CELL DEATH DURING FRUITING BODY FORMATION IN MYXococcus

E Gesellschaft für Biotechnologische Forschung mbH Braunschweig-Stöckheim ii j fe Aa ee Le 4 IT Abstracts of Lectures and Results Bibliothek of Technical Discussion of the Symposium August 1 to 5, 1975, Braunschweig-Stöckheim 2nd International Symposium on the Biology of Myxobacteria 1975 Schriftenreihe der GBF Nr. 2 (Dezember 1975) A first international symposium on " The Developmental Biology of Myxobacteria" took place August 16 to 20, 1974, at Cold Spring Harbour, New York, USA oes A SS Bibliothek “UB Ke Data from the abstracts must not be cited as published material, but should be treated as personal communications. - This booklet is distributed by: Gesellschaft fiir Biotechnologische Forschung mbH Mascheroder Weg 1 D-3300 Braunschweig-Stöckheim Federal Republic of Germany III I: NSTIRO DIUFCHT ION The participants of the very stimulating first symposium on myxobacteria last year in Cold Spring Harbour pronounced unaminously the opinion that repeated meetings of this kind would be of great help for their research. Therefore it was decided to have a second symposium on myxobacteria in 1975 at a place in Germany. This place later turned out to be Braunschweig. The Gesellschaft fiir Biotechnologische Forschung, one of the Research Centres of the Federal Government of Germany, was kind enough to be host to the symposium. Fortunately again many of the investigators working in the field of myxobacteria were able to assemble. The many fascinating discussions which developed in the course of the meeting fully justified all efforts made in arranging this symposium and encouraged the participants to continue with this kind of communication. The booklet presented here gives the summaries of all the lectures and a record of the technical discussions held at the end of the meeting. It is intended to pass the information to all those colleagues who were kept from participating personally in the symposium. Hans Reichenbach Braunschweig-Stöckheim December 1975 Fruiting body of Chondromyces apiculatus Thaxter 1897. The whole structure is about 400 pm high. 900 a PROGRAM Lecture Room of the G BF: Opening of the symposium J. Wireman, M. Dworkin? Minneapolis, Minnesota, USA: Cell death during fruiting body formation in Myxococcus Pecks Grilione* J. Pangborn, San José and Davis, California,USA: Scanning electron microscopy of fruiting body formation by Stigmatella aurantiaca and Chondromyces crocatus H. Reichenbach, Braunschweig, Federal Republic of Germany: Control of fruiting body differentiation in Chondromyces apiculatus by environmental factors Visit to the Fermentation Center of the G BF, guided by M. Reuß Lecture Room of the GBF: I. W. Sutherland, Edinburgh, Great Britain: Myxobacterial polysaccharides D. White, Bloomington, Indiana, USA: Coats from Myxococcus xanthus: characterization and synthesis during myxospore differentiation De Filer* E. Rosenberg, S.H. Kindler, Tel-Aviv, Israel: Biosynthesis of the capsular material of Myxococcus xanthus * indicates speaker 1: See ee Lecture Room of the G B F: K; Gerth? H. Reichenbach, Freiburg, Federal Republic of Germany: Myxospore induction in Stigmatella aurantiaca A. Kimchi? E. Rosenberg, Tel-Aviv, Israel: A correlation between initiation of DNA synthesis and cell division in Myxococcus xanthus R.P. Burchard? J.A. Kloetzel, Catonsville, Maryland, USA: Studies on fibrils produced by Myxococcus xanthus Lecture Room of the GB F: S.A. Dhalat P.K.R. Nair, Andheri, Bombay, India: Myxobacterial interaction with rhizobia Rees Burchard? A.C. Burchard, J.H. Parish, Catonsville, Maryland, USA and Leeds, Great Britain: Pigmentation phenotype instability in Myxococcus xanthus H.J. Hirsch, Braunschweig, Federal Republic of Germany: Bacteriocins in Myxococcus fulvus Symposium's excursion to Goslar, visit of the city and lunch in the Ratskeller. Address by H. Zeitträger, administrative director of the GBF. In the Afternoon excursion into the Harz mountains, walk along the Soese reservoir. Bible Room of the Herzog August Biliothek (library) in Wolfenbiittel: Discussion of general and technical problems connected with research on gliding bacteria. Guided tour through the treasure rooms of old books of the library. VII LIST OF PARTICIPANTS W. Bredt Institut fiir Medizin. Mikrobiologie der Universitat Mainz D-6500 Mainz Federal Republic of Germany Robert P. Burchard Biological Division University of Maryland Baltimore County Baltimore, MD 21228, US A Gavin A. Clark University of Toronto School of Hygiene, Dept. of Microbiology Toronto, Ontario M5S 1Al, Canada S.A. Dhala Bhavan's College, Dept. of Microbiology Andheri, Bombay-58, India Martin Dworkin University of Minnesota Medical School, Dept. of Microbiology Minneapolis, Minn. 55455, US A Erich Fautz Gesellschaft fiir Biotechnologische Forschung mbH Abteilung Mikrobiologie D-3300 Braunschweig-Stöckheim Federal Republic of Germany David Filer Tel-Aviv University Faculty of Life Sciences Dept. of Microbiology Tel-Aviv, Israel Klaus Gerth Institut für Biologie II der Universität Freiburg Lehrstuhl für Mikrobiologie D-7800 Freibur Federal rae of Germany Patricia L. Grilione San José State Universtiy, School of Science, Dept. of Biological Sciences San José, California 95192, US A Klaus Grimm Botanisches Institut der Universität Karlsruhe, Lehrstuhl I D-7500 Karlsruhe Federal Republic of Germany Heinz Josef Hirsch Gesellschaft für Biotechnologische Forschung mbH Abteilung Mikrobiologie D-3300 Braunschweig-Stöckheim Federal REpublic of Germany Ady Kimchi K.A. Malik Jose Arias Penalver Wolfgang Reichert Hans Reichenbach Carmen Rodriguez-Franco Eugene Rosenberg J. L. Stokes Ian W. Sutherland David White Tel-Aviv University Faculty of Life Sciences Dept. of Microbiology Tel-Aviv, Israel Institut fiir Mikrobiologie der Universtiät Göttingen D-3400 Göttingen-Weende Federal Republic of Germany Universidad de Granada Facultad de Ciencias Departamento de Microbiologia Granada, Spain Limnologisches Institut der Universität Freiburg D-7750 Konstanz Federal Republic of Germany Gesellschaft für Biotechnologische Forschung mbH Abteilung Mikrobiologie D-3300 Braunschweig-Stöckheim Federal Republic of Germany Universidad de Granada Facultad de Ciencias Departamento de Microbiologia Granada, Spain Tel-Aviv University Faculty of Life Sciences Dept. of Microbiology Tel-Aviv, Israel Washington State University Bacteriology and Public Health Pullman, Washington 99163, US A University of Edinburgh School of Agriculture, Dept. of Microbiology Edinburgh EH9 EJK Great Britain Indiana University, Dept. of Microbiology Bloomington, Indiana 47401, USA CELL DEATH DURING FRUITING BODY FORMATION IN MYXococcus J. WIREMAN and M. DWORKIN Department of Microbiology,University of Minnesota Minneapolis, Minnesota, USA Massive cell death occurs during fruiting construction by several species of Myxococcus . The events which occur lead us to the hypothesis that regulated senescence and cell death are integral part of myxobacterial development. During fruiting body formation by M. xanthus 60 - 8) % of the vegetative cells lyse. The majority of the survivina cells are eventually converted to myxospores in the fruiting body. This lysis has been measured both by the loss of 3y-methy1 thymidine label from DHA of the cells and by actual cell counts. Such lysis occurs under a variety of conditions leading to fruiting body formation. We have also demonstrated lysis during fruiting body formation in Myxococcus fulvus and Myxococcus virescens. If cells are removed at various times during fruiting body formation and replaced in a liquid growth medium the tendency to lyse is reversible until the fruiting bodies have formed; at that time the vegetative cells become irreversibly committed to lysis. We suggest that lysis in the organisms we have examined is a functional and necessary part of the developmental cycle. It is possible that the lysing cells are providing a source of biosynthetic precursors and/or a source of energy for the formation of myxospores and/or fruitina bodies. MYXOSPORE INDUCTION IN STIGMATELLA AURANTIACA K. GERTH and H. REICHENBACH Institut fuer Biologie II der Universitaet, Lehrstuhl fuer Mikrobiologie D-7800 Freiburg, Federal Republic of Germany The existence of different types of induction-resistant mutants allows one to classify all known inducers into one of 3 groups; 1) the glycerol group, 2) the phenethyl alcohol group, and 3) the t-butanol group. This pattern, and the occurrence of specific antagonists which competitively inhibit inducers of the glycerol group (e.g. oxindole, pyrrole), suqgest that the inducers work by interaction with group-specific receptors on the bacterial cell: receptor I for the glycerol aroup, receptor II for the phenethyl alcohol group, receptor III for the t-butanol group of inducers. There are only few inducers which may act on more than one receptor; as isopropanol (receptors I+III), or indole (receptors I+II). In order to become effective as an inducer, a compound needs an attachment site which is responsible for its affinity to the receptor, and a reaction site which determines the intrinsic activity of the inducer. The reaction site of inducers of the phenethyl alcohol group may be the aromatic ring, that of the glycerol group a positive charge or dipole moment, for those are the only functional groups common to all members of the respective groups. The conversion of t-butanol, which is a competitive inhibitor of glycerol induction, into t-butylamine, which induces at the glycerol receptor, seems to demonstrate that the amino group is the reaction site of the molecule. Indeed, introduction of different group-specific reaction sites into a certain molecule changes the receptor specificity of the latter: t-butanol - receptors (I+) III; phenylpropanol - receptor II;t-butylamine - receptors I. The dose-response curves of myxospore induction are sigmoidal. This indicates that induction is an all-or-nothing reaction. Inducers of different groups if applied at concentrations which lie below the lowest inducing concentration of each of them, add their individual effects. This suggests that 1) the stimulus for myxospore induction is the same with all types of inducers, and that 2) the stimulus has to reach a threshold value before sporulation can occur. It was possible to calculate the interaction of 2 different inducers, or of an inducer and an inhibitor. The exact agreement between predicted and experimentally determined effect indicates that our hypothesis and of inducer-receptor-stimulus may come close to reality. A CORRELATION BETWEEN INITIATION OF DNA SYNTHESIS AND CELL DIVISION IN MYXOCOCCUS XANTHUS A. KIMCHI and E. ROSENBERG Department of Microbiology, Tel Aviv University Tel Aviv, Israel Chromosome completion is a necessary condition for cell division in Myxococcus xanthus as in many other bacteria. By taking advantage of the unioue life cycle of M. xanthus we have been able to obtain evidence that an additional and earlier linkage exists between DNA replication and cell division in M. xanthus FBmpT . In the presence of nalidixic acid (NAL) glycerol induced myxospores germinate but do not divide (snake formation). In contrast , vegetative cells treated with chloramphenicol (CAP) undergo one subsequent cycle of division in the presence of NAL. This difference is surprising because during both treatments (alycerol and CAP) similar kinetics of cell division and DNA synthesis were obtained (immediate cessation of cell division;about 40 % increase in DNA content). One possible explanation for the failure of germinating myxospores to divide in the presence of NAL is that durina myxospore formation chromosomes are blocked just prior to completion. If this were the case, then cells in which chromosomes were allowed to complete in the presence of CAP and subsequently induced to form myxospores should divide in the presence of NAL. However, such germinating myxospores with completed chromosome do not divide in the presence of NAL. Kinetics of DNA synthesis during myxospores formation following release from amino-acid starvation showed that glycerol induction involves an immediate cessation of new cycles of DNA replication. During germination DNA synthesis started synchronous ly between 3 - 5 hrs, whereas the first round of synchronous cell division appeared between 4 - 6 hrs indicating that the first division follows a short interval of DNA synthesis. Treatment of germinating myxospores with NAL at different time intervals indicated that the critical time period in which DNA synthesis is necessary for the first division beqan at 3.5 hrs. This time period (3.5-5 hrs) was shown to involve only new initiations of DNA replication by the following 10 experiment: Myxospores induced by glycerol in the presence of NAL completed their chromosomes during the first 4 hr of germination. Hence, the potential for DNA elongation is present much earlier than the critical time period mentioned above. It is proposed that the first division following germination depends on DNA initiation. This requirement is not the synthesis of DNA per se,but involves the synthesis of specific RNA and protein triggered by DNA initiation: when germinating myxospores were released from inhibition of DNA synthesis by removal of NAL at 6 hrs, they failed to divide in the presence of CAP or rifampicin. These data support the hypothesis that during normal growth a component necessary for cell division is synthesized during initiation of DNA replication. This hypothetical component is necessary for subsequent cell division. During myxospore formation this component is destroyed. 11 STUDIES ON FIBRILS PRODUCED BY MYXOCOCCUS XANTHUS R. P. BURCHARD and J. A. KLOETZEL Department of Biological Sciences, University of Maryland Baltimore County Catonsville, Maryland/USA 4 - 5 rm diameter fibrils were originally observed in 2% phosphotungstate-negatively stained, 10* x g supernatant fractions of French Pressure Cell-disrupted M. xanthus FB vegetative cells. The fibrils are also present in the periplasmic fraction prepared by osmotic shock; treatment of cells with pronase (100 jig/ml; 30 min.) prior to osmotic shock increases the number of fibrils observed. They are also present in 10° Xie pellets of culture medium in which logarithmic phase cells had been growing. Aggregates of fibrils are observed in association with gliding cells on filmed electron microscope grids which have been pressed on the edges of colonies. Cells of at least one non-motile mutant also produce these structures. One hypothesis concerning the nature of the fibrils is that they are comparable to eukaryotic actin. This was based on their structure and on the presence of a major protein constituent (molecular weight of approximately 50,000) recovered in a procedure designed to extract actin from ™. xanthus. However, attempts to decorate the fibrils with rabbit heavy meromyosin have been unsuccessful. Not all fibrils are sedimented by centrifugation at 150,000 x g for 90 min. Fibrils are restitant to digestion by pronase and by lysozyme; treatment with sodium dedecyl] sulfate (1 %; 70 3 does not alter their morphology. We hypothesize that fibrils, produced by vegetative cells, serve as tracks on which cells glide. These tracks may account for the phase-bright trails left by gliders on agar. MYXOBACTERIAL INTERACTION WITH PHIZOBIA S.A. DHALA and P.K.R. NAIR Microbiology Department, Bhavan’s Colleae, Andheri, Bombay »India The predation of four soil myxobacteria - Myxococcus fulvus, “yxococcus virescens, Myxococcus stipitatus and Polyangium fuscum on several susceptible Rhizobium spp. was investiqated. No significant difference in the course of attack on living and heat killed rhizobial cells was seen in plate assays, but viable cells were resistant to the myxobacterial culture filtrate in tube assays. Some partially lysis-resistant rhizobia were rendered more susceptible when separated from their polysaccharide. On hydrolysis, the purified gum gave glucose, galactose, mannose and glucuronic acid; aminosugars were absent. This qum had no effect on the synthesis and activity of lytive. The partially purified lytic enzyme complex showed an N-acetyl hexosaminidase activity on purified Rhizobium leguminosarum cell walls. Viable cells, polysaccharide gum, purified cell walls and lipids of R. leguminosarum did not induce any chemotactic response in any of the myxobacters tested, while autoclaved cells and cell proteins attracted the migrating cells. These myxobacters were also found in the rhizosphere of several local legumes. When introduced in sterilized soil with added rhizobia, M. fulvus and P. fuscum suppresses root nodulation. = PIGMENTATION PHENOTYPE INSTABILITY IN MYXOCOCCUS XANTHUS R. P. BURCHARD, A. C. BURCHARD and J. H. PARISH Department of Biological Sciences ‚University of Maryland Baltimore County Catonsville, Maryland /USA and Department of Biochemistry University of Leeds Leeds, United Kingdom When M. xanthus FB.is plated on CTE or PTE agar, 2-10% of the colonies are yellow (y phenotype); the majority is tan (t). Cells from most t colonies produce t and y colonies; cells from most y colonies produce only those of the y phenotype. Apparently color-stable strains have been isolated; however, these de-stabilize during serial sub-culture. The y and t phenotype are also distinguishable by differences in colony morphology and fruiting behavior. Fluctuation test experiments demonstrate that conversion of t cells to the y phenotype occurs at random in suspension culture. These and other experiments indicate that the conversion to y is enhanced in low cell density culture ( <10°/m) and is suppressed at higher cell density. An extra-cellular inhibitor of expression of the y phenotype is present in hiah density FB cultures. Ultraviolet irradiation of FB results in an increase in the y:t ratio with exposure time. This is due to conversion of cells from the t to the y phenotype, as shown by sectored colonies and equal UV-sensitivity of temporarily stable t and y strains. Low level photoreactivation occurs. Exposure to mitomycin C (5 g/ml) or to nalidixic acid (20 pg/ml) and growth at 36.5 C all increase the y:t ratio. Acridine orange has no effect. Chloramphenicol (25 ig/ml) does not inhibit the mitomycin-induced conversion to y. A highly polar, yellow pigment (abs. max. 379 nm in methanol) accounts for the color difference. Synthesis of this pigment is inhibited at 36.5 C, the maximum growth temperature of the organism. Biblioth 5, 14 BACTERIOCINS IN MYXOCOCCUS FULVUS H.-J. HIRSCH Gesellschaft füer Biotechnologische Forschung mbH Braunschweig-Stoeckheim,Federal Republic of Germany During attempts to isolate temperate phages in Myxococcus fulvus , it was observed that three strains distinctly inhibited growth of other fulvus strains. Further investigations were carried out with Myxococcus fulvus 16 (Mxf 16) which produces larqe, clear inhibition-zones. Mxf 16 produces a bacteriocin ("fulvocin 16") which, according to the definition, is specific with respect to its action on the growth of other myxobacteria, while exerting no inhibitory effect on other bacterial genera. Maximal fulvocin 16 concentration is obtained when cultures are harvested at the end of the growth curve. Mitomycin fails to stimulate production of this bacteriocin. Ammonium sulfate precipitation, followed by column-chromatography on DEAE and Sephadex G 200 results in a ten-fold purification of this bacteriocin, is resistant to the actions of trypsin, pronase, DNase and RNase. No bacteriocin-activity is demonstrated after treatment with chloroform, while its activity gradually decreases when incubated at 100°C. It is presumed that fulvocin 16 is a bacteriocin of low molecular weight. CELLULOSE DECOMPOSING MYXOBACTERIA ow JAROSLAVA POKORNA Research Institute for Crop Production Praha-Ruzyne, CSSR 200 strains belonging to the order Myxobacterales were isolated from farm yard manure, compost, liquid manure and soil. These strains were tested for their ability to utilize cellulose. Most of the strains decomposed the cellulose. Myxobacteria decomposed 9-82 per cent of cellulose. They are mesophilic aerobic microorganisms. The highest degree of decomposition of cellulose by the different myxobacteria depends on the form of nitrogen in the substrate. The course of decomposition of cellulose is affected by the temperature.The highest degree of decomposition was at 28°C. The extreme temperatures (below 8°C, above 42°C) are not convenient for decomposition. The course of decomposition is affected by the moisture, too. Fairly good decomposition proceeded at 21 per cent moisture level. Moisture within 15-20 per cents accelerated the decomposition of cellulose. In the evaluation of the coenoses of cellulolytic microorgansms, myxobacteria are widely distributed microorganisms not only in farm yard manure but in the soil as well. Myxobacteria belong to typical soil flora and participate quite actively in the decomposition of cellulose in the soil. They are specially predominant cellulolytic microflora in manured soil. Therefore the biological activity of fertilized soil can be fairly estimated by the cellulolytic test. Unfortunately the author had to cancel her participation in the symnosium at the last moment for personal reasons. The summary of the lecture she intended to give in Braunschweig is included here nevertheless. RECORD OF THE TECHNICAL DISCUSSION AT THE SYMPOSIUM The majority of the people present voted for having also in future a symposium on myxobacteria every year. To allow sufficient time for planning and raising funds, however, the place of the meeting should be decided upon 2 years in advance. The meeting should alternate regularly between both sidesof the Atlantic. An invitation has been extended by our colleagues in California to arrange the next meeting at the Asilomar Conference Grounds, Pacific Grove, California. The invitation was gratefully accepted, and July 25 to 27 was suggested as the most desirable time for the meeting. (This date has been verified in the meantime. H.R.) Ian Sutherland proposed that he would contact Colin Clarke and Howard Parish for chances to arrange the 1977 meeting in the United Kingdom. This was approved. The majority decided to restrict also future symposia on the biology of myxobacteria and to exclude other gliding bacteria. However, people from other fields of research may be invited to participate in the meeting. There was agreement not to publish a detailed symposium's report. Only the summaries of the lectures should be distributed. In order to be able to trace back quickly experimental strains to their origin, it was agreed upon to suggest for general adaption the labelling system which was proposed at the Cold Spring Harbor meeting: Each strain should be identified by the initials of the individual who isolated the strain or, if this is not feasable, who first introduced it into the literature. Furthermore, different strains of one species coming from one person should be numbered consecutively, the numbering beginning anew with another species; e.g. Myxococcus xanthus RBl, strains is independent of designations given the strain in individual laboratories, and should always remain attached to the name of the strain whenever anything is published about the strain and regardless who works with it later on. When a strain is sent to another individual, care should be taken to supply the original labelling with the strain. The complete history of the strain should be given when the strain is 17 introduced into the literature for the first time. (Note added when writing down the Record: to avoid repeated labelling of one strain care should be taken to adopt as label the initials of the person who originally isolated and distributed the organism, for he might have sent the same strain to different laboratories. If necessary the label should be inquired from the original source of the strain. H.R.) Robert P. Burchard has been asked at the Cold Spring Harbor meeting to work out a system for the designation of mutants. His proposals have already been made public in the Myxobacterial Messenger. They were unanimously approved, suggested for general use, and may be cited here again: The system is based on that proposed by Demerec et al. (Genetics 54: 61 - 76, 1966). "Briefly, genus and species names should be followed by the initials of the describing investigator (first and last names). He or she would then assign a number to the strain, starting with 1. Each investigator should keep a log book with a list of his strains, their origin and history, and their genotype and/or phenotype. The latter could be accomplished with Demerec et al "3 letter plus" abbreviations. Using my strains as an example: M. xanthus RB1 derived from Dworkin's FB as a stable tan and called strain FB, to date. The phenotype nomenclature would be Tan-1. RB4 is my non-motile NM, derived from SM which in turn is derived from FB. The phenotype designation Cam-r25 means resistant to 25 ug chloramphenicol/ml. For genotype, lower case lettering is used. Thus, a methionine-requiring auxotroph might be designated as "met-1". The hyphen could be replaced by a letter (capital ) when mapping is accomplished". It was again urged to make better use of the Myxobacterial Messenger (M.M.) to our mutual benefit. Completed Ph.D. theses on gliding bacteria should be reported to the M.M. and a brief abstract supplied. Planned gatherings of myxobacteria people at ASM meetings should be announced early in the M.M.. As already suggested at the Cold Spring Harbor meeting each laboratory should sent a list of available strains, including mutants, to the M.M..