cyclic nucleotides and development ofmyxococcus xanthus: analysis of mutants

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CURRENT MICROBIOLOGY Vol. 12 (1985), pp. 289-294 Current Microbiology Springer-Verlag 1985 Cyclic Nucleotides and Development of Myxococcus xanthus: Analysis of Mutants Luciano Passador and Howard D. McCurdy Department of Biology, University of Windsor, Windsor, Ontario, Canada Abstract. Approximately 60 developmental mutants of Myxococcus xanthus M300 were ob- tained through nitrosoguanidine mutagenesis and placed into three operationally defined catego- ries. Type-I strains exhibited no aggregation or sporulation. Type-II strains were able to aggre- gate but did not sporulate. A strain classed as a type-III strain was a low-capacity fruiter. Each category displayed defects in cyclic nucleotide behavior that could be predicted from the current model. Most significantly, several aggregationless (type I) mutants lacking cGMP phos- phodiesterase aggregated in the presence of externally applied phosphodiesterase. A require- ment for cell-cell contact in sporulation has been confirmed. Evidence is presented that sug- gests the involvement of cAMP phosphodiesterase in sporulation and that sporulation may be a developmental pathway independent of aggregation. These results support a previously pub- lished hypothesis of the role of cyclic nucleotides in the development of M. xanthus. Myxococcus xanthus is a Gram-negative bacterium that possesses a complex life cycle [13]. Hence it provides an excellent prokaryotic system for stud- ies of morphogenesis and cellular differentiation. McCurdy et al. [14] proposed a dual role model for cyclic nucleotides in the development of M. xan- thus. First, that one cyclic nucleotide, most likely 3',5'-cyclic guanosine monophosphate (cGMP) functioned in aggregation possibly as a chemotactic signal. Second, they favored the suggestion of Zus- man [18] that 3',5'-cyclic adenosine monophos- phate (cAMP) may function as a derepressor of a differentiation-specific promoter site. Ho and McCurdy [7] have since added support to this pro- posal by demonstrating that cGMP is indeed a che- moattractant for M. xanthus under fruiting condi- tions. Subsequent reports on cyclic nucleotide levels [8], cyclic nucleotide phosphodiesterase (PD) activ- ities [8], adenylate and guanylate cyclase activities [4], and binding proteins [3, 16] were consistent with this proposal. If the current model is correct, then it should be possible to isolate developmental mutants deficient in one or more of the various aspects of the model. This article describes the isolation of morpho- genetic mutants of Myxococcus xanthus M300 and presents the results of studies of the involvement of cyclic nucleotides to support the current proposed model. Materials and Methods Organism and growth conditions. A wild-type strain M. xanthus M300 [14] obtained from the University of Windsor culture col- lection was used. All mutants used in this study were obtained from M. xanthus M300. Isolation of nonfruiting mutants. Mutagenesis was essentially by the procedure described by Hodgkin and Kaiser [10], with some minor modifications. Briefly, liquid cultures were grown at 30~ on a shaker in 125-mlflasks each containing 30 ml of CTT broth [10] for 36 h to obtain a sufficiently high cell density (-108 cells/ ml). Since this strain adheres to the sides of the flask, it is easily collected by mere decantation of the broth. Placed into the flask were 10 ml of TM buffer [10[ and the sides of the flask aseptically scraped to place the organisms into the buffer. The cells were washed by sedimentation (7000g) and resuspended in TM buffer. This washing was repeated twice more and the cells were resus- pended in TM buffer. This cell suspension was placed into an omnimixing vessel (Ivan SorvaU, Norwalk, CT) and omnimixed at a setting of 90 on the rheostat for 90 s. The omnimixed sample was placed into a sterile flask and fitter-sterilized N-methyl-N'- nitro-N-nitrosoguanidine (NG) was added to concentration of 100 ~g/ml. The flask was shaken at 30~ for 20-25 min. After treatment the cells were sedimented twice as above and resus- pended. This solution was diluted 1 : 10 into a fresh CTT broth flask and allowed to grow for 36 h. After growth, the cells were collected as before and suspended in dilution medium (DM) [12]. Address reprint requests to: Dr. Howard D. McCurdy, Department of Biology, University of Windsor, Windsor, Ontario, Canada N9B 3P4.

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CURRENT MICROBIOLOGY Vol. 12 (1985), pp. 289-294 Current Microbiology �9 Springer-Verlag 1985

Cyclic Nucleotides and Development of Myxococcus xanthus: Analysis of Mutants

Luciano Passador and Howard D. McCurdy

Department of Biology, University of Windsor, Windsor, Ontario, Canada

Abstract . Approximately 60 developmental mutants of Myxococcus xanthus M300 were ob- tained through nitrosoguanidine mutagenesis and placed into three operationally defined catego- ries. Type-I strains exhibited no aggregation or sporulation. Type-II strains were able to aggre- gate but did not sporulate. A strain classed as a type-III strain was a low-capacity fruiter. Each category displayed defects in cyclic nucleotide behavior that could be predicted from the current model. Most significantly, several aggregationless (type I) mutants lacking cGMP phos- phodiesterase aggregated in the presence of externally applied phosphodiesterase. A require- ment for cell-cell contact in sporulation has been confirmed. Evidence is presented that sug- gests the involvement of cAMP phosphodiesterase in sporulation and that sporulation may be a developmental pathway independent of aggregation. These results support a previously pub- lished hypothesis of the role of cyclic nucleotides in the development of M. xanthus.

Myxococcus xanthus is a Gram-negative bacterium that possesses a complex life cycle [13]. Hence it provides an excellent prokaryotic system for stud- ies of morphogenesis and cellular differentiation.

McCurdy et al. [14] proposed a dual role model for cyclic nucleotides in the development of M. xan- thus. First, that one cyclic nucleotide, most likely 3 ' ,5 '-cyclic guanosine monophosphate (cGMP) functioned in aggregation possibly as a chemotactic signal. Second, they favored the suggestion of Zus- man [18] that 3 ' ,5 '-cyclic adenosine monophos- phate (cAMP) may function as a derepressor of a differentiation-specific promoter site. Ho and McCurdy [7] have since added support to this pro- posal by demonstrating that cGMP is indeed a che- moattractant for M. xanthus under fruiting condi- tions.

Subsequent reports on cyclic nucleotide levels [8], cyclic nucleotide phosphodiesterase (PD) activ- ities [8], adenylate and guanylate cyclase activities [4], and binding proteins [3, 16] were consistent with this proposal.

If the current model is correct, then it should be possible to isolate developmental mutants deficient in one or more of the various aspects of the model.

This article describes the isolation of morpho- genetic mutants of Myxococcus xanthus M300 and presents the results of studies of the involvement of

cyclic nucleotides to support the current proposed model.

Materials and Meth od s

Organism and growth conditions. A wild-type strain M. xanthus M300 [14] obtained from the University of Windsor culture col- lection was used. All mutants used in this study were obtained from M. xanthus M300.

Isolation of nonfruiting mutants. Mutagenesis was essentially by the procedure described by Hodgkin and Kaiser [10], with some minor modifications. Briefly, liquid cultures were grown at 30~ on a shaker in 125-ml flasks each containing 30 ml of CTT broth [10] for 36 h to obtain a sufficiently high cell density (-108 cells/ ml). Since this strain adheres to the sides of the flask, it is easily collected by mere decantation of the broth. Placed into the flask were 10 ml of TM buffer [10[ and the sides of the flask aseptically scraped to place the organisms into the buffer. The cells were washed by sedimentation (7000 g) and resuspended in TM buffer. This washing was repeated twice more and the cells were resus- pended in TM buffer. This cell suspension was placed into an omnimixing vessel (Ivan SorvaU, Norwalk, CT) and omnimixed at a setting of 90 on the rheostat for 90 s. The omnimixed sample was placed into a sterile flask and fitter-sterilized N-methyl-N'- nitro-N-nitrosoguanidine (NG) was added to concentration of 100 ~g/ml. The flask was shaken at 30~ for 20-25 min. After treatment the cells were sedimented twice as above and resus- pended. This solution was diluted 1 : 10 into a fresh CTT broth flask and allowed to grow for 36 h. After growth, the cells were collected as before and suspended in dilution medium (DM) [12].

Address reprint requests to: Dr. Howard D. McCurdy, Department of Biology, University of Windsor, Windsor, Ontario, Canada N9B 3P4.

2 9 0 CURRENT MICROBIOLOGY Vol. 12 (1985)

Table 1. Categories of fruiting-defective isolates"

Type Phenotype N u m b e r of isolates

I agg- spo 9 II agg + spo - 51

III agg+spo + 1

a The single strain placed in category III is fruiting proficient but p roduces fewer fruiting bodies than the wild type. Hence , it is c lassed as a fruit ing-defective strain. All strains were obtained after isolation and replating on FM agar. The phenotypes de- scribed represent the phenotypes after ten days of growth on FM agar.

Omnimixing was done as previously described and a tenfold dilu- tion series (10 4-10 7) in DM prepared. Sets of plates o f C F agar with overlay [6] were made from each of the dilutions and al- lowed to incubate at 30~ Optimal plates were those with 100- 150 colonies. The plates were observed for fruiting body (FB) format ion after ten days .

Selection of mutants. Colonies visibly abnormal in fruiting behav- ior were isolated and placed on FM [14] or CF medium [6]. Stock cul tures o f isolated mutants were kept frozen at -76~

Harvesting of cells at different stages of development. FM plates were each spot- inoculated with approximately 7 x 106 cells and, after brief drying, incubated at 28~ After various time inter- vals, a number of replicate plates were harvested by the method described by Ho and McCurdy [8].

Cyclic nucleotide phosphodiesterase assay. The assay procedure used was essential ly that described by Ho and McCurdy [8] ex- cept that for separat ion of products 1.0 z 17.5-cm strips of po- lyethyleneimine cellulose (PEI-cel lulose) were developed with 1 N formic ac id-0 .5 M LiCI (1 : 1) for 90 min by ascending chroma- tography. Cells were s tarved for 38-40 h before being assayed as this has been shown to be the time of maximal phosphodies terase activity [8].

Extraction of radioactive labels from PEl-ce l lu iose . Radioactiv- ity was eluted from chromatographic plates according to the me thod reported by Ho et al. [9].

Effect of cyclic nucleotide phosphodiesterase on mutants. The technique used was that of McCurdy et al. [14].

Guanylate cyclase assays. The methods were essentially those of Devi and McCurdy [4]. Cells were s tarved for 8 h prior to being assayed as this is the time of m a x i m u m activity of guanylate cyclase [4].

Assay of cyclic nucleotides. Cyclic nucleotides were assayed us- ing the rad io immune assay of Steiner et al. [17]. The extracts to be a s sayed were treated as previously reported [9].

Mass induction of myxospores. The mass induction of myxos- pores was done by the method of Burchard [2].

Effect of exogenous cydic nucleotide. Exogenous cyclic nucleo- tide was added as per McCurdy et al. [14].

Table 2. Cyclic GMP-PD activity in type-I strains ~

Control (cpm) Tes t (cpm)

Strain cGMP GMP cGMP GMP

M 300 3110 + 267 42 • 29 492 • 150 2558 -+ 344 N F M 37 3042 • 102 95 • 15 2082 • 83 386 • 5 N F M 40 2708 • 92 42 • 18 1953 • 234 218 § 80 N F M 64 3776 • 66 35 • 2 1963 • 68 171 • 129 N F M 72 3279 --- 138 70 • 16 2183 • 72 498 • 30 N F M 84 2 6 4 4 • 341 114 • 10 1940• 520 359-+ 59 N F M 1 0 1 1 8 5 0 • 30 85 +- 15 1062• 38 3 5 5 • 45

a All cul tures were s tarved for 38-40 h prior to being assayed. The presence of activity was determined by the addition of triti- ated cGMP to boiled (control) and untreated (test) cell-free extract and measur ing the resulting tritiated GMP formed. Substrate and end-product were separated by thin-layer chromatography on PEI-cellulose strips. All strains were tested at least three t imes and the values given are expressed as average cpm followed by a s tandard deviation.

Protein determinations. The method of Lowry et al. [11] was used for protein est imations. Bovine serum albumin was used as s tandard.

Biochemicals. The cAMP and cGMP radioimmune assay (RIA) kits and Atomlight scintillation cocktail were obtained from New England Nuclear (Lachine, Quebec).

(8-3H) Guanos ine 3 ' ,5 ' -cycl ic monophospha te and (8-3H) adenosine 3 ' ,5 ' -cyclic monophospha te were obtained from A m e r s h a m (Oakville, Ontario).

Nonradioact ive nucleotides and nucleosides and reagents were obtained from Sigma (St. Louis , MO). For liquid scintilla- tion count ing, the filmware bags were obtained from Nalge (Rochester , New York). Samples were counted in a Beckman LS 3150 P counter .

The precoated plastic chromatographic sheets of PEI-ce l - lulose (Polygram Cel 300 PEI) were obtained from Brinkmann Ins t ruments (Canda).

Resul t s

In the clone fruiting procedure described by Hagen et al. [6], cells of M. xanthus were plated on CF agar with an overlay. This medium had sufficient nutrients to permit colony formation but did not allow colony growth. Under these conditions, sin- gle cells divided to form small colonies and the fruit- ing-proficient cells within the colony later formed several small fruiting bodies. If the cells were of a non-fruiting-proficient strain, then characteristic diffuse, flattened colonies resulted with no fruiting bodies being formed. This allowed for the selection of fruiting-defective suspects.

Cultures were mutagenized as described in Ma- terials and Methods. Through the above method, over 43,000 colonies were screened and over 150

L. Passador and H. D. McCurdy: Cyclic Nucleotides and Myxococcus xanthus Mutants 291

Table 3. Cyclic AMP-PD activity of type-I cells ~ Table 4. Cyclic GMP-PD activity of type-II strains a

Control (cpm) Test (cpm) Control (cpm) Test (cpm)

Strain cAMP AM P cAMP AMP Strain (cGMP) (GMP) (cGMP) (GMP)

M 3 0 0 2682-+ 321 22-+ 7 365_+ 20 2564-+ 148 M 3 0 0 3562-+ 751 17-+ 2 437-+ 76 3 2 4 0 + 749 N F M 40 3132-+ 35 392_+ 344 743-+ 128 2834-+ 141 N F M 1 3261 -+ 518 42 + 21 4 1 4 - + 9 8 3331 -+ 638 N F M 101 3560-+448 26-+ 15 1490-+ 125 2188-+ 194 N F M 3 3367--- 620 12-+ 1 281 -+ 17 3016 + 398 N F M 132 3761 -+ 245 453_+ 142 148-+ 67 3255-+ 139 N F M 65 3369-+ 247 31 -+ 6 418-+ 64 2961 _+ 50

N F M loo 3557-+ 661 34_+ 3 656 + 5 3 2894-+ 380 All cultures were s tarved for 38-40 h prior to being assayed.

The presence of activity was determined by addition of tritiated cAMP to boiled (control) and untreated (test) cell-free extracts and measur ing the tritiated AM P formed. The substrate and product were separated by thin-layer chromatography on PEI- cellulose strips. Each strain was tested three t imes and the val- ues given are expressed as average cpm followed by a s tandard deviation.

suspect colonies isolated. All suspected colonies were retransferred to CF agar twice more to estab- lish stability. Of these 150 colonies, approximately 60 stable fruiting-defective mutant strains were iso- lated.

Stable isolates were placed into three opera- tionally defined categories based on their colony morphology (Table 1). Type-I colonies showed no aggregation or sporulation. Type-II colonies were those that formed aggregates but did not sporulate. The type-III category contained only one isolate that did show aggregation and sporulation but was classed a fruiting-defective strain since it demon- strated a low fruiting capacity when compared with the wild-type M300.

Our current model assumes that cGMP is the chemoattractant involved in the aggregation pro- cess and thus a cGMP/cGMP-PD system was impli- cated [14]. According to the model then, aggrega- tion-deficient strains may not aggregate because of altered synthesis of cGMP, altered phosphodies- terase activity, or altered cGMP receptors. Phos- phodiesterase activity, being the simplest to mea- sure, was examined first. The assay was carried out as described in Materials and Methods.

All strains of the type-I, agg- phenotype tested showed severely reduced levels of cGMP-PD activ- ity when compared with the wild-type (Table 2). Furthermore, significant cAMP-PD activity was de- tected in these same agg strains (Table 3). For comparison, several strains of agg + phenotype cells were tested for cGMP-PD activity. All showed sig- nificant levels of cGMP-PD activity (Table 4).

If failure to aggregate is due to a defect in cGMP-PD, then addition of exogenous PD should correct the defect and produce fruiting. Indeed,

a All cul tures were s tarved for 38-40 h prior to being assayed. The presence of activity was determined as outlined in Table 2 note.

Table 5. Summary of results of phosphodies te rase application to isolates ~

Strain - PD + PD

M 3OO F A A , F

Type I N F M 37 - - - - N F M 40 - - F N F M 64 - - F N F M 72 - - - - N F M 101 - - F

Type H N F M 3 NA A A N F M 75 NA AA N F M 111 NA AA N F M 5 NA A A , F N F M 16 NA A A , F N F M 54 NA A A , F N F M 57 NA A A , F N F M 65 NA A A , F N F M 73 NA A A , F N F M 110 NA A A , F N F M 114 NA A A , F

Type 111 N F M 34 F A A , F

a NA, normal aggregation; AA, accelerated aggregation; F, com- pletes fruiting with sporulation; and - , fails to develop. A total of 25/zl of cell suspens ion containing approximately 106 cells was overlayed either on spots of bovine phosphodies te rase on FM agar or on the agar itself. Small 9 • 50-ram, tight-lid Petri d ishes were used. The presence and t ime of aggregation and/or sporula- tion were recorded. All results are for ten days after spotting.

approximately 50% of the aggregation-deficient, type-I strains tested showed induction of fruiting (Table 5).

Table-II strains aggregate with the same timing as the wild type. They also exhibited the same ac- celeration of aggregation as expected [14] (Table 5).

292 CURRENT MICROBIOLOGY VOI. 12 (1985)

50"

~25 " I 0

x

u~2 O" _J

~Ol5-

,.=, Q.

~10"

,.=,

0

LX

DAYS

Fig. 1. Induction of fruiting in NFM 34 by exogenous PD. The assay was carried out as described in Materials and Methods. The numbers of fruiting bodies on the M300 placebo plates were essentially equal to those on the control plates and thus, for the sake of simplicity, values for placebo plates were not plotted: A--A, M300; A--A, M300 + PD; 0 - - 0 , NFM 34 control; O--O, NFM 34 placebo; and Fq--[~, NFM 34 + PD.

Most surprisingly, however , some strains were ac- tually induced to sporulate (Table 5).

Addition of exogenous PD to N F M 34, the type-I l l , low-fruit ing-capacity strain, not only ac- celerated deve lopment but increased fruiting ap- proximate ly sevenfold (Fig. 1).

The addition of phosphodies terase to wild-type M300 resulted in accelerat ion of fruiting and a large increase in the number of fruiting bodies formed as reported previously by McCurdy et al. [14].

Two cGMP-PD-deficient , nonaggregating strains (NFM 37 and N F M 72) were not induced by exoge- nous PD (Table 5). This suggested that they were defect ive in some other capacity: for example, guanylate cyclase activity. When these strains were examined for guanylate cyclase, both exhibited sig- nificant activity (results not shown).

I f there were changes in the timing of the guany- late cyclase activity associated with morphogenesis then this might account for the lack of response. For this reason, a t ime course study of guanylate cyclase was done on several agg- and one agg § phe- no type strains. Results show that the agg § strain demonst ra ted a normal guanylate cyclase activity profile but two strains of the agg phenotype, N F M 37 and N F M 72, showed altered profiles (Fig. 2). Strain N F M 37 showed a delay in peak activity of

18-

z ~E '~. 16" z

0 ~12-

u.I 8"

4-

0 0

A

I i I l I ?o 4 I0 20 30 40 50 60

TIME (HR)

Fig. 2. Guanylate cyclase profile of mutant strains. Cells were harvested and processes as described in Materials and Methods. Guanylate cyclase activity was assayed using GTP as substrate and measuring cGMP formed by radioimmune assay: O--II, M300; O--O, NFM 3 (type 11); A--A, NFM 37 (type 1); and A--A, NFM 72 (type I).

approximate ly 22 h while for N F M 72 the delay was approximate ly 12 h.

Exogenous cGMP was added to cultures of agg § strains spotted on FM agar to determine whether the cGMP delays the appearance of aggre- gates as reported for the wild type [14]. In all cases, addition of exogenous 0.5 mM cGMP delayed the appearance of aggregates as expected (results not shown).

According to the current model, cAMP dere- presses development-specif ic genes. Thus, it should be possible to isolate strains having decreased lev- els of cAMP which when treated with exogenous cAMP should fruit. Only N F M 85, an aggregation- deficient strain, demonst ra ted fruiting after the ad- dition of 0.7 m M cAMP (results not shown).

Although the mutant strain N F M 34 does fruit, it forms fewer fruiting bodies (Fig. 1). The fact that this strain can actually complete the fruiting process implies that all the required genes are being ex- pressed and the only difference is in the efficiency of this gene expression.

The levels of cAMP were monitored in this mu- tant ove r its developmenta l period and, although the levels were found to peak at the same time as in the wild type, the actual levels were much lower than expected (Fig. 3).

To determine whether or not aggregation was required for sporulation to occur, aggregation-de- fective cells were placed in PO4-Mg buffer and cen- trifuged (7000 g) to pack the cells. Of all the strains tested, only one, N F M 84, was induced to sporu-

L. Passador and H. D. McCurdy: Cyclic Nucleotides and Myxococcus xanthus Mutants 293

20- _zz ua

1 5 -

w g~

u 5 - o

I I I I .q I I I 0 I0 2 0 30 4 0 6 0 70 8 0

TIME (HR)

Fig. 3. Levels of cAM P in N F M 34 (type IlI). The assay was carried out as descr ibed in Materials and Methods,. Cells were harves ted at the t imes indicated after s tarvation on FM medium. Cyclic nucleotide was measured by rad io immune assay: 0 - - 0 , M300; and O - - O , N F M 34.

late. When grown on a solid starvation medium (e.g., FM agar), there is no evidence of sporulation, even after ten days (results not shown).

Discussion

Although much is known about the sporulation pro- cess in the development ofM. xanthus, very little is known about the aggregation process. This study utilized developmental mutants of M. xanthus in an attempt to clarify the role of cyclic nucleotides in development.

The model put forward by McCurdy et al. [14] is supported by the results obtained in this study. First, the absence of cGMP-PD should prevent ag- gregation, according to the model, because its ab- sence should not allow cells of M. xanthus to main- tain the chemoattractant at detectable levels. It was found that all the aggregation-deficient strains ex- hibited severely reduced cGMP-PD activity (Table 2). The addition of exogenous bovine PD should therefore, have corrected this defect if the only de- fect was the absence of cGMP-PD itself. Only two agg- strains, NFM 37 and NFM 72, failed to re- spond to this addition of exogenous PD (Table 5). When tested for guanylate cyclase activity, both NFM 37 and NFM 72 exhibited a delay in the ap- pearance of enzyme activity (Fig. 2). In contrast, a type-II strain that aggregates normally but does not sporulate exhibited a guanylate cyclase profile com- parable to the wild type (Fig. 2). The above results are consistent with the roles assigned to each of the components in the current model and thus confirm

the requirement for a cGMP/cGMP-PD system in development as postulated by Ho and McCurdy [8].

The addition of exogenous cAMP caused NFM 85, an agg-spo- strain to fruit (Table 3). Further- more, studies on NFM 34, a low-capacity-fruiting strain, revealed decreased levels of cAMP although the time of appearance of peak levels were the same as for the wild type (Fig. 3). These results support the idea of cAMP being a derepressor of develop- ment-specific genes. In addition, as was originally suggested by Ho and McCurdy [8], cAMP-PD is confirmed to be a distinct enzyme from cGMP-PD. Its appearance in aggregation-defective strains (Ta- ble 3) suggested that its role in the aggregation re- sponse is to modulate the levels of the derepressor and that it is not required in the chemotactic system per se.

As an added confirmation of an earlier report by Burchard [2], close cell-cell contact seems to be required for sporulation. This is evidenced by the fact that an aggregation-defective, nonsporulating strain, NFM 84, was induced to sporulate when starved in liquid culture at high cell density. Fur- thermore, this result implies that aggregation and sporulation are two separate pathways since, by cir- cumventing the normal aggregation stage with artifi- cial packing, sporulation could take place (results not shown). The idea of two separate but linked pathways has also been suggested by Morrison and Zusman [15].

Finally, an extremely interesting and unex- pected result surfaced. The addition of exogenous bovine PD to some agg+spo - strains not only accel- erated development but actually induced sporula- tion (Table 5). This observation tentatively identi- fies a function for phosphodiesterase activity in sporulation. To date, no report has done so for M. xanthus but PD has been implicated in the sporula- tion of Dictyostelium discoideum [1], a cellular slime mold. An assumption that the phosphodies- terase may be cAMP-PD may be correlated to sev- eral observations. First, aggregation-capable, non- sporulating strains have been shown to possess a functional cGMP/cGMP-PD system. Thus cGMP- PD activity is present and still sporulation does not occur. The bovine heart phosphodiesterase used in the induction studies is known to hydrolyze both cAMP and cGMP [5] and hence may provide a source of cAMP-PD. Ho and McCurdy [8] have shown that there is a large increase in extracellular cAMP concentrations just before sporulation. Un- like levels of extracellular cAMP, these levels of

294 CURRENT MICROBIOLOGY Vol. 12 (1985)

cAMP are maintained. The high levels of cAMP may be correlated with the report of an increased activity of adenylate cyclase just before sporulation [4]. These observations support the assumption, but before it can be confirmed more work will need to be done.

ACKNOWLEDGMENT

This research was supported by grant AI022 from the National Science and Engineering Research Council of Canada.

Literature Cited

1. Brown SS, Rutherford CL (1980) Localization of cyclic nu- cleotide phosphodiesterase in the multicellular stages of Dic- tyostelium discoideum. Differentiation 16:173-183

2. Burchard RP (1975) Myxospore induction in a nondispersed growing mutant of Myxococcus xanthus. J Bacteriol 122:302-306

3. Devi AL, McCurdy HD (1984) Cyclic GMP and cyclic AMP binding proteins in Myxococcus xanthus. J Gen Microbiol 130:1845-1849

4. Devi AL, McCurdy HD (1984) Adenylate cyclase and guany- late cyclase in Myxococcus santhus. J Gen Microbiol 130:1851-1856

5. Goren EN, Rosen OM (1972) Purification and properties of a cyclic nucleotide phosphodiesterase from bovine heart. Arch Biochem Biophys 153:384-397

6. Hagen DC, Bretscher AP, Kaiser D (1978) Synergism be- tween morphogenetic mutants of Myxococcus xanthus. Dev Biol 64:284-296

7. Ho J, McCurdy HD (1979) Demonstration of positive che- motaxis to cyclic GMP and 5'-AMP in Myxococcus xanthus by means of a simple apparatus for generating practically

stable concentration gradients. Can J Microbiol 25:1214- 1218

8. Ho J, McCurdy HD (1980) Sequential changes in the cyclic nucleotide levels and cyclic nucleotide phosphodiesterase activities during development in Myxococcus xanthus. Curr Microbiol 3:197-202

9. Ho, J, Warner AH, McCurdy HD (1979) Recovery of quenched radioactivity from thin layer chromatographic plates: an improved assay for cyclic GMP phosphodiesterase in Myxococcus xanthus. J Biochem (Tokyo) 88:659-662

10. Hodgkin J, Kaiser D (1977) Cell to cell stimulation of move- ment in non-motile mutants of Myxococcus. Proc Natl Acad Sci USA 74:2938-2942

11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275

12. McCurdy HD (1963) A method for the isolation of myxobac- teria in pure culture. Can J Microbiol 9:282-285

13. McCurdy HD (1974) The gliding bacteria. In: Buchanan RE, Gibbons NE (eds) Bergey's manual of determinative bacteri- ology, 8th edn. Baltimore: Williams and Wilkins, pp 76-98

14. McCurdy HD, Ho J, Dobson WJ (1978) Cyclic nucleotides, cyclic nucleotide phosphodiesterase and development in Myxococcus xanthus. Can J Microbiol 24:1475-1481

15. Morrison CE, Zusman DR (1979) Myxococcus xanthus mu- tants with temperature-sensitive, stage-specific defects: evi- dence for independent pathways in development. J Bacteriol 140:1036-1042

16. Orlowski M (1980) Cyclic adenosine 3',5'-monophosphate binding protein in developing myxospores of Myxococcus xanthus. Can J Microbiol 26:905-911

17. Steiner AL, Parker CW, Kipnis DM (1972) Radioimmunoas- say for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J Biol Chem 247:1106-1113

18. Zusman DR (1978) A rapid batch assay for cyclic AMP phos- phodiesterase. Anal Biochem 84:551-558