The inner-core lipopolysaccharide biosynthetic waaE gene: function and genetic distribution among some Enterobacteriaceae

Departamento de Microbiología y Parasitología Sanitarias, División de Ciencias de la Salud, Facultad de Farmacia, Universidad de Barcelona, Av. Joan XXIII s/n, Barcelona 08028, Spain 1 Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain 2 Author for correspondence: Miguel Regué. Tel: +34 3 4024496. Fax: +34 3 4024498. e-mail: regue{at}farmacia.far.ub.es To determine the function of the waaE gene in the biosynthesis of the inner-core LPS of Klebsiella pneumoniae , a waaE non-polar mutant has been constructed. Data obtained from the comparative chemical analysis of LPS samples obtained from the wild-type, the mutant strain and the complemented mutant demonstrated that the waaE gene is involved in substitution of -L- glycero -D- manno -heptopyranose I (L,D-Hep p I) at the O-4 position by a ß-D-glucopyranose (ß-D-Glc p ) residue. In addition, DNA amplification and nucleotide sequence determination studies revealed that waaE homologues located between the waaA and coaD genes are present in clinical isolates of Enterobacteriaceae containing the structure ß-D-Glc p -(1 4)- -L,D-Hep p I ( K. pneumoniae , Proteus mirabilis and Yersinia enterocolitica ), as well as in strains of Serratia marcescens and Enterobacter aerogenes of unknown LPS-core structures. Complementation studies using non-polar waaE mutants prove that all the waaE homologues perform the same function. Furthermore, K. pneumoniae , Ser. marcescens and P. mirabilis non-polar waaE mutants showed reduced adhesion and pathogenicity. In addition, the Ser. marcescens and P. murabilis waaE mutants showed reduced swarming motility and ability to form biofilms in vitro . All these characteristics were rescued by reintroduction of the waaE gene independently of its origin. An easy DNA amplification method to detect this gene was established, which also helps in finding the potential presence of this structural feature [ß-D-Glc p -(1 4)- -L,D-Hep p I] in the inner-core LPS of Enterobacteriaceae members with unknown LPS-core structures. Keywords: LPS, waaE homologues, non-polar mutant characterization, cross-complementation Abbreviations: -D-Gal( p )A, -D-galacturonic acid; GlcN( p ), glucosamine; ß-D-Glc( p ), ß-D-glucopyranose; L,D-Hep( p ), -L- glycero -D- manno -heptopyranose; Kdo( p ), 3-deoxy-D- manno -octulopyranosonic acid; BATH, bacterial adherence to hydrocarbons; HIC, hydrophobic interaction chromatography; NHS, non-immune human serum b T