Phosphate Regulatory Protein PhoB-mediated Antimony Oxidation Regulation Mechanism In Agrobacterium Tumefaciens GW4

Antimony(Sb)is a toxic metalloid which can cause several human diseases,such as myocardial failure and hepatic necrosis.Some bacteria can oxidize the toxic antimonite[Sb(III)] to the less toxic antimonate [Sb(V)].Our previous studies have shown that in Sb(III)-oxidizing strain Agrobacterium tumefaciens GW4,Sb(III)oxidation is a co-metabolism process,including Sb(III)oxidase AnoA-catalyzed enzymatic Sb(III)oxidation and H2O2-mediated non-enzymatic Sb(III)oxidation.However,the regulatory mechanism of anoA remains unknown,and bacterial global response to Sb(III)has not been reported.In this study,global analysis of cellular responses to Sb(III)was performed using comparative proteomics with or without the addition of 50 ?M Sb(III)in strain GW4.In addition,the regulatory mechanism of anoA was investigated using a series of molecular biotechnologies.Using proteomics and MALDI-TOF-MS analyses,21 differentially expressed proteins were identified and all of them were up-regulated in response to Sb(III).As analyzed by KEGG pathway assignments,they were distributed in antimony oxidation and resistance,carbon metabolism,lipid metabolism,amino acid metabolism and phosphonate metabolism and phosphate transportation.Among these proteins,we found that the phosphate binding protein PstS was induced by Sb(III).It has been reported that PstS is regulated by phosphate transcriptional regulatory protein PhoB,which plays a global regulator role in various metabolism pathways.Thus,we proposed that the phosphate regulatory system may also involve in bacterial Sb(III)oxidation.Two phosphate regulatory systems were identified in the genome of strain GW4.The phosphate regulatory gene phoB1 is located upstream of the phosphate transport system pstBACS1 and transcripts in the opposite direction,whereas phoB2 gene is located downstream of the phosphate transport system pstBACS2 and transcripts in the same direction.The main results of this thesis are as follows:(1)lacZ reporter gene assay showed that phoB1 was not obviously induced by Sb(III),while phoB2 was significantly induced by Sb(III),suggesting that phoB2 appares to be more relevant toSb(III);(2)deletion of phoB2 significantly decreased the expression of anoA compared with that of wild-type strain GW4,while in strain GW4-?phoB1,the expression of anoA was only slightly decreased.In addition,the expression of anoA was almost disappeared in strain GW4-?phoB1/phoB2;(3)bacterial growth and Sb(III)oxidation experiments showed that Sb(III)had no effect on the growth of strains GW4-?phoB1,GW4-?phoB2and GW4-?phoB1/phoB2.Deletion of phoB1 and phoB2 respectively had no effect on the Sb(III)rate,while the Sb(III)oxidation rate was significantly decreased in double mutant strain GW4-?phoB1/phoB2;(4)The cellular H2O2 content in strain GW4-?phoB1 was higher than wild-type strain,and further increased in strain GW4-?phoB2.The phenotype of the complemented strain was recovered.Thus,the H2O2 mediated non-enzymatic Sb(III)oxidation was increased in strain GW4-?phoB1and further increased in GW4-?phoB2.However,the cellular H2O2 content was not high enough to compensate the function of anoA in strain GW4-?phoB1/phoB2.Thus,the Sb(III)oxidation rate was significantly decreased.(5)Bacterial one-hybrid system and EMSA assays demonstrated that PhoB1 and PhoB2 could bind with the promoter region of anoA respectively,both in vivo and in vitro.In addition,we also identified the consersed binding site.These results indicated that both two phosphate regulatory protein PhoB1 and PhoB2 could regulate Sb(III)oxidase gene anoA,however,PhoB2 plays a more important role in the regulation of bacterial Sb(III)oxidation.This study determined the regulatory mechanism of Sb(III)oxidase gene anoA in strain GW4 and analysed the global responses to Sb(III)in this heterotrophic bacterium.The results provides an important theoretical basis for the bioremediation of antimony pollution.