Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus.
The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh+ strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh+ strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh+ sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh+ strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.
Kamruzzaman
Muhammad
M
Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Bhoopong
Phuangthip
P
Vuddhakul
Varaporn
V
Nishibuchi
Mitsuaki
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D016386:Chromosome Walking; D004251:DNA Transposable Elements; D017353:Gene Deletion; D016680:Genome, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019895:Transposases / Q000235:genetics; D014736:Vibrio parahaemolyticus / Q000235:genetics*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004251:DNA Transposable Elements; D006460:Hemolysin Proteins; C068489:thermostable direct hemolysin; D019895:Transposases