Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in Myxococcus x a n t h u s ' H. D. MCCURDY, J . Ho,
AND
W. J. DOBSON
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Deptrrrtiretrr of'Biology, U11ir3ersir.vof' Witrtlsor, Witrdsor, O~lt.,Cnt~crtitrN9B 3P4 Accepted August 21, 1978 MCCURDY, H. D., J . Ho, and W. J . DOESON.1978. Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in My.\-ococcrrs xtrtrrlrrrs. Can. J . Microbiol. 24: 1475-1481. Exogenous cyclic nucleotide phosphodiesterase (PD) accelerated fruiting body (FB) formation and increased territory size of aggregates in M.v,\-ococcrr.\ st~t~thlts. Both gunnosine 3'5'monophosphate (cGMP) and gu21nosine 5'-monophosphate (GMP) were antagonistic to the PDeffect. Adenosine 3'5'-monophosphate (CAMP)increases FB numbers twofold in the absence but not in the presence of PD. PD induction is not affected by methionine or isoleucine, which inhibit, or by thl-eonine, which stimulates, FB formation. There is an increase and subsequent decrease in cAMP levels during early glycerol-induced rnicrocyst development but lOmM theophylline or caffeine not only inhibited microcyst development but induced germination in the presence of glycerol. On the basis of these results and the reports ofother investigators n tentative model is proposed bmed on a dual role for cyclic nucleotides in the developnlent in M . a a t ~ ~ h r r s . McCunny, H. D., J . Ho et W. J. DOESON.1968. Cyclic nucleotides, cyclic nucleotide phosphodiestel-ase, and development in My,vococc~rs.rrrtr~l~lr.s. Can. J . Microbiol. 24: 1475-1481. La phosphodiesterase nucleotide cyclique (PD) exogene accelere In fructification (FB) et augmente In dimension des agregitts de Mv,vococ.c~rssirr~fhos. La guanosine 3'5'-monophosphate (cGMP) et la guanosine 5'-monophosphate (GMP) sont antagonistes B I'action du PD. L'edenosine 3'5'-rnonophosphate (CAMP)augmente de deux fois le nombre de FB en I'absence de PD mais non en sa presence. L'induction du PD n'est pas affectee ni par la methionine ou I'isoleucine, lesquels inhibent la formation de FB, ni par la threonine, laquelle stimule la formation du PB. Aux premiers stades de developpement des microcystes lequel est induit par le glycerol, on observe une augmentation suivie d'une baisse dans les taux de CAMP. Cependant, lOmM de theophylline ou de cafeine inhibent le developpement des mycrocyste et induisent la germination en presence de glycerol. S'appuyant surces resultats et ceux d'autres chel-cheurs, on propose un modele sut. le double role que jouent les nucleotides cycliques dans le developpement de M. snt~rlzrrs. [Traduit par lejournal]
Introduction The myxobacteria are gliding prokaryotes which al-e capable of aggregation to form fruiting bodies ( ~ ~ 1966). ~ These ~ korganisms i ~ are thus considel-ed to be idea] pl-okal-yotes for mol-phogenetic and developmental studies (Wireman and Dworkin 1975). F1-uiting body (FB) formation in Myxococc~rs scrt7th~r.swas reported to be stimulated by 3'-5'cyclic adenosine monophosphate (CAMP), 5'adenosine diphosphate (ADP), and other adenine nucleotides (Campos and Zusman 1975). Subsequently, Parish et al. (1976) confirmed the presence of cAMP in vegetative cells of this organism. More recently Yajko and Zusman (1978) reported that during glycerol-induced sporulation, M . xrrnthus exhibited a sharp but transient rise in cAMP concentration. Cyclic AMP phosphodiesterase (PD) activity has also been detected in cell-free lSupported by NRCC Grant No. A 1022.
extracts of M. xcrtzth~rsvegetative cells (Zusman lg78). These I-ePol-ts suggest that there may be a significant involvement of CAMP and PD in morphogenesis in this organism. In the pl-esent work, We describe the effect of exogenous PD On FB formation in M. xcrnrh~rs. Materials and Methods Miooorgnnisms o t ~ d Grow111Cot~ditiotrs In glycerol induction and preliminary fruiting experiments, Myxococcrrsscir~tllrrsfb (M. Dworkin, University of Minnesota) was used. However, this strain yielded variable results in fruiting experiments. Subsequently, a new wild-type strain, M. xatrtklrs M300. was used, which had been isolated from soil samples collected from Point Pelee Provincial Pal-k, Ontario. Isolation and culture techniques used were essentially those of McCurdy (1963). Studies on Frriilit~gBody Fortncrtiotr Fruiting medium (FM) contained the following, per litre of distilled water: K,HP04, 1.4g; KHZP04, 0.35g; casitone (Difco), 0.4g; MgSO,.7H2O, I.Og; agar, 15g; pH 7.3. Three millilitre quantities of autoclaved FM were aseptically dis-
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1476
C A N . J . MICROBIOL. VOL. 24. 1978
pensed onto 50 x 9 mrn Falcon petri dishes (tight lid, type 1006). After the agar plates had solidified the condensate was allowed to dry o f f for 3 to 6 h with the covers pertially opened. When req~~ired the , appropl-iate nucleotide or cyclic n~~cleotide was added after sterilization. Each petri dish was divided into qu;idrants. On two were spotted 25 ~1 o f filte1.-sterilized PD in water ( 5 0 ~ protein, g 0.01 ~ ~ n i tcreating s) circular spots o f about 10-mm diameter. The two remaining quadrants sel-ved as controls end were spotted with 25 FI watel- placebos. Inocul?~ o f 25 PI, each containing about IOb cells, were overlayed on PD 01-placebo spots. The excess liquid was :illowed to evaporate before closing the petri dish and s ~ ~ b s e q ~ incuba~ent tion o f the plates at 22'C. Fr~~itin bodies g were examined with a dissecting microscope and counted with the aid o f n Nikon Profile Projector model 6C with a 10 x objective. Photomicrographs were taken with a Carl Zeis\ (Jena) autorn;rtic exposure device. Mrtr.s~i~.c,ttretrr itf'cAMP i t r Gl!cerol-i~rtlrrcerl Microcy.sts M~sococc.irsxtrtrr1rrr.s fb was grown for 48 h in 800ml C T medium (Dworkin 1962) in each o f five, 2.82 Fernbach flasks, shaken at 60cycles per minute (Northcott Instruments; Guelph. Ont.) (final cell density about IOH per millilitre). After the re~novao l f 200-ml samples from each flask (zero time), sterile glyce1.01was added to the remainder in the flasks to give a final concentration o f 0.5M glycerol in each (Dworkin and Sadler 1966). Subsequently, at the times indicated (Fig. 8) 200-ml a l i q ~ ~ o twere s withdrawn from different replicate flasks, and centrifi~gedat 8000g for 20min at 4°C. The pellets were then washed twice with equal volumes o f sterile distilled water. The washed pellets were resuspended in l 0 m l o f assay medium containing 8 m M theophylline. 6 m M 2-mercaptoethanol, and 5 0 m M Tris buffer at p H 7.5. The zero-time sample containing vegetative cells was sonicated for 5 min at full power (Biosonik, Brownwell Scientific. Rochester, N Y ) . Mic~.ocystswere dis~.uptedby omni-mixing (Servall Inc., Norwalk, CT) for7 min, at a rheostat setting o f 90, with an equal weight o f glass beads ( - 100 120. Flex-o-iite. St. Thomkrs, Ont.). This treatment gave 80-85% breakage. Cellular debris was [removed by centrifugation at 35 000g fol-20min. The supernatant fractions were deproteinized with 1.0N HCIO, and centrifuged at I0000g to remove the precipitate and neutlxlized with tric;~prylyl ;mine dissolved in chloroform (Warner and Finamore 1967). Cyclic A M P was assayed by the competitive binding-protein technique o f Brown el (11.(1971). Labelled C A M Pwas counted in a scintillation fluid consisting o f 10% (wlv) napthalene with 0.05% (wlv) PPO in 1,Cdioxane as specified by B D H Chemical Co. (publication no. 883Dl1.011072). Radioactivity was counted with 21 Nucle:~r Chicago Mark 11 liquid scintillation counter (Nuclear Chicago. Des Plaines, I L ) .
+
Detc,rttritrtrriotr of A4icrocyst N ~ o ~ r b c i;fier ~ r s T l ~ i ~ o p l ~ y l lon(/ ir~e Cr;fi,itre Tretrtttrrtlt T o study the effect o f theophylline and caffeine on glycerol induction o f microcysts. 7.5 ml o f 45 m M o f the appropriate inhibitors were added to 25 ml o f induction medium in a 125-ml Erlenmeyer flask (see above). At times indicated in Fig. 9, the number o f microcysts in 3-mI samples was detel-mined by plating out the sonication-resisti~nt fractions o n C T agar plates as described by Sudo and Dworkin (1969). Proteit~Deterttrit~trtiot~ Protein was determined according to L o w r y el (11. (1951). Bovine serum albumin was the standard.
Clrettrictrls The following compounds wel-e obtained from Sigma Chemical Co.. St. Louis, M O : 3'15'-cyclic nucleotide phosphodiestel-ase: 3':5'-CAMP; adenosine5'-monophosphoric acid. sodium salt (AMP); gu;rnosine 3'5'-cyclic monophosphoric acid, sodium salt (cGMP); gu:inosine 5'-monophosphoric acid, sodium salt (GMP); 2-mercaptoethanol; sodium azide; theophylline: caffeine; and tris(hyd~.oxymethyl);uninometh;tne. The radiochemical 'H-3':5'-CAMP (sp. act. 28 CilmM) ( I C i = 37GBq) was from Picker Nucleiir Cnnad;~,Montreal. P.Q. The 3':j'-CAMP binding protein (from bovine adrenil1 cortex) and Norit GSX were from B D H Chemiciils, Toronto. Ont. Tricaprylyl amine (Alamine 3363) was from General Mills Inc., Knnkakee. IL and 2.5-diphenyloxazole (PPO), napthalene, 1,4dioxane were fl-om Fisher Scientific Co., Don Mills. Ont.
Results
The Eflecf q f ' C y c l i c N ~ r c l e o t i c l eP h o ~ p l z o d i e s t e ~ ~ c r s e Preliminary experiments in our laboratory indicated that a commercial prepal-ation of PD induced FB formation by several my xobacteria including C y s t o h c r c t e ~ . firsc~rs, M y x - o c o c c ~ r s c o r c r l l o i d e s , Polyrrt7gir1tn c e l l r r l o s r r t ~ ,M y x o c o c c r r s s r r t ~ f / ~ r r sand , Stigt?lctte/lrr c r r ~ t . a n f i c r c ~ t .
Figure 1 illustrates the results obtained when M. on FM was treated with PD. It had previously been established that lo6-10' cells gave optimal results. Compared with the contl-01s without PD, the treatment resulted in (a) a very much earlier appearance of FB's; (12) a fourfold increase in numbel- for FB's obtained within 6 days, although the slow development of FB's on control plates ultimately resulted in a high final number of pool-ly formed FB's; and (c) FB's which were larger and more completely developed (Fig. 2). T o obtain I-eproducible numbers of FB's, it was important to use cultures that were not subjected t o more than three serial transfers in liquid culture although experiments utilizing cells subjected to up to two additional transfers (Figs. 3 and 4), yielded essentially the same relationships among the various experimental treatments. Attempts to use
s c r n t l z ~ r sinoculated
DAYS
FIG. I . The effect o f P D o n F B formation by M.xtrt~t1~1rs. Bars represent standard deviation o f eight replicates. -0PD, -0control, -0placebo.
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F I G . 2. PD-induced fruiting bodies. A. Natural FB's 10 days after inoculation. B. PD-induced FR's 10 days after with reflected light. inoculation. Squ;~t-esIrepresent 1 mm'. The samples were pho~ogr;~phed 400-
n
W LL
0 LL
n 100
.m
mmd
.m
-m
.Theophylbne
.m
-m
.Caffe~ne
F I G .3. The effect of theophylline end caffeine on FB formaInhibitor concentrations were 10mM. tion by M. .rcit~rlrr,~.
standa~d-sizepetri dishes led to excessive variation in FB formation. Because the commercial PD enzyme preparation used contained certain contaminating enzymes, each was tested for its effect on fruiting. These included 5'-nucleotidase. 5'-ATPase, inorganic pyrophosphatase, alkaline phosphatase. 2',3'cyclic nucleotide phosphodiesterase, and protein kinase. All were without effect. As a further confirmation that the obsel-vation depended upon PD, theophylline and caffeine, both inhibito~sof PD, were tested. Both counteracted the effect of PD a s well as delaying the appearance of FB's in the absence of PD (Fig. 3).
0
Dl 1 lo 100 PHOSPHATE CONCN mM
FIG.4. The effect of phosphate concentration on FB fol-mation by M . sotltll~rs.The number of FB's were recorded on the 6th day after inoculation. -0- PD, -0- control.
might have caused its production and influenced the results. Furthermore, Wireman and Dworkin (1975) stated that the need for high cell densities for FB formation could be lowered by the addition of phosphate to unsupplemented agar medium. However, no data were presented to substantiate this significant observation that suggests orthophosphate is a cell-density signal. As shown in Fig. 4, a definite optimum exists between 10 and 30 mM both in the presence and absence o f PD.
Efffct of Tlzreotzine, Isolerrcine, crnrl Methionine Rosenberg et NI.(1973) reported that methionine and isoleucine inhibit, while threonine stimulates, Effect of Phosphcrte of FB Fo1.m~rtior1 F B formation in M . xrrnthus. When these amino The effect of orthophosphate on fruiting was also acids were tested at the effective concentrations tested since contaminating enzymes in the PD (Campos and Zusman 1975) in the presence and
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absence of PD, none was found to alter significantly the stimulatory effect of PD. The FB's were essentially the same size and morphology and the kinetics of production essentially the same. In the absence of PD, however, our results duplicated those previously I-epurted, i.e. methionine and isoleucine depressed the numbers of fruits by about 80%; threonine slightly increased the numbers (Table 1). T11eEJrect of Nrlcleotides on PD Indlrction Campos and Zusman (1975) reported the stimulation of FB formation in M. scrntl7rw by 0.75 mM CAMP.The observations recorded in Fig. 5 confirm their results. Cyclic AMP at an optimum concentration of 0.7 mM increased FB numbers more than twofold in the absence of PD, but it did riot affect the time at which FB's appeared. In the presence of PD, cAMP had no significant effect. There was no significant effect of AMP either in the presence or absence of PD. Cyclic GMP was antagonistic to the PD effect (Fig. 6); indeed at concentl.ations above 0.3 mM the lag was similar to, and the numbers of fruiting bodies obtained were less even than those in, the controls without cGMP. Even in the absence of PD, cGMP markedly, and at 0.7 mM completely, suppressed FB formation. Similar inhibitory effects of GMPwel-e obsel-ved in the presence andabsence of PD (Fig. 7)' At rnM GMP with PD? FB's were formed later than the PD contr-ol but at about the same numbers. Levels oJcAMP in Glycerol-indrlced Microcysts Morphogenesis in M. xlrt~rhlrs ordinarily involves the convet-sion of cells to microcysts within a fruiting body. However, since it is possible to induce the convel.sion of vegetative cells to microcysts in liquid medium by the addition of 0.5 M glycerol (Dworkin and Gibson 1964) the degt-ee of linkage between cell differentiation and FB formation is yet unclear. Therefore we examined mici-ocysts formed in liquid suspension. TABLE 1. The effect of threonine, isoleucine, and methionine on FB formation Addition, m M
No. of FB's
None Threonine, 1.0 Methionine, 2.0 Isoleucine, 6.0
450 550 1 00 50
NOTE:Each amino acid was added to FM agar to give the final concentrations shown. Samples o f I D h cells In 25 p1 were spotted on the agar plates and the FB's were counted after 7 days as described in Materials and Methods.
-.-
DAYS
FIG.5. The effect of PD and cAMP on FB formation by M.
,trrtlrhir.s. Broken lines were the corresponding controls without
CAMP. -00.3 mM CAMP, -A0.5 mkl CAMP. 0.7 mM CAMP,-0l .O mM CAMP, -0- 0.3 mM CAMP + PD, -A0.5 mM c A M P + PD, -0- 0.7 mM cAMP + PD, -13- 1.0mM cAMP + PD. --- PDonly, +++control.
FIG.6 . The effect of PD and cGMP on FB formation by M. 0.3 m M cGMP + PD, -A-0.5 mM cGMP + PD, -0- 0.7 mM cGMP + PD. -3- 1.O mM cCMP + PD, 0,3M ,, cGMP, -AO ,m j M &Mp, -1- 0.7 m M c G M P .---P D o n l>v .. + + + c o n t l . o l ,
xrrnrllrrs. -0-
1m-
y
8W-
Y
*? 600-
mLL W
g
".
Loo-
LL
p
200-
5 1
2
3
4
5
6
7
8
DAYS
FIG.7. The effect of PD and G M P on FB formation by M . + PD, -0-0.7 mM GMP + PD. -A-0.5mM GMP + PD,-0-0.3mM G M P + ID ' .-.0.7mMGMP, A 0.5 m M G M P , --- P D only; +++ control.
xrrnllr!rs. -0- l .O mM GMP
Yajko and Zusrnan (1978) measured an increase followed by a decrease in cAMP concentrations after 45min of glycerol induction of M. xonthus cells. As shown in Fig. 8 we obtained similar results. The concentrationsof cAMPdetected in cells at various times after glycerol induction compared very well with the previously reported values.
I
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McCURDY ET AL.
1470
TABLE2. Effect of theophylline on microcysts of M. Eflkct o f P D Ilzhibitol..~017 Glycet.ol-it~cilicedMicroxar71llrrs f b" cyst Fot.~nntiotz Because cAMP levels changed during the conHours after resuspension No. of niicrocysts/ml version of vegetative cells to microcysts, it was in fresh medium of medium pertinent to examine whether the addition of PD or 0 1.3x108 PD inhibitors would affect microcyst differentia9 tion. Experiments in which PD was added to the CT 5 . 3 lo2 ~ glycerol-induction system indicate that there was CT + glycerol 7 . 6 lo7 ~ no effect by PD (data not shown). Inhibitors of PD, CT + glycerol + theophylline 3 . 4 lo5 ~ however, prevented the induction of microcysts. 'Six-hour, glycerol-induced microcysts were washed and resuspended in the media ind~cated and their numbers delermined a s As shown in Fig. 9, theophylline prevented the described in Materials and Methods. Final concentrations of glycerol and theophylline were 0.5 a n d 0.01 M respectively. stimulatol-y effect of glycerol on micl-ocyst formation. Similar results were obtained with caffeine Discussion (data not shown). Note that if microcysts were The results reported here strongly suggest a role maintained in the glycerol induction beyond 6 h there was a decline in numbers. To investigate this fol- cyclic nucleotides in the development of M . I I Lwhen I S . applied to cells plated upon FM, observation further, 6-h microcysts were harvested S L ~ ~ ~ ~PD, from the induction medium, washed, and resus- not only accelerated the appearance of FB's but pended in the media indicated in Table 2. In fresh produced larger and unusually well-formed FB's. C T medium there was a rapid convet-sion of micro- These results are strikingly similar to the shortencysts to vegetative cells which was prevented by ing of interphase produced by externally applied glycerol. Theophylline allowed microcyst germi- PD on fruiting in Dictyosteliri~n discoidelitn reported by Alcantal-a and Brazill (1976) and also nation in the presence of glycerol. Wier (1977). These authors argued that in Dictyostdililn, where cAMP is known to function in aggregation, there is an initial excess ofcAMP which, saturating its receptors, prevents the detection of a chemotactic gradient. PD decreases the cAMP concentration to levels which permit detection of the gradient and aggregation follows. A similar phenomenon is suggested to occut- in M. xnt7tlzris where PD not only hastens FB formation but incl.eases territory size or the area from which cells are mobilized to form fruits. However, in M . xnnFIG.8. The levels of cAMP in glycerol-induced rnicrocysts of thlis, the question is whether the cyclic nucleotide M. sctnl11rr.r,fh. involved is cGMP or CAMP. Both Yajko and Zusman (1978) and Parish ct 01. (1976) reported an increase in cAMP concentration when cells of M. xntzth~iswere induced to fruit. Zusman (1978) also detected the presence of an endogenous PD but its specificity was not established. We now show that neither cAMP nor AMP significantly affected the action of externally applied PD. Only the guanine nucleotides influenced the PD effect. Cyclic GMP both delayed the appearance and depressed the numbers of FB's (Fig. 6). GMP also delayed FB appearance although producing an anomalously large number of FB's at the 1 m M concentration (Fig. 7). Bovine heart PD hydrolyzes both cAMPand cGMP (Goren FIG. 9. The effect of theophylline on glycerol-induced miand Rosen 1972). It is worth noting that c ~ ~ ~ d o e s crosysts formation in M. . ~ ~ ~ jb. ~ l -c~ l ~ l0 s. 5 glycerol, ~ -0- 0.j glycerol + lOmM theoDhvlline. -0- l o m ~ not affect cGMP hydrolysis by this preparation but - . .theophylline, -A-control. that cGMP is an inhibitor of CAMP hydrolysis -
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Robert Woodruff Lib on 11/19/14 For personal use only.
C,4N. J . bIICI
AND
W. J. DOBSON
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Robert Woodruff Lib on 11/19/14 For personal use only.
Deptrrrtiretrr of'Biology, U11ir3ersir.vof' Witrtlsor, Witrdsor, O~lt.,Cnt~crtitrN9B 3P4 Accepted August 21, 1978 MCCURDY, H. D., J . Ho, and W. J . DOESON.1978. Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in My.\-ococcrrs xtrtrrlrrrs. Can. J . Microbiol. 24: 1475-1481. Exogenous cyclic nucleotide phosphodiesterase (PD) accelerated fruiting body (FB) formation and increased territory size of aggregates in M.v,\-ococcrr.\ st~t~thlts. Both gunnosine 3'5'monophosphate (cGMP) and gu21nosine 5'-monophosphate (GMP) were antagonistic to the PDeffect. Adenosine 3'5'-monophosphate (CAMP)increases FB numbers twofold in the absence but not in the presence of PD. PD induction is not affected by methionine or isoleucine, which inhibit, or by thl-eonine, which stimulates, FB formation. There is an increase and subsequent decrease in cAMP levels during early glycerol-induced rnicrocyst development but lOmM theophylline or caffeine not only inhibited microcyst development but induced germination in the presence of glycerol. On the basis of these results and the reports ofother investigators n tentative model is proposed bmed on a dual role for cyclic nucleotides in the developnlent in M . a a t ~ ~ h r r s . McCunny, H. D., J . Ho et W. J. DOESON.1968. Cyclic nucleotides, cyclic nucleotide phosphodiestel-ase, and development in My,vococc~rs.rrrtr~l~lr.s. Can. J . Microbiol. 24: 1475-1481. La phosphodiesterase nucleotide cyclique (PD) exogene accelere In fructification (FB) et augmente In dimension des agregitts de Mv,vococ.c~rssirr~fhos. La guanosine 3'5'-monophosphate (cGMP) et la guanosine 5'-monophosphate (GMP) sont antagonistes B I'action du PD. L'edenosine 3'5'-rnonophosphate (CAMP)augmente de deux fois le nombre de FB en I'absence de PD mais non en sa presence. L'induction du PD n'est pas affectee ni par la methionine ou I'isoleucine, lesquels inhibent la formation de FB, ni par la threonine, laquelle stimule la formation du PB. Aux premiers stades de developpement des microcystes lequel est induit par le glycerol, on observe une augmentation suivie d'une baisse dans les taux de CAMP. Cependant, lOmM de theophylline ou de cafeine inhibent le developpement des mycrocyste et induisent la germination en presence de glycerol. S'appuyant surces resultats et ceux d'autres chel-cheurs, on propose un modele sut. le double role que jouent les nucleotides cycliques dans le developpement de M. snt~rlzrrs. [Traduit par lejournal]
Introduction The myxobacteria are gliding prokaryotes which al-e capable of aggregation to form fruiting bodies ( ~ ~ 1966). ~ These ~ korganisms i ~ are thus considel-ed to be idea] pl-okal-yotes for mol-phogenetic and developmental studies (Wireman and Dworkin 1975). F1-uiting body (FB) formation in Myxococc~rs scrt7th~r.swas reported to be stimulated by 3'-5'cyclic adenosine monophosphate (CAMP), 5'adenosine diphosphate (ADP), and other adenine nucleotides (Campos and Zusman 1975). Subsequently, Parish et al. (1976) confirmed the presence of cAMP in vegetative cells of this organism. More recently Yajko and Zusman (1978) reported that during glycerol-induced sporulation, M . xrrnthus exhibited a sharp but transient rise in cAMP concentration. Cyclic AMP phosphodiesterase (PD) activity has also been detected in cell-free lSupported by NRCC Grant No. A 1022.
extracts of M. xcrtzth~rsvegetative cells (Zusman lg78). These I-ePol-ts suggest that there may be a significant involvement of CAMP and PD in morphogenesis in this organism. In the pl-esent work, We describe the effect of exogenous PD On FB formation in M. xcrnrh~rs. Materials and Methods Miooorgnnisms o t ~ d Grow111Cot~ditiotrs In glycerol induction and preliminary fruiting experiments, Myxococcrrsscir~tllrrsfb (M. Dworkin, University of Minnesota) was used. However, this strain yielded variable results in fruiting experiments. Subsequently, a new wild-type strain, M. xatrtklrs M300. was used, which had been isolated from soil samples collected from Point Pelee Provincial Pal-k, Ontario. Isolation and culture techniques used were essentially those of McCurdy (1963). Studies on Frriilit~gBody Fortncrtiotr Fruiting medium (FM) contained the following, per litre of distilled water: K,HP04, 1.4g; KHZP04, 0.35g; casitone (Difco), 0.4g; MgSO,.7H2O, I.Og; agar, 15g; pH 7.3. Three millilitre quantities of autoclaved FM were aseptically dis-
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1476
C A N . J . MICROBIOL. VOL. 24. 1978
pensed onto 50 x 9 mrn Falcon petri dishes (tight lid, type 1006). After the agar plates had solidified the condensate was allowed to dry o f f for 3 to 6 h with the covers pertially opened. When req~~ired the , appropl-iate nucleotide or cyclic n~~cleotide was added after sterilization. Each petri dish was divided into qu;idrants. On two were spotted 25 ~1 o f filte1.-sterilized PD in water ( 5 0 ~ protein, g 0.01 ~ ~ n i tcreating s) circular spots o f about 10-mm diameter. The two remaining quadrants sel-ved as controls end were spotted with 25 FI watel- placebos. Inocul?~ o f 25 PI, each containing about IOb cells, were overlayed on PD 01-placebo spots. The excess liquid was :illowed to evaporate before closing the petri dish and s ~ ~ b s e q ~ incuba~ent tion o f the plates at 22'C. Fr~~itin bodies g were examined with a dissecting microscope and counted with the aid o f n Nikon Profile Projector model 6C with a 10 x objective. Photomicrographs were taken with a Carl Zeis\ (Jena) autorn;rtic exposure device. Mrtr.s~i~.c,ttretrr itf'cAMP i t r Gl!cerol-i~rtlrrcerl Microcy.sts M~sococc.irsxtrtrr1rrr.s fb was grown for 48 h in 800ml C T medium (Dworkin 1962) in each o f five, 2.82 Fernbach flasks, shaken at 60cycles per minute (Northcott Instruments; Guelph. Ont.) (final cell density about IOH per millilitre). After the re~novao l f 200-ml samples from each flask (zero time), sterile glyce1.01was added to the remainder in the flasks to give a final concentration o f 0.5M glycerol in each (Dworkin and Sadler 1966). Subsequently, at the times indicated (Fig. 8) 200-ml a l i q ~ ~ o twere s withdrawn from different replicate flasks, and centrifi~gedat 8000g for 20min at 4°C. The pellets were then washed twice with equal volumes o f sterile distilled water. The washed pellets were resuspended in l 0 m l o f assay medium containing 8 m M theophylline. 6 m M 2-mercaptoethanol, and 5 0 m M Tris buffer at p H 7.5. The zero-time sample containing vegetative cells was sonicated for 5 min at full power (Biosonik, Brownwell Scientific. Rochester, N Y ) . Mic~.ocystswere dis~.uptedby omni-mixing (Servall Inc., Norwalk, CT) for7 min, at a rheostat setting o f 90, with an equal weight o f glass beads ( - 100 120. Flex-o-iite. St. Thomkrs, Ont.). This treatment gave 80-85% breakage. Cellular debris was [removed by centrifugation at 35 000g fol-20min. The supernatant fractions were deproteinized with 1.0N HCIO, and centrifuged at I0000g to remove the precipitate and neutlxlized with tric;~prylyl ;mine dissolved in chloroform (Warner and Finamore 1967). Cyclic A M P was assayed by the competitive binding-protein technique o f Brown el (11.(1971). Labelled C A M Pwas counted in a scintillation fluid consisting o f 10% (wlv) napthalene with 0.05% (wlv) PPO in 1,Cdioxane as specified by B D H Chemical Co. (publication no. 883Dl1.011072). Radioactivity was counted with 21 Nucle:~r Chicago Mark 11 liquid scintillation counter (Nuclear Chicago. Des Plaines, I L ) .
+
Detc,rttritrtrriotr of A4icrocyst N ~ o ~ r b c i;fier ~ r s T l ~ i ~ o p l ~ y l lon(/ ir~e Cr;fi,itre Tretrtttrrtlt T o study the effect o f theophylline and caffeine on glycerol induction o f microcysts. 7.5 ml o f 45 m M o f the appropriate inhibitors were added to 25 ml o f induction medium in a 125-ml Erlenmeyer flask (see above). At times indicated in Fig. 9, the number o f microcysts in 3-mI samples was detel-mined by plating out the sonication-resisti~nt fractions o n C T agar plates as described by Sudo and Dworkin (1969). Proteit~Deterttrit~trtiot~ Protein was determined according to L o w r y el (11. (1951). Bovine serum albumin was the standard.
Clrettrictrls The following compounds wel-e obtained from Sigma Chemical Co.. St. Louis, M O : 3'15'-cyclic nucleotide phosphodiestel-ase: 3':5'-CAMP; adenosine5'-monophosphoric acid. sodium salt (AMP); gu;rnosine 3'5'-cyclic monophosphoric acid, sodium salt (cGMP); gu:inosine 5'-monophosphoric acid, sodium salt (GMP); 2-mercaptoethanol; sodium azide; theophylline: caffeine; and tris(hyd~.oxymethyl);uninometh;tne. The radiochemical 'H-3':5'-CAMP (sp. act. 28 CilmM) ( I C i = 37GBq) was from Picker Nucleiir Cnnad;~,Montreal. P.Q. The 3':j'-CAMP binding protein (from bovine adrenil1 cortex) and Norit GSX were from B D H Chemiciils, Toronto. Ont. Tricaprylyl amine (Alamine 3363) was from General Mills Inc., Knnkakee. IL and 2.5-diphenyloxazole (PPO), napthalene, 1,4dioxane were fl-om Fisher Scientific Co., Don Mills. Ont.
Results
The Eflecf q f ' C y c l i c N ~ r c l e o t i c l eP h o ~ p l z o d i e s t e ~ ~ c r s e Preliminary experiments in our laboratory indicated that a commercial prepal-ation of PD induced FB formation by several my xobacteria including C y s t o h c r c t e ~ . firsc~rs, M y x - o c o c c ~ r s c o r c r l l o i d e s , Polyrrt7gir1tn c e l l r r l o s r r t ~ ,M y x o c o c c r r s s r r t ~ f / ~ r r sand , Stigt?lctte/lrr c r r ~ t . a n f i c r c ~ t .
Figure 1 illustrates the results obtained when M. on FM was treated with PD. It had previously been established that lo6-10' cells gave optimal results. Compared with the contl-01s without PD, the treatment resulted in (a) a very much earlier appearance of FB's; (12) a fourfold increase in numbel- for FB's obtained within 6 days, although the slow development of FB's on control plates ultimately resulted in a high final number of pool-ly formed FB's; and (c) FB's which were larger and more completely developed (Fig. 2). T o obtain I-eproducible numbers of FB's, it was important to use cultures that were not subjected t o more than three serial transfers in liquid culture although experiments utilizing cells subjected to up to two additional transfers (Figs. 3 and 4), yielded essentially the same relationships among the various experimental treatments. Attempts to use
s c r n t l z ~ r sinoculated
DAYS
FIG. I . The effect o f P D o n F B formation by M.xtrt~t1~1rs. Bars represent standard deviation o f eight replicates. -0PD, -0control, -0placebo.
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F I G . 2. PD-induced fruiting bodies. A. Natural FB's 10 days after inoculation. B. PD-induced FR's 10 days after with reflected light. inoculation. Squ;~t-esIrepresent 1 mm'. The samples were pho~ogr;~phed 400-
n
W LL
0 LL
n 100
.m
mmd
.m
-m
.Theophylbne
.m
-m
.Caffe~ne
F I G .3. The effect of theophylline end caffeine on FB formaInhibitor concentrations were 10mM. tion by M. .rcit~rlrr,~.
standa~d-sizepetri dishes led to excessive variation in FB formation. Because the commercial PD enzyme preparation used contained certain contaminating enzymes, each was tested for its effect on fruiting. These included 5'-nucleotidase. 5'-ATPase, inorganic pyrophosphatase, alkaline phosphatase. 2',3'cyclic nucleotide phosphodiesterase, and protein kinase. All were without effect. As a further confirmation that the obsel-vation depended upon PD, theophylline and caffeine, both inhibito~sof PD, were tested. Both counteracted the effect of PD a s well as delaying the appearance of FB's in the absence of PD (Fig. 3).
0
Dl 1 lo 100 PHOSPHATE CONCN mM
FIG.4. The effect of phosphate concentration on FB fol-mation by M . sotltll~rs.The number of FB's were recorded on the 6th day after inoculation. -0- PD, -0- control.
might have caused its production and influenced the results. Furthermore, Wireman and Dworkin (1975) stated that the need for high cell densities for FB formation could be lowered by the addition of phosphate to unsupplemented agar medium. However, no data were presented to substantiate this significant observation that suggests orthophosphate is a cell-density signal. As shown in Fig. 4, a definite optimum exists between 10 and 30 mM both in the presence and absence o f PD.
Efffct of Tlzreotzine, Isolerrcine, crnrl Methionine Rosenberg et NI.(1973) reported that methionine and isoleucine inhibit, while threonine stimulates, Effect of Phosphcrte of FB Fo1.m~rtior1 F B formation in M . xrrnthus. When these amino The effect of orthophosphate on fruiting was also acids were tested at the effective concentrations tested since contaminating enzymes in the PD (Campos and Zusman 1975) in the presence and
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absence of PD, none was found to alter significantly the stimulatory effect of PD. The FB's were essentially the same size and morphology and the kinetics of production essentially the same. In the absence of PD, however, our results duplicated those previously I-epurted, i.e. methionine and isoleucine depressed the numbers of fruits by about 80%; threonine slightly increased the numbers (Table 1). T11eEJrect of Nrlcleotides on PD Indlrction Campos and Zusman (1975) reported the stimulation of FB formation in M. scrntl7rw by 0.75 mM CAMP.The observations recorded in Fig. 5 confirm their results. Cyclic AMP at an optimum concentration of 0.7 mM increased FB numbers more than twofold in the absence of PD, but it did riot affect the time at which FB's appeared. In the presence of PD, cAMP had no significant effect. There was no significant effect of AMP either in the presence or absence of PD. Cyclic GMP was antagonistic to the PD effect (Fig. 6); indeed at concentl.ations above 0.3 mM the lag was similar to, and the numbers of fruiting bodies obtained were less even than those in, the controls without cGMP. Even in the absence of PD, cGMP markedly, and at 0.7 mM completely, suppressed FB formation. Similar inhibitory effects of GMPwel-e obsel-ved in the presence andabsence of PD (Fig. 7)' At rnM GMP with PD? FB's were formed later than the PD contr-ol but at about the same numbers. Levels oJcAMP in Glycerol-indrlced Microcysts Morphogenesis in M. xlrt~rhlrs ordinarily involves the convet-sion of cells to microcysts within a fruiting body. However, since it is possible to induce the convel.sion of vegetative cells to microcysts in liquid medium by the addition of 0.5 M glycerol (Dworkin and Gibson 1964) the degt-ee of linkage between cell differentiation and FB formation is yet unclear. Therefore we examined mici-ocysts formed in liquid suspension. TABLE 1. The effect of threonine, isoleucine, and methionine on FB formation Addition, m M
No. of FB's
None Threonine, 1.0 Methionine, 2.0 Isoleucine, 6.0
450 550 1 00 50
NOTE:Each amino acid was added to FM agar to give the final concentrations shown. Samples o f I D h cells In 25 p1 were spotted on the agar plates and the FB's were counted after 7 days as described in Materials and Methods.
-.-
DAYS
FIG.5. The effect of PD and cAMP on FB formation by M.
,trrtlrhir.s. Broken lines were the corresponding controls without
CAMP. -00.3 mM CAMP, -A0.5 mkl CAMP. 0.7 mM CAMP,-0l .O mM CAMP, -0- 0.3 mM CAMP + PD, -A0.5 mM c A M P + PD, -0- 0.7 mM cAMP + PD, -13- 1.0mM cAMP + PD. --- PDonly, +++control.
FIG.6 . The effect of PD and cGMP on FB formation by M. 0.3 m M cGMP + PD, -A-0.5 mM cGMP + PD, -0- 0.7 mM cGMP + PD. -3- 1.O mM cCMP + PD, 0,3M ,, cGMP, -AO ,m j M &Mp, -1- 0.7 m M c G M P .---P D o n l>v .. + + + c o n t l . o l ,
xrrnrllrrs. -0-
1m-
y
8W-
Y
*? 600-
mLL W
g
".
Loo-
LL
p
200-
5 1
2
3
4
5
6
7
8
DAYS
FIG.7. The effect of PD and G M P on FB formation by M . + PD, -0-0.7 mM GMP + PD. -A-0.5mM GMP + PD,-0-0.3mM G M P + ID ' .-.0.7mMGMP, A 0.5 m M G M P , --- P D only; +++ control.
xrrnllr!rs. -0- l .O mM GMP
Yajko and Zusrnan (1978) measured an increase followed by a decrease in cAMP concentrations after 45min of glycerol induction of M. xonthus cells. As shown in Fig. 8 we obtained similar results. The concentrationsof cAMPdetected in cells at various times after glycerol induction compared very well with the previously reported values.
I
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McCURDY ET AL.
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TABLE2. Effect of theophylline on microcysts of M. Eflkct o f P D Ilzhibitol..~017 Glycet.ol-it~cilicedMicroxar71llrrs f b" cyst Fot.~nntiotz Because cAMP levels changed during the conHours after resuspension No. of niicrocysts/ml version of vegetative cells to microcysts, it was in fresh medium of medium pertinent to examine whether the addition of PD or 0 1.3x108 PD inhibitors would affect microcyst differentia9 tion. Experiments in which PD was added to the CT 5 . 3 lo2 ~ glycerol-induction system indicate that there was CT + glycerol 7 . 6 lo7 ~ no effect by PD (data not shown). Inhibitors of PD, CT + glycerol + theophylline 3 . 4 lo5 ~ however, prevented the induction of microcysts. 'Six-hour, glycerol-induced microcysts were washed and resuspended in the media ind~cated and their numbers delermined a s As shown in Fig. 9, theophylline prevented the described in Materials and Methods. Final concentrations of glycerol and theophylline were 0.5 a n d 0.01 M respectively. stimulatol-y effect of glycerol on micl-ocyst formation. Similar results were obtained with caffeine Discussion (data not shown). Note that if microcysts were The results reported here strongly suggest a role maintained in the glycerol induction beyond 6 h there was a decline in numbers. To investigate this fol- cyclic nucleotides in the development of M . I I Lwhen I S . applied to cells plated upon FM, observation further, 6-h microcysts were harvested S L ~ ~ ~ ~PD, from the induction medium, washed, and resus- not only accelerated the appearance of FB's but pended in the media indicated in Table 2. In fresh produced larger and unusually well-formed FB's. C T medium there was a rapid convet-sion of micro- These results are strikingly similar to the shortencysts to vegetative cells which was prevented by ing of interphase produced by externally applied glycerol. Theophylline allowed microcyst germi- PD on fruiting in Dictyosteliri~n discoidelitn reported by Alcantal-a and Brazill (1976) and also nation in the presence of glycerol. Wier (1977). These authors argued that in Dictyostdililn, where cAMP is known to function in aggregation, there is an initial excess ofcAMP which, saturating its receptors, prevents the detection of a chemotactic gradient. PD decreases the cAMP concentration to levels which permit detection of the gradient and aggregation follows. A similar phenomenon is suggested to occut- in M. xnt7tlzris where PD not only hastens FB formation but incl.eases territory size or the area from which cells are mobilized to form fruits. However, in M . xnnFIG.8. The levels of cAMP in glycerol-induced rnicrocysts of thlis, the question is whether the cyclic nucleotide M. sctnl11rr.r,fh. involved is cGMP or CAMP. Both Yajko and Zusman (1978) and Parish ct 01. (1976) reported an increase in cAMP concentration when cells of M. xntzth~iswere induced to fruit. Zusman (1978) also detected the presence of an endogenous PD but its specificity was not established. We now show that neither cAMP nor AMP significantly affected the action of externally applied PD. Only the guanine nucleotides influenced the PD effect. Cyclic GMP both delayed the appearance and depressed the numbers of FB's (Fig. 6). GMP also delayed FB appearance although producing an anomalously large number of FB's at the 1 m M concentration (Fig. 7). Bovine heart PD hydrolyzes both cAMPand cGMP (Goren FIG. 9. The effect of theophylline on glycerol-induced miand Rosen 1972). It is worth noting that c ~ ~ ~ d o e s crosysts formation in M. . ~ ~ ~ jb. ~ l -c~ l ~ l0 s. 5 glycerol, ~ -0- 0.j glycerol + lOmM theoDhvlline. -0- l o m ~ not affect cGMP hydrolysis by this preparation but - . .theophylline, -A-control. that cGMP is an inhibitor of CAMP hydrolysis -
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C,4N. J . bIICI
