1 Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
2 School of Dentistry, University of California, Los Angeles, CA 90095, USA
3 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
Correspondence
Wenyuan Shi
wenyuan{at}ucla.edu
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ABSTRACT |
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INTRODUCTION |
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In P. aeruginosa and N. gonorrhoeae, TFP filaments are composed of a single structural protein, pilin. Crystal structures of full-length pilin have been obtained for the N. gonorrhoeae and P. aeruginosa proteins, revealing a highly conserved N-terminal hydrophobic tail that presumably serves as an oligomerization domain for fibre formation (Craig et al., 2003; Parge et al., 1995
). The M. xanthus pil operon shares substantial similarity with the components of the TFP biogenesis pathway in P. aeruginosa. Similar to the P. aeruginosa pilA gene which encodes pilin, the myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other type IV pilins (Wu & Kaiser, 1997
). However, direct evidence that pilA encodes the major pilin subunit in M. xanthus is still lacking.
Antibodies reactive with native pilin and pili would serve as an important tool for investigating the structure and function of TFP. Indeed, this has been the case for other bacteria such as N. gonorrhoeae (Forest et al., 1996; Merz et al., 2000
). Although there is an anti-PilA antibody available for M. xanthus (Wu & Kaiser, 1997
), its inability to recognize native pilin and pili limits its application. In this study, we successfully developed a new anti-PilA antibody which recognizes native pilin and pili. Using this antibody, we were able to further study the role of TFP in social motility of M. xanthus.
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METHODS |
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Overexpression and purification of truncated PilA.
The DNA sequence encoding residues 29220 of the mature M. xanthus PilA (herein referred to as PilA(29220)) was amplified by PCR. A sequence encoding five amino acid residues, Asp-Ile-Glu-Gly-Arg (numbered 2428 in this report; Ile-Glu-Gly-Arg serves as the Factor X protease recognition site), was fused to the N-terminus of PilA(29220) via PCR. The sequence encoding this fusion protein was then cloned into the BamHI and HindIII sites of the pQE30 expression vector (Qiagen), which fused a His6 tag to the N-terminus of PilA(24220). The resulting fusion protein was then overexpressed in Escherichia coli strain XL-1 Blue, which was pre-transformed with the repressor plasmid pREP4 (constitutively expressing the lac repressor protein encoded by the lacI gene). The cells were cultured in LB medium at 37 °C to an OD600 of 0·50·7, when expression was induced with 1 mM IPTG. After 3 h additional growth, the cells were harvested by centrifugation, lysed by sonication, and cell debris was removed by centrifugation. The supernatant was allowed to bind to Ni-NTA resin (Qiagen) for 1 h with agitation at 4 °C, and then transferred to a column. The column was washed at 4 °C with 20 bed volumes of washing buffer (Qiagen)/40 mM imidazole, and PilA was eluted with five bed volumes of elution buffer (Qiagen)/250 mM imidazole. The eluate was dialysed overnight against 50 mM sodium phosphate pH 8·0/100 mM NaCl and concentrated with a Vivaspin concentrator (Vivascience). The PilA appeared to be 80 % pure as determined by SDS-PAGE with Coomassie blue staining.
Generation and purification of anti-PilA antibody.
The purified PilA was used to immunize two rabbits to prepare polyclonal anti-PilA antibody. Immunizations were performed by Covance Research Products based on established protocols (Harlow & Lane, 1988). The antiserum was purified for specific anti-PilA antibody using acetone powder prepared from the pilA mutant DK10407 as well as an antigen blot as described by Harlow & Lane (1988)
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Western blots.
Western blotting on whole-cell lysates as well as cell-surface PilA was performed by standard protocols (Harlow & Lane, 1988). For whole-cell lysate, 5x107 DK1622 (wt) and DK10407 (pilA) cells were lysed by boiling in SDS-PAGE loading buffer. For cell-surface PilA, the extracellular pili/pilin were sheared off from 1010 DK1622 and DK10407 cells as described by Wu & Kaiser (1997)
, boiled in SDS-PAGE loading buffer and Western blotted.
Retraction blocking assay.
This assay was based on a cell mixing assay previously described (Li et al., 2003). Exponential-phase SW504 cells were harvested and resuspended to 5x109 cells ml1 in MOPS buffer (10 mM MOPS, 8 mM MgSO4, pH 7·6); 1, 5 or 10 µl anti-PilA serum was added to 100 µl cells and incubated for 1 h. The cells were then washed three times with MOPS buffer and mixed with equal amount of DK1622 cells to trigger retraction. After 30 min, the extracellular pilin of the mixture was sheared off as described by Wu & Kaiser (1997)
and analysed by Western blotting.
Fluorescence microscopy.
For immuno-fluorescence microscopy, exponential-phase M. xanthus cells were collected by centrifugation, washed in PBS buffer (8 g NaCl, 0·2 g KCl, 1·44 g Na2PO4 and 0·24 g KH2PO4 in 1000 ml distilled H2O, pH 7·4), and resuspended to 5x108 cells ml1. Fixing solution was prepared by mixing 100 µl 16 % paraformaldehyde, 0·2 µl 25 % glutaraldehyde (Electron Microscopy Sciences) and 20 µl 1 M sodium phosphate pH 7·4. Five hundered microlitres of cell suspension was added to the fixing solution and mixed. Ten microlitres of cells were dotted into one well of a 12-well Cel-Line glass slide (Erie Scientific) and incubated for 20 min in a covered Petri dish. The cells were then washed three times with PBS, blocked in PBS with 2 % (w/v) BSA and incubated with purified anti-PilA antibody (diluted 1 : 100 in PBS with 2 % BSA). Cells were washed five times with PBS and incubated with goat anti-rabbit antibody conjugated to FITC (Sigma-Aldrich). The samples were examined with a Nikon Eclipse E400 fluorescence microscope using a x40 objective, and images were acquired with a SPOT digital camera (Diagnostic Instruments, model 401-115).
Atomic force microscopy (AFM).
For direct imaging, exponential-phase M. xanthus cells were diluted 1 : 100 in MOPS buffer and 10 µl volumes of suspension were dotted onto a 12-well Cel-Line glass slide (Erie Scientific). The sample was air-dried and directly imaged with the atomic force microscope. For immuno-AFM, cells were prepared in a similar way as described above for immuno-fluorescence microscopy; the fixing step was eliminated and the secondary antibody was replaced with goat anti-rabbit IgG conjugated to microbeads (Miltenyi Biotec). Samples were imaged with a Nanoscope IV Bioscope (Veeco Digital Instruments). Olympus oxide sharpened cantilevers (OTR4) with spring constants of 0·02 N m1 and a tip radius of <10 nm were used in contact mode for all experiments.
ELISA for cell-surface pili.
This assay was developed based on the method of Harlow & Lane (1988). Exponential-phase M. xanthus cells were collected by centrifugation, washed in PBS buffer, and resuspended to 2·5x108 cells ml1. After adding 5 % BSA and incubating at room temperature for 15 min to block nonspecific binding, 1 µl or 10 µl of 100-fold diluted pre-absorbed anti-PilA antibody was added to 500 µl cell suspension and incubated for 1 h at room temperature. The suspension was then washed three times with PBS by centrifugation and resuspended to 500 µl. Ten microlitres of 100-fold diluted goat anti-rabbitalkaline phosphatase conjugated antibody (Sigma-Aldrich) was then added to the suspension and incubated for another 1 h at room temperature. After washing three times, the cells were suspended in 300 µl 1-Step PNPP (p-nitrophenyl phosphate, Pierce Biotechnology), and the reaction was stopped by centrifuging down the cells when yellow colour development became apparent (about 1015 min). The A405 of the supernatant was measured, which reflected the amount of antibody bound to the cell surface. DK10407 (pilA) was used as negative control.
Tethering assay.
This assay was based on a protocol published earlier (Sun et al., 2000) with the following modifications. A 24-well polystyrene plate was first coated with 2 % BSA for 30 min, and then with diluted (1 : 25 in PBS) anti-PilA serum for an additional 30 min. One microlitre of exponential-phase cells was diluted in 10 µl CYE and spotted into 250 µl 1 % methylcellulose (in CYE) in the coated 24-well plate. Tethering was monitored with a Nikon Eclipse TE200 inverted microscope using a x40 objective, captured with a Sony CCD-IRIS/RGB colour video camera and recorded with a Panasonic Time Lapse Video Cassette Recorder AG-6040. Recording was set at 60xslower than real-time, and the video was played back at normal speed to reveal cell movement. To calculate the percentage of tethered cells, 20 random movie frames were taken from the recording and the tethered cells were manually counted. For blocking of motility, M. xanthus cells were first incubated with anti-PilA antibody (1 : 10 diluted) and then added to a 24-well polystyrene plate, which was coated with antibody as described above. Cell motility was recorded as described above.
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RESULTS |
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We then performed immuno-fluorescence microscopy. M. xanthus cells were incubated with the antibody, probed with goat anti-rabbit IgG conjugated with FITC, and examined by fluorescence microscopy. Again, both DK10407 and anti-D-PilA antibody were used as controls. The fluorescence signal was clearly seen on the cell pole of wild-type cells (Fig. 3a), but was absent in the mutant lacking PilA (DK10407, Fig. 3b
) and in wild-type cells pre-incubated with anti-D-PilA (not shown). This observation strongly indicated that the antibody can recognize the native PilA protein in vivo. Additionally, in all the wild-type cells observed, fluorescence signals were seen on only one cell pole, confirming the earlier observation that M. xanthus pili appeared to localize unipolarly at a given time.
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Overpiliation of dif mutants
Since anti-PilA antibody could label pilus filaments, the immuno-fluorescence images were further examined. Previous electron microscopy studies indicated that around 30 % of wild-type cells were piliated at one end when the cells were taken from an agar plate (Wu et al., 1997). When fluorescent images of over 200 cells were analysed in this study, 31·5 % of the DK1622 cells had antibody labelling on one cell pole, correlating well with the data obtained by electron microscopy. Our earlier electron microscopy study found that dif mutants, a group of social motility mutants lacking extracellular fibril material, have longer pili at the cell poles (Li et al., 2003
; Sun et al., 2000
). When a dif mutant, SW504 (
difA), was examined by immuno-fluorescence microscopy, the majority of cells had a polar fluorescence signal, with the percentage reaching 76·9 % in the 200+ cells examined. This observation suggests that when the extracellular fibril material is missing, not only do individual cells have longer pili, but the percentage of piliated cells also dramatically increases. The fluorescent signals in SW504 were also unipolar, confirming that dif mutants are not defective in localization of pili.
Pilus retraction can be blocked with anti-PilA antibody
Direct observation and force measurement of pilus retraction in P. aeruginosa and N. gonorrhoeae (Merz et al., 2000; Skerker & Berg, 2001
) leave little doubt that pilus retraction powers twitching motility in these bacteria. In M. xanthus, extracellular polysaccharide-triggered pilus retraction has also been proposed (Li et al., 2003
). The methods employed in that study, however, did not allow direct assessment of pilus retraction. The anti-PilA antibody generated in the present study provides a useful tool to assess the retraction of pili directly.
The overpiliated dif mutant SW504 (difA) was incubated with different amounts of anti-PilA antibody prior to mixing with wild-type M. xanthus cells (see Methods for details), which triggers the retraction of pili on the dif mutant (Li et al., 2003
). Since the antibody can bind to pilus filaments, retraction should be blocked when enough antibodies are bound. Therefore, retraction will not occur when the mutant is then mixed with wild-type cells, and the level of cell-surface pilin will not decrease. As shown in the Western blot in Fig. 4
(a), when dif mutants were mixed with wild-type cells, the cell-surface pilin level of the mixture decreased, as reported before (Li et al., 2003
). However, when an increasing amount of antibody was incubated with dif mutant cells before the mixing, the cell-surface PilA level of the mixture increased, suggesting the blockage of pilus retraction. Since it is an obvious concern that an excess amount of anti-PilA antibody might contribute to the signal increase for the same amount of antigen, a control experiment was performed by incubating an identical amount of dif cells with increasing amounts of anti-PilA antibody. The cell-surface pili were then sheared off and Western blotted. Fig. 4(b)
shows that different antibody concentrations yielded similar signal intensity, dispelling this concern. These observations provided direct evidence for the extracellular polysaccharide-triggered pilus retraction hypothesis in M. xanthus.
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The tethering phenomenon was first reported in 1956 when E. coli cells were found tethered by their flagellum to a particle of debris or to the surface of the microscope slide (Stocker, 1956). Later, Silverman & Simon (1974)
developed an assay to tether a polyhook E. coli mutant to a solid surface using antisera to the polyhook. Under these conditions, the tethered flagellar hook cannot rotate, while the flagellar motor continues to rotate, causing the bacterial body to spin in the opposite direction (Silverman & Simon, 1974
). In the assay developed by Sun et al. (2000)
, M. xanthus cells were found to be tethered directly to polystyrene surface via their polar pili, and by adjusting the focal level of the microscope, it was observed that the tethered cells shortened over time, presumably due to the retraction of pili (Sun et al., 2000
). To further study the property of M. xanthus pili, we took an approach similar to that of the E. coli assay to tether M. xanthus cells with anti-PilA antibody.
Since polystyrene itself can tether pili, the wells were first coated with 2 % BSA for blocking and then with anti-PilA antibody for 1 h. Wild-type M. xanthus cells were spotted into the well with 1 % methylcellulose and monitored by time-lapse video microscopy (see Methods for details). The recording was then examined, and the percentage of tethered cells was calculated. In the wells without BSA coating, approximately 10 % cells were tethered; and in the wells covered with BSA alone, the percentage dropped to 1·5 % (Fig. 5a), confirming the blocking effect of BSA. In the anti-PilA antibody-coated wells (25x diluted anti-PilA serum; see Methods for details), however, an obviously higher percentage of cells was seen tethered (Fig. 5b
), averaging at 39·1 %. This observation further confirms that pili are the apparatus that tether M. xanthus cells to solid surfaces. When the tethered M. xanthus cells were observed over time, it was apparent that the cells often tilted and flopped from the vertical axis, leading to a rotation-like movement around the tethered end (Fig. 5e
). Since it has been reported that PilT mutants fail to retract pili, and tethered pilT cells showed no movement over time (Sun et al., 2000
), it is apparent that the movement of tethered wild-type cells was a result of pilus retraction and extension.
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DISCUSSION |
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Pilus retraction was originally proposed by David Bradley to account for the shortening of P. aeruginosa pili after phage attachment (Bradley, 1972). Using an anti-pilus antiserum, Bradley was able to stop not only this shortening effect, but also twitching motility in P. aeruginosa, suggesting that antibodies attached along the pilus fibre block retraction, and retraction plays an essential role in twitching motility (Bradley, 1974
, 1980
). In M. xanthus, retraction of pili was first reported by Sun et al. (2000)
, when the tethered cells (standing vertically on one cell pole) were found to move along the vertical axis over time. By quantifying the extracellular pilin level, a later study reported that the decrease of extracellular pilin, which is presumably caused by pilus retraction, can be triggered by extracellular polysaccharide (Li et al., 2003
). Using a similar approach to Bradley's, we report here that incubating M. xanthus cells with anti-PilA antibody could block this effect, confirming the pilus retraction hypothesis in M. xanthus. Furthermore, incubation with the antibody also blocked M. xanthus motility in 1 % methylcellulose, demonstrating the role of pilus retraction in M. xanthus S-motility.
The movement of tethered cells observed in Fig. 5(e) provided an interesting insight into how pili coordinate their retraction: electron microscopy studies showed that piliated wild-type cells have an average of 4 to 10 pili at one cell pole at a given time (Kaiser, 1979
). If all the pili in a tethered cell synchronized extrusion and retraction, the cell would only move up and down along the vertical axis; if pili extruded and retracted independently of each other, however, then elongation of certain pili and the shortening of others could contribute to the tilting of the cell body from the vertical axis as observed here. In P. aeruginosa, the independent extrusion and retraction of pilus filaments on one cell pole has been visualized on fluorescently labelled pili (Skerker & Berg, 2001
), providing supporting evidence that pili probably retract independently of each other in M. xanthus.
The anti-PilA antibody developed in this work provides a versatile tool for further study of TFP-dependent motility in M. xanthus. By coating an AFM tip with the antibody, or using antibody-coated beads with laser tweezers, the details of M. xanthus pilus retraction control as well as pilus mechanical properties can be further examined. Additionally, the truncated PilA protein can be used for crystallization and structural studies. In P. aeruginosa strain K122-4, the pilin protein truncated in a similar way was found to retain the same overall structure as full-length pilin (Keizer et al., 2001). In M. xanthus the amino-sugars in extracellular polysaccharide have been proposed to be involved in the trigger of pilus retraction (Li et al., 2003
). Therefore crystallography study of PilA in the presence of the amino-sugars should provide a structural basis for the extracellular polysaccharide-triggered pilus retraction, thus furthering our understanding of TFP-dependent motility in M. xanthus on a molecular basis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alm, R. A. & Mattick, J. S. (1996). Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178, 38093817.
Alm, R. A., Hallinan, J. P., Watson, A. A. & Mattick, J. S. (1996). Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue. Mol Microbiol 22, 161173.[Medline]
Binnig, G., Quate, C. F. & Gerber, C. (1986). Atomic force microscope. Phys Rev Lett 56, 930933.[CrossRef][Medline]
Bradley, D. E. (1972). Shortening of Pseudomonas aeruginosa pili after RNA-phage adsorption. J Gen Microbiol 72, 303319.[Medline]
Bradley, D. E. (1974). The adsorption of Pseudomonas aeruginosa pilus-dependent bacteriophages to a host mutant with nonretractile pili. Virology 58, 149163.[Medline]
Bradley, D. E. (1980). A function of Pseudomonas aeruginosa PAO polar pili: twitching motility. Can J Microbiol 26, 146154.[Medline]
Campos, J. M., Geisselsoder, J. & Zusman, D. R. (1978). Isolation of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus. J Mol Biol 119, 167178.[Medline]
Craig, L., Taylor, R. K., Pique, M. E. & 9 other authors (2003). Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol Cell 11, 11391150.[Medline]
Dufrene, Y. F. (2002). Atomic force microscopy, a powerful tool in microbiology. J Bacteriol 184, 52055213.
Forest, K. T., Bernstein, S. L., Getzoff, E. D., So, M., Tribbick, G., Geysen, H. M., Deal, C. D. & Tainer, J. A. (1996). Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies. Infect Immun 64, 644652.[Abstract]
Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Hodgkin, J. & Kaiser, D. (1979). Genetics of gliding motility in Myxococcus xanthus: two gene systems control movement. Mol Gen Genet 171, 177191.
Kaiser, D. (1979). Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc Natl Acad Sci U S A 76, 59525956.[Abstract]
Keizer, D. W., Slupsky, C. M., Kalisiak, M., Campbell, A. P., Crump, M. P., Sastry, P. A., Hazes, B., Irvin, R. T. & Sykes, B. D. (2001). Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili. J Biol Chem 276, 2418624193.
Li, Y., Sun, H., Ma, X., Lu, A., Lux, R., Zusman, D. & Shi, W. (2003). Extracellular polysaccharides mediate pilus retraction during social motility of Myxococcus xanthus. Proc Natl Acad Sci U S A 100, 54435448.
Merz, A. J., So, M. & Sheetz, M. P. (2000). Pilus retraction powers bacterial twitching motility. Nature 407, 98102.[CrossRef][Medline]
Notredame, C., Higgins, D. & Heringa, J. (2000). T-Coffee: a novel method for multiple sequence alignments. J Mol Biol 302, 205217.[CrossRef][Medline]
Paranchych, W., Frost, L. S. & Carpenter, M. (1978). N-terminal amino acid sequence of pilin isolated from Pseudomonas aeruginosa. J Bacteriol 134, 11791180.[Medline]
Parge, H. E., Forest, K. T., Hickey, M. J., Christensen, D. A., Getzoff, E. D. & Tainer, J. A. (1995). Structure of the fibre-forming protein pilin at 2·6 Å resolution. Nature 378, 3238.[CrossRef][Medline]
Russell, M. A. & Darzins, A. (1994). The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N-termini of type 4 prepilin proteins. Mol Microbiol 13, 973985.[Medline]
Silverman, M. & Simon, M. (1974). Flagellar rotation and the mechanism of bacterial motility. Nature 249, 7374.[Medline]
Skerker, J. M. & Berg, H. C. (2001). Direct observation of extension and retraction of type IV pili. Proc Natl Acad Sci U S A 98, 69016904.
Stocker, B. A. D. (1956). Bacterial flagella: morphology, constitution and inheritance. Symp Soc Gen Microbiol 6, 1940.
Sun, H., Zusman, D. R. & Shi, W. (2000). Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system. Curr Biol 10, 11431146.[CrossRef][Medline]
Wall, D. & Kaiser, D. (1999). Type IV pili and cell motility. Mol Microbiol 32, 110.[CrossRef][Medline]
Wu, S. S. (1997). The role of Type IV pili in social gliding motility of Myxococcus xanthus. PhD thesis, Stanford University.
Wu, S. S. & Kaiser, D. (1995). Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol 18, 547558.[Medline]
Wu, S. S. & Kaiser, D. (1997). Regulation of expression of the pilA gene in Myxococcus xanthus. J Bacteriol 179, 77487758.
Wu, S. S., Wu, J. & Kaiser, D. (1997). The Myxococcus xanthus pilT locus is required for social gliding motility although pili are still produced. Mol Microbiol 23, 109121.[Medline]
Yang, Z., Geng, Y., Xu, D., Kaplan, H. B. & Shi, W. (1998). A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility. Mol Microbiol 30, 11231130.[CrossRef][Medline]
Received 10 September 2004;
revised 8 November 2004;
accepted 9 November 2004.
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