1887

Abstract

NAD and NADP are ubiquitous in the metabolism of K-12. NAD auxotrophy can be rendered by mutation in any of the three genes , and . The and genes were defined as antivirulence loci in spp., as a mutation (mainly in ) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the gene, encoding -aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the gene of K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a mutant by inserting with a synthetic promoter, into the gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.

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2012-03-01
2020-07-14
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