sp.Arc7-635,a screened psychrophilic denitrifying bacterium,was selected for analysis of physiological and biochemical characteristics and genome-wide identification,.In addition,we disscussed its potential application of restoration under a stress of nitrogen pollution.Another bacterium Arc6-271,as well as a potential novel strain,was selected for preliminary multiphase taxonomic identification.Its classification status was preliminarily determined by the comparative analysis of the molecular genetic characteristics,the physiological characteristics and chemical composition characteristics.399 strains of bacteria were isolated from marine sediment samples collected from 11 stations during the 34th Chinese National Antarctic Expedition utilized Zobell2216E medium,nitrification medium and denitrification medium.The molecular identification and phylogenetic analysis of the 16S rRNA gene sequence demonstrated that these strains belonged to 4 phyla,6 classes,9 orders,15 families,28 genera and 58species of the bacterial domain.Among these strains,Halomonas,Pseudoalteromonas and Joostella were relatively abundant,accounting for 39%,25%and 10%of the total number of strains respectively.316 strains of bacteria were isolated from marine sediment samples collected from 11 stations during the 9th Chinese National Arctic Expedition.The results suggested that these 316 strains belonged to 4 phyla,5 classes,13 orders,18 families,30 genera and 64 species of the bacterial domain.And Shewanella,Pseudoalteromonas and Psychrobacter were more abundant,accounting for 32%,26%and 13%of the total number of strains respectively.Three strains with less than 98%similarity of 16S rRNA gene sequences to the model strain were obtained from polar sediment samples,which could be potential novel strain.The culturable bacteria in polar marine sediments may lay the foundation of polar resources utilization.The strain Colwellia sp.Arc7-635 can use NO₃^-or NH₄⁺as the only nitrogen source to grow at low temperature.Moreover,the growth temperature range of the strain is 0-20?and the NaCl tolerance concentration range is 0-9.0%.Thus the strain has a strong adaptive ability for the low temperature environment.The strain also have the capacity of synthesizing extracellular enzymes such as nitrate reductase?NapA and NapB?,alpha-glucosidase and protease.Furthrmore,the complete genome of Colwellia sp.Arc7-635 contains 4,741,350 bp with 38.41 mol%G+C content,3841protein-coding genes?CDSs?,91 tRNA genes,7 rRNA operons as 16S-23S-5S rRNA and 1 operons as 16S-23S-5S-5S rRNA.According to genome annotation,Colwellia sp.Arc7-635 encodes a complete denitrification pathway consisting of genes related to nitrate reductase,nitrite reductase,nitric oxide reductase and nitrous oxide reductase.The other genes were analysied to involve in nitrate reduction to ammonia include nitrate reductase?NapA and NapB?and nitrite reductase?NirA,NirB and NirD?.The complete genome sequence of Colwellia sp.Arc7-635 provides valuable information to further elucidate nitrogen metabolism and promote its potential application in nitrogen bioremediation.The strain Arc6-271 belonging to the class?-Proteobacteria and genus Colwellia.The highest similarity with Colwellia beringensis NB097-1?97.64%?based on 16S rDNA sequecing and the similarity with other model strains of Colwellia was in the range of 92.48-97.07%.Average nucleotide identity analysis showed that strain Arc6-271 shared similarity ranging from 72.63 to 82.53%with the other strains of genus Colwellia.Besides,the growth temperature of the strain on 2216E medium ranged from 0 to 37?and the optimal was 30?.The NaCl tolerance growth range was 1.5-6.0%,and the optimal was 3.0-4.5%.It can degrade gelatin,agar,carrageenan and starchrather than casein,lecithin,alginate and cellulose.The catalase reaction of the strain was positive,which suggest that it could reduce nitrate and hydrolyze heptachlor.And the reactions of alkaline phosphatase,leucine aryamylase and naphthol-AS-BI-phosphate hydrolase were all positive.The main respiratory quinone is Q-8and major fatty acids are C_15:1?8c,C_17:1?8c and C_16:1?7c and/or C_16:1?6c.The primary polar lipids of the strain are phosphatidyl glycerol?PG?,phosphatidyl ethanolamine?PE?and three unknown glycolipids.The complete genome contains one circular chromosome of 4,599,808 bp,4048 protein-coding genes?CDSs?,84 tRNA genes,as well as 15 gene islands.Furthermore,8 genes related to nitrogen metabolism and 9 genes related to antibacterial activities were defined.Through the comparative analysis of molecular genetic characteristics,physiological haracteristics and chemical composition characteristics and analysis of the whole genome,Arc6-271 was preliminarily judged to be a novel stain of Colwellia.It laid a foundation for the further development and utilization of the polar resources.