Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267

Abstract

The arsenate, arsenite, and antimonite resistance region of the Staphylococcus xylosus plasmid pSX267 was subcloned in Staphylococcus carnosus. The sequenced DNA region revealed three consecutive open reading frames, named arsR, arsB, and arsC. Expression studies in Escherichia coli with the bacteriophage T7 RNA polymerase-promoter system yielded three polypeptides with apparent molecular weights of 8,000, 35,000, and 15,000, which very likely correspond to ArsR, ArsB, and ArsC, respectively. ArsB was distinguished by its overall hydrophobic character, suggesting a membrane association. The arsenate, arsenite, and antimonite resistance was shown to be inducible by all three heavy metal ions. Inactivation of the first gene, arsR, resulted in constitutive expression of resistance. Similar results were obtained with transcriptional fusions of various portions of the ars genes with a lipase reporter gene, indicating a function of ArsR as a negative regulator of a putative promoter in front ofarsR. The inactivation ofarsR also resulted in reduction of resistance to arsenite and antimonite, while arsenate resistance was unaffected. The three ars genes conferred arsenite resistance in E. coli and arsenite as well as arsenate resistance in BaciUus subtilis.Plasmid-mediated resistance to arsenate, arsenite, and antimonite salts in Staphylococcus aureus was first described by Novick and Roth (29). The resistance genes are normally found on plasmids encoding penicillinase and various heavy metal resistance determinants, such as resistance to Cd2' and Hg'. The arsenical resistance genes on plasmid p1258 are composed of at least two loci (23), which are clustered (28). Studies on the arsenical resistance operon of the Escherichia coli conjugative R-factor plasmid R773 and the S. aureus plasmid pI258 revealed that the resistance determinants from both organisms are inducible by arsenate, arsenite, or antimonite. The mechanism of plasmid-mediated arsenate resistance in both E. coli and S. aureus is based on an efflux system (25,42), which, at least in E. coli, functions as an ATPase. It was demonstrated that, at least in E. coli, arsenite is translocated similarly to arsenate (32).Arsenate and arsenite resistances are common in other staphylococcal species. An investigation of a collection of Staphylococcus xylosus strains isolated from soybean oil meal showed that more than 90% were arsenate and arsenite resistant and that the resistance genes were plasmid encoded. One strain, S. xylosus DSM 20267, harboring a 29.5-kb plasmid, designated pSX267, was studied in more detail. In searching for a pSX267-associated resistance to different antibiotics, no indication of plasmid-bome resistance to any of 63 antibacterial drugs tested was detected in this strain. Heteroduplex analysis between pSX267 and p1258 revealed only high DNA homology within the arsenical resistance genes and the replication region (14).The arsenical resistance genes of pSX267 have been subcloned in Staphylococcus carnosus by using pC194. The resulting plasmid, p...

Dual mode of energy coupling by the oxyanion-translocating ArsB protein

“…Similar arsenite resistance operons have been identified in plasmids of the gram-positive bacteria Staphylococcus aureus(pI258) (12) and Staphylococcus xylosus(pSX267) (24). The staphylococcal ars operons do not contain an arsA gene, yet the resistant cells still actively extrude arsenite (27,28), suggesting that the ArsB protein can transport arsenite in the absence of an arsA gene product.…”

“…This motif is characteristic of secondary carrier proteins such as the lactose permease (13,16). Second, the ars operons of staphylococcal plasmids have no gene for an ATPase subunit (12,24), and yet they confer arsenite resistance and energy-dependent arsenite extrusion (4,12). Third, expression of the R773 arsB gene in the absence of the arsA gene confers an intermediate level of arsenite resistance in E. coli (Fig.…”

Structure and Biosynthesis of Staphyloxanthin from Staphylococcus aureus

“…S. carnosus and S. aureus were grown in tryptic soy broth medium (Invitrogen) or LB medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl). The plasmid vectors pCA44 (7,8) and the xylose-inducible pTX15 (9, 10) were used; for induction in the latter case, xylose was added to the medium to a final concentration of 0.5%. PCR was carried out with Vent polymerase (New England Biolabs) or Expand TM Long Template PCR System (Roche Applied Science).…”

Arsenate reduction and expression of multiple chromosomal ars operons in Geobacillus kaustophilus A1

“…These genes are often plasmid-encoded and are widespread in prokaryotes (Bruhn et al, 1996;Ji & Silver, 1992b;Oden et al, 1994;Rosenstein et al, 1992). In thoroughly studied systems, the ars operon has been reported to contain the five genes arsRDABC, as in Escherichia coli (Cai & DuBow, 1996;Rosen et al, 1992), or at least the three genes arsRBC, as in Staphylococcus aureus (Silver et al, 1993).…”