Identification of a virulence-associated protein homolog gene and IS Ra1 in a plasmid of Riemerella anatipestifer

Abstract

Riemerella anatipestifer is the causative agent of polyserositis of ducks and geese. We have previously reported that a 3.9-kb plasmid, pCFC1, carries protein genes ( vapD1 and vapD2) that are similar to virulence-associated genes of other bacteria. In the present study, we report the complete sequence of a second plasmid of 5.6 kb, pCFC2. pCFC2 has a 28% G-C content and three large open reading frames (ORFs). One of the ORFs (designated asVapD1) encodes a polypeptide that shares 53.9, 53.9, 48.3, 48.3 and 46.1% identity with virulence-associated proteins of Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae, Helicobacter pylori and Haemophilus influenzae, respectively. The second ORF encodes a putative DNA replication protein (RepA3) with 309 amino acids and a molecular mass of approximately 36 kDa. A novel insertion sequence (IS) element, designated IS Ra1, was found on the plasmid pCFC2. IS Ra1 was flanked by 15-bp imperfect inverted repeats (only one mismatched nucleotide). IS Ra1 contained an ORF encoding a putative transposase of 292 amino acids. Southern blot analysis indicated that in R. anatipestifer strains examined, IS Ra1 was present with 2–20 copies (at least). IS Ra1 displayed a sequence approximately 35% homologous to the putative IS 982 and RSBst-α from Lactococcus lactis ssp. cremoris SK11 and Bacillus stearothermophilus CU21. Three hybridization patterns of genomic DNA of eight R. anatipestifer strains with an IS Ra1 probe indicated that IS Ra1 might be a useful tool for epidemiological studies.