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. 2000 Jun;68(6):3368-76.
doi: 10.1128/iai.68.6.3368-3376.2000.

Transcriptional Organization and Function of Invasion Genes Within Salmonella Enterica Serovar Typhimurium Pathogenicity Island 1, Including the prgH, prgI, prgJ, prgK, orgA, orgB, and orgC Genes

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Free PMC article

Transcriptional Organization and Function of Invasion Genes Within Salmonella Enterica Serovar Typhimurium Pathogenicity Island 1, Including the prgH, prgI, prgJ, prgK, orgA, orgB, and orgC Genes

J R Klein et al. Infect Immun. .
Free PMC article

Abstract

Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together in Salmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH, -I, -J, and -K and orgA genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated orgA and orgB. Additionally, an opening reading frame downstream of orgB, which we have designated orgC, has been identified and partially characterized. Previously published work has indicated that the prgH, -I, -J, and -K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA. Here, we present experiments indicating that orgA expression is driven by the prgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the prgH, prgI, prgJ, prgK, orgA, and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.

Figures

FIG. 1
FIG. 1
Deletions in the prgH promoter disrupt expression and regulation of a Tn5lacZY transcriptional fusion in the orgA gene. Plasmid pBDJ149 is a HindIII-XhoI deletion derivative of pBDJ142. Serovar Typhimurium strains carrying either pBDJ142 or pBDJ149 were grown in high or low concentrations of oxygen before being assayed for β-Gal activity. Values are the means ± standard deviations from one experiment performed in triplicate and are representative of several experiments.
FIG. 2
FIG. 2
Complementation of a noninvasive polar Tn5::prgH serovar Typhimurium mutant. Serovar Typhimurium SL1344 is the invasive parent strain. Serovar Typhimurium EE656 carries a polar prgH::Tn5lacZY insertion. Plasmid pJK001 encodes prgH, -I, and -K and orgA and -B, while pJK007 encodes prgH, -I, -J, and -K and orgA, but lacks orgB. Data is presented as a percentage of wild-type (SL1344) tissue culture invasion which has been standardized to 100%. Values are the means ± standard deviations from one experiment performed in triplicate and are representative of several experiments.
FIG. 3
FIG. 3
RT-PCR analysis of the prg operon. (a) Genetic organization of the prg, org, and downstream genes. Location of 5′ and 3′ primers used in RT-PCR are indicated by numbered arrows below the map. Arrows above the map indicate the direction of transcription of each gene. (b) Results of RT-PCR amplification assays. Primers used in each reaction are listed above the bracket. Three reaction conditions were run for each primer pair. cDNA was amplified from serovar Typhimurium SL1344 whole-cell RNA by using the 3′ primer and PCR amplified with the same 3′ primer and the indicated 5′ primer (cDNA lanes). As a control that DNA was not the amplification template, each RT-PCR was run without the addition of RT (-RT lanes). Amplification of each band from SL1344 genomic DNA demonstrates the size of the expected band for each reaction. (DNA lanes). The size of each band is indicated.
FIG. 4
FIG. 4
Invasive phenotype of serovar Typhimurium mutants defective in individual genes. Each mutant strain was complemented with the indicated plasmid carrying the genes listed in parentheses. Data is presented as percent of wild type (SL1344) which has been standardized to 100%. Values are the means ± standard deviations from one experiment performed in triplicate and are representative of several experiments.
FIG. 5
FIG. 5
Secretion profile of serovar Typhimurium mutants defective in prgH, prgI, prgJ, prgK, orgA, orgB, and orgC. Proteins from culture supernatants were trichloroacetic acid precipitated and separated by SDS-PAGE. Strain phenotypes are listed above each lane and correspond to the following strains: WT, SL1344; prgH, JK11; prgI, JK17; prgJ, JK9; prgK, JK10 pBDJ143; orgA, BJ66; orgB, JK23; and orgC, TF78. Arrows indicate the positions of proteins not secreted from mutant strains. Molecular mass standards are indicated on the right in kilodaltons.

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