A Na+:H+ antiporter and a molybdate transporter are essential for arsenite oxidation in Agrobacterium tumefaciens

Abstract

Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na+:H+ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genome-sequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na+ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.

FIG. 1.

Cartoon illustration characterizing the Tn5-B22 insertions in each mutant and the genomic DNA PCR cloned from A. tumefaciens strain C58 for complementation experiments. For each mutant, the gray arrows represent specific genes and their orientation as they occur in the A. tumefaciens C58 genome and as they were PCR cloned into their respective mutants. Small horizontal bars underneath the gray arrows indicate regions of the respective mutant genome that were sequenced via primer walking experiments. (A) Tn5-B22 insertion in an mrpB homologue (annotated as mnh in strain C58). Point of insertion was estimated to be at nt 8 based on nucleotide alignments with the C58 strain mrpB/mnhB. (B) Tn5-B22 insertion that approximately bisects the modB gene (based on alignment with the A. tumefaciens C58 modB). Point of insertion was estimated to be at nt 299 based on nucleotide alignments with the C58 strain homologue. Tn5-B22 insertion points are indicated by the inverted arrowheads. Positions of the priming sites (identified in Results) used for amplification are shown.

FIG. 2.

Complementation of the As(III)-oxidase mutants. (A)MSUAt2. (B) MSUAt6. Culture growth is shown with closed symbols, and As(V) concentration is shown with open symbols. Symbols: squares and circles, MSUAt2 or MSUAt6; triangles, MSUAt2(pLB403) or MSUAt6(pLB404); diamonds, MSUAt2(pRK311) or MSUAt6(pRK311). Results are from one of two independent experiments demonstrating complementation. Error bars, where visible, represent 1 standard error of the mean calculated from two replicate cultures. Starting As(III) concentration was 50 μM.

FIG. 3.

Salt tolerance of A. tumefaciens as affected by the mrpB::Tn5-B22 mutation. (A) NaCl. (B) LiCl. Wild-type strain, □; mutant MSUAt2, ▪; mutant MSUAt2 complemented with the cloned C58 mrp/mnhABCDEFG operon (pLB403), •. Cultures were grown in modified LB broth amended with various amounts of NaCl or LiCl as shown. Results are the mean ± 1 standard error averaged from single cultures from two independent experiments (error bars are hidden by the symbols).

FIG. 4.

AsV reduction phenotype of the mrpB mutant. Shown is the AgNO₃ staining phenotype of MSUAt2 on MMN agar containing 1 mM As(V). The presence of As(V) is indicated by dark brown, whereas the presence of As(III) is indicated by yellow. Results with MSUAt6 were identical and are not shown.

FIG. 5.

Expression of modB and mrpB in response to As(III) exposure. (A) RT-PCR cDNA amplified from wild-type strain 5A RNA. Lane assignments: lane 1, molecular mass standards; sizes in kilobases are noted to the left of image; lane 2, early-log-phase, As(III)-naïve cells; lane 3, As(III)-exposed cells; lanes 4 and 5, amplicons derived from RT-PCRs using primers specific for the 16S rRNA as internal controls and corresponding to lanes 2 and 3, respectively. (B) mrpB::lacZ reporter gene activity recorded with MSUAt2 (closed symbols) and wild-type 5A (open symbols). Reporter enzyme units are ΔA₄₁₅ · min⁻¹ · culture optical density (measured as A₅₉₅)⁻¹. Cultures were incubated without As(III) (squares) or with 100 μM As(III) throughout the entire incubation (triangles) or spiked with 100 μM As(III) at mid-log phase (circles). Error bars represent 1 standard error of the mean calculated from triplicate cultures. Some standard errors are hidden by the symbols.