Application of In Vitro Mutagenesis to Identify the Gene Responsible for Cold Agglutination Phenotype of Streptococcus mutans

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold‐agglutination of the strain, since a correlation between this phenotype and expression...

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Bibliographic Details
Published in:Microbiology and immunology 2004-01, Vol.48 (6), p.449-456
Main Authors: Sato, Yutaka, Okamoto, Kazuko, Kagami, Akiyoshi, Yamamoto, Yasuhito, Ohta, Kazumasa, Igarashi, Takeshi, Kizaki, Harutoshi
Format: Article
Language:English
Subjects:
Agglutination - genetics
Amino Acid Motifs
Bacterial Adhesion - genetics
Cell Wall
cold agglutination
Cold Temperature
collagen adhesin homologue
DNA Transposable Elements
Electrophoresis, Polyacrylamide Gel
Genes, Bacterial
in vitro mutagenesis
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed
Phenotype
Streptococcus mutans
Streptococcus mutans - genetics
Streptococcus mutans - immunology
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Summary:A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold‐agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold‐agglutination‐negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR‐based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N‐terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.
ISSN:0385-5600
1348-0421
DOI:10.1111/j.1348-0421.2004.tb03535.x