JouRNAL OF BACTRIOLOGY, Mar. 1976, p. 1226-1228 Vol. 125, No. 3 Copyright X 1976 American Society for Microbiology Printed in USA. Cyclic Guanosine 3',5'-Monophosphate in the Dimorphic Fungus Mucor racemosus MICHAEL ORLOWSKI AND PAUL S. SYPHERD* Department ofMedical Microbiology, California College of Medicine, University of California, Irvine, California 92664 Received for publication 20 November 1975 The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3',5'-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3',5'-monophosphate has upon cell morphology. The cyclic nucleotide adenosine 3',5'-mono- N HCl, and quickly frozen in an ethanol-dry ice phosphate (cAMP) has been reported to effect slurry. After the cells were frozen and thawed the morphogenesis of many microorganisms, three times, they were suspended with a vortex including fungi (9, 10, 14, 15, 21, 22), proto- mixer. The membrane filters were removed, zoans (4), and bacteria (3, 17). High levels of and the cells were sedimented by centrifuga- cAMP were found in yeastlike cells of the di- tion. The supernatant fluid was assayed for morphic fungus Mucor racemosus, and low lev- cAMP and cGMP using radioimmunoassay kits els were found in the mycelial form (10). Fur- supplied by Schwarz/Mann (Orangeburg, thermore, the addition of exogenous dibutyryl N.Y.). The procedure was essentially that of cAMP influenced the cells to grow in a yeast- Steiner et al. (18-20), with the modification of like form under conditions otherwise conducive Weinryb et al. (24) used to collect the nucleotide to mycelial growth (10). antibody complex. The cGMP antibody showed Cyclic guanosine 3',5'-monophosphate no cross-reactivity with guanosine 5'-triphos- (cGMP) is the only other cyclic nucleotide thus phate or cAMP at levels obtained from the ex- far found in living cells (8). It has been proposed tracted cells. Complete destruction of all mate- that cGMP acts in an opposing direction to the rial reacting with the cGMP antibody occurred regulatory effects of cAMP (5, 6). Recent evi- during incubation of extracts with cyclic nucle- dence has been accumulated to support this otide phosphodiesterase under the conditions notion (1, 2, 12, 13, 16, 23). The present work described by Larsen and Sypherd (10). The as- establishes the presence of cGMP in cells ofM. say gave a linear response to increasing racemosus and tests the hypothesis that cGMP amounts of purified cGMP and material from acts in opposition to the effects of cAMP on the extracted cells. The cAMP assay was validated morphogenesis of the organism. earlier (11). Cellular protein was measured by a M. racemosus 1216B was used throughout procedure described previously (11). these experiments. The organism was grown in Essentially two kinds of experiments were a complex medium (YPG) described previously performed: (i) determination of the effects of (10) or in a defined minimal medium (DM; pH added exogenous cGMP upon cellular morphol- 4.5) consisting of glucose (20 g/liter), alanine ogy and (ii) measurement of intracellular cyclic (1.5 g/liter), sodium glutamate (1.5 g/liter), nucleotide concentrations. Added exogenous (NH 4) 2S04 (1.0 g/liter), and yeast nitrogen base cGMP had no apparent effect on Mucor di- (Difco) (0.5 g/liter). Inocula and growth condi- morphism. The substance neither antagonized tions were described previously (10). The mor- or mimicked the effects of cAMP on cellular phogenetic change from yeast to mycelia was morphology. The compound could not induce effected by shifting the atmosphere from CO 2 to CO2-grown cells to form mycelia or cells placed air (11). Cells were rapidly collected from cul- in air to grow as yeast. Cells responded to tures on membrane filters (Millipore Corp.; added cAMP in the presence of cGMP in a pore size, 0.45 ,um), immediately placed into 0.1 normal fashion, i.e., by growing as yeasts (Ta- 1226 VOL. 125, 1976 NOTES 1227 ble 1). Both the dibutyrated and free-acid forms ~ ~ ~ - cotn ofcGMP were tested over a 0.3 to 3 mM concen- 0 0 0 100 tration range in YPG and DM media with iden- a 0 AIR A so SHIFT CO0 tO AIR tical results. At present we have no evidence 60 that either form of the nucleotide penetrates , 2!O 0 -l I~- ~~~~~~~~~co, the cells. 0 40 -I10 cGMP has been detected in all mammalian 20 F 49 tissues examined and in many other phyla (7), i but has not been studied previously in any of 0 > J C~~~~~~~~~~Ocos I.- the phycomyces, including Mucor. Assay for A- 100 Z intracellular cGMP showed that the nucleotide o -A was present in all morphological forms of Mu- 0 AIR B a cor examined. No correlation could be made 0 60 2 4- SHIFT CO, TO AIR between cGMP levels and yeast-mycelial di- A. 40 morphism. The intracellular levels of cGMP 2 changed very little after a C02-to-air shift (Fig. 20 1). A transient increase in the amount of intra- ~~S~~==~~~A IR O cellular cGMP was observed during the germi- - -4 -2 0 2 4 6 6 0 nation ofsporangiospores in both YPG and DM TIME RELATIVE TO SHIFT (hrs) The mimics that re- FIG. 1. Intracellular concentration of cGMP dur- media (Fig. 2). pattern ing the morphogenetic conversion ofyeastlike cells to ported for intracellular cAMP during germina- mycelia in M. racemosus. The cells were grown under tion of Mucor sporangiospores (11) and may a CO2 atmosphere until the early exponential phase possibly reflect some regulatory phenomenon ofgrowth. The culture was then divided in half, and involved in morphogenetic processes occurring one part was shifted to an air atmosphere. (A) YPG during spore germination. The levels to which medium; (B) DM medium. Symbols: O, percentage of cGMP concentrations returned after germina- yeastlike cells; *, intracellular cGMP concentration. tion were roughly similar for all growing forms examined (Fig. 2). cAMP:cGMP ratios were cal- 0.4 culated in all of the above experiments but 7 10 indicated no meaningful pattern and are not 6 Q3 shown. On the basis ofthe data gathered in this 510 study, the hypothesis that cGMP acts in an 410 A 0.2 Z 3so hi. TABLE 1. Effect of exogenous cGMP on the cellular 2to 0.1 0 morphology of MucorP id I_ o -.3~~~~~~~~~~~~~.0I- Culture conditions Cell morphology E S Nlp E' CO2 Yeast a 10 1.0 0 5 CO2 + cGMP Yeast 0 410 B 0.6 z Air Mycelia s0 0.6 Air + cAMP Yeast hi2 m O02 2t0 0.4 0 Air + cGMP Mycelia It 0 110- 0.2 Air + cAMP + cGMP Yeast U) A.I a Cultures were grown under a CO2 atmosphere (yeastlike morphology) on YPG or DM until the 4to0 0.68 early exponential phase of growth. The cultures 10 0.6 were then divided into several parts, which were 2 I ~~~~~~~~~~~~~~0.4 varied in terms of atmospheric composition and cyclic nucleotide additions. The general culture con- 0- 0.2 ditions are summarized in the table. cAMP was -4 6 12 M 20 24 26 32 36 40 44 46 52 56 60 always in the dibutyrated form and used at a 3 mM TIME (hrs) concentration. cGMP was tested in the concentra- FIG. 2. Intracellular concentration of cGMP dur- tion range of0.3 to 3 mM in both the dibutyrated and ing germination of sporangiospores and vegetative free-acid forms. The cultures were incubated for an growth ofyeastlike and mycelial forms ofM. racemo- additional 24-h period, after which cell morphology sus. (A) Germination and (yeastlike) growth under was examined with a phase-contrast microscope. CO2 in YPG medium; (B) germination and (myce- The results listed were obtained in both growth lial) growth under air in YPG medium; (C) germina- media, with both the dibutyrated and free-acid tion and (mycelial) growth under air in DM medium. forms of cGMP, and at all concentrations of cGMP Symbols: 0, growth as cellular protein per culture used. volume; *, intracellular cGMP concentration. 1228 NOTES J. BACTERIOL. antithetical fashion with cAMP to control the capacity, 3',5'-cyclic adenosine monophosphate, and cellular morphology ofthe dimorphic fungus M. morphogenesis of Mucor racemosus. J. Bacteriol. 124:134-139. racemosus cannot be supported. 12. Perlman, R., B. Chen, B. de Crombrugghe, M. Emmer, M. Gottsman, H. Varmus, and I. Pastan. 1970. The This work was supported by grant GB25529 from the regulation oflac operon transcription by cyclic adeno- National Science Foundation and by Public Health Service sine 3':5'-monophosphate. Cold Spring Harbor Symp. grant GM18293 from the National Institute of General Med- Quant. Biol. 35:419-423. ical Sciences. 13. Rudland, P. S., M. Seeley, and W. Seifert. 1974. Cyclic GMP and cyclic AMP levels in normal and trans- LITERATURE CITED formed fibroblasts. Nature (London) 251:417-419. 1. Anderson, W. B., R. L. Perlman, and I. Pastan. 1972. 14. Schwalb, M. N. 1974. Effect of adenosine 3':5'-cyclic Effect ofadenosine 3':5'-monophosphate analogues on monophosphate on the morphogenesis of fruit bodies the activity of the cyclic adenosine 3':5'-monophos- of Schizophyllum commune. Arch. Microbiol. 96:17- phate receptor in Escherichia coli. J. Biol. Chem. 20. 247:2717-2722. 15. Scott, W. A., and B. Solomon. 1975. Adenosine 3',5'- 2.