The Spirochete FlaA Periplasmic Flagellar Sheath Protein Impacts Flagellar Helicity

The spirochetes are a phylogenetically and morphologically unique group of bacteria (42). This phylum contains not only many medically important species such as Treponema pallidum and Borrelia burgdorferi but others that are commensal with arthropods such as termites and some that are free-living and reside in soil and water (5, 17, 28, 42). The distinctive spirochete structure has been characterized in detail. Outermost is a membrane sheath, and within this sheath is the cell cylinder and periplasmic flagella (PFs). The PFs reside between the outer membrane sheath and cell cylinder in the periplasmic space. Each PF is subterminally attached to one end of the cell cylinder. Several lines of evidence indicate that the PFs are directly involved in spirochete motility and that these organelles rotate in a manner similar to that of flagella of other bacteria (6, 8, 30). The size of the spirochete, the number of PFs attached at each end of the cell cylinder, and whether the PFs overlap in the center of the cell vary from species to species (5). The protein composition of PFs is complex (see 29 for recent review). In contrast to the flagella of most bacterial species, PFs are comprised of two classes of proteins termed FlaA and FlaB (3, 8, 39). PFs consist of one to two FlaA proteins and three to four FlaB proteins. B. burgdorferi is an exception, as this species has one FlaA protein and only one FlaB protein (11, 14). These proteins share immunological and sequence similarity within a given class but not between classes. In addition, these classes show extensive conservation within the spirochete phylum (3, 8, 9, 14, 26, 33, 39, 40, 47, 52). Nucleotide sequence data and N-terminal amino acid sequence information indicate that FlaA is likely to be secreted into the periplasmic space via the general secretory pathway (4, 39, 40, 43). In contrast, FlaB proteins have significant homology to flagellin of other bacteria in the N-terminal and C-terminal regions. These proteins are not cleaved at the N terminus and are most likely secreted into the periplasmic space by a type-III secretion system (3, 8, 39, 40). The structure of the spirochete PF is atypical compared to the flagella of other bacteria. In fact, it is among the most complex of bacterial flagellar filaments so far studied (8). PFs consist of a core surrounded by a protein sheath (18, 38, 52). The intact PF has a diameter of 18 to 25 nm and a core of approximately 11 to 16 nm in diameter (18, 38, 52). Several lines of evidence indicate that FlaA comprises the sheath and that the several FlaB proteins form the core. The evidence includes immunoelectron microscopy of PFs from Spirochaeta aurantia, Leptospira interrogans, and Brachyspira (formerly Serpulina, Treponema) hyodysenteriae (3, 26, 31, 52), partially disrupted cells of T. pallidum analyzed by electron microscopy and Western blot analysis (9), and analysis of T. pallidum purified PFs by a Western blotting method termed epitope bridging (2). The function of the individual PF proteins is poorly understood, as gene transfer systems have only recently been established for spirochetes so that specific genes can be inactivated (19, 30, 49, 51). Recently, Rosey et al., using allelic exchange mutagenesis, inactivated two PF filament genes of the spirochete B. hyodysenteriae, the etiological agent of swine dysentery (44, 45). This spirochete has approximately eight to nine PFs subterminally attached at each end (5). Based on Western blot analysis of lysed cells, the PFs were reported to be comprised of a FlaA protein that migrated as a doublet and three FlaB proteins designated FlaB1, FlaB2, and FlaB3 (44). The flaA and flaB1 mutants were found to remain motile but were less virulent for mice than the wild-type strain, and their swimming behavior appeared altered (22, 44). In addition, using electron microscopic analysis of disrupted whole cells revealed that the diameter of the attached PFs of the flaA mutants were identical to that of the wild-type strain (44, 45). These latter results are in conflict with the model that FlaA comprises the PF sheath. In the work reported here, we constructed the additional mutants flaB2 and flaB3 and analyzed their purified PFs and those from the previous mutants in detail. We show that inactivation of one fla gene specifically inhibited the incorporation of the encoded protein into the assembled PF. In addition, we reexamined the PF diameter of the mutants and wild-type strain and made comparisons. Finally, because little is known about how each protein influences filament morphology, we examined the PFs of the wild type and mutants by high-magnification dark-field microscopy. We show that FlaA influenced the helical shape of the assembled PFs.