The virulence plasmid of Salmonella typhimurium is self-transmissible

Abstract

Most isolates of Salmonella enterica serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 x 10(-4) transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.

FIG. 1

Homologs of the F plasmid conjugation genes are physically located on the 90-kb virulence plasmid of S. typhimurium. DNA was prepared according to the methods described in reference . Pulsed-field gel electrophoresis utilized a 1% 0.5× Tris-borate-EDTA agarose gel with separation by a Bio-Rad CHEF DRII at 200 V. The separation was run for 5 h with a switching time that ramped from 6 to 150 s, 5 h at 6 to 12 s, and 10 h at 6 to 36 s. (A) The gel was stained with ethidium bromide. (B) Detection of the kanamycin resistance gene of MudJ by Southern hybridization after transfer to a positively charged nylon membrane obtained from Boehringer Mannheim. A random prime DNA labeling kit (Boehringer Mannheim) and [³²P]dCTP were used to label the 1.2-kb SmaI fragment from pUC4K-KIXX (Pharmacia), which contains the kanamycin resistance gene.

FIG. 2

Visualization of virulence plasmid transfer from Salmonella to E. coli. (A) Pulsed-field gel stained with ethidium bromide. (B) Southern hybridization of the pulsed-field gel with a probe specific to the kanamycin resistance gene of MudJ. The virulence plasmid is present in the donor Salmonella strain, BA1541 (spv::MudJ), and the three transconjugants but is not present in the recipient E. coli strain, BA769. Pulsed-field gel electrophoresis and Southern blotting were performed as described in the legend to Fig. 1.