Open AccessCloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin)
Author(s) -
Ehara Masahiko,
Ichinose Yoshio,
Hirayama Toshiya,
Kurazano Hisao,
Hidaka Yuji,
Morita Kouichi,
Igarashi Akira,
Shimodori Shouichi
Publication year - 1994
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1994.tb07220.x
Subject(s) - gene , biology , vibrio cholerae , nucleic acid sequence , microbiology and biotechnology , peptide sequence , open reading frame , genetics , molecular cloning , amino acid , sequence analysis , stop codon , bacteria
The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N‐terminus and that from a cyanogen bromide‐cleaved fragment of the fimbrillin. A PCR‐amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3′ and 5′ termini by RNA‐PCR using a primer containing a random hexamer at its 3′ end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fim A, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.