Characterization of a flagellar sheath component, PF60, and its structuralgene in marine Vibrio

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membra ne structure called a sheath. Five major proteins, whose molecular masses a re 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, resp ectively), have been detected in polar flagella, PF47 and PF45 have been id entified as flagellins while the other proteins are thought to be sheath-as sociated ones. In this study, we isolated and partially characterized a maj or 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 pe ptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunct ion with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was c loned by colony hybridization using the PCR fragment. Subsequent sequence a nalysis 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 bind ing periplasmic protein. The single pfsA gene constitutes an operon and enc odes a protein of 491 amino acids containing a putative signal peptide sequ ence at the N-terminal. A sequence database search revealed no homologous p rotein. However, PfsA seems to resemble lipoproteins in the N-terminal sign al 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 coo rdinately regulated with flagellar formation and similarly regulated to PF4 7 flagellin.