Classification Of Five Novel Marine Bacterial Speicies And Natural Products From Strain Pseudoaltermonas Sp.A6021

BackgroundCompared with the terrestrial environment,marine environment shows special characteristics including high salinity,high pressure,low temperature,oligotrophic and so on.The metabolic pathways of marine microorganisms are different from those of terrestrial microorganisms,and they can produce secondary metabolites with special structures and bioactivities,which have broad application prospects in the fields of medicine and health care.Recently,the results of high-throughput sequencing have shown that the vast majority(>99%)of marine microorganisms have not been cultured under present laboratory conditions,which are seen as"microbial dark matters".This restricts the research and development of natural active products.Objectives1.Enrich the culturable microorganism resources,isolate the novel species of uncultured or difficult to be cultured bacterium,and determine the taxonomic position of the novel species candidates by polyphasic taxonomy approach.2.Isolation and identification of the red pigments produced by strain Pseudoaltermonas sp.A6021.To find the natural product biosynthetic gene clusters from genome sequences of the strain A6021 by bioinformatics analysis using anti SMASH database.Methods1.Four kinds of media and two culture temperatures was used to separate and purify bacteria from the samples including seaweed and seawater collected from Rizhao,Shandong Province.The 16S rRNA genes of the purified strains were sequenced to determine the potential novel bacterial taxons.2.The taxonomic positions of the five potential novel bacterial species were determined by polyphasic taxonomy approach.3.The culture conditions of strain Pseudoaltermonas sp.A6021 for producing red pigment were optimized.Methanol supplemented with hydrochloric acid was used to extract red pigment.High performance liquid chromatography(HPLC)and LC-MS were used for initial separation and identification of the red pigments.4.The genome sequence of strain A6021 was submitted to anti SMASH database to predict the biosynthetic gene clusters of the natural products.The gene recombination method was used to construct gene engineering strains of the gene cluster N5.1NPRS(42,890 bp)that is a novel nature product biosynthetic gene cluster(might encode the small nitro molecules).Results1.In this study,a total of 204 strains of marine bacteria were isolated,and 94 strains of pure culture were obtained after 16S rRNA gene sequencing and replication,including 25potential novel taxons.2.16S rRNA gene sequence analysis demonstrated that strains C1424,C2222,H2322,H3510 and R2331 were most closely to model strains of Marinomonas,Marinomonas,Arcticiflavibacter,Labrenzia and Loktanella,with similarities 98.1%,98.1%,96.8%,98.2%,96.3%,respectively.Genomic DNA sequence analysis demonstrated that strains C1424,H2322,H3510 and R2331 showed highest ANI values with 75.69%,72.63%,74.69%and72.61%and showed highest DNA-DNA hybridization values with 19.8%,13.8%,17.8%and14.4%to phylogenetic related species,respectively.Combined with phenotypical,physiological,biochemical and chemical characterizations,strains C1424,C2222,H2322,H3510 and R2331 represented novel species of the genera Marinomonas,Marinomonas,Arcticiflavibacter,Labrenzia and Loktanella,respectively.3.Pseudoaltermonas sp.A6021,a red pigment producing strain with special phenotype,produced more red pigments when cultured in R2A medium at 37?.According to the analysis of liquid chromatography-tandem mass spectrometry(LC-MS/MS),the molecular weights of the red pigments were all about 350 Da,different from each other by 14 Da(about one CH₂ group).4.The results of anti SMASH analysis demonstrated that strain A6021 contain 10natural product biosynthetic gene clusters.Gene clusters N5.1NPRS has no similarity with the reported natural product biosynthetic gene clusters in the anti SMASH database.Combined with the reported literature,we speculated that N5.1NPRS may be a novel biosynthetic gene cluster encoding a new type of nitro small molecules.Genetic engineering strains of cluster N5.1NPRS(42,890 bp)were constructed.According to the results of HPLC,fermentation products of strain A6021-N5.1NPRS/BAP1 demonstrated 2 peaks different from control in LB supplemented with aspartic acid as substrate.Conclusions1.Twenty-five potential novel taxons were obtained.The taxonomic position of the five novel species were determined.The results enriched culturable marine bacterial resources database.2.Red pigments produced by Pseudoaltermonas sp.A6021 was a series of substances that was different from each other with 14 Da molecular weight.Strain A6021 contained 10natural product biosynthetic gene clusters.Genetic engineering strains of cluster were successfully constructed,which lied foundation for the future research of its function.