Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio.
The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.
Furuno
M
M
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. g44416a@nucc.cc. nagoya-u.ac.jp
Sato
K
K
Kawagishi
I
I
Homma
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002458:Cell Fractionation; D003001:Cloning, Molecular; D005407:Flagella / Q000737:chemistry* / Q000235:genetics*; D005408:Flagellin / Q000096:biosynthesis / Q000235:genetics; D005798:Genes, Bacterial; D008074:Lipoproteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010957:Plasmids / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000737:chemistry* / Q000235:genetics*
D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C406267:pFsA protein, Vibrio alginolyticus; D005408:Flagellin