Copvright 0 1999 bv the Genetics Society of Anlerica Perspectives

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

ROLANDTHAXTER’S Legacy and the Origins of Multicellular Development

Dale Kaiser

Departments of Biochemistry and of Developmental Biology, Stanford University, Stanford, Calijornia 94305

OLAND THAXTER published a bombshell in R December,1892. He reported that Chondro- myces crocatus, before then considered an imperfect fungus because of its complex fruiting bodv, was ac- tually a bacterium (Figure 1). THAXTERhad discov- ered theunicellular vegetative stage of C. crocatus; the cells he found were relatively short and they divided by binary fission. C. crocatus was, heconcluded, a “communal bacterium.” THAXTERdescribed the lo- comotion, swarming, aggregation and process of fruit- ing body formation of C. crocatus and its relatives, which are collectively called myxobacteria, with an accuracy that has survived 100 years of scrutiny. He recognized the behavioral similarity to the myxomv- cetes and the cellular slime molds, drawing attention in all three to the transition from single cells to an integrated multicellular state.He described the be- havior of myxobacteria in fructification in terms of a “course of development” because it was “a definitely recurring aggregation of individuals capable of con- certed action toward a definiteend” (THAXI‘ER1892). This essay will emphasize some implications of THAX- TER’S demonstrations, often apparently unrecognized. The striking similarities to cellular slime mold de- velopment probably led JOHN TYLERBONNER and KENNETHB. RAPER,50 years after THAXTER’Sdiscov- ery, to take independent forays into myxobacterial development. RAPER,an eminent mycologist, had in FIGURE1 .“Chondromyces crocatus fruiting bodv. Photograph by fact discovered Dictyostelium discoidium, recognizing it HANS KEICHENRACH.GBF, Braunschweig. as a superb subject for the study of morphogenesis, ments,intercellular integration and finally coordi- cellular differentiation and intercellular communica- nated differentiation that are in some ways cornpa- tion. BONNERwas fascinated by morphogenesis and rable to higher forms” (QUINLANand RAPER 1965). sought unifying principles behind the bewildering di- Both BONNERand RAPER soughtthe factors in C. versity (BONNER1952, 1974). Both RAPER and BON- crocatus that coordinated and guided the morphoge- NER seemed to be intrigued by the unusual example netic cell movements, noting thatindividual myxobac- of morphogenetic movements exhibited by the myxo- terial swarm cells retained their physical individuality bacteria as they formed fruiting bodies. RAPERsaw throughout the process of cooperative morphogene- “examples of interdependent cellular behavior that sis, and in that respect differed from the myxomycetes involve purposeful orientation, morphogeneticmove- but resembled the cellular slime molds.

Genetics 135: 249-254 (October, 1999) 250 D. Kaiser Their search was extended by HANSKUHLWEIN and are known which form spores, have a high G.C con- his students, particularly REICHENBACH,who prepared tent in their DNA, but do not build fruiting bodies, a series of time-lapse films of the behavior of different even though gliding bacteria have been systematically types of myxobacteria (REICHENBACH, HEUNERTand examined (REICHENBACHet al. 1988). That all the KUCZKA1965a,b,c,d; REICHENBACH,GALLE and HEU- myxobacteria arose fromthe same ancestor within the NERT 1976). Using time-lapse photography to con- 6 subgroup of purplebacteria is supported by an dense the roughly day-long process of fruiting body extensiveset of characters they hold in common: development into a few minutes of running time, as swarmingbehavior, closely related 16s ribosomal BONNERhad donefor D. discoideum, broughtthe RNA sequences (LUDWIGet al. 1983; WOESE1987; morphogenesis into a time scale more suggestive and SHIMKETS1993), high (66-72) mole % G.C content intriguing to the human psyche. The myxobacterial in their DNA (MANDEL and LEADBETTER1965; movies showed the simplest fruiting bodies to be MCCURDYand WOLF1967; BEHRENS,FLOSSDORF and mounds of myxospores covered by slime, while the REICHENBACH1976) and a set of notablechemo- more complex fruiting body structures enclosed systematic markers (REICHENBACHand DWORKIN myxospores within acellular skins of slime that either 1981). None of the other members of the 6 subgroup rested directly on the substratum or were raised on of purple bacteria form fruiting bodies or spores; in slime stalks. REICHENBACH(1 962) concludedthat the additiontothe myxobacteria, this phylogenetic formation of fruiting bodies generally passed through subgroup includes the bdellovibrios and the meso- several stages: vegetative growth of a multicellular philic sulfate-reducing bacteria (WOFSE 1987; STACK- swarm, inductionby starvation to begin development, EBRANDT 1992; WIDDELand BAK 1992). cell accumulation, rearrangement of cells within the In contrast to the monophyletic origin of myxobac- originally undifferentiated mass (includingproduc- teria, cell aggregation of eukaryotes leading to fruit- tion of slime stalks or sporangiole walls) and, finally, ing bodies appears, on cytostructural grounds,to have myxospore formation. REICHENBACH(1 965)also dis- evolved several times among the cellular slime molds covered thatthe ripples noted in myxobacterial (OLIVE1975). BONNER(1982) has argued that there swarms were traveling waves generated by many is likely to have been an independent originfrom myxobacteria during theaggregation phase. single-celled amoebaefor each group because of Work with dispersed (i.e., non-clumping) strains of uniqueaggregation attractants; he distinguishes at My3cococcus xanthus enabled DWORKIN,ROSENBERG least eight different attractants. and their students tostudy this bacterium’s nutrition No matter how many independent events there and metabolism, prerequisites for understanding the were among the cellular slime molds, it is clear that role of starvation in the induction phase of fruiting the changesfrom single to multicellular organism body development (summarized by DWORKIN1984). were taken independently fromthe myxobacteria. To Genetic studies of mutants defective in fruiting body date there has been no report of lateral gene transfer development becamepossible through theisolation of between myxobacteria and the slime molds. The cell transducing myxophages (CAMPOSand ZUSMAN 1975; biology of the slime molds and the myxobacteria are MARTINet al. 1978), the introduction of transposons very different, as THAXTERfirst discovered. The for- from Escherichia coli into M. xanthus (KUNER and mer are eukaryotic amoebae with a flexible cell mem- KAISER 1981) and the infusion of gene cloning tech- brane and a well defined cytoskeleton. We now rec- niques (GILLand SHIMKETS1993). ognize thatthe latter are rigid-walled, rod-shaped, THAXTER’Sdiscovery called attention to the transi- Gram-negative procaryotic cells that lack the struc- tion from single cells to an integrated multicellular tural anatomic features of a cytoskeleton. Molecular unit. There is general agreement that this step has studies of small ribosomal rRNA sequences as well as been taken many times in the course of organic evo- physiological and morphological studies show that the lution. For example,the sponges probably arose from myxobacteria arose among the purple sulfur eubac- solitary cells separately from all other animals, and teria, while the cellular slime molds arose among the the seed plants, the fungi, and the algae all gained protists. The two groups are thus separated by a wide their multicellular conditionindependently (WHIT- evolutionary gap (Figure 2) (WOESE1987). Features TAKER 1969). Comparing these independent experi- common to these two types of microorganisms prom- ments of nature should provide insight into the gen- ise insight into basic biological attributes of multicel- eral attributes of multicellular life. lular development.The width of the phylogenetic gap Myxobacteria, which belong to the 6 subgroup of decreases the effects of chance evolutionary “tinker- purple bacteria, are a well defined and unique exper- ing” UACOB1982), theaccidents of mutational history. iment in multicellularity. All myxobacteria construct The point is that features shared by different orga- multicellular fruiting bodies (LUDWIGet al. 1983), nisms will be the more robust andfunctionally inform- which is to say that no aerobic gliding bacterial species ative the less the organisms share a common descent. Perspectives 25 1 the aggregate in a species-specificpattern, thendiffer- entiation of individual cells into spores. .Both pass several chemical signals between their BACTERIA - purple bacteria cells. In D. discoideum, the signals include cAMP and - DIF (WILLIAMSand JERMYN 1991). cAMP seems not gram pOsitive bacteria cyanobacteria to be significant in myxobacteria in the way it is in D. deinococci discoideum. Instead, in M. xanthus a mixture of eight Thermotogales amino acids (called A-factor) is a signal early in devel- “ARCHAEA opment, and a 17-kDa surface protein known as C- factor is a signal later during the aggregation and sporulation phases (KIM, KAISER and KUSPA 1992; animals KAISERand KROOS 1993). EUCARYA The cells respond tosignal reception by expressing ~ cellular slime molds * new batteries of genes. ln D. discoideum thereare flagellates microsporidia CAMP-dependent andDIF-dependent genes(DE- VREOTES 1989; WILLIAMSand JERMYN 1991). In M. FIGUREZ.-Phylogeny of myxobacteria (8 in 8 purple bacteria) and the cellular slime molds (8 in Eucarya). Adapted from WOFSE xanthus there are A-factor-dependent and C-factor- (1 992) and STACKEBRANDT(1 992). dependent genes (reviewed in KROOS,KUSPA and KAI- SER 1986; KAISERand KROOS 1993). Common qualities: As understood today, cellular During aggregation, cells are swept into a fruiting slime molds (exemplified by D. discoideum) and myxo- body from neighboring regions. The mechanisms of bacteria have these similarities: sweeping are similar in several ways, including the Fruiting body development is asexual. The grow- generation of traveling waves. In D. discoideum, the ing cells are haploid and the fruitingbodies are filled traveling waves are generated by pulses of cAMP that with haploid spores. As expected, the genome sizes emanate from an aggregation center (TOMCHIKand are different. The sizes of several myxobacterial ge- DEVREOTES198 l), while in M. xanthus the traveling nomes have been determined by pulsed-field gel elec- waves are local accumulations of cells, which depend trophoresis and include the closed circular genome of for their formation onC-factor (SHIMKETSand KAISER M. xanthus at 9.4 Mb (CHENet al. 1991), Stipatella 1982). aurantiaca at 9.2-9.9 Mb and Stigmatella erecta at 9.7- Stalk formation differs. InC. crocatus, cells migrate 10 Mb (NEUMAN,POSPIECH and SCHAIRER1992). The upward inside a tube of “slime” (apparently mostly genome of D.discoideum consists of six (possiblyseven) polysaccharide), depositing more slime at the top and (DARCYet al. 1993) linkage groups that total 40 Mb elongating the stalk as they pass into the cellmass of DNA (KUSPAet al. 1992). resting on the top (QUINLANand RAPER1965; REI- Both move on surfaces, neither can swim. Slime CHENBACH, HEUNERT andKUCZKA 1965b; THAXTER mold cells translocate by amoeboid movement that 1892). In D. discoideum, prestalk cells move up the involves dynamic changes in their cytoskeleton. Myxo- outside of the preexisting cellulose tube (RAPERand bacteria move by gliding on surfaces without apparent FENNELL 1952).As these cells migrate over the lip of rotation or change in cell shape; they lack flagella or the tube, they deposit more cellulose, elongating the any other obvious organelles of movement. The tube (WILLIAMSand JERMYN 1991). Though Dictyos- mechanism of gliding is currently unexplained even telium stalks contain specialized, differentiated stalk though many bacteria cando so (MCBRIDE,HARTZELL cells, the stalk of another dictyostelid, Acytostelium, and ZUSMAN 1993). is acellular like those of the myxobacteria (RAPER Cell division is separate from development. Cells 1984). grow and divide when food is abundant. Starvation The fruiting bodies of the cellular slime molds and stops growth and induces development. Amino acid the myxobacteria coverthe same morphological range starvation appears to be a prime factor in the induc- of spheres and cylinders ordered and combined in tion of development. Addition of a complete set of various ways (REICHENBACHand DWORKIN 1981; the amino acids required for D. discoideum growth BONNER1982; RAPER1984). The fruiting bodies of delays the initiation of development (MARIN 1976). In D. discoideum and M. xanthus each containup to M. xanthus, limitation for any of theamino acids 100,000 cells. induces fruitingbody development (MANOILand KAI- Selective forces:Slime molds and myxobacteria are SER 1980). found in the same habitats, and are often isolated The program of morphological change and devel- fromthe same soil samples by enrichmentculture opment begins with recognition of starvation, aggre- (SINGH1947). Both feed on bacteria in the soil. How- gation of preexisting cells, arrangement of cells within ever, the slime molds ingest bacteria by endocytosis 252 D. Kaiser while the myxobacteria secretetheir digestive en- myxobacterial fruiting bodies (REICHENBACH 1984). zymes, thentake up theproducts of extracellular Survival under marginal conditions in a jluctuating digestion (Sum and DWORKIN 1972;LOOMIS 1975). environment: STEPHENBARCLAY (University ofWis- The observed evolutionary convergence of these two consin) has pointed out to me that soil amoebae often disparate groups is presumably a consequence of nat- find themselves in nutritional conditions thatare mar- ural selection in a common habitat. Both organisms ginal, neither rich enough for rapid growth nor poor are less insulated from their environment than flow- enough to trigger efficiently the encystment of indi- ering plants or higher animals and arehaploid, so that vidual cells. Marginal conditions may encourage slow natural selection can constantly play a role in shaping growth that would leave cells incapable of completing their development. What might the selective forces a final mitotic cycle, with death as the consequence. have been? Several have been suggested: One strategy to cope with marginal conditions may be Social feeding: The selective advantage for the evo- to aggregate andconstruct a fruiting body,withdraw- lution of multicellularity in myxobacteria is likely to ing cells from the ambiguous environment and allow- have been cooperative feeding. Myxobacteria feed on ing themto continue starvation-induced development. particulateorganic matter in the soil by means of Increased reliability of perceiving starvation: The A- extracellular bacteriolytic, proteolytic, cellulolytic and factor of M. xanthus, which is a mixtureof eight amino other digestive enzymes (REICHENBACH1984). Based acids, is a cell-density signal (KUSPA, PLAMANN and on their secretion of lytic enzymes, DWORKIN (1973) KAISER 1992b). The amount of A-factor released is proposed that myxobacteria feed like “packs of micro- proportional to the number of cells per unit volume, bial wolves.” ROSENBERG,KELLER and DWORKIN and acertain minimum quantity of A-factor is re- (1977)measured the growth rate when the only quired to continue development. Thus, the A-signal source of carbon and nitrogen for M. xanthus cells in ensures a cell density sufficient to complete a proper liquid culture was the polymeric substrate casein, so fruiting body (KUSPA, PLAMANN andKAISER 1992a). that proteolysis was required for growth. The growth A-factor, which is released about 2 hr after the begin- rate increased twofold as the cell density was raised ning of starvation, is also a way for cells to vote their above lo4 cells/ml. When intact casein was replaced individual assessment of nutritional conditions. Be- with enzymatically hydrolyzed casein, the cells grew cause new proteins must be made during aggregation at the more rapid rate independently of cell density. and sporulation, some protein synthetic capacity must Evidently, extracellular digestion of protein is en- be retained, and the cells must begin to aggregate hanced by cooperation between cells. before they have exhausted all their sources of amino A swarm may be the unit of efficient cooperative acids and energy. To initiate development or to grow feeding. REICHENBACHhas shown that a single ger- slowlyis animportant choice on which long-term minatingsporangiole of a Chondromyces apiculatus survival depends. An optimal choice is one that antic- fruiting body formsan active swarm that behaves ipates the future. When this decision is jointly made much like a swarm of bees (BONNER1952; QUINLAN by a population of cells, it is likely to be more reliable and RAPER1965; KUHLWEIN and REICHENBACH than that made by one cell. A protein, CMF, secreted 1968). Forming a multicellular fruiting body ensures by starved Dictyostelium cells playsa similar role UAIN that, when conditionsfavorable for growth are re- et al. 1992). stored, the myxospores can germinate and the new These forces, and others, can be discriminated by phase of growth can start as a preformed community experiment because myxobacteria and cellular slime of efficiently feeding cells. The success of the myxo- molds are microbes that can be conveniently handled bacterial design is evident in their distribution; they in largenumbers. Moreover, the set of molecular are common inhabitants of soils drawn from all over genetic tools currently available in both organisms the world regardless of climate (REICHENBACH1984). includes physical/genetic maps, tools for random in- Dispersal: BONNER(1982) has suggested thatthe sertional mutagenesis to identify genes and to provide cellular slime molds evolved from solitary soil amoe- genetic markers, methods for homologous gene re- bae to multicellular forms under selection foran placements including construction of null alleles, and efficient means of dispersal. He argues that “ . . . se- the capacity to clone genes in E. coli or Saccharomyces lection pressure for fruitingbodies in smallorganisms, cerevisiae for manipulation before returning them to be they amoebae, hyphae, plasmodia, swarms of bac- theirproper host for expression; see GILL and teria, or even ciliate protozoa (Olive, 1978), must be SHIMKETS(1993) for myxobacteria and KUSPA and enormous, and thescale of convergent evolutionvast” LOOMIS(1 992) forDictyostelium. (BONNER 1982).Rain water, wind, or movement of The selective forces, whichever may have been ef- small soil invertebrates could disperse fruiting bodies. fective, will have acted within a set of biological and Stalks, multiple sorogens and sporangioles might be physical constraints. Unexpected convergence on re- explained this way. Mites have been observed to carry lated body plans and systems for control of multicel- Perspectives 253 Mar development by cellular slime molds and myxo- phogenesis in Myxobacteria. Film C893/1965. Institut Wissen- bacteria suggests that the structural differences be- schaftliche Film, Gottingen. KUNER,J., and D. KAISER,1981 Introduction of transposon Tn5 tween eukaryotic and procaryotic cells may in fact be into Myxococcus for analysis of developmental and other non- secondary todeeper similarities. We are aware of selectable mutants. Proc. Natl. Acad. Sci. USA 78: 425-429. many metabolic similarities. We are becoming aware KUSPA,A,, andW. F. LOOMIS,1992 Taggingdevelopmental genes that there are also rules for the folding, structuring in Dictyostelium by restriction enzyme mediated integration of and assembly of proteins. Perhaps there arealso rules plasmid DNA. Proc. Natl. Acad. Sci. USA 89: 8803-8807. KUSPA, A., L. PLAMANN andD. KAISER, 1992a A-signalling and about the way development and morphogenesis are the cell density requirement for Myxococcus xanthus develop- regulated forreliability in relatively harsh or changing ment. J. Bacteriol. 174 7360-7369. environments. Use of cellular oscillators revealed by KUSPA,A,, L. PLAMANN andD. KAISER, 1992b Identification of traveling waves and the expression of genes in batter- heat-stable A-factor from Myxococcus xanthus. J. Bacteriol. 174 ies, triggered by differentextracellular signals, are 3319-3326. KUSPA, A,, D. MAGHAKAIN,P. BERGESCHand W. F. LOOMIS, cases in point. Part of ROLANDTHAXTER’S legacy is 1992 Physical mapping of genes to specific chromosomes in the notion that comparisons of eukaryotes and pro- Dictyostelium discoideum. Genomics 13: 49-61. karyotes may give insights that would comefrom LOOMIS,W. F., 1975 DictyosteliumDiscoideum. A Developmental neither examined alone. System. Academic Press, New York. LUDWIG,W., K. SCHLEIFER,H. REICHENBACHand E. STACKE- BRANDT, 1983 A phylogenetic analysisof the myxobacteria LITERATURECITED Myxococcus fulvus, Stigmatella aurantiaca, Cystobacter fuscus,Sor- angiumcellulosum and Nannocystisexedens. Arch. Microbiol. 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