Analysis of genes involved in arsenic resistance in Corynebacterium glutamicum ATCC 13032

Abstract

Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1') located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1', arsR2, arsB2, and arsC2 being inducible by arsenite.

FIG. 1.

Schematic representation of genes involved in arsenic resistance in C. glutamicum ATCC 13032 (A) and in other bacteria (B). (A) Operons ars1 and ars2 are indicated. Arrows represent open reading frames. Dashed boxes indicate the probes used for the cloning of internal fragments of the desired genes obtained by PCR amplification. (B) The three-gene operon (arsRBC), encoding the transcriptional repressor (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC), is present in the E. coli K-12 chromosome (accession number NC_000913), Staphylococcus plasmids pI258 (31) and pSX267 (48), C. glutamicum (NC_003450), C. efficiens (NC_004369), and R. erythropolis plasmid pBD2 (NC_005073). The five-gene operon (arsRDABC), encoding an arsenite-inducible repressor (arsR), a negative regulatory protein (arsD), an oxyanion-protruding ATPase, an arsenite efflux pump (arsA and arsB, respectively), and an arsenate reductase (arsC), is present in the E. coli plasmids R773 and R46 (10, 17) and in plasmid pKW301 from A. multivorum (57). The four-gene operon has been found in different species, such as the skin element of B. subtilis (52), A. ferrooxidans (11), plasmid R478 from S. marcescens (49), a Synechocystis sp. (36), and Pseudomonas putida (NC_002947). An isolated arsC gene is present in the genome of Haemophilus influenzae (NC_000907), and two operons involved in arsenic resistance have been identified on the chromosome of P. putida and C. glutamicum.

FIG. 2.

Complementation of C. glutamicum arsB mutants by the ars1 (pECAS1) and ars2 (pECAS2) operons. C. glutamicum RES167 (1), C. glutamicum ArsB1 (2), C. glutamicum ArsB2 (3), and C. glutamicum ArsB1-B2 (4) transformed with pECAS1 or pECAS2 were inoculated in TSA containing increasing amounts of arsenite (0 mM to 60 mM), incubated at 30°C for 36 h, and photographed. Please note that some strains containing plasmid pECAS2 were unable to grow in the presence of 20 mM arsenite and that none of them were able to grow in 60 mM arsenite.

FIG. 3.

Northern blot analysis of specific transcripts for arsB1, arsC1, arsC1′ arsB2, and arsC2 from C. glutamicum. Total RNA (15 μg) was isolated from C. glutamicum in the exponential phase (OD₆₀₀ = 1.5) in the absence (control) or presence of 5 mM arsenite, electrophoresed, transferred to nylon filters, and hybridized with internal fragments of arsB1, arsC1, arsC1′, arsB2, and arsC2 obtained by PCR (dashed boxes) using the primers described in Table S1 in the supplemental material. Size controls corresponding to the migration of bands in the gel are indicated.

FIG. 4.

Transcriptional analysis of arsB1-C1-C1′ by RT-PCR. Total RNA isolated from C. glutamicum in the exponential phase (OD₆₀₀ = 1.5) in the absence (lanes 1) or presence (lanes 2) of 5 mM arsenite was used as the template in a reverse transcriptase reaction with primer ars16 to generate cDNA and then amplified with primers (ars3/4, ars13/14, and ars15/16) internal to each gene. A positive control using C. glutamicum chromosomal DNA instead of cDNA was included (lanes 3), as well as RT-PCR of the 16S rRNA (rRNA) using primer rrn2 for the synthesis of cDNA and primers rrn1 and rrn2 for PCR amplification (see Table S1 in the supplemental material). DNA contamination of the RNA samples was ruled out by performing PCR directly on the RNAs (N).

FIG. 5.

The promoter of the arsB1 gene is induced in the presence of arsenite or arsenate. C. glutamicum [pGFP-EP] was grown in the presence of subinhibitory concentrations of arsenite (black bar) or arsenate (empty bar). C. glutamicum [pEGNC] (Table 1) was used as a negative control, and its fluorescence level was subtracted from all the values obtained. In all cases, the fluorescence level of each sample was divided by the OD₆₀₀ of the sample. The values are the means of four determinations, and the standard deviation is indicated on the bar top. The fluorescence level ratio of green fluorescent protein/OD₆₀₀ was measured on a Biotek Sinergy HT fluorimeter.