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Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the
1Department of Immunology and Medical Microbiology, University of Florida College of Medicine, Gainesville 32610.
Abstract:
We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.
Insights
The 28K protein, encoded by the virA gene on the Salmonella typhimurium virulence plasmid, is confirmed as a virulence factor. This protein is sufficient to restore virulence in bacteria with a specific mutation.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Salmonella typhimurium virulence plasmid carries genes essential for infection.
- A mutation (vir-22::Tn5) in this plasmid significantly reduces bacterial virulence.
- This mutation also inhibits the production of a 28,000-molecular-weight (28K) protein.
Purpose of the Study:
- To identify the specific gene responsible for virulence on the Salmonella typhimurium virulence plasmid.
- To confirm the role of the 28K protein in Salmonella virulence.
Main Methods:
- Complementation studies using plasmid pYA426 and its derivatives.
- Construction and analysis of nested deletions in the virulence plasmid.
- DNA sequence analysis of the relevant gene fragment.
Main Results:
- Plasmid pYA426 restored virulence and produced the 28K protein, complementing the vir-22::Tn5 mutation.
- Only deletion derivatives that retained the ability to produce the 28K protein complemented the mutation.
- DNA sequencing identified the virA gene encoding the 28K protein (27,572 MW).
Conclusions:
- The 28K protein encoded by the virA gene is a Salmonella typhimurium virulence factor.
- This protein is sufficient to restore virulence, indicating its critical role in pathogenesis.