Genetic analysis of phosphomannomutase/phosphoglucomutase from Vibrio furnissii and characterization of its role in virulence.
The pmm gene from Vibrio furnissii, which encodes phosphomannomutase (PMM), was cloned and sequenced. The open reading frame consisted of 1,434 bp, encoding a polypeptide of 477 amino acids with a molecular mass of 53,325 Da. The predicted amino acid sequence of V. furnissii PMM showed high similarity with PMMs from other enteric bacteria, such as V. cholerae, Salmonella sp. and Escherichia coli. The PMM protein was overexpressed in E. coli as a His(6)-tagged recombinant protein. The estimated apparent K(m )and k(cat) values of the purified recombinant protein for mannose 1-phosphate were about 60 microM and 800 min(-1), respectively. To investigate the biochemical functions and the role of pmm in the virulence of V. furnissii, a pmm knock-out mutant was constructed by homologous recombination mutation. Under the various physical conditions, cell numbers of the wild-type and the mutant did not differ. Oral introduction of bacterial suspensions to a mouse model showed that the pmm-deficient mutant decreased in viability at the intestine. Microscopy of the isolated intestines from mice revealed significant damage after 3 days in intestinal mucosa infected with the wild-type as compared with the mutant. The pmm-deficient mutant caused a reduction of virulence in mice and the loss of O-antigen polysaccharide, and showed low resistance relative to the wild-type when incubated with normal human serum.
Kim
Sun-Hoi
SH
Department of Biotechnology and Bioengineering, Pukyong National University, 608-737, Busan, Republic of Korea.
Ahn
Sun-Hee
SH
Lee
Jong-Hee
JH
Lee
Eun-Mi
EM
Kim
Nam-Hyun
NH
Park
Kee-Jai
KJ
Kong
In-Soo
IS
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D051379:Mice; D023421:Models, Animal; D008969:Molecular Sequence Data; D010733:Phosphoglucomutase / Q000235:genetics / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology* / Q000472:pathogenicity / Q000502:physiology; D014774:Virulence
D008070:Lipopolysaccharides; D017875:Phosphotransferases (Phosphomutases); D010733:Phosphoglucomutase; C052799:phosphomannomutase