Proc. Nati. Acad. Sci. USA Vol. 76, No. 11, pp. 5952-5956, November 1979
Microbiology
Social gliding is correlated with the presence of pili in Myxococcus xanthus (motility/swarming/cell interactions/fimbriae/Myxobacterales)
DALE KAISER Department of Biochemistry, Stanford University, Stanford, California 94305
Contributed by A. Dale Kaiser, August 20, 1979
ABSTRACT Myxococcus xanthus, an organism whose motility involves cell interactions, normally bears pili. Myxococcal pili are found only at cell poles, are less than 10 nm in diameter, and may be longer than a cell. Myxococcus has two basic patterns of cell movement, adventurous (A-motility) and social (S-motility). Pili are found to be completely correlated with the presence of S-motility. (The S-motility pattern has many groups of cells, almost no single cells, and is governed by a set of genes called system S.) On the other hand, A-motility is independent of piliation. (The A-motility pattern has many single, isolated cells and it is governed by a second set of genes called system A.) Electron microscopic examination of more than 40 genetically different strains shows that all A+S+ (wild-type) and A-S+ strains have pili, but A+S- and A-S- strains lack them. Mutations in four different loci belonging to system S were tested and were found to stop production of pili: the loci sglA, sglB, sglG, and tgl. When brought into contact with tgl+ cells, cells of a tgl - strain, which lack pili, become phenotypically S+ produce pili, and become S-motile. Both motility and the prouction of pili are transient when initiated in this way. Thus it appears that pili permit cells that are close to one another to move.
mutation in any one of the loci of system S, exhibit (pure) Amotility. A-S+ mutants, which have a mutation in any one of the loci of system A, exhibit (pure) S-motility. A-S- mutants, which have a mutation in any locus of system A and a mutation in any locus of system S, are nonmotile. There is evidence that pili (fimbriae), first reported on myxobacteria by MacRae and McCurdy (8), are associated with motility. Many, but significantly not all, motile myxobacteria and other gliding bacteria that have been examined have polar pili (8-11) and several nonmotile mutants of M. xanthus were shown to lack them. But if pili are associated with motility, how can there be nonpiliated motile strains? The existence of two patterns of movement in M. xanthus, controlled by different sets of genes, suggested the possibility that pili might be associated with only one of the two patterns. The experiments reported here were designed to test that possibility.
MATERIALS AND METHODS Bacteria. The origin and description of strains DK1200DK1292 may be found in refs. 6 and 7 and of strains DK801-DK898 in ref. 12. DZ2 was described by Campos and Zusman (13). DK1050 is a single-colony isolate of the motile prototype M. xanthus strain FB (14). DK1622 and DK1253SR are A+S+ transductants of DK1217 and DK1253, respectively, isolated by David Morandi. DK1805, DK1808, DK1811, DK1813, DK1818, and DK1819 are A+S- transductants of the A-S- strains DK1246, DK1251, DK1252, and DK1292, produced by using phage Mx8 grown on the A+S- strain DK1253. A+ transductants were selected by their capacity to glide through a Millipore membrane filter 150 tm thick with 0.45-,gm pores. Transductants were picked and purified and their A-motility was confirmed by microscopic examinations of the halo of cells around single colonies. The general conditions for growth of bacterial cultures, transduction, and stimulation have been described (15). The symbol A+ indicates that all loci of system A are wild-type and A- indicates that at least one locus in system A is mutant. S+ and S- have analogous meanings for the loci of system S. Assay of Pili. Bacterial cultures were grown in 1% casitone/8 mM MgSO4/10 mM Tris.HCl, pH 7.6/1 mM potassium phosphate, pH 7.6 (CTT broth) (15) at 250C with rotary shaking to cell densities of 1-4 X 108 cells per ml. (Several cultures were also grown at 330C, but no differences in piliation from 25'C cultures were noted, except for temperature-sensitive mutants.) The cells were sedimented, the supernatant fluid was poured off, and the cell pellet was resuspended in 10 mM Tris-HCl, pH 7.6, at a density of 2 X 108 cells per ml. One drop of cell suspension was placed on a carbon-coated electron microscope grid
Myxobacteria are rod-shaped cells that move by gliding on surfaces, an activity that helps them carry out their primitive kind of multicellular development (1, 2). When starved, thousands of cells assemble to form a fruiting body whose shape is genetically determined (3). Not only when fruiting but also when growing in the presence of ample nutrient, myxobacteria move in multicellular units (4, 5). A thin halo of cells forms at the edge of a colony on agar and this halo continually expands outward. When viewed at higher magnification the edge of the halo is seen to consist of a single-layer filigree of cells in which single cells and groups of cells move and reassort (5). The work to be reported here is aimed at understanding how myxobacterial cells interact to regulate and coordinate their movement. An investigation of more than 100 mutants of Myxococcus xanthus has revealed that its movement on agar is composed of two distinct patterns of movement, named A (for adventurous) and S (for social). In A-motility, single cells move (6), resulting in a spatial distribution with many single cells. In Smotility, isolated cells do not move, but cells that are close to one another do move (7). The net result is a spatial distribution of cells with many clusters and very few isolated single cells (4, 7). The two patterns are governed by different sets of genes: A-motility by one set of 21 loci, called gene system A and Smotility by another set of 9 loci, called gene system S (6, 7). One motility locus, mgl, is necessary for both kinds of movement. Wild-type M. xanthus has both gene systems and shows a mixture of the two patterns. However, because all but one of the motility loci affect either one or the other of the two patterns but not both, by using mutations it is possible to separate the two patterns from each other. Thus A+S- mutants, which have a
Abbreviations: A-motility, adventurous movement; S-motility, social
movement.
5952
Microbiology:
Kaiser
and cells were allowed to settle and adhere to the carbon for 1-2 min. The liquid droplet on the grid was washed by addition of several successive drops of the negative stain- 1% uranyl acetate mixed with 30 Mg of bacitracin per ml, serving as a
spreading agent (16). The stain solution was immediately removed with blotting paper and the grid was dried in air. Grids were imaged at an electron optical magnification of X12,600 and the image was viewed through a X10 telescope. Only those cell ends were scored that were free of adhering extracellular material and for which the quality of staining was such that in the judgment of the observer it could have revealed pili. Each cell end was scored individually because very often cells were joined by one end to a chain or clump of cells or partly overlapped the metallic portion of the grid. To the extent that cells adhere to other cells by their piliated ends, the number of pihated ends will be underestimated. However, nonpiliated cells disperse well and offer no problems for counting. Generally, only piliated strains form clumps but cultures of these strains also contain many free piliated cells. Therefore, enumeration of cell ends with pili is most accurate at low degrees of piliation and may underestimate the high degrees of piliation. RESULTS Pili of M. xanthus Fig. 1 shows a piliated cell of M. xanthus, illustrating several characteristic features. Pili arise from cell ends. Whenever the anchor point of a pilus in the cell surface was clearly visible in the negatively stained image, it was found in the curved region at the end of a cell. Sometimes pili appeared to emerge from the side of a cell, but closer examination showed that these filaments lie beneath cells. No unambiguous lateral insertions have been observed. Typically, 4-10 pili emerge from the same end. The majority of cells have pili at one end, but in heavily piliated strains many cells have pili at both ends. For example, in one sample of strain DK1217, 73 cells bore pili at one end, 59 cells had pili at both ends, and 2 cells had no pili. Bipolarly piliated cells usually had more than five pili at one end and
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. Piliation of wild-type strains Cell ends with pili* Strain Number % Origin DK801 Soil (Tracy, CA) 35 28/79 DK804 Soil (Yosemite, CA) 83 44/53 DK805 Soil (Palo Alto, CA) 65 49/76 DK806 Soil (Merced, CA) 36 12/33 DK813 Soil (South Dakota) 61 45/74 DK816 Soil (Ontario, Canada) 67 20/30 DK854 Soil (St. Louis, MO) 58 40/69 DK879 Soil (King City, CA) 47 16/34 DK891 Soil (Ames, IA) 79/110 72 DK897 Soil (Maryhill, WA) 42 17/41 DK898 Soil (Fiji) 61 35/57 DZ2 FB (13) 54 53/98 DK1050 FB (From M. Dworkin in 1973) 68/153 44 DK1622 Transduction of DK1217 41/70 58 DK1253SR Transduction of DK1253 157/373 42 * Under "Number" is given a fraction whose numerator is the number of cell ends with pili and whose denominator is the total number of ends scored.
fewer than three at the other. The two poles of a cell thus differ from each other in a way that might be explained by the difference in their ages. Pili observed on negatively stained cells from liquid culture vary in length. Most are around one cell length long,-3-5 Am-but some pili are at least 10 ttm long. The elementary pilus filament appears to be less than 10 nm in diameter. On stained carbon films the pili are cohesive and are often seen adhering to each other. The cohesiveness of pili for each other or of pili for cells may account for the tendency of piliated strains to grow nondispersed in liquid culture. Cultures of piliated strains growing in CTT broth with rotary agitation at 25°C form a macroscopic ring of cells adhering to the culture flask at the uppermost intersection of the meniscus of culture liquid with the surface of the vessel. Also, in the bulk phase of the liquid, spherical clumps of hundreds of cells form. The piliation of wild-type strains of Myxococcus are reported in Table 1. These strains are highly motile and fruit well. The list includes 11 Myxococcus species recently isolated from soil by the method of Singh (17), a procedure that selects for strains that fruit. Strain FB of M. xanthus, which has been propagated in the laboratory since 1963 (14), retains the capacity to fruit. The distribution of cells at the edge of a colony of FB on agar includes many single cells and many groups of cells, which is diagnostic of the A+S+ state (7). Two cultures of FB, DK1050 and DZ2, that had been propagated separately for more than 10 years in the laboratories of M. Dworkin and of D. Zusman show the A+S+ phenotype, and both strains have pili (Table 1). Finally, two A+S+ strains, DK1622 and DK1253SR, generated Table 2. Piliation of A-motile (A+S-) strains
FIG. 1. Electron micrograph of a piliated M. xanthus cell negatively stained with uranyl acetate. Bar, 0.5 ,m.
5953
Strain Genotype sglAl DK101 sglB3 DK1818 sglB46 DK1805 sglB46 DK1808 sglGl DK1300 tgl-1 DK1253 tgl-3 DK1817 Data were tabulated as in Table 1.
Cell ends with pili Number %
23/97 1/112 6/112 0/116 0/77 2/97 1/129
23 1 5
Microbiology
Social gliding is correlated with the presence of pili in Myxococcus xanthus (motility/swarming/cell interactions/fimbriae/Myxobacterales)
DALE KAISER Department of Biochemistry, Stanford University, Stanford, California 94305
Contributed by A. Dale Kaiser, August 20, 1979
ABSTRACT Myxococcus xanthus, an organism whose motility involves cell interactions, normally bears pili. Myxococcal pili are found only at cell poles, are less than 10 nm in diameter, and may be longer than a cell. Myxococcus has two basic patterns of cell movement, adventurous (A-motility) and social (S-motility). Pili are found to be completely correlated with the presence of S-motility. (The S-motility pattern has many groups of cells, almost no single cells, and is governed by a set of genes called system S.) On the other hand, A-motility is independent of piliation. (The A-motility pattern has many single, isolated cells and it is governed by a second set of genes called system A.) Electron microscopic examination of more than 40 genetically different strains shows that all A+S+ (wild-type) and A-S+ strains have pili, but A+S- and A-S- strains lack them. Mutations in four different loci belonging to system S were tested and were found to stop production of pili: the loci sglA, sglB, sglG, and tgl. When brought into contact with tgl+ cells, cells of a tgl - strain, which lack pili, become phenotypically S+ produce pili, and become S-motile. Both motility and the prouction of pili are transient when initiated in this way. Thus it appears that pili permit cells that are close to one another to move.
mutation in any one of the loci of system S, exhibit (pure) Amotility. A-S+ mutants, which have a mutation in any one of the loci of system A, exhibit (pure) S-motility. A-S- mutants, which have a mutation in any locus of system A and a mutation in any locus of system S, are nonmotile. There is evidence that pili (fimbriae), first reported on myxobacteria by MacRae and McCurdy (8), are associated with motility. Many, but significantly not all, motile myxobacteria and other gliding bacteria that have been examined have polar pili (8-11) and several nonmotile mutants of M. xanthus were shown to lack them. But if pili are associated with motility, how can there be nonpiliated motile strains? The existence of two patterns of movement in M. xanthus, controlled by different sets of genes, suggested the possibility that pili might be associated with only one of the two patterns. The experiments reported here were designed to test that possibility.
MATERIALS AND METHODS Bacteria. The origin and description of strains DK1200DK1292 may be found in refs. 6 and 7 and of strains DK801-DK898 in ref. 12. DZ2 was described by Campos and Zusman (13). DK1050 is a single-colony isolate of the motile prototype M. xanthus strain FB (14). DK1622 and DK1253SR are A+S+ transductants of DK1217 and DK1253, respectively, isolated by David Morandi. DK1805, DK1808, DK1811, DK1813, DK1818, and DK1819 are A+S- transductants of the A-S- strains DK1246, DK1251, DK1252, and DK1292, produced by using phage Mx8 grown on the A+S- strain DK1253. A+ transductants were selected by their capacity to glide through a Millipore membrane filter 150 tm thick with 0.45-,gm pores. Transductants were picked and purified and their A-motility was confirmed by microscopic examinations of the halo of cells around single colonies. The general conditions for growth of bacterial cultures, transduction, and stimulation have been described (15). The symbol A+ indicates that all loci of system A are wild-type and A- indicates that at least one locus in system A is mutant. S+ and S- have analogous meanings for the loci of system S. Assay of Pili. Bacterial cultures were grown in 1% casitone/8 mM MgSO4/10 mM Tris.HCl, pH 7.6/1 mM potassium phosphate, pH 7.6 (CTT broth) (15) at 250C with rotary shaking to cell densities of 1-4 X 108 cells per ml. (Several cultures were also grown at 330C, but no differences in piliation from 25'C cultures were noted, except for temperature-sensitive mutants.) The cells were sedimented, the supernatant fluid was poured off, and the cell pellet was resuspended in 10 mM Tris-HCl, pH 7.6, at a density of 2 X 108 cells per ml. One drop of cell suspension was placed on a carbon-coated electron microscope grid
Myxobacteria are rod-shaped cells that move by gliding on surfaces, an activity that helps them carry out their primitive kind of multicellular development (1, 2). When starved, thousands of cells assemble to form a fruiting body whose shape is genetically determined (3). Not only when fruiting but also when growing in the presence of ample nutrient, myxobacteria move in multicellular units (4, 5). A thin halo of cells forms at the edge of a colony on agar and this halo continually expands outward. When viewed at higher magnification the edge of the halo is seen to consist of a single-layer filigree of cells in which single cells and groups of cells move and reassort (5). The work to be reported here is aimed at understanding how myxobacterial cells interact to regulate and coordinate their movement. An investigation of more than 100 mutants of Myxococcus xanthus has revealed that its movement on agar is composed of two distinct patterns of movement, named A (for adventurous) and S (for social). In A-motility, single cells move (6), resulting in a spatial distribution with many single cells. In Smotility, isolated cells do not move, but cells that are close to one another do move (7). The net result is a spatial distribution of cells with many clusters and very few isolated single cells (4, 7). The two patterns are governed by different sets of genes: A-motility by one set of 21 loci, called gene system A and Smotility by another set of 9 loci, called gene system S (6, 7). One motility locus, mgl, is necessary for both kinds of movement. Wild-type M. xanthus has both gene systems and shows a mixture of the two patterns. However, because all but one of the motility loci affect either one or the other of the two patterns but not both, by using mutations it is possible to separate the two patterns from each other. Thus A+S- mutants, which have a
Abbreviations: A-motility, adventurous movement; S-motility, social
movement.
5952
Microbiology:
Kaiser
and cells were allowed to settle and adhere to the carbon for 1-2 min. The liquid droplet on the grid was washed by addition of several successive drops of the negative stain- 1% uranyl acetate mixed with 30 Mg of bacitracin per ml, serving as a
spreading agent (16). The stain solution was immediately removed with blotting paper and the grid was dried in air. Grids were imaged at an electron optical magnification of X12,600 and the image was viewed through a X10 telescope. Only those cell ends were scored that were free of adhering extracellular material and for which the quality of staining was such that in the judgment of the observer it could have revealed pili. Each cell end was scored individually because very often cells were joined by one end to a chain or clump of cells or partly overlapped the metallic portion of the grid. To the extent that cells adhere to other cells by their piliated ends, the number of pihated ends will be underestimated. However, nonpiliated cells disperse well and offer no problems for counting. Generally, only piliated strains form clumps but cultures of these strains also contain many free piliated cells. Therefore, enumeration of cell ends with pili is most accurate at low degrees of piliation and may underestimate the high degrees of piliation. RESULTS Pili of M. xanthus Fig. 1 shows a piliated cell of M. xanthus, illustrating several characteristic features. Pili arise from cell ends. Whenever the anchor point of a pilus in the cell surface was clearly visible in the negatively stained image, it was found in the curved region at the end of a cell. Sometimes pili appeared to emerge from the side of a cell, but closer examination showed that these filaments lie beneath cells. No unambiguous lateral insertions have been observed. Typically, 4-10 pili emerge from the same end. The majority of cells have pili at one end, but in heavily piliated strains many cells have pili at both ends. For example, in one sample of strain DK1217, 73 cells bore pili at one end, 59 cells had pili at both ends, and 2 cells had no pili. Bipolarly piliated cells usually had more than five pili at one end and
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. Piliation of wild-type strains Cell ends with pili* Strain Number % Origin DK801 Soil (Tracy, CA) 35 28/79 DK804 Soil (Yosemite, CA) 83 44/53 DK805 Soil (Palo Alto, CA) 65 49/76 DK806 Soil (Merced, CA) 36 12/33 DK813 Soil (South Dakota) 61 45/74 DK816 Soil (Ontario, Canada) 67 20/30 DK854 Soil (St. Louis, MO) 58 40/69 DK879 Soil (King City, CA) 47 16/34 DK891 Soil (Ames, IA) 79/110 72 DK897 Soil (Maryhill, WA) 42 17/41 DK898 Soil (Fiji) 61 35/57 DZ2 FB (13) 54 53/98 DK1050 FB (From M. Dworkin in 1973) 68/153 44 DK1622 Transduction of DK1217 41/70 58 DK1253SR Transduction of DK1253 157/373 42 * Under "Number" is given a fraction whose numerator is the number of cell ends with pili and whose denominator is the total number of ends scored.
fewer than three at the other. The two poles of a cell thus differ from each other in a way that might be explained by the difference in their ages. Pili observed on negatively stained cells from liquid culture vary in length. Most are around one cell length long,-3-5 Am-but some pili are at least 10 ttm long. The elementary pilus filament appears to be less than 10 nm in diameter. On stained carbon films the pili are cohesive and are often seen adhering to each other. The cohesiveness of pili for each other or of pili for cells may account for the tendency of piliated strains to grow nondispersed in liquid culture. Cultures of piliated strains growing in CTT broth with rotary agitation at 25°C form a macroscopic ring of cells adhering to the culture flask at the uppermost intersection of the meniscus of culture liquid with the surface of the vessel. Also, in the bulk phase of the liquid, spherical clumps of hundreds of cells form. The piliation of wild-type strains of Myxococcus are reported in Table 1. These strains are highly motile and fruit well. The list includes 11 Myxococcus species recently isolated from soil by the method of Singh (17), a procedure that selects for strains that fruit. Strain FB of M. xanthus, which has been propagated in the laboratory since 1963 (14), retains the capacity to fruit. The distribution of cells at the edge of a colony of FB on agar includes many single cells and many groups of cells, which is diagnostic of the A+S+ state (7). Two cultures of FB, DK1050 and DZ2, that had been propagated separately for more than 10 years in the laboratories of M. Dworkin and of D. Zusman show the A+S+ phenotype, and both strains have pili (Table 1). Finally, two A+S+ strains, DK1622 and DK1253SR, generated Table 2. Piliation of A-motile (A+S-) strains
FIG. 1. Electron micrograph of a piliated M. xanthus cell negatively stained with uranyl acetate. Bar, 0.5 ,m.
5953
Strain Genotype sglAl DK101 sglB3 DK1818 sglB46 DK1805 sglB46 DK1808 sglGl DK1300 tgl-1 DK1253 tgl-3 DK1817 Data were tabulated as in Table 1.
Cell ends with pili Number %
23/97 1/112 6/112 0/116 0/77 2/97 1/129
23 1 5
