The chromate-inducible chrBACF operon from the transposable element TnOtChr confers resistance to chromium(VI) and superoxide

Abstract

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

FIG. 1.

(A) Growth of O. tritici 5bvl1 and E117 mutant on LB plates containing the indicated chromate concentrations. (B) Southern hybridization of total DNA from mutant E117 digested with SphI and SalI and strain 5bvl1 digested with SphI as a control. An internal transposon fragment labeled with dioxigenin-dUTP was used as a probe. Both 5bvl1 and E117 samples were analyzed on the same blot.

FIG. 2.

(A) Physical map of the chr locus in O. tritici 5bvl1 strain. The Tn5 insertion in chrA is indicated by the small black vertical arrowhead. (B) Alignment of the inverted repeat sequences of TnOtChr, Tn21, and Tn3. Identical bases are indicated by dark shading.

FIG. 3.

(A) Sensitivity of O. tritici strains to chromate. Cultures of O. tritici 5bvl1 (♦), O. tritici^T (▪), O. tritici^T chrFCAB (▴), O. tritici^T chrCAB (×), O. tritici^T chrAB (□), and O. tritici^T chrB (○) strains were grown in Tris-glucose medium for 15 h at 37°C in the presence of the indicated concentrations of chromate. (B) Clonogenic survival at different chromate concentrations. The cultures were plated on LB plates, and the colonies were counted after 3 days of incubation at 37°C. Strain symbols are as defined above for panel A. (C) Chromate uptake by O. tritici strains. Exponential-phase cells were incubated at 37°C with chromate for 3 h, and cellular chromium concentrations were measured as described in Materials and Methods. (D) Survival of O. tritici 5bvl1 (♦), O. tritici (○), E117 mutant (▴), and E117:chrA (×) strains at different chromate concentrations. The cultures were also plated on LB plates, and the colonies were counted after 3 days of incubation at 37°C. (E) Chromate uptake. Exponential growing cells were exposed to 1 mM chromate for 3 h. Values are means ± standard deviations (error bars) from three independent experiments. (F) Chromate efflux by O. tritici 5bvl1 (○), E117 mutant (▪), and E117:chrA (▴) strains. Cells were first preincubated with 0.1 mM chromate for 1 h to induce the chr operon and then pulse-loaded for 15 min with high chromate concentrations (1 mM chromate for E117 mutant and 5 mM chromate for strains 5bvl1 and E117:chrA), resulting in approximately equal initial levels of cellular chromium. Cells were washed and then incubated in fresh medium for 0 to 30 min at 37°C to assess efflux of cellular chromium. Results are the percentages of remaining cellular concentrations of Cr after normalization for protein content (means ± standard deviations [error bars] from three independent experiments).

FIG. 4.

Induction of chrB expression in 5bvl1 cells by Cr(VI). The presence of chrB cDNA was detected by RT-PCR. Total RNA was isolated from control and 0.5 mM chromate-treated cells. The chrB RT-PCR product was amplified from total RNA extracted from mid-log-phase control cells and Cr(VI)-exposed cells. Control PCRs for DNA contamination contained Taq polymerase but lacked reverse transcriptase (RT). The quality of isolated RNA was verified by RT-PCR of 16S rRNA. All samples were analyzed on the same gel.

FIG. 5.

Regulation of the chr promoter. (A) Reporter activity of the promoterless lacZ-carrying pSJ3 (○) and chrp::lacZ vectors (▪) following treatment of the cells with Cr(VI) for 1 h. (B) Reporter activity using the chrBp::lacZ construct. Cultures at mid-log phase were incubated with or without chromate (○), dichromate (▪), or Cr(III) (▴) for 1 h. (C) Activity of the chrBp::lacZ reporter in cells incubated with different concentrations of sulfate in the presence and absence of 10 μM chromate. (D and E) chrBp::lacZ reporter activity in cells incubated for 1 h in the presence of different concentrations of oxyanions (D) or oxidants (E). Values are means ± standard deviations (error bars) from at least two experiments.

FIG. 6.

Sensitivity of E. coli strains to superoxide-producing paraquat (A) and menadione (B). Cell suspensions were plated on LB plates, and the colonies were counted after 24 h of incubation at 37°C. The wild-type E. coli AB1157 (•) strain and the mutant strain JI132 carrying construct pTrc99A (♦), pTrc_chrF (□), pTrc_chrC (▴), pTrc_chrFC (○), or pTrc_chrB (▪) are shown.

FIG. 7.

Growth of E. coli strains in M9 mineral medium under different aerobic conditions. (A to C) The bacteria were grown in the presence of all 20 amino acids (A); all amino acids, except phenylalanine, tryptophan, and tyrosine (B); and all amino acids, except cysteine and methionine (C). Wild-type E. coli AB1157 (•) strain and the mutant strain JI132 carrying construct pTrc99A (♦), pTrc_chrF (□), pTrc_chrC (▴), pTrc_chrFC (○), or pTrc_chrB (▪) are shown. Growth was monitored at 600 nm by examining the OD₆₀₀. (D) Detection of SOD activity on native gels. Protein extracts (50 μg) were separated on 10% polyacrylamide, and the presence of proteins with SOD activity was visualized by illumination of gels impregnated with a riboflavin-nitroblue tetrazolium mixture. JI132 is a sodA sodB double null E. coli mutant. WT, wild-type E. coli AB1157.