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.

Efflux Permease CgAcr3-1 of Corynebacterium glutamicum Is an Arsenite-specific Antiporter

“…It is not clear why CgAcr3-1 and CgAcr3-2 confer different levels of As(III) resistance because they are very similar to each other (72% sequence identity) (26). The results of immunoblots (Fig.…”

“…Arsenite Tolerance-Previously, we constructed three C. glutamicum mutant strains in which each Cgacr3 gene was disrupted (26). The observed As(III) resistance levels obtained for the mutants indicate that Cgacr3-1 and Cgacr3-2 are involved in arsenite resistance in C. glutamicum, whereas Cgacr3-3 is not (Fig.…”

“…Heterologous and homologous complementation analyses of Cgacr3s expressed under their own promoters were performed either in E. coli AW3110 (corynebacterial promoters are frequently functional in E. coli) (26,28), using the pKars1up derivative vectors pKacr3-1, pKacr3-2, and pKacr3-3, or in C. glutamicum 2⌬ars, in the latter case using integrative suicide (pKacr3-1, pKacr3-2, and pKacr3-3) or bifunctional (pECacr3-1, pECacr3-2, and pECacr3-3) vectors. Integrative pKars1up derivatives were incorporated into a 0.5-kb chromosomal region located at the 5Ј-upstream region of the ars1 C. glutamicum operon by means of an identical 0.5-kb fragment present in all of the pKars1up derivatives.…”

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

“…After gel purification, the DNA fragment was ligated into vector plasmid pTrcHis2A accordingly digested, generating plasmid pTrcHis2A-AmAcr3-His. A similar strategy was used to clone the Cgacr3 gene using chromosomal DNA from C. glutamicum strain ATCC 13032 (19) and the corresponding primers indicated in supplemental Table 3. The PCR-amplified band (1,113 bp) was subcloned into pGEM-T Easy (Promega, Madison, WI) and sequenced.…”

“…glutamicum is a Gram-positive soil bacterium that is used for commercial production of glutamate, lysine, and other amino acids, nucleotides, and vitamins and from which the genome sequence has been described (NCBI accession number NC_006958). It is highly arsenic-resistant and has three genes encoding Acr3 homologues (19). Two of the homologues are in ars operons regulated by ArsRs (arsR 1 B acr3-1 C 1 C 1Ј and arsR 2 B acr3-2 arsC 2 ) and a third orphan gene (arsB acr3-3 ) that is not in an operon and may not be expressed to the same extent as the other two.…”