1 CREST, Japan Science and Technology Agency (JST), 1064-18 Takahori, Hirata, Takanezawa, Shioya-gun, Tochigi 329-1206, Japan
2 Graduate School of Natural Sciences, Nagoya City University, 1 Yamanohata, Mizuho, Nagoya 467-8501, Japan
3 Division of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
Correspondence
S.-I. Aizawa
aizawa{at}softnano.org
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The flagellar structure is anchored in the cytoplasmic membrane via the MS ring structure (DePamphilis & Adler, 1971). In Salmonella typhimurium the FliF protein is the sole component of the MS ring complex (Ueno et al., 1992
). Although both terminal regions of FliF, which form the cytoplasmic domain, are cleaved by a tryptic digest, the major part of the basal bodies is considerably stable against this treatment in vitro (Ueno et al., 1994
). Jenal & Shapiro (1996)
showed by immunoblot and pulsechase assays that the C. crescentus FliF protein is proteolytically turned over during the SW-to-ST cell differentiation, suggesting that degradation of the FliF anchor protein is coupled to flagellar ejection.
To understand how this proteolytic digestion of FliF and consequently the destruction of the MS ring complex occurs, we developed a method for purification of intact flagella from C. crescentus and examined the physico-chemical properties of the basal structure. We found that the C. crescentus MS ring structure, in contrast to the same structure isolated from enteric bacteria, is very susceptible to protease treatment. This is in line with the idea that the physico-chemical properties of the C. crescentus HBB structure are adjusted to the developmentally regulated process of flagellar ejection.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
All reagents used were special grade. Trypsin and chymotrypsin were purchased from Wako Pure Chemical Industries and Pronase E was from Sigma.
Observations of cell types.
Cells were analysed in a concave well on a hole-slide glass (Toshinriko, Oosaka), observed with a phase-contrast microscope (Olympus CH-2), and recorded on VTR through a CCD camera (Panasonic BL200) attached to the microscope. Sixty to one hundred cells in each scene of 1020 s duration were counted, and the ratio of cell types (SW cells : ST cells : PD cells) was calculated.
Purification method.
The hookbasal body (HBB) complexes of C. crescentus were purified by a modification of the original protocol used for S. typhimurium (Aizawa et al., 1985). The modified protocol was as follows (see Results and Discussion for details).
Spheroplasts were formed by adding lysozyme and 0·5 mM EDTA, and lysed with 1 % AM-3130N (Nihon Surfactant Kogyo K. K.). AM-3130N is a non-ionic detergent and its major constituent is myristic acid betaine. Chromosomal DNA present in the lysate was digested by adding a small amount of DNase. Bundles of flagella formed after addition of 2 % polyethylene glycol (PEG) were collected by low-speed centrifugation, and resuspended in 0·2 M KCl/KOH buffer (pH 12). The sample was resuspended in TET buffer (10 mM Tris/HCl pH 8, 1 mM EDTA and 0·1 % Triton X-100) for further analysis.
SDS-PAGE and Western blotting.
SDS-PAGE was carried out using the mini-gel kit from Bio-Rad. Acrylamide concentrations of gels were 10 %, 12·5 % or 15 % depending on the molecular range of the proteins of interest in each experiment. Gels were stained by silver. Molecular mass standard protein markers were obtained from Bio-Rad. To confirm the degradation of FliF by trypsin, Western blotting was carried out, using anti-C. crescentus FliF antibody (Jenal & Shapiro, 1996).
Electron microscopy.
Samples were negatively stained with 2 % phosphotungstic acid (pH 7·0) and observed with a JEM-1200EXII electron microscope (JEOL). Micrographs were taken at an accelerating voltage of 80 kV.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Flagella ejected into the medium during the SW-to-ST cell transition were collected by PEG precipitation from an overnight culture. Electron microscopy revealed that the ejected structure consisted of the filament, the hook and part of the rod (Fig. 1). The length of the sheared rod was about 18 nm (Table 1
). Acid treatment (pH 4·4) of these isolated flagellar structures gave rise to a complex consisting of the hook and rod structure. This complex consists of four major protein bands visible in SDS gels, 70, 68, 33 and 28 kDa; these were identified by N-terminal amino acid sequencing as FlgK (hook-associated protein HAP1), FlgE (hook), ribosomal protein L2 and FlgG (distal rod), respectively (Table 2
).
|
|
|
Purification method for C. crescentus flagellar HBBs
To examine the physico-chemical properties of the C. crescentus basal body structure, we had to develop a purification method that allowed the isolation of intact flagella in high enough quantities. This is a general problem encountered with mono-flagellated bacteria, and was aggravated by the fact that due to the intrinsic properties of the C. crescentus developmental cycle, more than half of the population is non-flagellated. This problem was overcome by using two regulatory mutants that fail to eject the flagellum during cell differentiation: the first has a mutation in the pleC gene, which encodes a polar sensor histidine kinase (strain UJ248; Aldridge et al., 2003); the second has a deletion in pleD, which encodes a response regulator involved in polar development (strain UJ506; Paul et al., 2004
). The basal body structure and composition isolated from both mutants was compared with that of flagella isolated from wild-type cells. There were no discernible differences between them, arguing that the failure of these mutants to eject the flagellum is not caused by an altered structure of the HBB itself.
The original protocol used for S. typhimurium was modified as follows.
Spheroplast formation.
EDTA is necessary for lysozyme to digest the peptidoglycan layer of Gram-negative bacteria and to form spheroplasts. While spheroplast formation was easily achieved above 0·5 mM EDTA, an excess of chromosomal DNA was released under these conditions and could not be removed efficiently by endogenous DNase. Therefore, we maintained the EDTA concentration at 0·5 mM during spheroplast formation.
Cell lysis.
For C. crescentus cells, the non-ionic detergent Triton X-100 did not effectively remove membranous material. We surveyed a collection of detergents (kindly provided by Nihon Surfactant Kogyo K. K., Utsunomiya) and found that the non-ionic detergent AM3060N was best suited to solubilize most of the membranous remnants, leaving the flagella intact.
CsCl density gradient.
CsCl density-gradient centrifugation is a final step for further purification of intact flagella to separate the basal structure from membranous contaminants for S. typhimurium, Bacillus subtilis and Rhodobacter sphaeroides (Aizawa et al., 1985; Kubori et al., 1997
; Kobayashi et al., 2003
). However, CsCl effectively destroyed the basal structure of C. crescentus flagella. Therefore, we omitted this step. Instead, we employed polyethylene glycol (PEG); bundles of flagella formed after addition of 2 % PEG were collected by low-speed centrifugation.
Depolymerization of filaments.
To purify HBBs at a high yield, it is necessary to remove flagellar filaments, which constitute 99 % of the material of intact flagella. In conventional methods, acidic buffers (pH 23) were employed for this purpose. C. crescentus HBBs were fragile to acid and started to disintegrate at pH 4. We found that at pH 4·5, filaments dissociated into flagellin monomers, leaving the HBB structure intact.
Analysis of purified HBBs
The overall structure of C. crescentus HBBs purified as described above was similar to the structure of S. typhimuriun HBBs, with four rings and a central penetrating rod connected to the curved hook structure (Fig. 2). Although C. crescentus HBBs have been reported to contain an extra ring between the inner MS ring and the distal PL ring complexes, called the E ring (Stallmeyer et al., 1989
), we were unable to detect such an extra ring structure in our preparations. There are several possible reasons for this discrepancy. First, the E ring could have detached from the HBBs in our preparation. Second, the E ring reported by Stallmeyer et al. (1989)
could be an artifact of negative staining. It is important to note that so far a gene encoding an E ring structure has not been identified in C. crescentus (Wu & Newton, 1997
).
|
|
|
When flagella were incubated with 1 µg Pronase E ml1 at 30 °C for 30 min, both the MS and PL ring structures were degraded. The remaining rods were apparently longer than those of ejected flagella (Fig. 4d). The mean length of rods of ejected flagella was 18 nm, while rods of Pronase E-treated flagella had a mean length of 31 nm. The distance between the S and P ring of intact flagella was 10 nm, while the thickness of the P and L ring complex was 14 nm (Fig. 5
, Table 1
), indicating that a few nanometres of the rod might penetrate into the MS ring complex. From this we conclude that the rod exposed after partial digestion by Pronase E is intact. Interestingly, the naked rod appeared as a uniform rod with no trace of the E ring or a narrow gap along the axis, indicating that the E ring is not functionally linked to flagellar ejection.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aldridge, P. & Jenal, U. (1999). Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator. Mol Microbiol 32, 379391.[CrossRef][Medline]
Aldridge, P., Paul, R., Goymer, P., Rainey, P. & Jenal, U. (2003). Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol Microbiol 47, 16951708.[CrossRef][Medline]
DePamphilis, M. L. & Adler, J. (1971). Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J Bacteriol 105, 384395.[Medline]
Ely, B., Ely, T. W., Crymes, W. B., Jr & Minnich, S. A. (2000). A family of six flagellin genes contributes to the Caulobacter crescentus flagellar filament. J Bacteriol 182, 50015004.
Jenal, U. & Shapiro, L. (1996). Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell. EMBO J 15, 23932406.[Abstract]
Judd, E. M., Ryan, K. R., Moerner, W. E., Shapiro, L. & McAdams, H. H. (2003). Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter. Proc Natl Acad Sci U S A 100, 82358240.
Kobayashi, K., Saitoh, T., Shah, D. S. H., Ohnishi, K., Goodfellow, I. G., Sockett, R. E. & Aizawa, S.-I. (2003). Purification and characterization of the flagellar basal body of Rhodobacter sphaeroides. J Bacteriol 185, 52955300.
Kubori, T., Okumura, M., Kobayashi, N., Nakamura, D., Iwakura, M. & Aizawa, S.-I. (1997). Purification and characterization of the flagellar hook-basal body complex of Bacillus subtilis. Mol Microbiol 24, 399410.[Medline]
Paul, R., Weiser, S., Amiot, N. C., Chan, C., Schirmer, T., Giese, B. & Jenal, U. (2004). Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 18, 715727.
Schoenhals, G. L. & Macnab, R. M. (1996). Physiological and biochemical analysis of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium. J Bacteriol 178, 42004207.
Shapiro, L. & Maizel, J. (1973). Synthesis and structure of Caulobacter crescentus flagella. J Bacteriol 113, 478485.[Medline]
Stallmeyer, M. J. B., Hahnenberger, K., Sosinsky, G. E., Shapiro, L. & DeRosier, D. J. (1989). Image reconstruction of the flagellar basal body of Caulobacter crescentus. J Mol Biol 205, 511518.[CrossRef][Medline]
Stephens, C., Reisenauer, A., Wright, R. & Shapiro, L. (1996). A cell cycle-regulated bacterial DNA methyltransferase is essential for viability. Proc Natl Acad Sci U S A 93, 12101214.
Ueno, T., Oosawa, K. & Aizawa, S.-I. (1992). The M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF. J Mol Biol 227, 672677.[Medline]
Ueno, T. K., Oosawa, & Aizawa, S.-I. (1994). Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium. J Mol Biol 235, 546555.[CrossRef]
Wu, J. & Newton, A. (1997). Regulation of the Caulobacter flagellar gene hierarchy; not just for motility. Mol Microbiol 24, 233239.[Medline]
Received 8 June 2004;
revised 24 September 2004;
accepted 1 November 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |