**2. Biology and Phylogeny of Myxobacteria**

Myxobacteria are soil dwelling deltaproteobacteria and are distributed all over the world. Temperate zones, tropical rain forests, arctic tundra, deserts, acidic soils [1–3], marine and other saline environments [4–7], and even caves [8], for example, are appropriate habitats. Myxobacteria can be isolated from various natural sources as soil, bark, rotting wood, leaves of trees, compost [9], or dung of herbivores [1,10]. They live aerobically, except the only described facultative anaerobic genus and species, *Anaeromyxobacter dehalogenans* [11]. Nevertheless, it is highly likely that further facultative or even strictly anaerobic myxobacteria exist, which hitherto withstand the common isolation efforts. Currently the monophyletic order Myxococcales comprises 3 suborders, 10 families, 29 genera, and 58 species (Figure 1).

**Figure 1.** Monophyletic order Myxococcales (delta-proteobacteria), suborders, families, and genera of myxobacteria (status May 2018). The number of species within the genera is mentioned in brackets (original graphic from Corinna Wolf, modified by K. I. Mohr).

Myxobacteria are fascinating because of their extraordinary social lifestyle, which is unique in the bacterial domain. Under appropriate environmental conditions, vegetative cells move in swarms by gliding over solid surfaces [12]. Myxobacteria do not have flagella, but two motility systems, used for locomotion, and well studied in *Myxococcus xanthus*, are known: social (S-) motility, powered by retraction of type IV pili is responsible for the movement of cells which travel in groups [13]. In addition, extracellular matrix polysaccharide (EPS), also referred to as fibrils, are used. Therefore, the intrinsic polarity of rod-shaped cells lays the foundation, and each cell uses two polar engines for gliding on surfaces. It sprouts retractile type IV pili from the leading cell pole and secretes capsular polysaccharide through nozzles from the trailing pole [14]. On the other hand, slime secretion enables cell movements, when cells were isolated from the group (adventurous A-motility) [15]. For a detailed description of myxobacterial gliding mechanisms see Nan et al. [13] and Faure et al. [16].

Due to their nutritional behavior and based on their specialization in degradation of biomacromolecules, members of the order Myxococcales can be divided into two groups: predators (the majority), which are able to lyse whole living cells of other microorganisms by exhausting lytic enzymes, and cellulose-decomposers, the latter are represented by the genera *Sorangium* and *Byssovorax* [12]. But, as mentioned for the (facultative) anaerobic myxobacteria, it is also highly likely that further cellulose-degrading genera exist, which successfully resisted standard cultivation attempts.

If nutrients become rare, the cells undergo an impressive process of cooperative morphogenesis. Cells agglomerate and form species-specific fruiting bodies by directed cell movement [12]. These fruiting bodies consist of one to several sporangioles [1]. The architecture of these fruiting bodies ranges from simple, single sporangioles (*M. xanthus*, *Cystobacter* spp.), stalked sporangioles (*M. stipitatus*), or

even delicate tree-like structures of high complexity (*Chondromyces* spp.; *Stigmatella* spp.) [10]. Colors of cells/fruiting bodies vary from milky, yellow, orange, red, brown to even black (Figure 2) [1].

**Figure 2.** Variation of myxobacterial fruiting bodies. Genus/species, strain designation, (agar medium) are mentioned. (**a**) *Myxococcus xanthus* Mxx42 (P); (**b**) *Cystobacter ferrugineus* Cbfe48 (VY/2); (**c**) *Archangium* sp. Ar7747 (VY/2); (**d**) *Chondromyces* sp. (Stan 21 with filter); (**e**) *Sorangium* sp. Soce 1462 degrading filter paper on Stan 21 agar; (**f**) *Polyangium* sp. Pl3323 (VY/2); (**g**) *Cystobacter fuscus* Cbf18 (VY/2); (**h**) *Corallococcus coralloides* Ccc379 (VY/2).

A known function of these mainly carotenoid or melanoid pigments is to provide protection against photo-oxidation [17]. Within the fruiting bodies, most of the vegetative cells die and serve as food for the remaining cells, which convert into short and hardy myxospores, especially resistant to desiccation [18,19]. These spores are not as heat-resistant as *Bacillus* spores, but they can survive in the environment and are able to germinate under appropriate conditions even after decades of resting [1]. Therefore, it is possible to isolate myxobacteria from dried environmental samples, which were stored for several years at room temperature [10]. A fruiting body consists of 105–106 cells [18]. This ensures that the new cycle starts with a sufficient amount of cells, necessary for the typical collaborative feeding [20]. A further very interesting feature of myxobacteria is their ability to produce a large number and variety of secondary metabolites, as described in the next section.

In 1892, Thaxter was the first who described myxobacteria in literature [19]. He found out that *Chondromyces crocatus* was a bacterium and he had discovered its unicellular vegetative stage. This was spectacular, because until such time, *C. crocatus* had been considered a slime mold for more than 20 years [14]. Studies by Bauer [21], Kofler [20], Jahn [22,23], and Kühlwein [24] followed in the early 20th century. Myxobacteria have always fascinated scientists due to their social behavior, including cooperative swarming, group predation, and multicellular fruiting body formation. *Myxococcus xanthus* for example has become one of the model systems for the study of prokaryotic development [25]. Today, beside their capabilities to produce promising bioactive secondary metabolites, myxobacteria are of utmost importance in elucidating multicellular behavior in bacteria, as well as working out social evolution theory.

#### **3. Current Status of Antibiotics and Myxobacterial Secondary Metabolites**

Before the first antibiotics were commercially available in the early 20th century, people were delivered helplessly to various kinds of infections like pest, cholera, and tuberculosis, which often reached epidemic proportions and have cost the lives of millions of people [26]. In 1940, quinine was used against malaria, the arsenic derivative arsphenamine, Salvarsan, was used against syphilis, and sulfa drugs like Prontosil were used against mainly Gram-positive cocci infections. However, most agents of infectious diseases were still untreatable. The situation improved radically with the

detection of the first beta-lactam antibiotic, penicillin, produced by the mold *Penicillium rubens* [27]. Henceforth, soil organisms like fungi [28] and bacteria [29] as producers of secondary metabolites with bioactive properties moved into the focus of research. The Golden Age of Antibiotics started. Aminoglycosides [30], tetracyclines [31], and macrolides [32] are only some examples of important antibiotic classes, discovered in those days. Numerous pharmaceutical companies participated on large-scale screening activities of antibiotic producing organisms, mainly actinobacteria [33]. However, in most cases, it took only a few years from the launch of a new antibiotic to the detection of the first resistant germs [34]. Incorrect use in human medicine, incorrectly prescribed antibiotics, extensive agricultural use and fast spread of resistant bacteria caused by increasing mobility led to substantial problems with multi-drug resistant bacteria. Some of the most problematic germs belong to the so-called ESKAPE-panel: *Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter* spp, are mainly responsible for nosocomial infections. Since the 1960s more and more companies retracted from the time- and cost-consuming screening procedures. Of the 18 largest pharmaceutical companies, 15 abandoned the antibiotic field [35]. Indeed, from the late 1960s through the early 1980s, the pharmaceutical industry introduced many new antibiotics to solve the resistance problem. After that the antibiotic pipeline began to dry up and fewer new drugs were brought to market [36]. This led to a dangerous bottleneck of currently available reserve-antibiotics and a widely held concern over the lack of innovation and productivity in the research and development of novel bioactive substances [37]. Eligible countermeasures include the development of synthetic and semi-synthetic drugs, evaluation of rediscovered drugs and the classical screen of natural secondary metabolite producers. Here, especially new genera and species are of great interest [38]. But for natural production of secondary metabolites in large-scale fermentation processes the corresponding producer strains have to be isolated from nature. Maintenance, cultivation, and upscale are challenging. Beside the appropriate expertise and equipment for fermentation and isolation of substances from the fermenter broth, for every producer strain the specific biotic and abiotic conditions need to be determined. Myxobacteria for instance are one of the most promising natural product producers, but demanding with regard to isolation and large-scale cultivation. Successful handling of these organisms places special challenges to microbiologists and biotechnologists in equal measure.

Myxobacteria are among the best natural product producers, together with actinomycetes [39], *Bacillus* species [40], and fungi [31]. Even shortly after their discovery, scientists described predatory and cellulolytic action of myxobacteria. Already in 1947, Singh complained that many antibiotics were isolated from various groups of microorganisms, except myxobacteria [41]. He observed that some species of the Myxococcaceae lyse living bacteria, including Gram-negatives such as *Pseudomonas fluorescens* and *Bacterium (Escherichia*) *coli,* and concluded that a detailed study of myxobacteria may be profitable in discovering new antibiotics. In 1955, Mathews and Dudani investigated the lysis of human pathogenic bacteria by myxobacteria [42] and in 1962, Noren and Raper described the antibiotic activity of myxobacteria in relation to their bacteriolytic capacity [43]. But, it took another 15 years until the first antifungal metabolite, ambruticin, was isolated from a *Sorangium* strain (Figure 3) [44].

**Figure 3.** *Sorangium* sp. strain Soce 1014, an ambruticin-producer, swarming on VY/2-agar and the structure of ambruticin A, the first secondary metabolite which was isolated and described from myxobacteria.

The majority of myxobacterial compounds are polyketides, non-ribosomal polypeptides, and their hybrids, terpenoids, phenyl-propanoids, and alkaloids [45]. Many of these substances show promising activities against bacteria [46,47], viruses [48], fungi [49], cancer cells [50] immune cells [51], and malaria [52], respectively, as well as unusual modes of action [53]. Many strains produce metabolites belonging to multiple structural classes, as well as a number of chemical variants on each basic scaffold [48]. Whole-genome sequencing of several myxobacterial strains like *Sorangium cellulosum* [54] and *Myxococcus xanthus* [55] has revealed that the secondary metabolite potential is far greater than that suggested by fermentation under standard laboratory conditions.

It is, of course, possible to isolate new substances from known (myxobacterial) species [51,52]. But again: the low hanging fruit have long been harvested and it is more likely to find new substances in new families, genera and species [53,56–61]. The study of Hoffmann et al. confirmed this [41]. The authors found a correlation between taxonomic distance and the production of distinct secondary metabolite families, and supported the idea that the chances of discovering novel metabolites are greater by examining strains from new genera rather than additional representatives within the same genus. For comprehensive overviews about secondary metabolites produced by myxobacteria and their mode of action, I recommend Weissman and Müller [48] and Herrmann et al. [49].

#### **4. The Great Plate Count Anomaly and Microbial Biogeography**

Based on cultivation, approximately only 1% of the naturally occurring bacterial community is known and characterized so far [62]. Most bacterial groups remain uncultured and uncharacterized, because appropriate culture conditions are lacking [63]. This Great Plate Count Anomaly is the oldest unresolved microbiological challenge. The Austrian microbiologist Heinrich Winterberg was the first who described this phenomenon in 1898 [64]. Winterberg observed that the number of microbial cells in his samples did not match the number of colonies formed on nutrient media. Since Winterberg, numerous authors who investigated bacterial communities in different habitats confirmed this phenomenon. The establishment of culture independent analytical methods in the early 1990s greatly expanded the dimension of knowledge about the bacterial diversity again [65]. Estimations, that about 80% of bacteria resist standard laboratory cultivation approaches were obsolete after publication of the first culture-independent analyses of bacterial communities, which were based on 16S rRNA-coding genes. Now, the estimated amount of uncultivable species has increased to 90–99% and it can be assumed that many of these uncultured bacteria could be probably a source for new antibiotics [66].

Notwithstanding the frequent discovery and description of new species/genera, the real number of myxobacteria is unknown. The current knowledge about the diversity of organisms is always just a snapshot. However, several (NCBI) 16S rRNA-sequences of cultures belong to the order Myxococcales, but are only distantly related to valid type strains (up to 12% distance) and therefore probably belong to new species, genera, or even families. One example: "*Anaeromyxobacter dehalogenans*" strain WY75 (Acc. no. KC921178) was isolated from ginger foundation soil and shows highest similarity (87.4%) to the type strain of *Sandaracinus amylolyticus.* It is therefore at least a representative of a new myxobacterial family (Figure 4). Nevertheless, as long as a valid publication of such strains in taxonomic journals as for example *IJSEM* or *Antonie van Leeuwenhoek* is absent, even the current diversity of cultivable myxobacteria is not fully reflected.

**Figure 4.** Neighbour joining tree with myxobacterial type strains shows the phylogenetic position of strain WY75, cultivated from ginger foundation soil, within the Sorangiineae suborder. Comparison of 16S rRNA sequences revealed only 87.4% similarity to the next myxobacterial type strain *S. amylolyticus*. Accession numbers are in brackets. Bar, 0.1 substitutions per nucleotide position.

Although there are numerous reports about cultivable myxobacteria in soils and other habitats [1], it has to be considered that myxospores may tolerate considerable environmental extremes. Most isolation techniques involve the cultivation of extensively dried samples [10]. Species which are present in the sample as vegetative cells will probably not survive this process and therefore will not grow on the isolation plates. Also, and irrespective of the detection method used, it is difficult to determine whether myxobacteria were present as dormant spores or metabolically active vegetative cells in the environmental sample taken [12]. The standard procedure to isolate myxobacteria is drying the sample (soil, plant material, etc.) at 30 ◦C to reduce growth of undesired bacteria and fungi, and subsequent placement on water agar with *E. coli*-bait (to attract predators) and on Stan 21 agar with filter paper (for cellulose decomposers), respectively (Figure 5). As the degradation of biomacromolecules like microbial cells (*E. coli* bait) or cellulose requires a sufficient amount of viable myxobacterial cells in the sample, underrepresented species will probably not be able to start growing.

**Figure 5.** Common isolation procedure for myxobacteria: Soil/environmental sample is placed on **a.** Stan 21 with filter paper to enrich cellulose decomposing strains and **b.** on water agar with *E. coli* bait (cross) for predators. Numerous transfers of fruiting bodies/swarm edge material to fresh plates are necessary to purify myxobacteria.

As was mentioned at the beginning, myxobacteria live in various habitats. It is recommended, but not mandatory, to investigate uncommon habitats from different geographic regions with regard to new secondary metabolite producers. But, already within microscale areas of environmental samples, different strains of one myxobacterial species show surprising genetic differences, as biogeographical studies of myxobacteria revealed. Biogeography is the study of the distribution of organisms across space and time [67]. As mentioned by Ramette and Tiedje, prokaryotic biogeography is "the science that documents the spatial distribution of prokaryotic taxa in the environment at local, regional, and continental scales" [68]. Hanson et al. propose that four processes, selection, drift, dispersal, and mutation, create and maintain microbial biogeographic patterns on inseparable ecological and evolutionary scales [69]. For example, Bacteria and Archaea are globally distributed [70]. At the class level, the β-proteobacteria, cyanobacteria, actinobacteria, and flavobacteria have been shown to display worldwide distribution in marine or terrestrial ecosystems [71–73]. According to Hedlund and Staley, at the genus level, many prokaryotes have a cosmopolitan distribution in their respective habitats [74]. Recent global surveys indicate that most bacteria are restricted to broad habitat types, as there is little overlap among bacterial taxa found in soils, sediments, freshwater, and seawater [75,76]. Dawid gave a comprehensive overview about the ecology and global distribution of myxobacteria in the macroscale range [1]. The study was based on data given in the literature as well as on his own analyses of almost 1400 soil samples from 64 countries and all continents. The study found that an exceptionally high average species number was determined for soils from countries that belong to the winter rain climates of the Mediterranean type, the permanent wet rain forest climates and the tropical semi-desert climates. However, soils of countries with cold temperate coniferous forest climates and cool temperate intermediate climates with peat mosses and coniferous forests harbor a low average number of species. Jiang et al. determined biogeographic patterns of myxobacterial taxa in deep-sea sediments [77]. They screened DNA from four different depths for myxobacteria-like 16S rRNA genes and provided the first evidence, that marine myxobacteria are phylogenetically distinct from terrestrial species. Brinkhoff et al. studied the biogeography and phylogenetic diversity of marine myxobacteria and found a deep-branching monophyletic cluster of exclusively marine myxobacteria within the Myxococcales [78]. Wielgoss et al. sequenced the genomes of 22 *Myxococcus xanthus* isolates from a 16 × 16-cm-scale patch of soil. They found out "that two closely related *M. xanthus* clades inhabiting the same centimeter-scale patch of soil, display strong sexual isolation, with homologous recombination occurring frequently between members within each clade, but with almost no detectable levels of genetic exchange occurring across clades" [79]. Kraemer et al. resolved the micro biogeography of social identity and genetic relatedness in local populations of *M. xanthus* at small spatial scales [80]. The study comprises samples taken from fruiting bodies, neighboring fruiting

bodies separated by millimeters, neighborhoods of fruiting bodies separated by centimeters and finally soil patches separated by meters and kilometers. They found out that "relatedness decreases greatly with spatial distance even across the smallest scale transition and that both, social relatedness and genetic relatedness are maximal within individual fruiting bodies at the micrometer scale but are much lower already across adjacent fruiting bodies at the millimeter scale." What will this mean with regard to myxobacteria and natural product research? The cellulose degrading genus/species *Sorangium cellulosum* serves as an example: already in 2003, the myxobacterial strain collection of the HZI (former GBF) comprises 7000 strains from which 23.2% belong to *S. cellulosum.* On the other side, *S. cellulosum* strains produced 48.4% of all known secondary metabolites described so far from myxobacteria [81]. This means that closely related strains also have huge potential to produce different chemical and biological bioactive metabolites [48,49] and that the search for new antibiotic producers can be successful in both, small and large scale. For a comprehensive overview about biogeographic patterns of myxobacteria, I refer to Velicer et al. [82]. For a deeper insight to prokaryotic biogeography, see the study of Ramette and Tiedje [73] and the review of Hanson et al. [74].

With regard to numerous studies based on cultivation-dependent approaches, the number of publications that focus on the non-cultivable myxobacteria is comparatively small. Nevertheless, there are about 4000 (often unpublished) 16S rRNA sequences deposited at the NCBI database which are mentioned to be "uncultured Myxococcales". Under consideration of further myxobacteria-related sequences which are just deposited as "uncultured (delta) proteobacterium" [83] or even "uncultured bacterium", the true extent of uncultivated myxobacteria can just be surmised. Most of the deposited sequences are "by-products" from cultivation-independent studies of bacterial communities in general, without special focus on Myxococcales.

To give an impression about the diversity of cultivable and uncultivable myxobacteria in different habitats, published and unpublished 16S rRNA sequences from NCBI are compared with each other and the results are summarised subsequently.
