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Isolation,Bioactivity Screening And Identification Of Actinomycetes From Extreme Habitats

Posted on:2020-01-30Degree:DoctorType:Dissertation
Country:ChinaCandidate:Full Text:PDF
GTID:1360330590954111Subject:Biochemistry and Molecular Biology
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Extreme environments are rich sources of novel actinobacteria with the unique capacity to produce novel bioactive compounds.This present study was focused on the actinomycetes from two extreme habitats,deep-sea and Arctic region.The deep sea is an extreme and unique environment,considered to start at about 200 m depth,and divided into the bathyal zone(depths between 200-2,000 m),the abyssal(depths between 2,000-6,000 m)and the hadal zone(depths below 6,000 m).It is an excellent spot for hunting marine actinomycetes and secondary metabolites.A total of 43 actinomycete strains were isolated from the different depth(34-3,235 m)of 14 marine sediments,among which 32 were from the deep-sea samples(1,645-3,235 m).Non-heat pretreatment was effective for actinomycete isolation.RH and M6 were more effective media regarding the number and diversity of isolates recovered.A higher percentage of actinomycete colonies were obtained from the plate stamping technique(15.18%)than the dilution plate method(3.85%).Moreover,low-temperature incubation(10 °C)was also significantly effective on actinomycetes isolation from deep-sea samples with a higher percentage of the ratio of actinomycetes to other total viable bacteria.Higher number and diversity of actinomycete genera were found in deep-sea zones(depth 1,675-3,235 m)with 6 different genera comparing to shallow marine sediments(depth < 200 m)with 3 different genera.Partial 16 S r RNA gene sequence data revealed that the isolates belong to the Class Actinobacteria,with genetic affiliations to three orders,six families,and six genera.Micromonospora(n=23)and Streptomyces(n=14)were the dominant genera,followed by Dietzia(n= 2),Tsukamurella(n= 2),Blastococcus(n=1)and Microbacterium(n=1).Among 43 marine actinomycetes,18 isolates exhibited antimicrobial activities against at least one of the pathogens,Staphylococcus aureus ATCC 5165(37.21%)and Candida albicans ATCC 10231(27.91%).The bioactive substance-producing isolates belonged to the genera Micromonospora(n=8),Streptomyces(n=8)and one isolate for each belonged to the genera Tsukamurella and Microbacterium.The higher number of bioactive substanceproducing isolates was recovered from deep-sea samples(14 out of 32 isolates;43.75%)when compared to the active isolates from shallow marine sediments(4 out of 11 isolates;36.36%).Thirty-one isolates(72.09%)possessed at least one of the three biosynthetic gene clusters.Among these,24 isolates(75% of all deep-sea isolates)were obtained from deep-sea sediments(2,017-3,235 m).PKS-II pathways were found to be the most distributed(44.19% of all isolates),followed by PKS-I pathways(37.21%)and NRPS pathways(11.63%).PKS and NRPS gene screening positive strains belonged to genera Micromonospora(n=21)Streptomyces(n=8),Blastococcus(n=1)and Microbacterium(n=1).Our preliminary studies showed that the deep South China Sea in the Abyssal zone(2,017-3,235 m)represent an important extreme habitat that has remarkably high genera richness which could provide the potential resources for secondary metabolite production.The Arctic region has been considered as extremely cold,lacking liquid water and having high levels of UV radiation all year round.In this study,strain ZLN712 T,which had been isolated from the foreland of a glacier in the High Arctic(78°55'847 " N 012°13'323" E),at a depth of 12 m in July 2014,was identified using a polyphasic approach.The 16 S r RNA gene sequence of strain ZLN712 T showed the highest similarity to Streptomyces polygonati NEAUG9T(99.17%),followed by Streptomyces yanglinensis 1307T(98.10%).Result from 16 S r RNA gene sequencing and phylogenetic tree supported the classification of the strain ZLN712 T in the genus Streptomyces.DNA–DNA relatedness(54.4 ± 0.0%)between the strain and its close phylogenetic neighbours showed that it belonged to distinct species.Multilocus sequence analysis(MLSA)using four housekeeping genes(atp D,gyr B,rec A,and rpo B)for comparing Streptomyces type strains showed that the MLSA distance of strain ZLN712 T to the closely related species was greater than the 0.007 threshold.The DNA G+C contents of the genomic DNA of stain ZLN712 T was 72.77 mol%.The results of genotypic,phenotypic and chemotypic studies allowed genotypic,phenotypic and chemotypic differentiation of strain ZLN712 T from phylogenetically closely related type strains.Therefore,strain ZLN712T(=CCTCC AA 2018011 T =DSM 107266T)represents a type strain of a novel species of the genus Streptomyces,for which the names Streptomyces septentrionalis sp.nov.is proposed.It indicated that the Arctic;an important extreme habitat could have been pursued as a source of novel actinobacteria and have an opportunity to obtain novel natural products from this habitat.
Keywords/Search Tags:Deep-sea, Marine actinomycetes, Isolation and diversity, Bioactivity, Secondary metabolism genes, Arctic, Novel species, Polyphasic taxonomy
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