Summary auto-generated
This study identified homologous DNA sequences between the Salmonella enteritidis virulence plasmid and the S. typhi chromosome using Southern blot hybridization with small DNA probes. Researchers cloned a 13 kb chromosomal fragment from S. typhi that showed sequence similarity to virulence plasmid regions adjacent to fimbrial operons. The cloned regions contained genes related to the pef fimbrial operon of S. typhimurium and the sef operon of S. enteritidis. Notably, researchers identified two open reading frames (dlp and dlt) that encode proteins belonging to the DsbA family of thiol:disulfide oxidoreductases, which are essential for protein secretion and folding. The dlp gene product successfully complemented multiple defective phenotypes of a dsbA mutant in E. coli, including DTT sensitivity, inability to metabolize glucose 1-phosphate, and low alkaline phosphatase activity. The dlt gene product partially restored alkaline phosphatase activity but not other functions. Both proteins were detected by SDS-PAGE with estimated molecular masses of 23 kDa (Dlp) and 26 kDa (Dlt), and the Dlp protein exhibited the characteristic double-band pattern indicating oxidized and reduced forms.
Key findings
- A homologous region between the S. enteritidis virulence plasmid and S. typhi chromosome encodes genes related to fimbrial operons (sef and pef), providing evidence that S. typhi chromosome carries virulence-plasmid-related sequences despite lacking the plasmid itself.
- Two genes (dlp and dlt) identified in these homologous regions encode proteins with the characteristic Cys-X-X-Cys motif of DsbA-family thiol:disulfide oxidoreductases, suggesting roles in protein secretion and folding.
- The dlp gene product fully complemented DsbA deficiency in E. coli, restoring DTT resistance, glucose 1-phosphate utilization, and alkaline phosphatase activity, indicating functional equivalence to bacterial DsbA proteins.
- The dlt gene product only partially complemented E. coli DsbA deficiency, suggesting differential functional capabilities between the chromosomal and plasmid-encoded versions.
- Southern blot analysis revealed dlp-related sequences in multiple Salmonella serovars, including S. enteritidis and S. dublin, but notably absent from S. typhimurium and S. montevideo.
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Abstract
The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol:disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.