Polyphasic Taxonomy Investigations and Functional Genomic Analysis of Two New Species of Arsenic-resistant Bacteria

Author:Mu Yao

Supervisor:zeng xian chun

Database:Doctor

Degree Year:2019

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Pages:149

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The groundwater in some areas in Jianghan Plain was found to be contaminated by arsenic.However,little is known about the germplasm resources of the arsenic-resistant microbes present in the arsenic-containing sediments.In this work,two novel arsenic-resistant bacterial species,referred to as Luteimonas arsenica 26-35T and Pseudaminobacter arsenicus CB3T,were isolated from the arsenic-contaminated sediments of Jianghan Plain using enrichment culture and random sequencing of microbial 16S rRNA gene.The multi-phase taxonomic investigations and functional genomic analysis of the two novel bacterial species were thus conducted.A Gram-staining-negative,rod shaped bacterium that forms yellow and viscous colonies,was isolated from the arsenic-contaminated sediments collected from Jianghan plain,Hubei Province,China,and it was designated 26-35T.This strain can tolerate arsenate and arsenite up to MICs of 40 mM and 20 mM,respectively.The 16S rRNA gene of this novel isolate showed 96.7%–94.2%sequence similarities to those of other known species belonging to the genus Luteimonas.The respiratory quinone was ubiquinone-8(Q-8).The DNA G+C content was 71.4%.The predominant cellular fatty acids were iso-C15:0,iso-C16:0,iso-C17:0,iso-C11:0,iso-C11:0 3-OH,and iso-C17:1ω9c.The major polar lipids were diphosphatidylglycerol(DPG),phosphati-dylethanolamine(PE)and phosphatidylglycerol(PG).Phylogenetic and physiological analysis indicated that the isolate represent a novel species of the genus Luteimonas,of which the name Luteimonas arsenica sp.nov.is proposed.The type strain is 26-35T(KCTC 42824T=CCTCC AB2014326T).An arsenic-resistant strain,CB3T,was also isolated from arsenic-contaminated sediments sampled from Jianghan Plain.Phylogenetic and biochemical analyses suggested that it represents a new species belonging to the genus Pseudaminobacter(family Phyllobacteriaceae).The 16S rRNA gene of CB3T shared the highest sequence similarities to those of the type strains Pseudaminobacter defluvii THI 051T(97.8%identity)and Pseudaminobacter salicylatoxidans BN12T(97.4%).The DNA–DNA relatedness values of CB3T with respect to strains belonging to the genus Pseudaminobacter were less than 70%.The fatty acid profile of CB3T consisted of C16:0,cyclo-C19:0ω8c and summed feature 8(C18:1ω7c and/or C18:1ω6c)as major components.The major polar lipids were phosphatidylcholine(PC),phosphatidylglycerol,phosphatidyldimethylethanolamine(PDE),phosphatidylmonomethylethanolamine(PME),phosphatidylethanolamine(PE)and diphosphatidylglycerol(DPG).The DNA G+C content was 61.4%.On the basis of phenotypic,chemotaxonomic and phylogenetic data,strain CB3T was distinct from previously described Pseudaminobacter species.Therefore,we propose that strain CB3T represents a novel species belonging to the genus Pseudaminobacter,and Pseudaminobacter arsenicus sp.nov.,strain CB3T(=CCTCC AB2016116T=KCTC 52625T)is designated as the type strain.This work suggested that Pseudaminobacter arsenicus CB3T possesses the abilities to reduce nitrate into ammonium and to denitrify under anaerobic conditions.The denitrification efficiency of CB3T was significantly affected by carbon-nitrogen ratio,and was inhibited by As(V).Functional genomic analysis provided the molecular basis for the arsenic resistance and nitrate reduction of this strain.We found that CB3T possesses three arsenic resistance operons,two types of intracellular arsenate reductase genes,and multiple As()excretion proteins;this is responsible for the high arsenic resistance ability of the strain.Furthermore,the strain bears nitrate reductase(Nap),nitrite reductase(Nir)and nitrous oxide reductase(Nos),suggesting that it has a relatively complete denitrification system;however,the key genes involving in dissimilatory nitrate reduction to ammonium(DNRA),nrfA and nirB,are absent in the bacterial cells,indicating that bacteria may use other unknown metabolic pathways for DNRA.This study also analyzed the presence and organization of arsenic related genes within the genomes of P.arsenicus CB3T and 180 strains belonging to family Phyllobacteriaceae.We identified 89 unique arsenic-resistant operons structures from Phyllobacteriaceae family.Unlike most arsenic-resistant bacteria,which use ArsB as the As(III)efflux protein,Phyllobacteriaceae family bacteria employ ACR3,AqpS and ArsK efflux proteins for detoxification.Comparing to other Phyllobacteriaceae species,P.arsenicus CB3T contains more diverse arsenic-catalyzing genes.This work offered valuable germplasm resources of arsenic-resistant bacteria for further investigations of the interactions between bacteria and arsenic,and gained insights into the mechanisms for bacteria to adapt themselves to high arsenic environment.