Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T

Abstract

Background: Arsenic (As) is a natural metalloid, widely used in anthropogenic activities, that can exist in different oxidation states. Throughout the world, there are several environments contaminated with high amounts of arsenic where many organisms can survive. The most stable arsenical species are arsenate and arsenite that can be subject to chemically and microbiologically oxidation, reduction and methylation reactions. Organisms surviving in arsenic contaminated environments can have a diversity of mechanisms to resist to the harmful effects of arsenical compounds.

Results: The highly metal resistant Ochrobactrum tritici SCII24 was able to grow in media with arsenite (50 mM), arsenate (up to 200 mM) and antimonite (10 mM). This strain contains two arsenic and antimony resistance operons (ars1 and ars2), which were cloned and sequenced. Sequence analysis indicated that ars1 operon contains five genes encoding the following proteins: ArsR, ArsD, ArsA, CBS-domain-containing protein and ArsB. The ars2 operon is composed of six genes that encode two other ArsR, two ArsC (belonging to different families of arsenate reductases), one ACR3 and one ArsH-like protein. The involvement of ars operons in arsenic resistance was confirmed by cloning both of them in an Escherichia coli ars-mutant. The ars1 operon conferred resistance to arsenite and antimonite on E. coli cells, whereas the ars2 operon was also responsible for resistance to arsenite and arsenate. Although arsH was not required for arsenate resistance, this gene seems to be important to confer high levels of arsenite resistance. None of ars1 genes were detected in the other type strains of genus Ochrobactrum, but sequences homologous with ars2 operon were identified in some strains.

Conclusion: A new strategy for bacterial arsenic resistance is described in this work. Two operons involved in arsenic resistance, one giving resistance to arsenite and antimonite and the other giving resistance to arsenate were found in the same bacterial strain.

Figure 1

Genetic organization of the two arsenic resistance clusters in strain O. tritici SCII24. Gene orientations are shown by arrows. Within the predicted structure of the promoters, the -35, -10 regions and ribosome binding sites (RBS) are boldfaced and ATG codons are in boxes.

Figure 2

Alignment of ArsR (A) and ArsC (B) proteins. The three ArsR sequences from O. tritici were aligned with ArsR of E. coli pR773 (P15905). Both ArsCs from O. tritici were aligned with ArsC homologues from E. coli pR773 (AAA21096) and Staphylococcus aureus pI258 (AAA25638). The multiple alignment was calculated with CLUSTAL W.

Figure 3

Expression of the O. tritici ars genes in E. coli AW3110 under control of ptrc promoter. The gel used was a SDS-12% polyacrylamide gel. Lane1, marker proteins, lane2, plasmid pTRC 99A without an insert; lanes 3, 4 and 5, construct parsRDAcbsB in absence of any oxyanion, in presence of 1 mM As(III) and 1 mM Sb(III), respectively; lanes 6, 7 and 8, construct parsDAcbsB without any oxyanion, in presence of 1 mM As(III) and 1 mM Sb(III), respectively.

Figure 4

Growth of E. coli AW3110 containing different constructs in the presence of arsenite (A and B), arsenate (C) and antimonite (D). Arsenite and antimonite resistance assays were performed in LB medium and arsenate growths were performed in low-phosphate medium. Each data point represents the results of three independent assays. The error bars indicate standard deviations. O.D. 600 nm, optical density at 600 nm.

Figure 5

RT-PCR analysis of ars1 genes of O. tritici SCII24. Total RNA isolated from O. tritici cells in the exponential phase was used as template in a reverse transcriptase reaction using the reverse primer from arsB to generate cDNA. Then, the several intergenic regions were amplified: arsR-arsD (ane1), arsD-arsA (lane2), arsD-cbs (lane3) and cbs-arsB (lane 4).

Figure 6

Southern blot analysis of ars genes in Ochobactrum strains. Panels contain hybridization results for arsA, gene coding for CBS domain, arsB, arsC1, Acr3, arsC2 and arsH genes. Lanes:1, O. tritici SCII24; 2, O. tritici 5bvl1; 3, O. grignonense OgA9a; 4, O. anthropi LMG 3331; 5. O. intermedium LMG 3301