ISOLATION OF EXTRACYTOPLASMIC PROTEINS FROM SERPULINA-HYODYSENTERIAE B204 AND MOLECULAR-CLONING OF THE FLAB1 GENE ENCODING A 38-KILODALTON FLAGELLAR PROTEIN

Extracytoplasmic proteins were released from Serpulina (Treponema) hyo dysenteriae (strain B204) by treatment of whole cells with a nonionic detergent (Tween 20), Centrifugation of the Tween 20-released proteins at 100,000 x g sedimented 10 major extracytoplasmic proteins with app roximate molecular masses of 44, 43.5, 42, 39, 38, 34, 33.5, 33, 31, a nd 29 kDa, Treatment of the sedimented fraction with 6 M urea solubili zed all of the proteins except the 39-kDa protein, Peptide sequences w ere obtained for the purified 42-, 39-, 38-, 34-, 31-, and 29-kDa prot eins, The peptide sequences of the 42-, 38-, and 31-kDa proteins indic ate that they likely are components of the periplasmic flagella. The a mino-terminal peptide sequence of the 38-kDa protein was used to desig n an oligonucleotide probe and to clone an S. hyodysenteriae DNA fragm ent containing the gene encoding this protein, The predicted 290-amino -acid protein sequence derived from the cloned gene was highly homolog ous to those of several other bacterial flagellar proteins and is prec eded by consensus sigma(D) nucleotide sequences found upstream of othe r flagellar genes, On the basis of its similarity to the FIaB proteins of other spirochetes, we propose to designate the cloned S. hyodysent eriae gene flaB1 and its encoded protein FlaB1, Vaccination of pigs wi th FlaB1 or its recombinant counterpart did not protect them from an e xperimental challenge.