The intA element and the vrl are found in almost all virulent strains (Rood et al., 1996; Cheetham et al., 2006) selleck inhibitor and are absent from the majority of benign strains. The intA, intB and intD elements integrate into a tRNA-ser gene immediately downstream from pnpA. The pnpA product, polynucleotide

phosphorylase (PNPase), has 3′ to 5′ exoribonuclease activity and is involved in mRNA decay initiated by endoribonucleases (Deutscher & Li, 2001). In Salmonella enterica, PNPase is a global regulator of virulence (Clements et al., 2002) that negatively regulates the expression of genes from the pathogenicity islands SPI-1 and SPI-2 (Ygberg et al., 2006). By contrast, in Yersinia spp., the PNPase S1 domain is important for the optimal functioning of the type III secretion system and a mutant lacking the S1 domain was found to be less virulent than the wild-type strain (Rosenzweig et al., 2007). We have proposed that these integrated elements modulate the expression of pnpA, thereby

altering the activity of PnpA, which acts as a global repressor of virulence (Whittle et al., 1999). To investigate the hypothesis that pnpA encodes a virulence regulator, we created C-terminal deletions in PNPase in several benign and virulent strains, and determined the effect on extracellular protease thermostability and twitching motility, two characteristics associated with virulence in D. nodosus. This deletion increased this website the twitching motility of benign strains, consistent with the hypothesis that PNPase acts as a virulence repressor in these D. nodosus strains. Methods for the growth of D. nodosus, preparation of genomic DNA, cloning and analysis of DNA, Southern blotting, DNA sequencing and DNA sequence analysis, together with the source of D. nodosus strains, have been reported previously (Katz et al., Cobimetinib nmr 1994; Bloomfield et al., 1997; Whittle et al., 1999). Transformation of D. nodosus used the method described by Kennan et al. (1998). Tetracycline (1 μg mL−1)

or kanamycin (10 μg mL−1) was used to select transformants. The sequence of the D. nodosus pnpA gene was extracted from the D. nodosus genome sequence (GenBank accession no. CP000513) and analysed using NCBI-ORF finder. Comparison of pnpA sequences of D. nodosus, Escherichia coli and Vibrio cholerae (GenBank accession nos. ZP_00924446 and ZP_00755444) showed that the predicted D. nodosus PnpA was very similar, consisting of 693 amino acids with five conserved domains (Fig. 1). The construction of plasmids pCF5 and pCF7 is described in Fig. 1, and the primers are shown in Table 1. These plasmids are unable to replicate in D. nodosus, but homologous recombination at the pnpA locus may insert part or all of the plasmid into the D. nodosus chromosome. A double crossover event at pnpA would interrupt the pnpA coding region by introducing the tetM gene after nt 891 (pCF7) or nt 1718 (pCF5) as shown in Fig. 1.