The ecology of the myxobacteria

Myxobacteria are Gram-negative eubacteria with relatively large (0.6–1.2 by 3–15 μm) rod-shaped cells. They live in soil and related habitats and are famous for three capabilities: (i) they move by gliding and their colonies are therefore thin, film-like swarms that spread gradually over the culture plate; (ii) they have sophisticated intercellular communication systems and a highly developed social life; and (iii) they show a remarkable morphogenetic potential, which is expressed on two levels. In a co-operative morphogenesis involving 105–106 cells, induced by starvation conditions, they may produce a fruiting body. Within the maturing fruiting body, a cellular morphogenesis takes place, during which the vegetative cells convert into desiccation-resistant myxospores. It might also be mentioned that myxobacteria have the largest genomes of all bacteria (9500–10 000 kbp) and a DNA with a G + C content of 66–72 mol%. Details can be found in several reviews (Reichenbach and Dworkin, 1992; Dworkin and Kaiser, 1993; Dworkin, 1996). These peculiarities of the myxobacteria clearly must have a biological, and most likely an ecological, meaning. It appears that most of the phenomena mentioned above are best explained by the nutritional requirements and the habitats preferentially colonized by myxobacteria. All myxobacteria specialize in the degradation of biomacromolecules. Most species are proteolytic and bacteriolytic, attacking dead organic matter and dead and living bacteria, yeasts and other microorganisms with a host of excreted enzymes. One group of myxobacteria decomposes cellulose. Gliding motility may be useful to find and approach these non-diffusing substrates, particularly in a relatively dry and complex environment such as soil. The enzymatic attack, which involves the investment of substantial energy into the production of exoenyzmes, is more efficient if a community is at work, because then higher enzyme levels can be reached, and losses by diffusion can be minimized. A cell may profit from the efforts made by another cell and vice versa. The following experiment appears to support this assumption. When a medium containing 0.2% casein as the only nutrient is inoculated with Myxococcus xanthus cells, growth only occurs above a certain cell density (around 103 cells ml−1). Below that level, the cells cannot produce sufficient enzyme activity for casein hydrolysis (Rosenberg et al., 1977). Revealingly, the culture would grow at the same low cell density on a medium with hydrolysed casein (Casitone). Fruiting body formation, too, may be explained by the need to live in a community. While myxospores are already resistant to harmful environmental factors, the fruiting body guarantees that a new life cycle is started with a population rather than a single cell. Further, in the swarm colony, chemical and physical mechanisms (pheromones, pili, slime tracks) keep the cells together, although they can move much faster than the swarm expands. On the other hand, it is not trivial to produce single-cell colonies with myxobacteria. Individual cells normally do not grow readily into colonies even on agar media that allow luxuriant growth when inoculated massively. In some cases, results improve if spent medium of a liquid culture is added to the plating agar. This could mean that some quorum-sensing mechanism (Gray, 1997) is involved that prevents the futile growth of individual cells. Myxobacteria produce many different growth inhibitors. In fact, they have been shown to be one of the few bacterial groups that are a rich source of secondary metabolites (Reichenbach and Höfle, 1998). Conceivably, the organisms use that strategy to kill prey microorganisms and to defend their niche in the habitat, because their coherent colonies are essentially stationary, in a similar manner to the mycelia of the Actinomycetales. With their specific lifestyle, myxobacteria are obviously very successful. They are found nearly everywhere, from Antarctica to the tropics, and from sea level to high altitudes, in all vegetation belts from tropical rainforests to deserts. They may not live in all these places very conveniently and may simply have been blown to some of the more austere environments and survive there for some time, judging from experimental data for at least 10–20 years, owing to their desiccation and temperature-resistant myxospores. The richest myxobacterial floras are found in warm, semiarid areas, e.g. in the south-west of the USA, in northern India or in Egypt. A single spoonful of soil from such areas may yield 5–10 different species. Their myxospores and undegraded biomacromolecules in the habitat may give the myxobacteria a selection advantage in these areas. But they are also very common in central Europe, the mid-west of the USA and similar places. In more restrictive environments, such as the Sahara, in northern forests and at high altitudes, only few species are normally found, mostly Myxococcus, Corallococcus, Archangium and Nannocystis strains. The typical habitat of myxobacteria is soil, from dune and desert sands to rich black chernozem. All known myxobacteria are strictly aerobic and live in the topmost layers of the soil. In addition to soil, myxobacteria colonize decaying plant material including rotting wood and bark from dead and living trees, decomposing lichens and insects, and dung, especially of certain herbivorous mammals, such as wild rabbits, hares, deer, sheep and goats. Rabbit dung is a classical source for the isolation of myxobacteria. When incubated in a moist chamber at room temperature or at 30°C for a couple of days, fruiting bodies will almost always appear on the surface of some dung pellets and can be used for isolation (technical details will be found in Reichenbach and Dworkin, 1992). Where are the myxobacteria on dung coming from? As aerobic organisms, myxobacteria are not likely to live within animals. But myxobacteria on dung collected on thick layers of snow suggest that they may pass through the digestive track of the animal (Rückert, 1975a; personal observation). This has indeed been shown by pipette feeding fruiting bodies of Chondromyces to white mice in the laboratory and demonstrating the organism, certainly absent in that environment, on the faeces of the animals (Kühlwein, 1950). Myxobacteria have also been isolated regularly from the gut content of carp, and it has been concluded that they pass through rather than inhabit the fish (Till, 1982). Myxobacteria can also be isolated from fresh water, which is perhaps not so astonishing, because soil organisms notoriously exchange into water bodies, being regularly washed or blown in and often surviving there periodically or permanently (Brauss et al., 1967; Gräf, 1975; Trzilováet al., 1981). They may be found on drippling bodies in sewage plants. One species, Polyangium parasiticum, has been described as a parasite of the green alga, Cladophora, in the environments of Vienna (Geitler, 1925). It made holes in the cell wall of the alga, entered the cells and lysed its contents. Fruiting bodies were produced inside and on the surface of the algal filaments. Pl. parasiticum may have been a facultative parasite, but was never reported again. In this connection, the reader must be warned that, for some time, Cytophaga and Cytophaga-like bacteria were classified as myxobacteria, among them many water bacteria including fish pathogens. We know today that those organisms are not related to the myxobacteria at all, but belong to the Cytophaga–Flavobacterium–Bacteroides phylum. The myxobacteria are found in the delta branch of the Proteobacteria. Considering the life cycle of myxobacteria with their desiccation-resistant myxospores, aquatic habitats appear not to be very typical for them. Yet myxobacteria have been isolated from marine sources. They have been found repeatedly in sand and debris taken from the intertidal range of the Atlantic Ocean (Brockman, 1967; Rückert, 1975b), and once even from samples collected at 50 m depths around the island of Helgoland (Rückert, 1984). But all those strains were isolated on low-salt media, and none of them tolerated the salt concentrations of seawater. My own experience is equally negative. Experiments to isolate myxobacteria on seawater agar from samples collected in the salt marshes on the island of Sylt in the North Sea were a complete failure. When isolating marine Cytophaga-like bacteria at the marine station of Roscoff in Brittany, we started hundreds of cultures on seawater agar with all kinds of samples from near the station, using techniques that should have yielded myxobacteria as well. But there were none. I obtained many myxobacteria from marine debris collected on the shores of the island of Fynen in Denmark, when low-salt media were used for isolation, just as described in the articles cited above. On the other hand, Japanese colleagues reported recently at a symposium on myxobacteria in Greece that they had isolated truly marine, halotolerant myxobacteria (R. Fudou et al., personal communication). Using molecular taxonomy, they found that those organisms belonged to the Nannocystis complex. Does this mean that there are myxobacteria in the Pacific and not in the Atlantic? This may sound weird, but many algae and seaweeds also show a restricted distribution. Dust and soil are transported by air currents and so are desiccation-resistant soil organisms. Air contamination by myxobacteria has been reported occasionally, if rarely (Wu et al., 1968). About half the leaves taken from various shrubs and trees in Germany yielded myxobacteria (Rückert, 1981). However, not all tree species seem to harbour myxobacteria. Myxobacteria have also been found in samples taken deep within caves in areas apparently not touched by man before (Menne and Rückert, 1988). Only a few species have been discovered, as is typical of unfavourable habitats. Somewhat unexpectedly, myxobacteria have also been isolated from bogs. Many myxobacteria were obtained from various parts of an alkaline bog (pH 6–8.7) near Vestaburg, MI, USA (Hook, 1977). Of particular interest was the abundant occurrence of Angiococcus (Cystobacter) disciformis specifically in the fossa, i.e. the ditch surrounding the bog (Hook et al., 1980). While the organism is also found in other places, it may be particularly well adapted to that habitat. Really astonishing was the discovery of myxobacteria (Myxococcus, Corallococcus and Polyangium sp.) in peat bogs in the Rhön mountains in Germany (pH 3.2–4.8) and of the Hautes Fagnes/Hohe Venn at the Belgian–German border (pH 3.7, water content 91%, oxygen 2.58 mg l−1; Dawid, 1984). The samples were taken from the bog at a depth of 5–10 cm below the plant cover of about 10 cm. The myxobacteria were isolated at a neutral pH. It is not clear whether the organisms really grow, perhaps very slowly, in the bog or are washed down from the plant cover or from soil blown into the bog. The rather narrow spectrum of species may speak for the former assumption. Very little is known about specific habitats of the various myxobacterial species. A reliable and convenient source of Myxococcus species is the dung of herbivorous mammals. Mx. fulvus is often found on rotting wood, and all species can also be isolated from soil. While it is plausible that harsh environments, such as high altitudes, deserts or peat bogs, select for tough species, mainly those with spherical myxospores (e.g. Norén, 1952), the distribution patterns of species are still enigmatic. Almost all myxobacteria grow well at 30°C, although their temperature range is much wider. Many strains are psychrotrophic and grow, albeit slowly, at low temperatures, e.g. in the refrigerator at 4–8°C (Zhukova, 1963; Menne and Rückert, 1988; personal observation). Also, isolation at 10°C brought forth many different myxobacteria (Krzemieniewska and Krzemieniewski, 1927). Myxobacteria have been isolated from soil collected along the Arctic coast of North America (Brockman and Boyd, 1963). Only enrichment cultures incubated for 6 weeks at 25–26°C, but not those incubated at 6–8°C, yielded myxobacteria. But the incubation time for isolating psychrophiles may simply have been too short. Myxobacteria were also obtained from Antarctic soils. In one study, the crude cultures were kept at 30°C and at room temperature and, understandably, only mesophilic myxobacteria were obtained (Rückert, 1985). In a second study, psychrophilic myxobacteria could be isolated after incubation times of between 8 weeks and 9 months at 4°C (Dawid et al., 1988). These organisms would not grow at 18°C. Survival of fruiting bodies for a long time in dry environments at low temperatures can be expected from the behaviour of myxobacteria in the laboratory. Vegetative cells also survive freezing but remain living for a longer time only at temperatures below −50 to −60°C. In the laboratory, the useful temperature range for cultivating myxobacteria is 28–34°C, with generation times between 4 and 14 h. In nature, the organisms certainly grow normally at lower temperatures and probably much more slowly. Many strains still grow at 38–40°C (e.g. McCurdy, 1969; Gerth et al., 1994; unpublished data), but cultures get increasingly unstable at these high temperatures and quickly break down and lyse at the end of the growth cycle. Vegetative cells are killed at temperatures above 45°C, but myxospores suspended in water tolerate 58–60°C, which can be used to purify strains during isolation (for technical details, see Reichenbach and Dworkin, 1992). Dry myxospores may survive much higher temperatures (G. Rückert, personal communication). We obtained growing cultures from fruiting bodies of various genera that had been heated on filter paper in the oven at 140°C for 30 min, in some cases even for 45 min (unpublished data). In culture, the pH range of myxobacteria is normally 6.8–7.8. The cellulose degraders may still grow at a somewhat lower pH but, even for them, the limit is 6.0–6.4. This may reflect in part the practice of using media with a pH of around 7 for isolation. Alkalophilic myxobacteria that do not grow below a pH of 9 have indeed been obtained from alkaline lakes of east Africa (sample pH 9.5), when alkaline media were used for isolation (W. Dawid, personal communication). So far, no acidophilic myxobacteria have been reported, although myxobacteria can be isolated from soils with an acid pH as low as 2.5 (e.g. Krzemieniewska and Krzemieniewski, 1927; Rückert, 1972; 1979; Dawid, 1979). The salt tolerance of myxobacteria appears to be rather low. As already mentioned, myxobacteria may be isolated from marine habitats but, until recently, all strains thus obtained were not halotolerant. Soils from salt marshes or certain desert habitats with a high salt content have to be desalted before myxobacteria can be isolated from them (Rückert, 1983). Apparently some myxobacteria tolerate salt better than others. The addition of 0.5% NaCl to the isolation agar stimulates the development of Mx. virescens and Ar. gephyra (Rückert, 1978). On water agar with streaks of living Escherichia coli, a standard for the isolation of myxobacteria, I obtained only Mx. virescens and when NaCl was added to the A in the NaCl content to also Mx. fulvus and Corallococcus to (unpublished an myxobacteria of need Vegetative cells are rather to so that is not for their In myxospores are desiccation resistant and may be kept dry at room e.g. in fruiting bodies on filter for years, the we have been to so It appears that the of substantial of in myxospores of Mx. xanthus is for desiccation and In to myxospores only their to harmful environmental in the dry It is not to get a clear about the of myxobacteria in soil. the organisms are to by excreted slime and, in they are probably among by pili, so that a of individual cells for plating cannot be most myxobacteria would not readily produce colonies from single cells. Yet some have been In between and 000 myxobacteria have been of soil. The in 000 cells of Mx. fulvus as well as of other myxobacteria, have been found A of various of soil in 000 myxobacteria The were in and soils with a high the in and which may be in to pH and organic In the of was to be between and 000 soil In soils on the the was with to 10 cells (Zhukova, All these are probably too low and, in only to a selection of species. We may that myxobacteria are in soil in substantial several different species can be isolated from a soil the of a which that many species are common and to the myxobacterial It has been known for many that most myxobacteria are and and lyse other bacteria, whether living or dead (e.g. it has been concluded that myxobacteria should have a on the soil and could perhaps soil In fact, when and Mx. xanthus were inoculated into soil, the of very during incubation et al., to soil, a of three bacteria was The to appear was a with spherical myxospores, perhaps a Myxococcus species and 1983). the myxospores in soil for a time but not until the pH was to neutral and was The of has been repeatedly in the that myxobacteria could be used to water et al., et al., 1985). on agar as well as in liquid In liquid media, the Myxococcus strains used and the in layers of excreted with a of 9 at were As few as 50 Myxococcus cells with cells were sufficient to a myxobacterial In the myxobacteria on or on the wall of the and a of the was activity was in the culture Also, of various yeasts has been many (e.g. and an medium for cultivating all myxobacteria is agar with 0.5% of green algae in a habitat by Polyangium parasiticum has already been very similar to the has been reported for and the had been in soil for 4 they many in their and a apparently a could be isolated from them. This organism produced the same holes in the cell in them, groups of myxobacteria would the cells, lyse their and or produce fruiting bodies inside 1984). It should be mentioned in this that, thus far, no myxobacteria have been are fish especially in that were classified for some time in the as myxobacteria the of In fact, these organisms are Cytophaga-like It has been repeatedly in the that certain myxobacteria, particularly those with more complex fruiting bodies, may live in with other of this has been until recently, because of myxobacteria was not well until the When to purify it that, with one single all strains would only grow as long as a rod-shaped was in the of the All other Chondromyces species, at least the several strains in can be as strains. The too, not grow well its Chondromyces Very colonies on various media after weeks of growth was only obtained when the was in a with the culture in the other The strains can readily be with the in large as the two were their development et al., 1994; When the of the two were it was that the was always the same species, of whether the strains from samples from or et al., The to be a species, probably to the one a in its also a from the of the other strains. This that the have for a long find its when in a With the of it was shown that the is already within the of the Chondromyces fruiting body et al., This is very as myxobacterial fruiting bodies are not normally when A between and a specific was described the of that it must be that the has not the that the is probably related to well on various media and is lysed by the all of which not the found in Myxobacteria with one The mechanisms are (e.g. by as well as In a has been during fruiting body et al., and its has been recently et al., et al., et al., 1998). cultures often produce an in is by et al., 1981). As myxobacteria are so common in soil, they may to its which a in water of have an of which is so and typical that the organism can be by it at As already mentioned, myxobacteria also produce and many secondary metabolites (Reichenbach and Höfle, 1998). These often show very mechanisms of different new on complex I or complex have been discovered, with the or with the with high at and on acid Most of these appear to be developed to in the probably and In the gliding motility has often been taken as a of a between organisms. But this is not the Gliding are known from most bacterial (e.g. In many cases, a for the organism can be in gliding organisms living in and such as and organisms in the degradation of non-diffusing substrates, such as Cytophaga and Cytophaga-like bacteria, and the myxobacteria. In of many efforts over of the mechanism of gliding is still not a of by has been in the surface of myxobacteria and et al., This appears to that could a in the cell and could thus the of gliding