Requirement Motility Myxobacteria · (E) MD120(A- S+), no antibiotic. (F) Sameas E, but with 10...
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JOURNAL OF BACTERIOLOGY, Nov. 1989, P. 6093-6096 Vol. 171, No. 11 0021-9193/89/116093-04$02.00/0 Copyright C 1989, American Society for Microbiology Calcium Requirement for Gliding Motility in Myxobacteria BELINDA J. WOMACK,t DAVID F. GILMORE,t AND DAVID WHITE* Department of Biology, Indiana University, Bloomington, Indiana 47405 Received 8 May 1989/Accepted 1 August 1989 The ability to glide on a solid surface was inducible by calcium ion in Stigmatella aurantiaca. The induction of motility but not motility itself was prevented by chloramphenicol and erythromycin. Calcium ion was also required for cells to glide, even when they were previously induced. The ability of Myxococcus xanthus to glide in groups using the S motility system but not as single cells (A system) was prevented by chloramphenicol and erythromycin. Myxobacteria are unicellular gram-negative bacteria that glide as single cells and as groups on solid surfaces. When nutrients are depleted, the cells move into aggregation centers consisting of several thousands of cells and form multicellular fruiting bodies (10, 21, 25, 26, 30). The mech- anistic basis for gliding motility in bacteria has not been established, although several hypotheses have been sug- gested, including some based upon contractile fibers, the secretion of surfactive agents, rotating disks in the cell surface, and surface adsorption sites moving along tracks in the cell envelope complex (4, 5, 7-9, 11, 14, 15, 22-24, 28). Myxobacteria move both as groups and as single cells; in Myxococcus xanthus, single-cell and group motility are determined by two gene systems called the A and S system, respectively (17-19). Kaiser reported that the presence of polar fimbriae is correlated with cells that glide by using the S system (20). The A and S systems share a common locus, mgl, which is required for both kinds of motility and may code for a protein in the gliding motor (31). In this paper we report that gliding motility in Stigmatella aurantiaca is inducible with calcium ion (i.e., the capability to glide is acquired only after incubation with calcium ion) and that the induction can be prevented by inhibitors of protein synthe- sis. In addition, calcium was required for motility per se in both M. xanthus and S. aurantiaca. The S motility system but not the A system in M. xanthus strains was prevented from functioning by inhibitors of protein synthesis. MATERIALS AND METHODS Strains. M. xanthus MD2 (agl+ sgo, MD120 (cglCJ sgl+), MD901 (cglDl agl-l sgl+), and MD207 (agl+ sgl+) were obtained from Martin Dworkin, University of Minnesota. S. aurantiaca DW135 was described previously (12). Growth conditions. M. xanthus and S. aurantiaca were grown in 1% Tryptone (Difco Laboratories, Detroit, Mich.) and 8 mM MgSO4. When required, sterile CaCl2 was added after autoclaving at a concentration of 1 mM. Preparation of agarose. Ultrapure electrophoresis-grade agarose (Bethesda Research Laboratories, Inc., Gaithers- burg, Md.) was washed with 0.1 M disodium EDTA (Sigma Chemical Co., St. Louis, Mo.) as follows. Agarose (5 g) was added to 150 ml of cold EDTA (pH 7.1). The mixture was stirred continuously with a magnetic bar for 2 h at room * Corresponding author. t Present address: National Cancer Institute, Frederick, MD 21701. t Present address: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06268. temperature. After 2 h the agarose-EDTA was filtered through Whatman no. 1 filter paper. The agarose was then rinsed three times with 200 ml of reverse-osmosis double- distilled water and then dried overnight in a desiccator under a vacuum. Preparation of slide cultures. The medium used for M. xanthus consisted of 1.5% EDTA-washed agarose, Al de- fined medium (2), 10 mM HEPES (N-2-hydroxyethylpipera- zine-N'-2-ethanesulfonic acid) (pH 7.2), and 8 mM MgSO4. The medium for S. aurantiaca was identical, except that 0.1% Tryptone was used instead of Al medium. CaCl2 at various concentrations was added to both media before autoclaving. Exponentially growing cells were washed twice in cold 10 mM HEPES buffer containing 8 mM MgSO4 and suspended in the same buffer to 5 x 108 cells per ml. Slide cultures were prepared as described by Reichenbach and Dworkin (26). Approximately 1 x 106 cells were placed in a 2-,ul drop in each slide culture. Slide cultures were incubated in a humid 30°C incubator and examined with a Zeiss Photomicroscope III (Hitschfel Optical Instruments, St. Louis, Mo.). Antibiotics. Chloramphenicol and erythromycin were pur- chased from Sigma. Control experiments demonstrated that gliding motility was restored when the cells were washed free of antibiotics, showing that the antibiotics did not cause irreversible cell damage. RESULTS Induction of gliding motility in S. aurantiaca DW135 with calcium ion. S. aurantiaca DW135 cells grown in liquid medium with 1 mM CaCl2 were capable of gliding when subsequently placed into slide culture (Fig. 1A). In cells that were not grown with Ca2+ little or no gliding took place (Fig. 1B). Careful observation of the center of the swarms failed to reveal slime trails or any organizational pattern of cells that would suggest motility. Calcium added to the cells at the time of harvesting was not effective in inducing gliding motility, suggesting that the cell washing procedure effec- tively removed free calcium ion (data not shown). Four hours of incubation with calcium in the growth medium was sufficient to induce gliding (Fig. 1C), and the induction was prevented by very small amounts of chloramphenicol (Fig. 1D). Chloramphenicol did not prevent induced cells from gliding (Fig. 1E). Induced cells also glided when the chlor- amphenicol was incorporated into the agarose (data not shown). Similar results were obtained with erythromycin (data not shown). This suggests that the incubation with calcium stimulated the synthesis of proteins required for gliding motility. The minimum amount of calcium ion in the 6093 on April 19, 2020 by guest http://jb.asm.org/ Downloaded from
Transcript of Requirement Motility Myxobacteria · (E) MD120(A- S+), no antibiotic. (F) Sameas E, but with 10...
JOURNAL OF BACTERIOLOGY, Nov. 1989, P. 6093-6096 Vol. 171, No. 110021-9193/89/116093-04$02.00/0Copyright C 1989, American Society for Microbiology
Calcium Requirement for Gliding Motility in MyxobacteriaBELINDA J. WOMACK,t DAVID F. GILMORE,t AND DAVID WHITE*
Department ofBiology, Indiana University, Bloomington, Indiana 47405
Received 8 May 1989/Accepted 1 August 1989
The ability to glide on a solid surface was inducible by calcium ion in Stigmatella aurantiaca. The inductionof motility but not motility itself was prevented by chloramphenicol and erythromycin. Calcium ion was alsorequired for cells to glide, even when they were previously induced. The ability of Myxococcus xanthus to glidein groups using the S motility system but not as single cells (A system) was prevented by chloramphenicol anderythromycin.
Myxobacteria are unicellular gram-negative bacteria thatglide as single cells and as groups on solid surfaces. Whennutrients are depleted, the cells move into aggregationcenters consisting of several thousands of cells and formmulticellular fruiting bodies (10, 21, 25, 26, 30). The mech-anistic basis for gliding motility in bacteria has not beenestablished, although several hypotheses have been sug-gested, including some based upon contractile fibers, thesecretion of surfactive agents, rotating disks in the cellsurface, and surface adsorption sites moving along tracks inthe cell envelope complex (4, 5, 7-9, 11, 14, 15, 22-24, 28).Myxobacteria move both as groups and as single cells; inMyxococcus xanthus, single-cell and group motility aredetermined by two gene systems called the A and S system,respectively (17-19). Kaiser reported that the presence ofpolar fimbriae is correlated with cells that glide by using theS system (20). The A and S systems share a common locus,mgl, which is required for both kinds of motility and maycode for a protein in the gliding motor (31). In this paper wereport that gliding motility in Stigmatella aurantiaca isinducible with calcium ion (i.e., the capability to glide isacquired only after incubation with calcium ion) and that theinduction can be prevented by inhibitors of protein synthe-sis. In addition, calcium was required for motility per se inboth M. xanthus and S. aurantiaca. The S motility systembut not the A system in M. xanthus strains was preventedfrom functioning by inhibitors of protein synthesis.
MATERIALS AND METHODSStrains. M. xanthus MD2 (agl+ sgo, MD120 (cglCJ sgl+),
MD901 (cglDl agl-l sgl+), and MD207 (agl+ sgl+) wereobtained from Martin Dworkin, University of Minnesota. S.aurantiaca DW135 was described previously (12).Growth conditions. M. xanthus and S. aurantiaca were
grown in 1% Tryptone (Difco Laboratories, Detroit, Mich.)and 8 mM MgSO4. When required, sterile CaCl2 was addedafter autoclaving at a concentration of 1 mM.
Preparation of agarose. Ultrapure electrophoresis-gradeagarose (Bethesda Research Laboratories, Inc., Gaithers-burg, Md.) was washed with 0.1 M disodium EDTA (SigmaChemical Co., St. Louis, Mo.) as follows. Agarose (5 g) wasadded to 150 ml of cold EDTA (pH 7.1). The mixture wasstirred continuously with a magnetic bar for 2 h at room
* Corresponding author.t Present address: National Cancer Institute, Frederick, MD
21701.t Present address: Department of Molecular and Cell Biology,
University of Connecticut, Storrs, CT 06268.
temperature. After 2 h the agarose-EDTA was filteredthrough Whatman no. 1 filter paper. The agarose was thenrinsed three times with 200 ml of reverse-osmosis double-distilled water and then dried overnight in a desiccator undera vacuum.
Preparation of slide cultures. The medium used for M.xanthus consisted of 1.5% EDTA-washed agarose, Al de-fined medium (2), 10 mM HEPES (N-2-hydroxyethylpipera-zine-N'-2-ethanesulfonic acid) (pH 7.2), and 8 mM MgSO4.The medium for S. aurantiaca was identical, except that0.1% Tryptone was used instead of Al medium. CaCl2 atvarious concentrations was added to both media beforeautoclaving. Exponentially growing cells were washed twicein cold 10 mM HEPES buffer containing 8 mM MgSO4 andsuspended in the same buffer to 5 x 108 cells per ml. Slidecultures were prepared as described by Reichenbach andDworkin (26). Approximately 1 x 106 cells were placed in a2-,ul drop in each slide culture. Slide cultures were incubatedin a humid 30°C incubator and examined with a ZeissPhotomicroscope III (Hitschfel Optical Instruments, St.Louis, Mo.).
Antibiotics. Chloramphenicol and erythromycin were pur-chased from Sigma. Control experiments demonstrated thatgliding motility was restored when the cells were washedfree of antibiotics, showing that the antibiotics did not causeirreversible cell damage.
RESULTSInduction of gliding motility in S. aurantiaca DW135 with
calcium ion. S. aurantiaca DW135 cells grown in liquidmedium with 1 mM CaCl2 were capable of gliding whensubsequently placed into slide culture (Fig. 1A). In cells thatwere not grown with Ca2+ little or no gliding took place (Fig.1B). Careful observation of the center of the swarms failed toreveal slime trails or any organizational pattern of cells thatwould suggest motility. Calcium added to the cells at thetime of harvesting was not effective in inducing glidingmotility, suggesting that the cell washing procedure effec-tively removed free calcium ion (data not shown). Fourhours of incubation with calcium in the growth medium wassufficient to induce gliding (Fig. 1C), and the induction wasprevented by very small amounts of chloramphenicol (Fig.1D). Chloramphenicol did not prevent induced cells fromgliding (Fig. 1E). Induced cells also glided when the chlor-amphenicol was incorporated into the agarose (data notshown). Similar results were obtained with erythromycin(data not shown). This suggests that the incubation withcalcium stimulated the synthesis of proteins required forgliding motility. The minimum amount of calcium ion in the
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FIG. 1. Induction of gliding motility in S. aurantiaca. Photographs were taken after 15 h of incubation. Bar, is 30 pm. (A) Cells grown with1 mM calcium chloride. (B) Cells grown without the addition of calcium to the growth medium. (C) Same as B, except that 1 mM calciumwas added 4 h before harvesting. (D) Same as C, except that 1 ,ug of chloramphenicol per ml was added with the calcium. (E) Same as A,except that 1 p.g of chloramphenicol per ml was added 4 h before harvesting.
growth medium that was sufficient for the induction was 0.5mM. Similar experiments were done with M. xanthus MD2and MD207 grown in defined medium without the addition ofcalcium ion. In contrast to results with S. aurantiaca,previous growth in Ca2'-supplemented media was not re-quired for M. xanthus cells to glide (data not shown).Calcium requirement for functioning of the gliding machin-
ery. As described below, calcium ion was required forgliding motility in Myxococcus cells and in Stigmatella cellspreviously induced by calcium. However, the amounts ofcalcium required for gliding were less than those required forinduction. Previously induced Stigmatella cells and Myxo-coccus cells were capable of gliding on agarose to which weadded 0.1 to 0.3 mM CaCl2. Two other cations in the calciumseries, i.e., Ba2+ and Sr2', did not substitute for Ca2+ whenadded as chloride salts, even at a concentration of 1 mM. Allof the agarose media were supplemented with 8 mM MgSO4;hence, Mg2' also did not substitute for calcium. It could beargued that the small amounts of calcium in the agarose werenot required for motility per se but rather kept the cells in theinduced state. The data do not rule out this possibility.However, as discussed above, induced cells glided when 1,ug of chloramphenicol per ml, an amount sufficient toprevent induction in cell suspension (Fig. 1), was incorpo-rated into the agarose.
Effects of protein synthesis inhibitors on gliding motility ofM. xanthus MD207, MD2, MD120, and MD901. During thecourse of the experiments, it was discovered that chloram-phenicol and erythromycin inhibited gliding motility in the Ssystem of M. xanthus but not in the A system. StrainsMD207 (A+ S+), MD2 (A+ S-), MD120 (A- S+), andMD901 (A S+) were spotted on slide cultures prepared withAl defined medium and 1 mM CaCl2 with or without
chloramphenicol. Chloramphenicol prevented motility ofA-S' strains (Fig. 2E and F) but did not inhibit motility of A'S- strains (Fig. 2C and D). Since MD2 is a leaky S-systemmutant, the chloramphenicol experiment was repeated withMD123, which is a nonleaky A' S- strain. As with MD2,chloramphenicol did not prevent gliding motility (data notshown). Similar results were obtained with 5 jig of erythro-mycin per ml (data not shown).
DISCUSSION
Both S. aurantiaca and M. xanthus required 0.1 to 0.3 mMCa2+ for gliding motility. Calcium ion could not be replacedwith Mg2+, Ba2+, or Sr2. These results confirm those ofBurchard, who concluded from his experiments with both aCa2+ ion chelator and an ionophore that M. xanthus requiresCa2+ for gliding motility (3). Calcium ion is also required forgliding motility in Oscillatoria sp., Mastigocladus lamino-sus, Phormidium unicinatum, and Spirulina subsalsa (1, 3,6, 13, 16).We were surprised to find that gliding motility was an
inducible phenotype in S. aurantiaca. This was unexpected,since there is no obvious advantage for S. aurantiaca to benonmotile in its natural habitat. We examined several Stig-matella strains, and they all showed the calcium-induciblephenotype. We are currently purifying two proteins whoseamounts greatly increase in calcium-grown cells.We reported previously that intercellular cohesion is also
a calcium-inducible phenotype in S. aurantiaca and that theinduction was prevented by inhibitors of RNA and proteinsynthesis (12). Furthermore, the cohesion process itself isprevented by inhibitors of energy metabolism, suggestingthat cohesion is an energy-dependent process and not due
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Ca REQUIREMENT FOR GLIDING MOTILITY IN MYXOBACTERIA 6095
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FIG. 2. Effect of chloramphenicol on gliding motility in M. xanthus. Cells were spotted on Al agarose medium containing 1 mM CaCl2.Photographs were taken after 24 h of incubation. Bar, 30 ,um. (A) MD207 (A' S+), no antibiotic. (B) Same as A, but with 10 ,ug ofchloramphenicol per ml. (C) MD2 (A' S-), no antibiotic. (D) Same as C, but with 10 ,ug of chloramphenicol per ml. (E) MD120 (A- S+), noantibiotic. (F) Same as E, but with 10 ,g of chloramphenicol per ml.
simply to nonspecific ionic or hydrophobic interactionsbetween spatially fixed molecules on the cell surfaces. Sinceboth cohesion and motility are calcium inducible and sinceboth require metabolic energy to function, it is possible thatcohesion and motility are related phenomena ip S. auranti-aca.We were not able to demonstrate a similar induction of
gliding motility for M. xanthus. Either motility is constitu-tive in M. xanthus, or the small amounts of calcium in theagarose (0.1 mM) were sufficient to induce motility.However, we did learn that group motility in M. xanthus,
but not single cell motility, appears to require proteinsynthesis. The reason why inhibitors of protein synthesisprevented motility in the S system of Myxococcus strains isnot clear. The implication is that certain proteins that arerequired for S motility are either not present in cells grown insuspension culture or are present but then easily lost duringgliding. The former possibility implies an induction on theagar of the synthesis of proteins required for S motility. Thelatter possibility suggests the existence of an unstable pro-tein or one lost from the cells during the gliding process.Kaiser reported that pili are correlated with S motility in M.xanthus (20). However, pili are present on cells grown insuspension, and at this time there is no evidence that theyare lost during gliding motility. The proteins in question mayalso be involved in cohesion, since Shimkets reported acorrelation between cohesion and the S motility system inM. xanthus (29).
ACKNOWLEDGMENT
This work was supported by National Science Foundation grantDCB 88-01934.
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