Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica

Abstract

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3' end of the tRNA genes pheV or pheR The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO₄ compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na₂HAsO₄ compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Keywords: Salmonella enterica serovar Typhimurium; Salmonella genomic island 3; arsenic; copper; integrative and conjugative element; metal resistance.

FIG 1

Schematic view of full-length SGI3 of Salmonella 4,[5],12:i:– L-3841. The diagram shows the predicted classification of each gene, which is represented by arrows according to the following scheme: violet, genes for conjugative transfer; green, genes for heavy metal resistance; orange, genes for DNA replication or partitioning; and gray, genes with other functions.

FIG 2

Pairwise alignment of SGI3 of Salmonella 4,[5],12:i:– L-3841 with the relevant mobile genetic elements, pAPEC-01-R of Escherichia coli APEC-O1, pECL_A of Enterobacter cloacae ATCC 13047, and ICE-like chromosomal island of Edwardsiella icutaluri MS-17-156.

FIG 3

PCR and PFGE-Southern blot hybridization images demonstrating the chromosomal location and/or generation of the circular form of SGI3 in Salmonella 4,[5],12:i:– strains and Salmonella Typhimurium LT2 transconjugants. Lane 1, L-3838; lane 2, L-3841; lane 3, LT2; lane 4, LT2TCpheV; lane 5, LT2TCpheR; lane M1, 100-bp DNA ladder; lane M2, XbaI-digested Salmonella Braenderup. (a) SNP genotyping and PCR to detect junction regions of integrated or circular form of SGI3. (b) PFGE separation of I-CeuI-digested genomic DNA from S. enterica strains, followed by Southern blot hybridization with 23S rRNA, integrase, and pcoA gene probes.