Regulation Of Arsenite Oxidation By The Phosphate Regulatory System PhoBR In Arsenite Oxidizing Halomonas Sp.HAL1

Arsenic is a kind of toxic metalloid,and its contamination is serious in China.Some bacteria can oxidize arsenite[As(Ⅲ)]to less toxic arsenate[As(Ⅴ)]using As(Ⅲ)oxidase AioBA,which could be used in bioremediation.Understanding of bacterial As(Ⅲ)oxidation regulatory mechanism would provide theoretical foundation and technology support for the bioremediation of arsenic contamination.So far,the research found that the expression of As(Ⅲ)oxidase AioBA is regulated by a three component regulatory system AioXSR.However,the As(Ⅲ)oxidation regulatory mechanism of other bacteria without AioXSR,such as Halomonas sp.HAL1 is still unknown.Thus,in this study,we used strain HAL1 as a model to elucidate the As(Ⅲ)oxidation regulatory mechanism of the As(Ⅲ)-oxidizing bacteria without AioXSR.Moreover,during the study of bacterial resources of mining soil,a heavy metal resistant novel species Arenimonas metalli CF5-1T was found and identified.In addition,the comparative genomics analysis were conducted within the genus Arenimonas.The major results of this dissertation were as follows:A,Regulation of arsenite oxidation by the phosphate two component system PhoBR in Halomonas sp.HAL1Transposon mutagenesis in combination with qualitative KMnO4 screening method were used to isolate As(Ⅲ)oxidation mutant of strain HAL1.Five mutants disabling for As(Ⅲ)oxidation were screened from about 2000 tranformates.One mutant was interrupted in phoR which coding for the sensory histidine kinase of phosphate two component regulatory system PhoBR.Since PhoB is the response regulator in the two component system PhoBR,the mutant HAL1-△phoB and the complementary strain HAL1-△phoB-C were constructed.Growth and As(Ⅲ)oxidation tests of HAL 1,HAL1-phoR931,HAL1-△phoB and HAL1-△phoB-C in low Pi(Pi=0.1 mM)and normal Pi(Pi=1.0 mM)condition found that:the As(Ⅲ)oxidation phenotype of two mutants were negative only in low Pi condition.Therefore,the phosphate two component regulatory system PhoBR was essential for As(Ⅲ)oxidation of strain HAL1 in low Pi condition.Quantitative lacZ reporter gene analysis of the expression of PhoBR and AioBA in strain HAL1 at low Pi and normal Pi condition found that:the expression of PhoBR was induced by As(Ⅲ)in low Pi condition,not in normal Pi condition;while the expression of AioBA was induced by As(Ⅲ)in both low and normal Pi conditions.Moreover,in low Pi condition,the expression of AioBA was up-regulated by As(Ⅲ)significantly in HAL1 and HAL1-△phoB-C,but not in HAL1-phoR931 and HAL1-△phoB;in normal Pi condition,the expression of AioBA was up-regulated by As(Ⅲ)significantly in all strains.PhoBR could respond to As(Ⅲ)and activate the As(Ⅲ)inducing expression of AioBA in strain HAL1 at low Pi condition.One putative Pho box was predicted in the regulatory region of aioBA.The results of bacterial one-hybrid assay and EMSA indicated PhoB could bind to the putative Pho box of aioBA promoter region in vivo and in vitro,respectively.The mutation of Pho box will lead to the interaction disappeared.Based on above results,in low Pi condition,the AioBA expression in strain HAL1 was regulated by PhoBR.However,in normal Pi condition,the expression of AioBA was not influenced by PhoBR.B.Taxonomic identification of a heavy metal resistance novel species Arenimonas metalli CF5-1T and genome analysisThe bacterium CF5-1 was isolated from iron mine of Hongshan Iron Mine of Daye city,Hubei province,China.Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CF5-1 was most closely related to Arenimonas strains(94.5%-95.3%).Polyphasic taxonomic studies showed that strain CF5-1 represents a novel species of the genus Arenimonas,for which the name Arenimonas metalli sp.nov.is proposed based on isolation site.The major characteristics of strain CF5-1 were that:gram-negative;aerobic;rod-shaped;without flagella;the genomic DNA G+C content was 70.5 mol%;the major respiratory quinone was Q-8;the major cellular fatty acids were iso-C16:0,iso-C15:0,C16:1ω7c alcohol and iso-C17：1ω9c;the major polar lipids were DPG,PG and PE.Strain CF5-1 could resistant to Sb3+,As3+,Zn2+,Cu2+,Ni2+,Co2+,Cr6+and Fe3+.The type strain is CF5-1T(=CGMCC 1.10787T=KCTC 23460T=CCTCC AB 2010449T).The genomic sequencing of Arenimonas strains provided the genomic information of this genus for the first time.Comparative analysis of different Arenimonas species was conduct.The basic metabolic pathways analysis indicated that:TCA cycle is complete in five Arenimonas strains;hexokinase which catalyze the first step of glycolysisis absent in these five strains,this is consistent with the negative phenotype of glucose assimilation of Arenimonas strains;enzymes responsible for the oxidative phase of pentose phosphate pathway are incomplete in five Arenimonas strains,this may be part of the reason that only several single carbon sources could be utilized by the Arenimonas strains.These results confirmed the reliability of phenotypic characteristics in genomic level for the genus Arenimonas.In conclusion,a novel As(Ⅲ)oxidation regulatory mechanism of bacteria was illustrated in this dissertation,which could provide theoretical foundation for bioremediation of arsenic contamination.Taxonomic identification of a novel species Arenimonas metalli CF5-1T enriched bacterial diversity of the genus Arenimonas.Comparative genomics analysis provided genomic information of the genus Arenimonas for the first time,confirmed the reliability of phenotypic characteristic at genomic level,could lay the foundation for using genomic information in polyphasic taxonomy and other application in the future study.