Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange

Abstract

Serpulina hyodysenteriae, the etiologic agent of swine dysentery, contains complex periplasmic flagella which are composed of multiple class A and class B polypeptides. To examine the role these proteins play in flagellar synthesis, structure, and function and to develop strains which may provide insight into the importance of motility in the etiology of this pathogen, we constructed specific periplasmic flagellar mutations in S. hyodysenteriae B204. The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and/or kanamycin gene cassettes. Following delivery of these suicide plasmids into S. hyodysenteriae, homologous recombination and allelic exchange at the targeted chromosomal flaA1 and flaB1 genes was verified by PCR, sequence, and Southern analysis. The utility of a chloramphenicol resistance gene cassette for targeted gene disruption was demonstrated and found more amenable than kanamycin as a selective marker in S. hyodysenteriae. Immunoblots of cell lysates of the flagellar mutants with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath or core protein. Both mutations selectively abolished expression of the targeted gene without affecting synthesis of the other flagellar polypeptide. flaA1 and flaB1 mutant strains exhibited altered motility in vitro and were less efficient in movement through a liquid medium. Paradoxically, isogenic strains containing specifically disrupted flaA1 or flaB1 alleles were capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. This is the first report of specific inactivation of a motility-associated gene in spirochetes.