CLONING AND GENETIC-CHARACTERIZATION OF THE HELICOBACTER-PYLORI AND HELICOBACTER-MUSTELAE FLAB FLAGELLIN GENES AND CONSTRUCTION OF HELICOBACTER-PYLORI FLAA-NEGATIVE AND FLAB-NEGATIVE MUTANTS BY ELECTROPORATION-MEDIATED ALLELIC EXCHANGE

Helicobacter pylori is one of the most common human pathogens. It caus es chronic gastritis and is involved in the pathogenesis of gastroduod enal ulcer disease and possibly gastric carcinoma. Helicobacter mustel ae is a bacterium closely related to H. pylori that causes gastritis a nd ulcer disease in ferrets and is therefore considered an important a nimal model of gastric Helicobacter infections. Motility, even in a vi scous environment, is conferred to the bacteria by several sheathed fl agella and is regarded as one of their principal virulence factors. Th e flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the m ajor flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin gene s from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequen ce of the H. pylori gene proved that it encoded the second flagellin m olecule found in H. pylori flagellar filaments. The genes were named f laB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respec tively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flag ellin was much lower (58%). Both flaB genes were preceded by sigma54-l ike promoter sequences. Mapping of the transcription start site for th e H. pylori flaB gene by a primer extension experiment confirmed the f unctional activity of the sigma54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. py lori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility t esting. Both mutations selectively abolished the expression of the tar geted gene without affecting the synthesis of the other flagellin mole cule. Whereas flaA mutants were completely nonmotile, flaB mutants ret ained motility.