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.
Microbiology (Reading). 2012 Mar;158(Pt 3):736-745. doi: 10.1099/mic.0.052043-0. Epub 2011 Dec 15.
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.
NAD 和 NADP 在大肠杆菌 K-12 的代谢中普遍存在。nadB、nadA 和 nadC 这三个基因中的任何一个突变都会导致 NAD 营养缺陷型。nadB 和 nadA 基因被定义为志贺氏菌属的抗病毒基因座，因为破坏喹啉酸盐合成的突变（主要在 nadB 中）是毒力所必需的。来自急性膀胱炎患者的尿路致病性大肠杆菌 (UPEC) 分离株表现出烟酰胺营养缺陷型，血清型为 O18：K1：H7。模型尿路致病性和 O18：K1：H7 菌株 UTI89 需要烟酰胺或喹啉酸盐才能生长。证明编码 L-天冬氨酸氧化酶的 nadB 基因突变是 UTI89 对烟酰胺需求的原因。这进一步通过用含有大肠杆菌 K-12 的 nadB 基因的重组质粒互补 UTI89 得到证实。重组质粒的 Ala28Val 点突变体未能支持 UTI89 在最小培养基中的生长。这证明了 UTI89 的 NadB 基因中的 Ala28Val 突变完全阻止了烟酰胺的从头合成。在 UTI89 的自发原养回复突变体中，nadB 基因有一个 Val28Ala 突变。这两种分析都表明，UTI89 的烟酰胺营养缺陷型是由 NadB 中的单个 Ala28Val 突变引起的。我们表明，相同的突变也存在于其他 NAD 营养缺陷型大肠杆菌 O18 菌株中。在小鼠上行尿路感染模型中，同基因 NAD 营养缺陷型和原养型菌株的毒力没有观察到显著差异。考虑到这些数据，我们将 nadB 基因座作为细菌染色体中 DNA 插入的中性位点。我们成功地通过将带有合成 em7 启动子的 fimH 插入 nadB 基因，恢复了 fimH 突变体的亲本表型。这个中性插入位点对于进一步研究 UPEC 的致病性具有重要意义。
