Cloning and genetic characterization of a Helicobacter pylori flagellin gene.
Helicobacter pylori produces polar sheathed flagella, which are believed to be essential for the bacterial colonization of the human gastric mucosa. Here we report on the cloning and genetic characterization of a H. pylori gene encoding the subunit of the flagellar filament, the flagellin. Screening of a genomic library of H. pylori with an oligonucleotide probe derived from the N-terminal amino acid sequence of purified flagellin resulted in a recombinant plasmid clone carrying the flagellin-encoding gene flaA on a 9.3 kb Bg/II fragment. The nucleotide sequence of flaA revealed an open reading frame of 1530 nucleotides, encoding a protein with a predicted molecular mass of 53.2 kDa, which is similar in size with the purified flagellin protein in SDS-polyacrylamide gel electrophoresis. Sequence alignment of H. pylori flagellin (FlaA) with other bacterial flagellins demonstrates a high degree of similarity in the amino-terminal and carboxy-terminal regions, including those of the closely related genus Campylobacter (56% overall identity with Campylobacter coli flaA), but little homology in the central domain. Southern hybridizations of chromosomal DNA with flaA-specific probes did not reveal the presence of additional homologous flagellin genes in H. pylori. Sequence analysis of the flaA flanking regions and mapping of the flaA mRNA start site by a primer extension experiment indicated that transcription of the gene is under the control of a sigma 28-specific promoter sequence in H. pylori. The region upstream of the flaA promoter is subject to local DNA modification, resulting in the masking of two out of three closely linked HindIII restriction sites in the chromosome of strain 898-1. Escherichia coli strains harbouring the recombinant plasmid did not produce full-length flagellin and data obtained with FlaA fusion proteins using an E. coli plasmid expression system suggest that a distinct nucleotide sequence in the gene interferes with productive translation of this protein in E. coli.
Leying
H
H
Max-Planck-Institut für Biologie, Abteilung Infektions-biologie, Tübingen, Germany.
Suerbaum
S
S
Geis
G
G
Haas
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005407:Flagella / Q000378:metabolism; D005408:Flagellin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D005408:Flagellin; C067138:flaA protein, bacteria