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Cell-cell interactions in developmental lysis of Myxococcus xanthus
DEVELOPMENTAL
BIOLOGY
112,194-202
(19%)
Cell-Cell Interactions in Developmental Lysis of Adyxococcus xanthus GARY R. JANSSEN~ AND MARTIN DWORKIN Department
of Microbiology,
University
of Minnesota,
Minneapolis,
Minnesota
55.455
Received March 20, 1985; accepted in revised form May 7, 1985 The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously 0 1985 Academic Press, Inc. supplied glucosamine or mannosamine.
When cells of the bacterium Myxococcus xanthus are on a solid surface and are at a high cell density, they respond to nutritional starvation by shifting the mode of their life cycle from vegetative feeding to development. The developmental sequence involves aggregation of cells into centers that eventually form raised, slimecovered fruiting bodies. The majority of cells lyse during the developmental process while most of the surviving cells differentiate to myxospores within the fruiting bodies (Wireman and Dworkin, 1975,1977). The lysis is an autolytic process (Wireman and Dworkin, 1977) and, while its function is unknown, its occurrence just prior to and concomitant with myxospore induction and maturation suggests a possible causal relationship between the two developmental events. Observations that lysis occurs during development of other species of myxobacteria (Wireman and Dworkin, 1977) and that, under certain conditions, autolysis may be required for myxospore induction (Wireman, 1979) suggest that cell death is an important event in myxobacterial development. Furthermore, the dependence of subsequent development on the cell density of the lysing population (Wireman and Dworkin, 1977) suggests that the development is dependent on a product of the autolysis. Nevertheless, while there is abundant evidence that M xanthus does indeed manifest a variety of cell interactions (Rosenberg, 1984), no endogenous signals have thus far been isolated. Hagen et al. (1978) have described a set of conditional nonsporulating mutants of M. xanthus that can be divided into four groups that undergo extracellular, phenotypic complementation. Mutants within each group i Present address: Department of Genetics, Colney Lane, Norwich, NR4 7UH, England. 0012-1606/85 $3.00 Copyright All rights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
John Innes Institute,
194
are sporulation (spo) deficient but can be synergized to sporulate when mixed with members of other complementation groups or with the developmentally proficient parental strain. It has been suggested (Hagen et aZ.,1978) that this complementation reflects the normal exchange of signals during multicellular development. We have used this system of extracellular complementation to examine the process of developmental autolysis. In this paper we shall describe a partially purified extract that contains a cell-associated substance which acts as the signal of developmental autolysis. Furthermore, we show that the effect of the endogenous signal can be replaced by mannosamine and glucosamine. MATERIALS
AND
METHODS
Bacterial strains and media. M. xanthus strains MD2 and DKlOl were developmentally proficient, yellow, swarming variants of Al. xanthus FB (Wireman and Dworkin, 1975). DKlOl and MD2 were both derived from M. xanthus FB, and under the conditions used here their developmental responses were similar; DKlOl is the parental strain of the synergy mutants described below. The following strains were obtained from D. Kaiser (Dept. of Biochemistry, Stanford University) and represent single colony isolates of synergy groups A, B, C, and D: DK476 (Group A), DK468 (Group B), DK731 and DK741 (Group C), DK429 (Group D), DK2453 (kan’group B), DK2634 (contains the spoC allele of group C mutant DK731). DKlOl was also obtained from D. Kaiser. Cells were vegetatively cultured in liquid, Casitone growth medium (CT), as described (Orndorff and Dworkin, 1980). Vegetative growth on solid medium was on CTT agar (Hagen et al., 1978). Liquid-grown cultures were started from isolated yellow or tan colonies (Bur-
JANSSEN AND DWORKIN
Cell Interactims
195
in M~XOCOCCUS
chard et al., 1977) picked from CTT agar; yellow and tan phase variants were distinguished by viewing colonies under a long wave (379 nm) ultraviolet light (Ultra-Violet Products, Inc.). Development was induced on clone fruiting (CF) agar (Hagen et al, 1978) or CF agar supplemented with glucosamine-HCl, mannosamine, or a mixture of N-acetylglucosamine, D-alanine, and diaminopimelic acid (Sigma Chemical Co.; St. Louis, MO.).
error of less than 10% .The number of viable myxospores was determined by heating harvested cell suspensions to 51°C for 2 hr, followed by aseptic, extended sonication (as above) and plating for colony-forming units on CTT agar. The combined heat and sonication treatments resulted in less than lo-’ survival for vegetative cells while not significantly affecting mature myxospore viability.
Induction of development and measurement of cell number. Vegetative cultures, grown in CT broth to 4-10
Cells were harvested from CF agar after 3 days of incubation at 32°C (inoculated at a to cell density of l-2 X lo7 cells cm-‘). Harvested cells, on ice in TPM buffer, were sonicated twice at 70 W for 1 min each time, followed by sedimentation of cell debris at 27,OOOg for 15 min. The supernatant fraction was vigorously extracted with an equal volume of chloroform, followed by centrifugation at 12,000g for 10 min. The resulting aqueous phase was acidified to 1 N HCl for approximately 8 hr at room temperature, followed by centrifugation at 27,000g for 15 min. The supernatant fraction, containing DS activity, was neutralized by dialysis in Spectrapor 3 tubing (mol wt cutoff approximately 3500) against TPM buffer or H20. These extractions resulted in the removal, by precipitation or phase partitioning, of a large amount of material while not seriously affecting the amount of DS activity present.
X lOa cells ml-‘, were harvested by centrifugation (12,000g for 10 min) at 4”C, washed and resuspended in cold TPM buffer [IO mM Tris-HCl (pH 7.6), 1 mM potassium phosphate (pH 7.6), and 8.3 mM magnesium sulfate], spread uniformly onto the surface of CF agar at a starting density of 10’ cells cm-’ and incubated at 32°C. Cells harvested from developmental plates were fixed in 2.5% glutaraldehyde for at least 18 hr at 4°C. Cell clumps were dispersed by sonication (5 set at 65 W; Sonifier Cell Disrupter, Heat Systems-Ultrasonics, Inc.). Extended sonication (two 30-set pulses at 65 W with intermittent cooling) was used for quantitation of ovoid, refractile myxospores. Cell numbers were determined in a Petroff-Hausser counting chamber with a maximum
Production (0s) activity.
and extraction
of development-stimulating
----
Sporulotion - - -Net Lyrir
r
6
-----_
IOf
- - --Aggregation
INet
0
Growth
I
5
20
40
Fruiting Body Formatton
60 fi0 Time(h)
I I
1. 100
120
4 140
FIG. 1. Sequence of developmental events during fruiting body formation by M. zanthus. Cells were prepared as described under Materials and Methods and spread uniformly on development-inducing CF agar at 3.1 X lo6 cells cm-*. (A) Morphological changes during M. zanthus MD 2 development. (B) Quantitation of cell number during M. zanthus development. At the times indicated, duplicate plates were harvested for determination of cell numbers. Symbols: (W - W) MD 2; (W --- n ) MD 2 myxospores; (0 - 0) DK468; (0 - 0) DK741; (Cl - 0) mixture of DK468 X DK741; (0 --- 0) DK468 X DK741 myxospores.
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DEVELOPMENTAL BIOLOGY
Screening for develqwnumtal rescue by agar well assays. Plugs were removed from the central region of petri plates containing developmental cells on CF agar. The resultant wells were then filled with cell extracts or sugar solutions that had been mixed with molten agar. The developmental effect of a cell extract or sugar was determined by comparison to agar wells containing TPM buffer. The assay allowed for a concentration gradient (i.e., through diffusion) of a substance to be tested at selected times during development. RESULTS
Development of M. xanthus Strain MD2
VOLUME 112, 1985 TABLE 1 CORRELATION OF SPORULATION WITH SYNERGIZED DEVELOPMENTAL LYSIS’
Strain DK476 DK468 DK741 DK429 DKlOl 476 476 476 468 468 741
X X X x X x
468 741 429 741 429 429
Sporulation (% of parental)”
% decrease in maximum vegetative numberb
Direct counts
Viable counts
A B C D fru +
22% 32% 29% 30% 93%
AB AC AD BC BD CD
25% 70% 22% 96% 17% 82%
0.2% 25% 0.2% 94%
0.03% 7.5% 0.07% 96% 0.02% 13%
Synergy grow
Figure la illustrates the changes in colonial morphology during fruiting body development. At about 28 hr cells began moving into aggregates; this process was completed at about 60 hr, at which time the aggregates “Each value represents the average of two or three independent are referred to as immature fruiting bodies. Lysis began determinations. b Decrease in vegetative cell number is determined by a direct count at about 60 hr and ended at about 80 hr; at this point, the surviving cells converted to myxospores and the determination of cell number at the point of maximum growth (i.e., hr, see Fig. la) and at the developmental endpoint time of 168 hr mound of cells acquired the appearance of the mature 66 (i.e., normal and synergized development are essentially complete). fruiting body. Figure lb illustrates the changes in de’ Sporulation is expressed relative to the level of sporulation meavelopment and cell number that occurred with time when sured in the developmentally proficient (fru+) parental strain DKlOl. the developmentally proficient M. xanthus strain MD2 Direct counts of optically refractile myxospores were done with a Pewas placed under appropriate conditions. The cells un- troff-Hausser counting chamber. Viability counts were measured as forming units after heat treatment and sonication (see Materials derwent an initial period of growth resulting in the ac- colony and Methods). cumulation of a number of cells sufficient for the initiation of development (Shimkets and Dworkin, 1981). Cell number reached maximal value and leveled off, presum- followed by a developmental lysis phase in which greater ably in response to depletion of essential nutrients in than 80% of the cells lysed. The developmental lysis the medium. The growth and aggregation phases were phase slightly preceded and was then concomitant with
FIG. 2. Rescue by DS of fruiting body formation in DK741. The group C synergy mutant DK741 was spread uniformly on CF agar at 10’ cells/cm*. After about 30-hr incubation at 32°C crude DS extract or TPM buffer were assayed for development stimulating activity by the agar-well assay method. Pictures were taken after 5 days incubation.
JANSSEN AND DWORKIN
the differentiation myxospores. Developmental
of the remaining
Lysis in Sporulation
Cell Interactions
cells to resting
Synergy Mutants
Representative strains of groups A, B, C, and D sporulation synergy mutants, described by Hagen et al. (19’78), were examined for developmental lysis. When placed under developmental conditions, the mutants displayed the usual growth phase followed by a plateau in cell number, but the characteristic lysis phase normally observed during development of M. xanthus (Fig. lb) was absent. The strains tested were also deficient in sporulation (Table 1). Sporulation in this set of mutants is phenotypically complemented when a mutant of one synergy group is mixed with a mutant from a different synergy group, as described by Hagen et al. (1978). Pairwise mixtures of these lysis-deficient, sporulation-deficient strains were tested to determine if lysis was complementable. Table 1 shows the lysis phenotype of various pairwise mixtures when tested under developmental conditions. Cell number was quantitated at the time of maximal growth (approximately 66 hr under these conditions) and at an endpoint time of 168 hr. Mixtures of DK476 X DK468 (i.e., group A and group B), DK476 X DK429 (i.e., group A and group D), and DK468 X DK429 (i.e., group B and group D) showed negligible complementation of developmental lysis. Mixtures of DK468 X DK741 (i.e., group A and group C) and DK741 X DK429 (i.e., group C and group D) showed significant complementation of lysis. Mixtures of DK468 X DK741 (i.e., group B and group C) showed levels of developmental lysis comparable to that observed with the developmentally proficient MD2 and the parental strain DKlOl (Table 1 and Fig. lb). Therefore, among the strains tested, phenotypic complementation of developmental lysis fell into two groups, with strains DK476, DK468, and DK429 comprising one group (A, B, and D) and DK741 (C) representing the other group. The lysis-deficient strains and strain mixtures showed a slight, gradual decrease in cell number with extended incubation (Table l), but the extent and rate was qualitatively distinct from the rapid, synchronous, developmental lysis that is tightly associated with myxosporulation and fruiting body formation (Fig. lb and Wireman and Dworkin, 1975, 1977). Sporulation synergy was also measured in the pairwise mixtures tested above (Table 1). Extracellular complementation of sporulation was greatest in pairwise mixtures where developmental lysis was complemented (i.e., in mixtures of group A with C, group B with C, and group D with C); minimal amounts of sporulation synergy occurred in mixtures showing minimal complementation of developmental lysis (i.e., in mixtures of group A with B, group A with D, and group B with D).
197
in Myxococcus
Cell-Free Complementation of Lysis and Development in Group C Synergy Mutants (1) Complementation by a cell-free extract. Developmental lysis in the mixture of DK468 (group B) and DK741 (group C) closely paralleled the lysis observed in the developmentally competent il4 xanthus strain MD2 (Fig. lb). Thus, it was appropriate to use one member of this pair as the source of a cell-free complementing factor and the other to assay for complementation of developmental lysis. The close association of developmental lysis with fruiting body formation (Wireman and Dworkin, 1975) suggested that complementation of fruiting body formation would be an appropriate parameter of lysis-stimulating activity as an initial screening step. Cell extracts that complemented fruiting TABLE 2 DISSOCIATION OF DS ACTIVITY FROM DK2453 CELLS
Extraction
conditions“
1. Vigorous vortexing of developmental DK2453 cell suspension in TPM buffer a. Supernatant fraction b. Crude DS extract of sedimented cells 2. Developmental DK2453 cell suspension stirred at 4°C for 12-14 hr in TPM buffer a. Supernatant fraction b. Crude DS extract of sedimented cells 3. Developmental DK2453 cell suspension subjected to four cycles of freeze-thaw in TPM buffer a. Supernatant fraction b. Crude DS extract of sedimented cells 4. Developmental DK2453 cell suspension stirred at 4’C in 1 M NaCl for 12-14 hr a. Supernatant fraction b. Crude DS extract of sedimented cells 5. Developmental DK2453 cell suspension stirred at 4°C in 10% TCA for 12-14 hr a. Supernatant fraction b. Crude DS extract of sedimented cells 6. Crude DS extract of DK2453 vegetative cells (mid-log growth in liquid CT)
Relative DS activityb
<5Y0 95-100%
<5% 95-100%
30-60% 20-40%
20-60% 30-50%
50-90% O-20% 0%
a Extraction conditions l-5 used DK2453 cells cultivated on CF agar for production of DS extract, as described under Materials and Methods. After the various extraction procedures, cells were sedimented by 27,1009 centrifugation; the supernatant fraction and sedimented cells, after crude DS extraction, were assayed for DS activity. Extraction condition 6 used an equivalent number of mid-log growth phase vegetative cells. b DS activity was measured as number of fruiting bodies stimulated in an agar-well assay on DK741. Table 2 presents results from multiple experiments and, because of variability from experiment to experiment, DS activity is expressed relative to the DS activity observed in the crude DS extracted control for that experiment. A range of values for “Relative DS Activity” represents the range of results obtained in multiple testing. The crude DS extract of untreated cells generally stimulated about 300-600 fruiting bodies in these experiments.
DEVELOPMENTAL
BIOLOGY
VOLUME 112. 1985
I
*’ t ,+ f
i 0
#
20
40
0’ :
I
60 60 Time (h)
loo
20
40
60 Time
80
.’
/’
/’
100
.’
9
120
(h)
FIG. 3. Rescue of developmental lysis in DK741 and DK731 by DS extract. Cells were spread uniformly on CF agar at an initial density of lo7 cells cm-’ (12 ml agar in 60-mm diameter plate). Lyophilized DS extract was added under the agar of a set of DK741 and DK731 plates and incubation continued. Duplicate plates were harvested at the times indicated for determination of cell number. The amount of DS extract applied to each assay plate represented DS extract from 2.6 to 2.8 X 10 in. DK2453 developmental cells (containing 0.24 mg protein and 4.23 mg carbohydrate when assayed in B). (A) Rescue of DK741 by DS extract. Symbols: (0 - 0) DK741; (A - A) DK2453; (0 - 0) mixture of DK741 X DK2453; (0 - 0) DK741 + DS extract; (m --- n ) myxospores from DK741 X DK2453 mixture; (0 --- 0) myxospores from DK741 + DS extract. (B) Rescue of DK731 by DS extract. Symbols: (0 - 0) DK731; (0 - 0) DK2634; (0 - 0) DK731 + DS extract; (B - W) DK2634 + DS extract; (O - 0) myxospores from DK731 + DS extract. Sporulation by DK2634 + DS extract, approximately 2% of that observed with DK731 + DS extract, is not shown on this graph.
could then be further analyzed for complementation of the lysis deficiency. We have obtained a cell-free extract from developing cells of group B (DK468 or DK2453) that complemented fruiting body formation in the group C mutants DK’741 and DK’731. The effect of this development-stimulating (DS) extract on DK741 development is shown in Fig. 2; DK741 development was usually arrested at the stage of aggregate formation but addition of the DS extract allowed the aggregates to mature into darkened, myxospore-filled fruiting bodies (cf. Fig. la). The DS activity in this extract was tightly cell associated (i.e., nondiffusible), but readily released by a variety of methods that damaged or lysed the cells (Table 2). The DS extract did not rescue development in the group B strains DK468 or DK2453 (data not shown). DS rescue of sporulation and fruiting body formation in group C suggested that developmental lysis might
also be complemented. Rescue of the lysis deficiency in DK741 and DK731 was determined by quantitating the effect of the DS extract under developmental conditions. Figure 3a shows the developmental kinetics of DK2453 and DK741 when plated separately and in mixture. After 24 hr of incubation, DS extract was added under the agar of a parallel set of plates containing DK741 cells. Continued incubation in the presence of DS extract resulted in complementation of developmental lysis, sporulation, and fruiting body formation in a manner similar to the synergized mixture. Figure 3b shows the kinetics of DK731 when incubated under developmental conditions. Strain DK731 shows the characteristic growth phase but is deficient in developmental lysis. Addition of DS extract under the agar of a parallel set of DK731 plates resulted in complementation of developmental lysis, sporulation, and fruiting body formation. The group C synergy mutants contain a mutation,
JANSSEN AND DWORKIN
Cell Interactions
1
IO Monnosomme
Concentrotlon
(mM)
199
in M~XOCOCCUS
Glucosomlne
I
I
20
30
Concentration
40 (mM)
FIG. 4. Dose response of DK741 sporulation to glucosamine and mannosamine. DK’741 was spread uniformly on CF agar containing various concentrations of glucosamine or mannosamine. Duplicate plates were harvested after 10 days incubation and myxospore numbers were determined by direct counts. (The extent of complementation by glucosamine or mannosamine is directly dependent on the presence of tan variants in the assay population; the DK741 cells used in this experiment were approximately 70-80% yellow phase variants and 20-30% tan phase variants at the time of plating.)
called spoC, that has been linked to an identified Tn5 insertion site (LaRossa et al, 1983). The spoC locus, when transduced into a wild-type genetic background, defines a requirement for cell wall components during developmental lysis and sporulation (Shimkets and Kaiser, 1982). The spoClocus of DK731 has been transductionally introduced into DK2634 (see strain designations under Materials and Methods and Shimkets and Kaiser, 1982). Addition of DS extract to DK2634 revealed negligible complementation of development (Fig. 3b), suggesting that the DK731 mutation complemented by DS extract differs from the isolated spoC mutation present in DK2634. (2) Rescue of group C development by glucosamine or mannosamine. Biochemical analysis of partially purified DS extract suggested the involvement of carbohydrate in the DS biological activity (submitted for publication). Several sugars present in the surface layers of iV. xanthus (Sutherland, 1976; Sutherland and Smith, 1973; Sutherland and Thomson, 1975) were therefore tested for their effect on development in the group C mutants DK741 and DK731. The sugars ribose, rhamnose, glucose, glucosamine, N-acetylglucosamine, mannose, mannosamine, and muramic acid were screened for developmental effects by the agar well assay method. Glucos-
amine and mannosamine were found to stimulate fruiting body formation in the group C strain DK741. Figure 4 relates sporulation of DK741 to various concentrations of glucosamine and mannosamine; the optimal response to mannosamine occurred at approximately 2-3 m&i while that to glucosamine was at approximately 20 mM. Glucosamine was found to complement development fully (i.e., lysis, sporulation, and fruiting body formation) in DK741 and DK731 (Figs. 5, 6, 8). Mannosamine fully complemented development in DK741 but only developmental lysis and partial complementation of sporulation in DK731 (Figures 5, 6, 8). The spoC locus, present in DK2634, can be complemented by exogenously supplied N-acetylglucosamine, D-alanine, and diaminopimelic acid (Shimkets and Kaiser, 1982). Addition of these compounds to DK2634 under developmental conditions resulted in complementation of lysis and sporulation (Fig. 7). Incubation of the group C synergy mutants DK741 and DK731 in the presence of the cell wall components that rescued DK2634 resulted in negligible complementation of developmental lysis, sporulation, or fruiting body formation (Figures 5, 6, 8), suggesting that other mutations present in the group C strains are interfering with the response observed by the isolated spoC locus (cf. response by DK2634
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DEVELOPMENTAL BIOLOGY
VOLUME 112, 1985
ergized response (data not presented). (Experiments in this paper showing developmental complementation of DK741 and DK731 typically contained approximately 1525% tan variants at the time of plating for development.) The developmental response to glucosamine and mannosamine was also affected by the presence of tan cells in the population as was the complementation of DK741 and DK731 development by the DS extract (manuscript in preparation). DISCUSSION
In this report, developmental lysis was observed only when development culminated in sporulation or fruiting body formation; likewise, efficient sporulation occurred only when developmental lysis was present. This close association of developmental lysis with sporulation and fruiting body formation suggests a possible role for lysis in the development of M. xanthus and other species of myxobacteria (Wireman and Dworkin, 1975,1977).
0’
,/
20
40
60 80 Time (h)
,,
100
120
I
140
FIG. 5. Rescue of DK741 development by glucosamine and mannosamine. DK731 cells, containing approximately 78% yellow and 22% tan phase variants, were inoculated on CF agar (k supplements) and harvested at the indicated times for determination of cell number. Symbols: (0 - 0) CF agar; (0 - 0) CF with 15 mM glucosamine; (A - A) CF with 3 mM mannosamine; (0 - 0) CF with a mixture of 2.5 mMeach N-acetylglucosamine, diaminopimelate, and D-alanine; (M --- W) myxospore number on CF + glucosamine; (A --- A) myxospore number on CF + mannosamine.
containing spoC, Fig. 7). Addition of glucosamine to DK2634 resulted in delayed complementation of lysis, sporulation, and normal fruiting body formation; mannosamine showed no complementation of DK2634 development (Fig. 7). Phase Variation and Complementation Group C Development
of
Strains DK741 and DK731 displayed a pigment phase variation typical of that described for other strains of M. xanthus (Burchard et ak, 1977; Burchard and Dworkin, 1966); the mechanism and function of this pigment phase variation is unknown. Complementation of development in the group C strains DK741 and DK731 was significantly influenced by the phase variant composition of the population (data not presented). A population of yellow DK741 or DK731 showed only slight, if any, synergy of development when mixed with the group B strains DK468 or DK2453; inclusion of a few percent of tan cells in the population greatly stimulated the syn-
/-
t
I 0
: I 20
40
60 Time
80
rdo
120
(h)
FIG. 6. Rescue of DK731 development by glucosamine and mannosamine. DK’731 cells, containing approximately 76% yellow and 24% tan phase variants, were inoculated onto CF agar (k supplements) and harvested at the indicated times for determination of cell number. Symbols: (0 - 0) CF agar; (0 - Cl) CF with 15 mM glucosamine; (A - A) CF with 3 mM mannosamine; (0 - 0) CF with a mixture of 2.5 mM each N-acetylglucosamine, diaminopimelate, and D-alanine; (W --- W) myxospore number on CF + glucosamine.
JANSSEN AND DWORKIN
Cell Interackms
in
201
Myxococcus
ficient complementation of sporulation in the strains examined. We have identified a DS activity, contained in a cellfree extract of developing cells, that complements developmental lysis, sporulation, and fruiting body formation in two group C mutants. The DS activity was tightly cell associated (i.e., nondiffusible) but was released by various extraction procedures that damaged or lysed the cells. Three differences distinguish the group C mutants tested here from the spoC locus that has been identified and isolated from the group C mutants (e.g., compare group C strain DK731 with DK2634 containing the spoC locus of DK731, Figs. 6-8). First, the group C mutants were developmentally rescued by the DS extract but the .spoC mutant was not. Second, the spoC mutant was developmentally rescued by exogenous cell wall components but the group C strains were not. Third, the group
I
Strain
I I
I
1
Lr
1 GlcNH3
1 MonNH3
1 C.W.C.
,\
0
20
40
ko Time
80
100
120
(h)
FIG. ‘I. Rescue of spoC DK2634 development. DK2634 cells, containing approximately 75% yellow and 25% tan phase variants, were inoculated onto CF agar (k supplements) and harvested at the indicated times for determination of cell number. Symbols: (0 - 0) CF agar; (0 - 0) CF with 15 mM glucosamine; (A - A) CF with 3 mM mannosamine; (0 - c\) CF with a mixture of 2.5 mMeach N-acetylglucosamine, diaminopimelate, and D-alanine; (U---m) myxospore number on CF + glucosamine; (* --- +) myxospore number on CF with N-acetylglucosamine, diaminopimelate and D-alanine.
DK 741
DK 731
The results presented here identify two phenotypic complementation groups for developmental lysis and sporulation. The inability of certain group mixtures (i.e., groups AB, AD, and BD; see Table 1) to show synergized sporulation was unexpected in view of the previous report (Hagen et al., 1978) that identified four such groups. Recently, complementation groups A, B, C, and D have also been shown to be distinguishable by genetic and biochemical criteria (LaRossa et al., 1983). It is possible that mixture of strains defining two different genetic or biochemical complementation groups, but representing only one lysis complementation group, could result in low levels of sporulation sufficient for detection by the qualitative screening method initially used (Hagen et al., 1978). Our data suggest, however, that the group A, B, C, and D mutants define only two phenotypically complementable groups with regard to developmental lysis and that developmental lysis is correlated with ef-
DK 2634
FIG. 8. Developmental morphologies of DK741, DK731, and DK2534 in the presence of glucosamine, mannosamine, and cell wall components. DK741,731, and 2634 cells, containing yellow and tan variants, were spread uniformly on CF agar or CF agar supplemented with 15 mM glucosamine, 3 mM mannosamine, or a mixture of cell wall components (2.5 mM each of N-acetylglucosamine, diaminopimelate, and D-alanine; denoted in figure as C.W.C.). Developmental morphologies are those observed after 6 days incubation at 32°C.
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DEVELOPMENTAL BIOLOGY
C mutants were developmentally rescued by mannosamine but the spoC mutant was not. Both group C and spoC, however, were developmentally rescued by glucosamine suggesting that the developmental defects present are related and that glucosamine by itself is sufficient for spoC rescue. The observed differences between spoC and group C suggests that an additional mutation may be present in the group C strains examined or that the spoC mutation behaves differently when transduced into a wild-type genetic background. [An additional difference between the group C mutants and strain DK2634 which contains the spoC allele should be pointed out. The group C mutants are derived from a parent strain (DKlOl) that contains the motility mutation &Al, while the parent strain of DK2634 is the fully motile (AS+) wild type strain (Kaiser, 1979)]. DS extract rescue of a secondary mutation in group C strains could act to initiate developmental lysis and thereby provide the cell wall components required by the resident spoC locus. Glucosamine and mannosamine rescue of group C development could reflect the inability of group C strains to generate free glucosamine and mannosamine. The relatively high levels necessary for complementation (i.e., 15 mMglucosamine and 3 mMmannosamine) make it unlikely that they function in a catalytic or secondary messenger capacity (e.g., analogous to CAMP) but rather as developmental substrates. Alternatively, they could be involved in the assembly of molecules necessary for developmental interactions; the absence or improper configuration of such molecules would result in development being blocked at an intermediate stage. Biochemical analysis of semipurified DS extract indicates the presence of carbohydrate (submitted for publication), leading us to suggest that the glucosamine/mannosamine effect may be related to complementation by the DS extract and may occur through a carbohydratecontaining cell-associated molecule involved in the regulation of developmental lysis, sporulation, and fruiting body formation in M. xanthus. The cell-associated nature of DS activity and of the contact-stimulated A and S motility systems (Hodgkin and Kaiser, 1979a,b; Kaiser, 1979) suggests that contactmediated interactions may be an important general method of intercellular communication of M. xanthus. The close proximity of cells throughout the life cycle of M. xanthus may obviate the need for diffusible signaling molecules; instead, cell-associated signals may be more
VOLUME 112. 1985
effective and economical. Analysis of such interactions in ill xanthus would be facilitated by the relative genetic and biochemical convenience of prokaryotic ikf. xanthus. We thank Dorothy Marquis, Todd Starich, Ed Stellwag, and James Zissler for many helpful discussions. This work was supported by National Institutes of Health Grant GM-19957. G.R.J. acknowledges receipt of a University Foundation Doctoral Dissertation Fellowship. REFERENCES BURCHARD, R. P., BURCHARD, A. C., and PARISH, J. H. (1977). Pigmentation phenotype instability in Myxococcus xanthus. Can&. J. Microbiol. 23,1657-1662. BURCHARD, R., and DWORKIN, M. (1966). A bacteriophage for Myxococcus zanthus: Isolation, characterization and relation of infectivity to host morphogenesis. J. Bactetiol. 91, 535-545. HAGEN, D., BRETSCHER, A. P., and KAISER, D. (1978). Synergism between morphogenetic mutants of M~xococcus xanthus. Dev. BioL64, 284-296. HODGKIN, J., and KAISER, D. (1979a). Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement. Mol. Gen. Genet. 171,177-191. HODGKIN, J., and KAISER, D. (1979b). Genetics of gliding motility in Myxococm xunthus (Myxobacterales): Genes controlling movements of single cells. Mol. Gen. Genet. 171, 167-176. KAISER, D. (1979). Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 76,5952-5956. LAROSSA, R., KUNER, J., HAGEN, D., MANOIL, C., and KAISER, D. (1983). Developmental cell interactions of Myxococcus xanthus: Analysis of mutants. J. Bacterial. 153, 1394-1404. ORNDORFF, P., and DWORKIN, M. (1980). Separation and properties of the cytoplasmic and outer membranes of vegetative cells of M~XCF coccus xanthus. J BacterioL 141,914-927. ROSENBERG, E. (1984). “Myxobacteria: Development and Cell Interactions,” Springer-Verlag, New York. SHIMKETS, L., and DWORKIN, M. (1981). Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxe coccus xanthus. Dev. BioL 84,51-60. SHIMKETS, L., and KAISER, D. (1982). Induction of coordinated cell movement in Myxococcus xanthus. J Bactetiol. 152,462-470. SUTHERLAND, I. (1976). Novel surface polymer changes in development of Myxococcus xanthus. Nature (London) 259,46-47. SUTHERLAND, I., and SMITH, M. (1973). The lipopolysaccharides of fruiting and non-fruiting myxobacteria. J. Gen. Microbial 74, 259266. SUTHERLAND, I., and THOMSON, S. (1975). Comparison of polysaccharides produced by Myxococcus strains. J. Gen. MicrobioL 89,124-132. WIREMAN, J. (1979). Developmental induction of Myxococcus xanthus myxospores. J. Bactetiol. 140, 147-153. WIREMAN, J., and DWORKIN, M. (1975). Morphogenesis and developmental interactions in myxobacteria. Science (Washington, D. C.) 189,516-523. WIREMAN, J., and DWORKIN, M. (1977). Developmentally induced autolysis during fruiting body formation by Myxococcus xanthus. J. BacterioL 129, 796-802.
BIOLOGY
112,194-202
(19%)
Cell-Cell Interactions in Developmental Lysis of Adyxococcus xanthus GARY R. JANSSEN~ AND MARTIN DWORKIN Department
of Microbiology,
University
of Minnesota,
Minneapolis,
Minnesota
55.455
Received March 20, 1985; accepted in revised form May 7, 1985 The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously 0 1985 Academic Press, Inc. supplied glucosamine or mannosamine.
When cells of the bacterium Myxococcus xanthus are on a solid surface and are at a high cell density, they respond to nutritional starvation by shifting the mode of their life cycle from vegetative feeding to development. The developmental sequence involves aggregation of cells into centers that eventually form raised, slimecovered fruiting bodies. The majority of cells lyse during the developmental process while most of the surviving cells differentiate to myxospores within the fruiting bodies (Wireman and Dworkin, 1975,1977). The lysis is an autolytic process (Wireman and Dworkin, 1977) and, while its function is unknown, its occurrence just prior to and concomitant with myxospore induction and maturation suggests a possible causal relationship between the two developmental events. Observations that lysis occurs during development of other species of myxobacteria (Wireman and Dworkin, 1977) and that, under certain conditions, autolysis may be required for myxospore induction (Wireman, 1979) suggest that cell death is an important event in myxobacterial development. Furthermore, the dependence of subsequent development on the cell density of the lysing population (Wireman and Dworkin, 1977) suggests that the development is dependent on a product of the autolysis. Nevertheless, while there is abundant evidence that M xanthus does indeed manifest a variety of cell interactions (Rosenberg, 1984), no endogenous signals have thus far been isolated. Hagen et al. (1978) have described a set of conditional nonsporulating mutants of M. xanthus that can be divided into four groups that undergo extracellular, phenotypic complementation. Mutants within each group i Present address: Department of Genetics, Colney Lane, Norwich, NR4 7UH, England. 0012-1606/85 $3.00 Copyright All rights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
John Innes Institute,
194
are sporulation (spo) deficient but can be synergized to sporulate when mixed with members of other complementation groups or with the developmentally proficient parental strain. It has been suggested (Hagen et aZ.,1978) that this complementation reflects the normal exchange of signals during multicellular development. We have used this system of extracellular complementation to examine the process of developmental autolysis. In this paper we shall describe a partially purified extract that contains a cell-associated substance which acts as the signal of developmental autolysis. Furthermore, we show that the effect of the endogenous signal can be replaced by mannosamine and glucosamine. MATERIALS
AND
METHODS
Bacterial strains and media. M. xanthus strains MD2 and DKlOl were developmentally proficient, yellow, swarming variants of Al. xanthus FB (Wireman and Dworkin, 1975). DKlOl and MD2 were both derived from M. xanthus FB, and under the conditions used here their developmental responses were similar; DKlOl is the parental strain of the synergy mutants described below. The following strains were obtained from D. Kaiser (Dept. of Biochemistry, Stanford University) and represent single colony isolates of synergy groups A, B, C, and D: DK476 (Group A), DK468 (Group B), DK731 and DK741 (Group C), DK429 (Group D), DK2453 (kan’group B), DK2634 (contains the spoC allele of group C mutant DK731). DKlOl was also obtained from D. Kaiser. Cells were vegetatively cultured in liquid, Casitone growth medium (CT), as described (Orndorff and Dworkin, 1980). Vegetative growth on solid medium was on CTT agar (Hagen et al., 1978). Liquid-grown cultures were started from isolated yellow or tan colonies (Bur-
JANSSEN AND DWORKIN
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195
in M~XOCOCCUS
chard et al., 1977) picked from CTT agar; yellow and tan phase variants were distinguished by viewing colonies under a long wave (379 nm) ultraviolet light (Ultra-Violet Products, Inc.). Development was induced on clone fruiting (CF) agar (Hagen et al, 1978) or CF agar supplemented with glucosamine-HCl, mannosamine, or a mixture of N-acetylglucosamine, D-alanine, and diaminopimelic acid (Sigma Chemical Co.; St. Louis, MO.).
error of less than 10% .The number of viable myxospores was determined by heating harvested cell suspensions to 51°C for 2 hr, followed by aseptic, extended sonication (as above) and plating for colony-forming units on CTT agar. The combined heat and sonication treatments resulted in less than lo-’ survival for vegetative cells while not significantly affecting mature myxospore viability.
Induction of development and measurement of cell number. Vegetative cultures, grown in CT broth to 4-10
Cells were harvested from CF agar after 3 days of incubation at 32°C (inoculated at a to cell density of l-2 X lo7 cells cm-‘). Harvested cells, on ice in TPM buffer, were sonicated twice at 70 W for 1 min each time, followed by sedimentation of cell debris at 27,OOOg for 15 min. The supernatant fraction was vigorously extracted with an equal volume of chloroform, followed by centrifugation at 12,000g for 10 min. The resulting aqueous phase was acidified to 1 N HCl for approximately 8 hr at room temperature, followed by centrifugation at 27,000g for 15 min. The supernatant fraction, containing DS activity, was neutralized by dialysis in Spectrapor 3 tubing (mol wt cutoff approximately 3500) against TPM buffer or H20. These extractions resulted in the removal, by precipitation or phase partitioning, of a large amount of material while not seriously affecting the amount of DS activity present.
X lOa cells ml-‘, were harvested by centrifugation (12,000g for 10 min) at 4”C, washed and resuspended in cold TPM buffer [IO mM Tris-HCl (pH 7.6), 1 mM potassium phosphate (pH 7.6), and 8.3 mM magnesium sulfate], spread uniformly onto the surface of CF agar at a starting density of 10’ cells cm-’ and incubated at 32°C. Cells harvested from developmental plates were fixed in 2.5% glutaraldehyde for at least 18 hr at 4°C. Cell clumps were dispersed by sonication (5 set at 65 W; Sonifier Cell Disrupter, Heat Systems-Ultrasonics, Inc.). Extended sonication (two 30-set pulses at 65 W with intermittent cooling) was used for quantitation of ovoid, refractile myxospores. Cell numbers were determined in a Petroff-Hausser counting chamber with a maximum
Production (0s) activity.
and extraction
of development-stimulating
----
Sporulotion - - -Net Lyrir
r
6
-----_
IOf
- - --Aggregation
INet
0
Growth
I
5
20
40
Fruiting Body Formatton
60 fi0 Time(h)
I I
1. 100
120
4 140
FIG. 1. Sequence of developmental events during fruiting body formation by M. zanthus. Cells were prepared as described under Materials and Methods and spread uniformly on development-inducing CF agar at 3.1 X lo6 cells cm-*. (A) Morphological changes during M. zanthus MD 2 development. (B) Quantitation of cell number during M. zanthus development. At the times indicated, duplicate plates were harvested for determination of cell numbers. Symbols: (W - W) MD 2; (W --- n ) MD 2 myxospores; (0 - 0) DK468; (0 - 0) DK741; (Cl - 0) mixture of DK468 X DK741; (0 --- 0) DK468 X DK741 myxospores.
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Screening for develqwnumtal rescue by agar well assays. Plugs were removed from the central region of petri plates containing developmental cells on CF agar. The resultant wells were then filled with cell extracts or sugar solutions that had been mixed with molten agar. The developmental effect of a cell extract or sugar was determined by comparison to agar wells containing TPM buffer. The assay allowed for a concentration gradient (i.e., through diffusion) of a substance to be tested at selected times during development. RESULTS
Development of M. xanthus Strain MD2
VOLUME 112, 1985 TABLE 1 CORRELATION OF SPORULATION WITH SYNERGIZED DEVELOPMENTAL LYSIS’
Strain DK476 DK468 DK741 DK429 DKlOl 476 476 476 468 468 741
X X X x X x
468 741 429 741 429 429
Sporulation (% of parental)”
% decrease in maximum vegetative numberb
Direct counts
Viable counts
A B C D fru +
22% 32% 29% 30% 93%
AB AC AD BC BD CD
25% 70% 22% 96% 17% 82%
0.2% 25% 0.2% 94%
0.03% 7.5% 0.07% 96% 0.02% 13%
Synergy grow
Figure la illustrates the changes in colonial morphology during fruiting body development. At about 28 hr cells began moving into aggregates; this process was completed at about 60 hr, at which time the aggregates “Each value represents the average of two or three independent are referred to as immature fruiting bodies. Lysis began determinations. b Decrease in vegetative cell number is determined by a direct count at about 60 hr and ended at about 80 hr; at this point, the surviving cells converted to myxospores and the determination of cell number at the point of maximum growth (i.e., hr, see Fig. la) and at the developmental endpoint time of 168 hr mound of cells acquired the appearance of the mature 66 (i.e., normal and synergized development are essentially complete). fruiting body. Figure lb illustrates the changes in de’ Sporulation is expressed relative to the level of sporulation meavelopment and cell number that occurred with time when sured in the developmentally proficient (fru+) parental strain DKlOl. the developmentally proficient M. xanthus strain MD2 Direct counts of optically refractile myxospores were done with a Pewas placed under appropriate conditions. The cells un- troff-Hausser counting chamber. Viability counts were measured as forming units after heat treatment and sonication (see Materials derwent an initial period of growth resulting in the ac- colony and Methods). cumulation of a number of cells sufficient for the initiation of development (Shimkets and Dworkin, 1981). Cell number reached maximal value and leveled off, presum- followed by a developmental lysis phase in which greater ably in response to depletion of essential nutrients in than 80% of the cells lysed. The developmental lysis the medium. The growth and aggregation phases were phase slightly preceded and was then concomitant with
FIG. 2. Rescue by DS of fruiting body formation in DK741. The group C synergy mutant DK741 was spread uniformly on CF agar at 10’ cells/cm*. After about 30-hr incubation at 32°C crude DS extract or TPM buffer were assayed for development stimulating activity by the agar-well assay method. Pictures were taken after 5 days incubation.
JANSSEN AND DWORKIN
the differentiation myxospores. Developmental
of the remaining
Lysis in Sporulation
Cell Interactions
cells to resting
Synergy Mutants
Representative strains of groups A, B, C, and D sporulation synergy mutants, described by Hagen et al. (19’78), were examined for developmental lysis. When placed under developmental conditions, the mutants displayed the usual growth phase followed by a plateau in cell number, but the characteristic lysis phase normally observed during development of M. xanthus (Fig. lb) was absent. The strains tested were also deficient in sporulation (Table 1). Sporulation in this set of mutants is phenotypically complemented when a mutant of one synergy group is mixed with a mutant from a different synergy group, as described by Hagen et al. (1978). Pairwise mixtures of these lysis-deficient, sporulation-deficient strains were tested to determine if lysis was complementable. Table 1 shows the lysis phenotype of various pairwise mixtures when tested under developmental conditions. Cell number was quantitated at the time of maximal growth (approximately 66 hr under these conditions) and at an endpoint time of 168 hr. Mixtures of DK476 X DK468 (i.e., group A and group B), DK476 X DK429 (i.e., group A and group D), and DK468 X DK429 (i.e., group B and group D) showed negligible complementation of developmental lysis. Mixtures of DK468 X DK741 (i.e., group A and group C) and DK741 X DK429 (i.e., group C and group D) showed significant complementation of lysis. Mixtures of DK468 X DK741 (i.e., group B and group C) showed levels of developmental lysis comparable to that observed with the developmentally proficient MD2 and the parental strain DKlOl (Table 1 and Fig. lb). Therefore, among the strains tested, phenotypic complementation of developmental lysis fell into two groups, with strains DK476, DK468, and DK429 comprising one group (A, B, and D) and DK741 (C) representing the other group. The lysis-deficient strains and strain mixtures showed a slight, gradual decrease in cell number with extended incubation (Table l), but the extent and rate was qualitatively distinct from the rapid, synchronous, developmental lysis that is tightly associated with myxosporulation and fruiting body formation (Fig. lb and Wireman and Dworkin, 1975, 1977). Sporulation synergy was also measured in the pairwise mixtures tested above (Table 1). Extracellular complementation of sporulation was greatest in pairwise mixtures where developmental lysis was complemented (i.e., in mixtures of group A with C, group B with C, and group D with C); minimal amounts of sporulation synergy occurred in mixtures showing minimal complementation of developmental lysis (i.e., in mixtures of group A with B, group A with D, and group B with D).
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in Myxococcus
Cell-Free Complementation of Lysis and Development in Group C Synergy Mutants (1) Complementation by a cell-free extract. Developmental lysis in the mixture of DK468 (group B) and DK741 (group C) closely paralleled the lysis observed in the developmentally competent il4 xanthus strain MD2 (Fig. lb). Thus, it was appropriate to use one member of this pair as the source of a cell-free complementing factor and the other to assay for complementation of developmental lysis. The close association of developmental lysis with fruiting body formation (Wireman and Dworkin, 1975) suggested that complementation of fruiting body formation would be an appropriate parameter of lysis-stimulating activity as an initial screening step. Cell extracts that complemented fruiting TABLE 2 DISSOCIATION OF DS ACTIVITY FROM DK2453 CELLS
Extraction
conditions“
1. Vigorous vortexing of developmental DK2453 cell suspension in TPM buffer a. Supernatant fraction b. Crude DS extract of sedimented cells 2. Developmental DK2453 cell suspension stirred at 4°C for 12-14 hr in TPM buffer a. Supernatant fraction b. Crude DS extract of sedimented cells 3. Developmental DK2453 cell suspension subjected to four cycles of freeze-thaw in TPM buffer a. Supernatant fraction b. Crude DS extract of sedimented cells 4. Developmental DK2453 cell suspension stirred at 4’C in 1 M NaCl for 12-14 hr a. Supernatant fraction b. Crude DS extract of sedimented cells 5. Developmental DK2453 cell suspension stirred at 4°C in 10% TCA for 12-14 hr a. Supernatant fraction b. Crude DS extract of sedimented cells 6. Crude DS extract of DK2453 vegetative cells (mid-log growth in liquid CT)
Relative DS activityb
<5Y0 95-100%
<5% 95-100%
30-60% 20-40%
20-60% 30-50%
50-90% O-20% 0%
a Extraction conditions l-5 used DK2453 cells cultivated on CF agar for production of DS extract, as described under Materials and Methods. After the various extraction procedures, cells were sedimented by 27,1009 centrifugation; the supernatant fraction and sedimented cells, after crude DS extraction, were assayed for DS activity. Extraction condition 6 used an equivalent number of mid-log growth phase vegetative cells. b DS activity was measured as number of fruiting bodies stimulated in an agar-well assay on DK741. Table 2 presents results from multiple experiments and, because of variability from experiment to experiment, DS activity is expressed relative to the DS activity observed in the crude DS extracted control for that experiment. A range of values for “Relative DS Activity” represents the range of results obtained in multiple testing. The crude DS extract of untreated cells generally stimulated about 300-600 fruiting bodies in these experiments.
DEVELOPMENTAL
BIOLOGY
VOLUME 112. 1985
I
*’ t ,+ f
i 0
#
20
40
0’ :
I
60 60 Time (h)
loo
20
40
60 Time
80
.’
/’
/’
100
.’
9
120
(h)
FIG. 3. Rescue of developmental lysis in DK741 and DK731 by DS extract. Cells were spread uniformly on CF agar at an initial density of lo7 cells cm-’ (12 ml agar in 60-mm diameter plate). Lyophilized DS extract was added under the agar of a set of DK741 and DK731 plates and incubation continued. Duplicate plates were harvested at the times indicated for determination of cell number. The amount of DS extract applied to each assay plate represented DS extract from 2.6 to 2.8 X 10 in. DK2453 developmental cells (containing 0.24 mg protein and 4.23 mg carbohydrate when assayed in B). (A) Rescue of DK741 by DS extract. Symbols: (0 - 0) DK741; (A - A) DK2453; (0 - 0) mixture of DK741 X DK2453; (0 - 0) DK741 + DS extract; (m --- n ) myxospores from DK741 X DK2453 mixture; (0 --- 0) myxospores from DK741 + DS extract. (B) Rescue of DK731 by DS extract. Symbols: (0 - 0) DK731; (0 - 0) DK2634; (0 - 0) DK731 + DS extract; (B - W) DK2634 + DS extract; (O - 0) myxospores from DK731 + DS extract. Sporulation by DK2634 + DS extract, approximately 2% of that observed with DK731 + DS extract, is not shown on this graph.
could then be further analyzed for complementation of the lysis deficiency. We have obtained a cell-free extract from developing cells of group B (DK468 or DK2453) that complemented fruiting body formation in the group C mutants DK’741 and DK’731. The effect of this development-stimulating (DS) extract on DK741 development is shown in Fig. 2; DK741 development was usually arrested at the stage of aggregate formation but addition of the DS extract allowed the aggregates to mature into darkened, myxospore-filled fruiting bodies (cf. Fig. la). The DS activity in this extract was tightly cell associated (i.e., nondiffusible), but readily released by a variety of methods that damaged or lysed the cells (Table 2). The DS extract did not rescue development in the group B strains DK468 or DK2453 (data not shown). DS rescue of sporulation and fruiting body formation in group C suggested that developmental lysis might
also be complemented. Rescue of the lysis deficiency in DK741 and DK731 was determined by quantitating the effect of the DS extract under developmental conditions. Figure 3a shows the developmental kinetics of DK2453 and DK741 when plated separately and in mixture. After 24 hr of incubation, DS extract was added under the agar of a parallel set of plates containing DK741 cells. Continued incubation in the presence of DS extract resulted in complementation of developmental lysis, sporulation, and fruiting body formation in a manner similar to the synergized mixture. Figure 3b shows the kinetics of DK731 when incubated under developmental conditions. Strain DK731 shows the characteristic growth phase but is deficient in developmental lysis. Addition of DS extract under the agar of a parallel set of DK731 plates resulted in complementation of developmental lysis, sporulation, and fruiting body formation. The group C synergy mutants contain a mutation,
JANSSEN AND DWORKIN
Cell Interactions
1
IO Monnosomme
Concentrotlon
(mM)
199
in M~XOCOCCUS
Glucosomlne
I
I
20
30
Concentration
40 (mM)
FIG. 4. Dose response of DK741 sporulation to glucosamine and mannosamine. DK’741 was spread uniformly on CF agar containing various concentrations of glucosamine or mannosamine. Duplicate plates were harvested after 10 days incubation and myxospore numbers were determined by direct counts. (The extent of complementation by glucosamine or mannosamine is directly dependent on the presence of tan variants in the assay population; the DK741 cells used in this experiment were approximately 70-80% yellow phase variants and 20-30% tan phase variants at the time of plating.)
called spoC, that has been linked to an identified Tn5 insertion site (LaRossa et al, 1983). The spoC locus, when transduced into a wild-type genetic background, defines a requirement for cell wall components during developmental lysis and sporulation (Shimkets and Kaiser, 1982). The spoClocus of DK731 has been transductionally introduced into DK2634 (see strain designations under Materials and Methods and Shimkets and Kaiser, 1982). Addition of DS extract to DK2634 revealed negligible complementation of development (Fig. 3b), suggesting that the DK731 mutation complemented by DS extract differs from the isolated spoC mutation present in DK2634. (2) Rescue of group C development by glucosamine or mannosamine. Biochemical analysis of partially purified DS extract suggested the involvement of carbohydrate in the DS biological activity (submitted for publication). Several sugars present in the surface layers of iV. xanthus (Sutherland, 1976; Sutherland and Smith, 1973; Sutherland and Thomson, 1975) were therefore tested for their effect on development in the group C mutants DK741 and DK731. The sugars ribose, rhamnose, glucose, glucosamine, N-acetylglucosamine, mannose, mannosamine, and muramic acid were screened for developmental effects by the agar well assay method. Glucos-
amine and mannosamine were found to stimulate fruiting body formation in the group C strain DK741. Figure 4 relates sporulation of DK741 to various concentrations of glucosamine and mannosamine; the optimal response to mannosamine occurred at approximately 2-3 m&i while that to glucosamine was at approximately 20 mM. Glucosamine was found to complement development fully (i.e., lysis, sporulation, and fruiting body formation) in DK741 and DK731 (Figs. 5, 6, 8). Mannosamine fully complemented development in DK741 but only developmental lysis and partial complementation of sporulation in DK731 (Figures 5, 6, 8). The spoC locus, present in DK2634, can be complemented by exogenously supplied N-acetylglucosamine, D-alanine, and diaminopimelic acid (Shimkets and Kaiser, 1982). Addition of these compounds to DK2634 under developmental conditions resulted in complementation of lysis and sporulation (Fig. 7). Incubation of the group C synergy mutants DK741 and DK731 in the presence of the cell wall components that rescued DK2634 resulted in negligible complementation of developmental lysis, sporulation, or fruiting body formation (Figures 5, 6, 8), suggesting that other mutations present in the group C strains are interfering with the response observed by the isolated spoC locus (cf. response by DK2634
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DEVELOPMENTAL BIOLOGY
VOLUME 112, 1985
ergized response (data not presented). (Experiments in this paper showing developmental complementation of DK741 and DK731 typically contained approximately 1525% tan variants at the time of plating for development.) The developmental response to glucosamine and mannosamine was also affected by the presence of tan cells in the population as was the complementation of DK741 and DK731 development by the DS extract (manuscript in preparation). DISCUSSION
In this report, developmental lysis was observed only when development culminated in sporulation or fruiting body formation; likewise, efficient sporulation occurred only when developmental lysis was present. This close association of developmental lysis with sporulation and fruiting body formation suggests a possible role for lysis in the development of M. xanthus and other species of myxobacteria (Wireman and Dworkin, 1975,1977).
0’
,/
20
40
60 80 Time (h)
,,
100
120
I
140
FIG. 5. Rescue of DK741 development by glucosamine and mannosamine. DK731 cells, containing approximately 78% yellow and 22% tan phase variants, were inoculated on CF agar (k supplements) and harvested at the indicated times for determination of cell number. Symbols: (0 - 0) CF agar; (0 - 0) CF with 15 mM glucosamine; (A - A) CF with 3 mM mannosamine; (0 - 0) CF with a mixture of 2.5 mMeach N-acetylglucosamine, diaminopimelate, and D-alanine; (M --- W) myxospore number on CF + glucosamine; (A --- A) myxospore number on CF + mannosamine.
containing spoC, Fig. 7). Addition of glucosamine to DK2634 resulted in delayed complementation of lysis, sporulation, and normal fruiting body formation; mannosamine showed no complementation of DK2634 development (Fig. 7). Phase Variation and Complementation Group C Development
of
Strains DK741 and DK731 displayed a pigment phase variation typical of that described for other strains of M. xanthus (Burchard et ak, 1977; Burchard and Dworkin, 1966); the mechanism and function of this pigment phase variation is unknown. Complementation of development in the group C strains DK741 and DK731 was significantly influenced by the phase variant composition of the population (data not presented). A population of yellow DK741 or DK731 showed only slight, if any, synergy of development when mixed with the group B strains DK468 or DK2453; inclusion of a few percent of tan cells in the population greatly stimulated the syn-
/-
t
I 0
: I 20
40
60 Time
80
rdo
120
(h)
FIG. 6. Rescue of DK731 development by glucosamine and mannosamine. DK’731 cells, containing approximately 76% yellow and 24% tan phase variants, were inoculated onto CF agar (k supplements) and harvested at the indicated times for determination of cell number. Symbols: (0 - 0) CF agar; (0 - Cl) CF with 15 mM glucosamine; (A - A) CF with 3 mM mannosamine; (0 - 0) CF with a mixture of 2.5 mM each N-acetylglucosamine, diaminopimelate, and D-alanine; (W --- W) myxospore number on CF + glucosamine.
JANSSEN AND DWORKIN
Cell Interackms
in
201
Myxococcus
ficient complementation of sporulation in the strains examined. We have identified a DS activity, contained in a cellfree extract of developing cells, that complements developmental lysis, sporulation, and fruiting body formation in two group C mutants. The DS activity was tightly cell associated (i.e., nondiffusible) but was released by various extraction procedures that damaged or lysed the cells. Three differences distinguish the group C mutants tested here from the spoC locus that has been identified and isolated from the group C mutants (e.g., compare group C strain DK731 with DK2634 containing the spoC locus of DK731, Figs. 6-8). First, the group C mutants were developmentally rescued by the DS extract but the .spoC mutant was not. Second, the spoC mutant was developmentally rescued by exogenous cell wall components but the group C strains were not. Third, the group
I
Strain
I I
I
1
Lr
1 GlcNH3
1 MonNH3
1 C.W.C.
,\
0
20
40
ko Time
80
100
120
(h)
FIG. ‘I. Rescue of spoC DK2634 development. DK2634 cells, containing approximately 75% yellow and 25% tan phase variants, were inoculated onto CF agar (k supplements) and harvested at the indicated times for determination of cell number. Symbols: (0 - 0) CF agar; (0 - 0) CF with 15 mM glucosamine; (A - A) CF with 3 mM mannosamine; (0 - c\) CF with a mixture of 2.5 mMeach N-acetylglucosamine, diaminopimelate, and D-alanine; (U---m) myxospore number on CF + glucosamine; (* --- +) myxospore number on CF with N-acetylglucosamine, diaminopimelate and D-alanine.
DK 741
DK 731
The results presented here identify two phenotypic complementation groups for developmental lysis and sporulation. The inability of certain group mixtures (i.e., groups AB, AD, and BD; see Table 1) to show synergized sporulation was unexpected in view of the previous report (Hagen et al., 1978) that identified four such groups. Recently, complementation groups A, B, C, and D have also been shown to be distinguishable by genetic and biochemical criteria (LaRossa et al., 1983). It is possible that mixture of strains defining two different genetic or biochemical complementation groups, but representing only one lysis complementation group, could result in low levels of sporulation sufficient for detection by the qualitative screening method initially used (Hagen et al., 1978). Our data suggest, however, that the group A, B, C, and D mutants define only two phenotypically complementable groups with regard to developmental lysis and that developmental lysis is correlated with ef-
DK 2634
FIG. 8. Developmental morphologies of DK741, DK731, and DK2534 in the presence of glucosamine, mannosamine, and cell wall components. DK741,731, and 2634 cells, containing yellow and tan variants, were spread uniformly on CF agar or CF agar supplemented with 15 mM glucosamine, 3 mM mannosamine, or a mixture of cell wall components (2.5 mM each of N-acetylglucosamine, diaminopimelate, and D-alanine; denoted in figure as C.W.C.). Developmental morphologies are those observed after 6 days incubation at 32°C.
202
DEVELOPMENTAL BIOLOGY
C mutants were developmentally rescued by mannosamine but the spoC mutant was not. Both group C and spoC, however, were developmentally rescued by glucosamine suggesting that the developmental defects present are related and that glucosamine by itself is sufficient for spoC rescue. The observed differences between spoC and group C suggests that an additional mutation may be present in the group C strains examined or that the spoC mutation behaves differently when transduced into a wild-type genetic background. [An additional difference between the group C mutants and strain DK2634 which contains the spoC allele should be pointed out. The group C mutants are derived from a parent strain (DKlOl) that contains the motility mutation &Al, while the parent strain of DK2634 is the fully motile (AS+) wild type strain (Kaiser, 1979)]. DS extract rescue of a secondary mutation in group C strains could act to initiate developmental lysis and thereby provide the cell wall components required by the resident spoC locus. Glucosamine and mannosamine rescue of group C development could reflect the inability of group C strains to generate free glucosamine and mannosamine. The relatively high levels necessary for complementation (i.e., 15 mMglucosamine and 3 mMmannosamine) make it unlikely that they function in a catalytic or secondary messenger capacity (e.g., analogous to CAMP) but rather as developmental substrates. Alternatively, they could be involved in the assembly of molecules necessary for developmental interactions; the absence or improper configuration of such molecules would result in development being blocked at an intermediate stage. Biochemical analysis of semipurified DS extract indicates the presence of carbohydrate (submitted for publication), leading us to suggest that the glucosamine/mannosamine effect may be related to complementation by the DS extract and may occur through a carbohydratecontaining cell-associated molecule involved in the regulation of developmental lysis, sporulation, and fruiting body formation in M. xanthus. The cell-associated nature of DS activity and of the contact-stimulated A and S motility systems (Hodgkin and Kaiser, 1979a,b; Kaiser, 1979) suggests that contactmediated interactions may be an important general method of intercellular communication of M. xanthus. The close proximity of cells throughout the life cycle of M. xanthus may obviate the need for diffusible signaling molecules; instead, cell-associated signals may be more
VOLUME 112. 1985
effective and economical. Analysis of such interactions in ill xanthus would be facilitated by the relative genetic and biochemical convenience of prokaryotic ikf. xanthus. We thank Dorothy Marquis, Todd Starich, Ed Stellwag, and James Zissler for many helpful discussions. This work was supported by National Institutes of Health Grant GM-19957. G.R.J. acknowledges receipt of a University Foundation Doctoral Dissertation Fellowship. REFERENCES BURCHARD, R. P., BURCHARD, A. C., and PARISH, J. H. (1977). Pigmentation phenotype instability in Myxococcus xanthus. Can&. J. Microbiol. 23,1657-1662. BURCHARD, R., and DWORKIN, M. (1966). A bacteriophage for Myxococcus zanthus: Isolation, characterization and relation of infectivity to host morphogenesis. J. Bactetiol. 91, 535-545. HAGEN, D., BRETSCHER, A. P., and KAISER, D. (1978). Synergism between morphogenetic mutants of M~xococcus xanthus. Dev. BioL64, 284-296. HODGKIN, J., and KAISER, D. (1979a). Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement. Mol. Gen. Genet. 171,177-191. HODGKIN, J., and KAISER, D. (1979b). Genetics of gliding motility in Myxococm xunthus (Myxobacterales): Genes controlling movements of single cells. Mol. Gen. Genet. 171, 167-176. KAISER, D. (1979). Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 76,5952-5956. LAROSSA, R., KUNER, J., HAGEN, D., MANOIL, C., and KAISER, D. (1983). Developmental cell interactions of Myxococcus xanthus: Analysis of mutants. J. Bacterial. 153, 1394-1404. ORNDORFF, P., and DWORKIN, M. (1980). Separation and properties of the cytoplasmic and outer membranes of vegetative cells of M~XCF coccus xanthus. J BacterioL 141,914-927. ROSENBERG, E. (1984). “Myxobacteria: Development and Cell Interactions,” Springer-Verlag, New York. SHIMKETS, L., and DWORKIN, M. (1981). Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxe coccus xanthus. Dev. BioL 84,51-60. SHIMKETS, L., and KAISER, D. (1982). Induction of coordinated cell movement in Myxococcus xanthus. J Bactetiol. 152,462-470. SUTHERLAND, I. (1976). Novel surface polymer changes in development of Myxococcus xanthus. Nature (London) 259,46-47. SUTHERLAND, I., and SMITH, M. (1973). The lipopolysaccharides of fruiting and non-fruiting myxobacteria. J. Gen. Microbial 74, 259266. SUTHERLAND, I., and THOMSON, S. (1975). Comparison of polysaccharides produced by Myxococcus strains. J. Gen. MicrobioL 89,124-132. WIREMAN, J. (1979). Developmental induction of Myxococcus xanthus myxospores. J. Bactetiol. 140, 147-153. WIREMAN, J., and DWORKIN, M. (1975). Morphogenesis and developmental interactions in myxobacteria. Science (Washington, D. C.) 189,516-523. WIREMAN, J., and DWORKIN, M. (1977). Developmentally induced autolysis during fruiting body formation by Myxococcus xanthus. J. BacterioL 129, 796-802.