The virulence region of the 55 kb plasmid pNL2001 was identified by Tn-insertion mutagenesis, DNA hybridization studies, and Western blot analysis of proteins encoded in the virulence region of the plasmid. DNA hybridization studies showed that the pNL2001 plasmid contained a 6.4 kb I-RI fragment homologous to the 6.4 kb I-RI plasmid virulence () region of the 50 kb plasmid (pKDSC50). One of the 247 Tn-insertion mutants of , designated strain TA19, showed a reduced mouse lethality, and the Tn-insertion of strain TA19 was located within this homologous 6.4 kb region, suggesting that the 6.4 kb I-RI fragment of pNL2001 contained the region. Two contiguous I-RI (2.3 kb) and RI-RI (4.1 kb) fragments derived from the 6.4 kb I-RI fragment were cloned into the expression vectors. By Western blot analysis using four Spv peptide antisera, each specific for individual proteins encoded in the and genes of pKDSC50, four proteins encoded in the 6.4 kb I-RI fragment of pNL2001 were identified. Protein SpvR with an apparent molecular mass of 32 kDa was produced from the 2.3 kb I-RI fragment, and proteins SpvA, SpvB and SpvC with apparent molecular masses of 32, 70 and 29 kDa, respectively, were produced from the 4.1 kb RI-RI fragment. From the 4·1 kb RI::Tn fragment of the TA19 plasmid, proteins SpvA and SpvB were expressed, but not SpvC. It was therefore suggested that the gene may contribute to the expression of virulence of . Furthermore, the nucleotide sequence of the 6.4 kb I-RI fragment encoding these four proteins was determined. Four open reading frames which encoded the four proteins with deduced molecular masses of 33906, 28200, 65349 and 27646 Da were detected. Deduced amino acid sequences of each protein showed a high degree of identity to corresponding sequences in the virulence region of and virulence plasmids. Therefore, we confirmed that the virulence plasmids of Salmonellae including share the highly conserved region responsible for virulence.


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