title: Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis.
abstract: Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced into Aeromonas salmonicida 449 which produces a surface protein array known as the A-layer. Kanamycin-resistant exconjugants of 449 with altered ability to produce the A-layer were selected by virtue of their altered colonial morphology and color on medium containing the dye Congo red. Analysis of culture supernatants, periplasmic shock fluid, outer membranes, and whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to A-protein revealed five classes of single-insertion mutations that affected the ability of cells to produce and export A-protein and to assemble the A-layer. These studies suggest that A-protein is produced from a single chromosomal gene. The subunits subsequently pass through the periplasm and across the outer membrane. At least one gene product is required for this export. Assembly of A-layer on the cell surface then requires the presence of O polysaccharide chains on the lipopolysaccharide. In one case, insertion of Tn5 resulted in loss of ability to produce both A-protein and lipopolysaccharide with O polysaccharide chains, suggesting that synthesis of A-protein and synthesis of lipopolysaccharide may involve coordinate regulation.
authors.forename: R J
authors.initials: RJ
authors.lastname: Belland
authors.forename: T J
authors.initials: TJ
authors.lastname: Trust
mesh_terms: D000333:Aeromonas / Q000235:genetics* / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003090:Coliphages / Q000235:genetics; D003227:Conjugation, Genetic; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D008070:Lipopolysaccharides / Q000096:biosynthesis / Q000302:isolation & purification; D009154:Mutation; D010957:Plasmids
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D008070:Lipopolysaccharides; D004262:DNA Restriction Enzymes