title: Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF.
abstract: Neuroinvasive Escherichia coli K1 synthesizes and assembles a polysialic acid capsule virulence factor on the external leaflet of the outer membrane. This capsule functions in pathogenesis by blocking non-immune host defence mechanisms and acting as a relatively non-immunogenic molecular mimic of the polysialic acid chains found in high concentrations on neural cell adhesion molecules of the human embryo and neonate. The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps 'pathogenicity island'. These regions are organized as two convergently transcribed operons inserted into the monocistronic tRNA gene, pheV. The six genes of the so-called region 1 operon are transcribed in the same direction as pheV, and at least four of these genes are required for polysialic acid export. Expression of this operon is thermoregulated by transcriptional control of its first gene, kpsF. To investigate the function of region 1 further, two independent chromosomal disruptions were engineered by inserting promoterless, terminatorless kanamycin or chloramphenicol resistance cassettes into the HindIII site of the kpsF coding sequence. The chromosomal insertions were regulated by temperature in the same way as the wild-type operon, demonstrating that this control mechanism remained intact in these mutants. Chemical, immunological and ultrastructural microscopical methods demonstrated that full-length polysialic acid chains were synthesized but not exported by the kpsF mutants. This phenotype was correlated with decreased plaque diameter when the mutants were infected with the capsule-specific bacteriophage K1F. The export defect could not be complemented in trans with kpsF+ containing its cis-regulatory region because of titration of an apparent positive regulator of region 1 expression, whereas complementation was observed with a plasmid expressing kpsF from a physiologically irrelevant promoter. An N-terminal polyhistidine peptide was attached to KpsF and used to purify the overproduced polypeptide. Antibodies raised against KpsF identified at least one of its paralogues in E. coli, GutQ, suggesting that KpsF and its homologues are membrane associated. The results indicate the requirement for a precise balance between region 1 components of the capsule export machinery, and that KpsF plays a positive role in the assembly, operation or regulation of this apparatus.
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana 61802, USA.
authors.forename: M
authors.initials: M
authors.lastname: Cieslewicz
authors.forename: E
authors.initials: E
authors.lastname: Vimr
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D002876:Chromosomes, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009154:Mutation; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012794:Sialic Acids / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C102536:KpsF protein, E coli; D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; C021319:polysialic acid