Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.

NAD and NADP function ubiquitously in the metabolism of Escherichia coli. NAD auxotrophy can be rendered by mutation in any of the three genes, nadB, nadA, and nadC. The nadB and nadA genes are anti-virulence loci in Shigella spp., as a mutation disrupting the synthesis of quinolinate is required for virulence. We found that the uropathogenic E. coli strain UTI89 requires quinolinate for growth. An Ala28Val mutation in the nadB gene, encoding L-aspartate oxidase, was proven to be responsible for the nicotinamide requirement of UTI89. Prototrophic derivatives of UTI89 were obtained by P1-mediated transduction of nadB from E. coli K-12 and by Val28Ala reversion mutations. No significant difference was observed between the virulence of wild type NAD auxotrophic UTI89 and its prototrophic derivatives in the murine ascending urinary tract infection model. Considering that the function of nadB gene is impaired in UTI89, we applied this locus as a neutral site for DNA insertions in the bacterial chromosome. We restored the parental phenotype by insertion of fimH and phoA separately, with a synthetic em7 promoter, into the nadB gene of a fimH or phoA mutant. This neutral insertion site is of significance for further research on the pathogenicity of UTI89.