title: Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.
abstract: NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-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 nadB gene of E. coli 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 de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB 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 E. coli 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 nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, HVRI, Harbin 150001, PR China.|Viral Genetics Laboratory, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Zhaoli
authors.initials: Z
authors.lastname: Li
authors.affiliation: Unité de Glycobiologie Structurale et Fonctionelle, UMR8576 du CNRS, Université de Lille 1, Avenue Mendeleiev, 59655 Villeneuve d'Ascq, France.|Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Julie
authors.initials: J
authors.lastname: Bouckaert
authors.affiliation: Viral Genetics Laboratory, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Francine
authors.initials: F
authors.lastname: Deboeck
authors.affiliation: Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, B-1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Henri
authors.initials: H
authors.lastname: De Greve
authors.affiliation: Viral Genetics Laboratory, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Hernalsteens
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism*; D017354:Point Mutation; D017378:Quinolinic Acid / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014552:Urinary Tract Infections / Q000382:microbiology; D056689:Uropathogenic Escherichia coli / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D009536:Niacinamide; D017378:Quinolinic Acid