Identification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks

Abstract

From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described.

FIG. 1

Numbers of chicks from groups of approximately 30 animals (expressed as percentages) excreting mutants of Nal^r S. typhimurium F98 in the feces after oral inoculation with approximately 10⁸ CFU. ▪, mutant 14/12/90-14 (positive control); ○, mutant 14/12/90-7; •, mutant 8/5/91-309; ▵, mutant 14/12/90-43; □, mutant 30/11/90-85; ×, mutant 21/12/90-82; ▴, mutant 10/4/91-86; ◊, mutant 17/12/90-117.

FIG. 2

LPS profiles of S. typhimurium F98, both wild type and mutants carrying mutations in genes required for colonization of the chicken alimentary tract. LPS was extracted from the strains and visualized by SDS-PAGE. Lanes: 1, wild type; 2, dksA; 3, hupA; 4, rfbK; 5, ptsC; 6, sipC; 7, clpB; 8 to 11, T14/12/90-7i, -ii, -iii, and -iv, respectively (rfaY::Tn5-TC1 transductants); 12, 14/12/90-7 (rfaY::Tn5-TC1 transposon mutant); 13, 12/6/91-86 (rfaK::Tn5-TC1 transposon mutant); and 14, 30/11/90-85 (rfbK::Tn5-TC1 transposon mutant).

FIG. 3

RNA contents of the S. typhimurium F98 parental strain (lane 1) and defined dksA::km (lane 2) and hupA::km (lane 3) mutants. The bands are stable low-molecular-weight RNA, which is probably rRNA (upper three bands) and tRNA (lower band).