The ars operon in the skin element of Bacillus subtilis confers resistance to arsenate and arsenite

Abstract

The Bacillus subtilis skin element confers resistance to arsenate and arsenite. The ars operon in the skin element contains four genes in the order arsR, ORF2, arsB, and arsC. Three of these genes are homologous to the arsR, arsB, and arsC genes from the staphylococcal plasmid pI258, while no homologs of ORF2 have been found. Inactivation of arsR, arsB, or arsC results in either constitutive expression of ars, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, respectively. These results suggest that ArsR, ArsB, and ArsC function as a negative regulator, a membrane-associated protein need for extrusion of arsenite, and arsenate reductase, respectively. Expression of the ars operon was induced by arsenate, arsenite, and antimonite. Northern hybridization and primer extension analysis showed that synthesis of a full-length ars transcript of about 2.4 kb was induced by arsenate and that the ars promoter contains sequences that resemble the -10 and -35 regions of promoters that are recognized by E sigmaA.

FIG. 1

Genetic construction of the ars mutants designated ars-R3, ars-23, ars-B3, and ars-C. The relevant portions of the genomes of the mutant strains are shown. Pars, ars promoter; Pspac, spac promoter; Em^r, erythromycin resistance gene; Cm^r, chloramphenicol resistance gene.

FIG. 2

Inhibition of growth of various B. subtilis strains by arsenate, arsenite, and antimonite. (A) Resistance of strains JH642 (○), skin-1 (•), ars-C (▴), and QUA2 (▵). (B) Resistance of strain ars-R3 with IPTG (•) (ORF2, arsB, and arsC are induced) or without IPTG (○) (ORF2, arsB, and arsC are not induced). (C) Resistance of strain ars-23 with IPTG (•) (arsB and arsC are induced) or without IPTG (○) (arsB and arsC are not induced). (D) Resistance of strain ars-B3 with IPTG (•) (arsC is induced) or without IPTG (○) (arsC is not induced). Overnight cultures of JH642, skin-1, ars-R3, ars-23, ars-B3, QUA2, and ars-C were inoculated into fresh LB broth (5 Klett units) with or without 1 mM IPTG (for panels B, C and D) and increasing amounts of arsenate, arsenite, or antimonite. Cultures were incubated for 8 h at 37°C, and then the turbidity (Klett units) was measured.

FIG. 3

Induction of the ars operon. Cells of strains ars-R3 (A), ars-23 (B), and ars-B3 (C) were grown in LB broth at 37°C to the exponential phase of growth (50 Klett units), and then the ars operon was induced by the addition of the indicated amount of arsenite (▵), antimonite (□), or arsenate (○). β-Galactosidase activity was measured 1 h after induction.

FIG. 4

Analysis of the transcript of the ars operon. S1 nuclease mapping and Northern hybridization experiments were performed as described previously (25). (A) Structure of the ars operon and nucleotide sequence of the upstream region of the ars operon (24). Sizes of ORFs are shown to scale. Solid lines indicate the sizes and positions of the probes used. Dashed lines indicate the sizes and deduced positions of the S1 product and the observed mRNA. The site of initiation of transcription is indicated by an arrow. Putative −10 and −35 RNA polymerase-binding regions of the ars promoter are underlined, and the putative translation initiation codon of arsR is boxed. The converging arrows indicate the region of inverted repeats. b, bases. (B) Northern blotting analysis of the ars transcript. Lanes 1 and 2, transcript hybridized with the 2,804-bp probe. Lane 3, transcript hybridized with the 573-bp probe. Total RNA was isolated from strains skin-1 (lane 1) and JH642 (lanes 2 and 3) 20 min after induction by 1 mM arsenate. (C) S1 nuclease mapping of the ars transcript. The ³²P-labelled 573-bp PCR product (20,000 cpm; approximately 10 ng) was hybridized with 50 μg of RNA that had been prepared as described previously (9). Lanes 1 through 7, S1 nuclease-protected fragments of the probe that had been hybridized with RNA isolated from JH642 cells which had been incubated for 0, 2, 5, 10, 20, 30, and 60 min, respectively, after the addition of 1 mM arsenate. Lane 8, ³²P-labelled HpaII fragments of phage M13mp18 as size markers. (D) Primer extension mapping of the ars transcript. The primer extension reaction was performed as described previously (27). Dideoxynucleotide sequencing reactions, performed with the same primer as used to map the 5′ end of the ars transcript, were included in the analysis. The complementary base at the initiation site is indicated by an asterisk.

FIG. 5

Comparison of the B. subtilis ars operon and the probable ars operon of M. tuberculosis. aa, amino acid residues.