Interaction of the Salmonella typhimurium transcription and virulence factor SlyA with target DNA and identification of members of the SlyA regulon.
The SlyA protein from Salmonella typhimurium is a transcription factor that contributes to virulence. It is shown that a slyA mutant is attenuated in the presence of murine macrophages compared with the parent strain. Moreover, after growth in minimal medium, survival of the slyA mutant was reduced. Altered levels of flagellin (fliC), PagC, IroN, and outer membrane proteins suggest that the slyA mutation affects the surface properties of Salmonella. The isolated SlyA protein is a cofactor-free homodimer that recognizes five sites within the promoter region of the slyA gene. One of these sites contained a near perfect inverted repeat TTAGCAAGCTAA. The other four sites contained related sequences. Occupation of the SlyA sites in the slyA promoter prevented open-complex formation, consistent with the pattern of slyA::lacZ expression parental and slyA mutant strains. By combining the footprinting data with potential SlyA binding sites recovered from a pool of random DNA sequences, a consensus was defined and used to probe the NIH Salmonella unfinished genomes data base. These searches revealed the presence of consensus SlyA sites upstream of omp, ispA, xseB, slyA, and a gene encoding a protein with homology to a hemagglutinin. Accordingly, transcription of an omp::lacZ fusion was reduced in a slyA mutant. Given the difficulties in obtaining a comprehensive picture of intracellular gene expression, the definition of the DNA sequence recognized by a transcription factor (SlyA) that is essential for survival in the macrophage environment should allow a complete regulon of genes with altered expression upon exposure to macrophages to be determined once the S. typhimurium genome annotation is complete.
Stapleton
Melanie R
MR
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
Norte
Valia A
VA
Read
Robert C
RC
Green
Jeffrey
J
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005408:Flagellin / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D018085:Regulon; D012486:Salmonella typhimurium / Q000235:genetics*; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D014157:Transcription Factors; C056785:salmolysin; D005408:Flagellin; C066766:pagC protein, Salmonella typhimurium