The Preliminary Research On The Mechanism Associated To Arsenic Resistance Of Pseudomonas Sp. E003

Arsenic is a virulent metalloid. Due to the geochemical origin and and human activities, arsenic has became a widespread contamination which is dangerous for people’s health. With the development of the bioremediation using microorganism, more and more arsenic resistant bacteria and their arsenic resistant mechanisms have been found and studied. However, the researches have been almost focused on the hot spots related the resistance mechanisms with arsenate reducase and arsenite oxidase as the core. The mechanisms of arsenic intake, extrusion, and transfering etc. were less investigated. The objective of this study was to inverstigate novel arsenic resistant mechanisms by study of an arsenic resistant Pseudomonas strain.In this study a high arsenic resistant bacteria was isolated and named as E003. It was characterized as an arsenate-reducing bacteria by KMnO4 screening method. Its MIC for arsenite was 36 mmol/L, which was at the high rank in the known bacteria. Based on the results of morphology, 16S rRNA gene （99%） and biophysical/biochemical tests, the strain is most probable belonged to Pseudomonas alcaligenes and named as Pseudomonas sp. E003. The arsenite transporter protein arsB gene and ACR3（2） gene were both cloned. Containing both arsB and ACR3（2） genes could be a reason for the high arsenite tolerance of E003 which is in agreement with previous researches.Using transposon mutagenesis techniques, by tri-parental mating, pTnMod-OTc was transducted into strain E003 to obtain transposon mutations with the help of the plasmid pRK2013. A random insertion mutant library of E003 was generated and a mutant strain named as M48 was isolated. Its MIC for arsenite was 45 mmol/L, increasing by 9 mmol/L compared to the wild type. Southern blot analysis further proved the mutant site was caused by a single insertion. The increase of the arsenite resistance was caused by the insertion fragment.At the cellular level, the increase of the total amount of arsenic in the cells and the increase of the cell volume were both observed. The increase rate of the arsenic in the cells was not as high as the increase rate of the cell dimension, so the arsenic concentration in the mutant was lower than that in the wild type. Thus the mutant strain M48 could grow in the medium with higher arseniite concentration.At moleculer level, an about 16 kb gene cluster was isolated using plasmid rescue and PCR amplification with degenerate primers. The gene cluster was classified as type II secretion system after the blast analysis. The insertion sequence was just inserted in the promoter region and did not destroy other genes. Except for the conventional intra-membrane protein genes xcpPQ and xcpSRTUVWXYZ , two genes （orfV and orfX） with unknown functions were also found. The protein encoded by orfV showed a 43% identity with a MalT-like regulator belonging to LuxR family （YP₀02586356）, which is probably a positive gene regulatory. The result of the Real-time quantification RT-PCR showed the expression of the xcpPQ gene, orfV gene and xcpR gene in the M48 mutant strain were all raised that were about 1.5-folds of the gene expression levels of the wild type E003. The insertion sequence probably changed the transcription efficiency of the original promoter or replaced it with stronger promoters. The increase of the expression led the enhancement of the typeⅡsecretion system, which may speed up the extrusion of the arsenic from the cell that is in agreement with the hypothesis at the cellular level.Remarkably, the expression of the orfX gene was 4-folds of the wild type. The protein encoded by the orfX gene showed a 65% identity with an animal heme peroxidase in peroxidase family （YP₀02584780）. It probably had similar functions of glutathione peroxidase and cytochrome c peroxidase to increase arsenic resistance by elimination of free radicals and association with electron transportation during arsenic metabolism.In this study, for the first time, the typeⅡsecretion system was found to associate with bacterial arsenite resistance. The results provide preliminary knowledges of the arsenic transportation mechanisms and gene regulation of bacterial arsenic resistance.