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1 Marine Actinobacteria

1.1 Introduction

Over the last years, the commercial natural product research came back into focus because “the pipeline for new antibiotics is running dangerously low” (Fenical and Jensen 2006). While the rate of newly discovered antibiotics from soil inhabiting Actinobacteria decreased, the rate of re-isolation increased (Fenical et al. 1999; Lam 2006). However, to further explore this promising source of novel bioactive secondary metabolites, new strains had to be isolated with alternative methods or in unexplored environments (Lam 2006). Therefore, not only isolation techniques, but also the sampling sites had to be altered. With 70 % of the earth surface and a microbial abundance of 106 per mL in sea water and 109 per mL in ocean bottom, oceans are the world’s biggest environment (Fenical and Jensen 2006). However, not only sea water and sediments are microbial rich environments, also marine organisms, flora and fauna like sponges harbor abundant communities (Ward and Bora 2006).

1.2 Marine Actinobacteria?

Over a long time, it was not clear whether truly “marine” Actinobacteria really exist because of the fact that there has been a lot of re-isolation of terrestrial strains and known compounds (Moore et al. 2005). The explanation for this assumption was the wash-in from terrestrial spores into the sea (Goodfellow and Haynes 1984) and the sampling problems of marine samples which were taken mostly close to the coast (Fenical and Jensen 2006). However, in the year 1984, the first marine Actinobacterium was found: Rhodococcus marinonscens (Helmke and Weyland 1984). 7 Years later, in 1991, the first marine Actinobacteria genus “Salinispora spp.” which obligately requires seawater for growth (Jensen et al. 1991) was published. But even with the application of DNA sequence-based methods and the corresponding ability to analyze the relationships between this genus and their terrestrial relatives, the first seawater-obligate Actinobacteria genus “Salinospora” (grammatically incorrect; corrected to Salinispora) was described in 2005 (Mincer et al. 2005) and its two species Salinispora tropica and Salinispora arenicola were published (Maldonado et al. 2005a). Moreover, with the help of the type strains of these species, which are actively growing in sediment samples, the metabolic activity in the natural marine environment was demonstrated (Mincer et al. 2005). In addition to this finding, Fenical and Jensen (2006) also detected uncommon secondary metabolites produced by Salinispora strains and were inspired to further search for new groups of marine Actinobacteria. With the help of 16S analysis of the phylogenetic diversity as well as new cultivation approaches, Stach and Bull (2005) demonstrated that deep-sea sediments contained more than 1300 different actinobacterial operational taxonomical units which led to the assumption that there is a great opportunity to find novel species and genera. Fenical and Jensen (2006) cultivated diverse strains within six Actinobacteria families and many of them seemed to represent new taxa like Salinispora and Mariniphilus (Fig. 1). In addition to the taxonomic findings and therefore the demonstration of the existence of marine Actinobacteria, these strains turned out to be “excellent producers for secondary metabolites”.

Fig. 1
figure 1

Tree illustrating the phylogenetic relationships of 13 groups of marine-derived actinobacteria within six different families. The taxonomic status of the MAR groups is not really clear whereas it is known that they include numerous of new species. However, the MAR1 group was described as the genus Salinispora and the MAR2 group as the genus Marinispora (modified after Fenical and Jensen 2006)

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1.3 Where Can One Find Marine Actinobacteria?

The geographical origin of the new actinobacterial producer strains, published compounds and bioactivities of 67 % of marine natural products (up to 2003) was restricted to Japan, the Mediterranean as well as the Western Pacific Ocean (Blunt et al. 2007; Bull and Starch, 2007). However, because of the growing focus on the research on marine natural products, a dramatic rise of the published data from the China Sea was observed just one year later (Bull and Starch 2007). Marine Actinobacteria are present in diverse marine habitats, which are widespread over the ocean. These habitats are influenced by numerous geographical as well as physical parameters like temperature and salinity. Furthermore, they underlie geochemical impacts and ocean currents. But also ecosystems like salt marshes, wetlands, estuaries, continental shelves as well as the open ocean and the deep sea are habitats for specialized marine Actinobacteria (Ward and Bora 2006). The marine habitat starts with the sea surface microlayer, followed by the water column, from a few millimeters below the surface to more than 10,000 m depth, down to the sea floor with the micro- and macro-fauna and-flora, which were used as host for epibiosis and symbiosis, as well as the sea subfloor and deep biosphere. Within the habitat of the sea floor, varying sediments of varying geology, mineral nodule fields, carbonate mounds, cold seeps, hydrocarbon seeps, saturated brines, and hydrothermal vents were observed (Ward and Bora 2006).

The sea surface microlayer is an environment which is to date poorly characterized. However, some studies showed the existence of Actinobacteria and Proteobacteria within this habitat (Ward and Bora 2006). In the water column, together with the β- and δ-Proteobacteria, Firmicutes, Cytophaga-Flavobacter-Bacteriodes (CFGs) and Chlorobia, Actinobacteria belong to the mid-range of abundance. α- and γ-Proteobacteria dominated this habitat. Interestingly, theses compositions of strain collectives were also found in coastal and pelagic waters, despite differences in isolation techniques used and scale (Ward and Bora 2006).

Marine sediments are the best known source for the isolation of Actinobacteria from marine habitats. Studies on the 16S rRNA, to detect the phylogenetic diversity of Actinobacteria in marine sediments, showed that some deep-sea sediment contained up to 13,000 different actinobacterial operational taxonomic units which are forecast to belong to a large number of novel species and genera (Stach and Bull 2005). Also cultivation approaches of a range of depths and sediments (Maldonado et al. 2005b; Kim et al. 2004; Takami et al. 1997) illustrate the diversity and new insights in Actinobacteria classification (Maldonado et al. 2005a) and biogeography. These insights were used as inspirations for the isolation and recognition of novel marine Actinobacteria (Magarvey et al. 2004; Ward and Bora 2006). Furthermore, molecular studies on three different structural types of stromatolites (“organisms that have been present in the fossil record from greater than 3.5 billion years ago,” cf. Ward and Bora 2006) ensured the presence 6–9 % of actinobacterial clones in the their complex mat community (Papineau et al. 2005).

However, there are not only the upper centimeters of marine sediments harboring Actinobacteria but also the below sea subfloor and the deep biosphere were found to be a natural habitat for these bacteria. Up to a depth of 800 m, a minor fraction of Actinobacteria were isolated (Stach et al. 2003; Inagaki et al. 2003) and molecular studies with specific primers showed a high actinobacterial diversity in clone libraries (Stach et al. 2003).

Moreover, marine Actinobacteria even live in marine organisms like many free swimming as well as sessile vertebrates and invertebrates. These organisms are mostly known to produce bioactive metabolites but these substances are often produced by symbiotic living microorganisms. The pufferfish, for example, was for a long time supposed to be the producer of the potent neurotoxin tetrodotoxin (TTX). However, to date this substance is known to be produced by numerous of marine organisms. Additionally, the production could also be correlated to many taxa of marine bacteria which live in symbiotic relationships with these organisms. In case of the pufferfish, a TTX-producing Actinobacteria (closely related to Nocardiopsis dassonvillei) and some TTX producing Bacillus strains were isolated out of its ovaries which are known to harbor high levels of TTX (Wu et al. 2005; Ward and Bora 2006). With the help of a sodium channel blocker assay using a mouse neuroblastoma cell culture bioassay, toxicity levels of 0.1–1.6 MU/g bacteria cells were observed [MU: amount of toxin which was expressed, calculated from the observed cell ratio (relationship between survival cell ratio and authentic TTX amount)] (Wu et al. 2005; Ward and Bora 2006). Furthermore, some TTX producing Actinobacteria like Micrococcus spp. and Streptomyces spp. were isolated out of deep sea and marine sediment (Do et al. 1990, 1991) which support the assumption that the symbiotic living organisms are responsible for the TTX production. However, to date the biosynthesis of TTX in bacteria or other organisms has not been described (Chau et al. 2011).

Furthermore, marine invertebrates such as sponges were described as natural habitat for marine Actinobacteria. They are known over a long time to be a prolific source of bioactive substances. Because of the reason that they are sessile organisms, they use the bioactive metabolites as a kind of chemical defence (Hill 2004; Ward and Bora 2006). However, up to 35 % of sponge biomass is comprised of microorganisms, which build an abundant microbial community (Hentschel et al. 2012; Webster and Taylor 2012; Steinert et al. 2014). Sponge-associated bacteria have been also frequently described as producers of bioactive natural products (Blunt et al. 2011, 2012, 2013). These communities include amongst other Actinobacteria also Salinispora and related strains (Hentschel et al. 2002).

1.4 Isolation of Marine Actinobacteria

To date, the most common sources for the isolation of marine Actinobacteria are sediments and sponges. However, as mentioned above, Actinobacteria were found almost everywhere in the marine environment. Because of the large amount of bacteria living in the marine sediment (109 bacteria per mL, Fenical and Jensen 2006), the cells have to be separated using diverse dilution or stamp techniques.

The common method for the isolation of marine Actinobacteria is very similar to the methods used to isolate terrestrial ones. On the one hand, sea mud can be directly spread over the agar media (Okami and Okazaki 1972) or the sediment samples may be diluted and treated with diverse methods as described below.

Some sediment samples were mixed with sterile sea water (Jensen et al. 1991; Mincer et al. 2002) or used as dried samples (Takizawa et al. 1993) before they were heated up between 6 and 60 min (Mincer et al. 2002; Jensen et al.1991, Takizawa et al. 1993) at about 50 °C. This heat-shock treatment should dispatch most non-spore forming bacteria to provide the slow growing Actinobacteria a selective benefit for growth. Such measures had to be taken to avoid that, as described in the concurrent paper of Karwehl and Stadler (2016) for fungi, the fast-growing strains in soil samples would inadvertently overgrow the more interesting, hitherto unexploited ones. Afterwards, the samples were diluted in several dilution steps and plated on different types of nutrient rich agar plates. Pathom-Aree et al. (2006) used sterile saline solution (Ringer’s solution) to pre-incubate the sediment samples for 30 min before the dilution and the following inoculation on a range of different media and an incubation temperature of 55 °C for the isolation of new thermophilic Actinobacteria taxa.

Another dilution technique was described by Mincer et al. (2002) for the isolation of the first Salinispora species. In this study, in addition to the heat-shock dilution series approach, the wet sediment was air dried and afterwards pressed into a sterile form plug. This plug was used as a stamp to inoculate the agar plates by stamping the plug several times in a circular fashion onto the plate to cause a dilution of the sediment. This approach was used with and without a previously described heat-shock treatment.

For the isolation of “novel marine-derived Actinobacteria taxa” Magarvey et al. (2004) used a modification of the medium Stan21, which is normally used to isolate Myxobacteria from soil samples (Shimkets et al. 2004). Therefore, the yeast extract was eliminated and the distilled water was replaced by artificial sea water. After the media preparation and the addition of cycloheximide, filter paper disks were placed on the agar plates and the wet sediment was spotted on the surface of the cellulose. The plates were incubated afterwards for 30–90 days at 30 °C in a humidified chamber. With this method they isolated some unknown marine Actinobacteria which cluster between the Salinispora clade and the genus Micromonospora.

One possibility for the isolation of marine Actinobacteria out of seawater is a simple filter technique. Okami and Okazaki (1972) described a method by which the seawater was centrifuged and filtered through a 0.3 µl pore size filter. The concentrated microorganisms on the filter were afterwards suspended in filtered sea water and plated on different isolation media.

To isolate Actinobacteria out of sponges several approaches are described. In classical isolation techniques, the sponge samples were directly stamped on isolation media or, in the dilution series method, sponge samples are crashed with a mortar, placed into sea water and after sedimentation, plated in different dilutions on specific sponge isolation agar (Mantalvo et al. 2005; Jiang et al. 2007). However, there are also some modifications of this method. Abdelmohsen et al. (2010) added the supernatant of crashed sponges (sponge extract) into the isolation media to rebuilt the natural environment. A further approach is the addition of both, “aqueous extract” like described before and “organic extract” where the sponge tissue was extracted with the help of hexane, dichlormethane and methanol. Finally, both studies demonstrated an enhanced number of novel isolated strains (Webster and Hill 2001; Selvin et al. 2004; Abdelmohsen et al. 2010). However, for all of these approaches, the sponges had to be harvested. To keep the sponges alive, Steinert et al. (2014) constructed a so called diffusion growth chamber (DGC). This chamber was built out of two combined centrifuge microfilter sections and was inoculated with different types of media. The media compositions differed in the amount of nutrients. Furthermore, every medium includes a small amount of sponge homogenate which was prepared out of a homogenized sponge sample in sterile sea water. After inoculation, the DGCs were directly inserted in the living sponge and retaken after 4 weeks of incubation (Fig. 2). Subsequently, one part of the inoculated media was plated on isolation media and the other part was used to inoculate new DGCs which were inserted into the same sponges. In this study, the authors showed that they were able to detect and cultivate more bacteria than using the classical direct plating method while the sponges stayed in their natural environment.

Fig. 2
figure 2

Diffusion growth chamber (DGC) for in vivo cultivation of sponge-associated bacteria. Build-up of DGC out of two combined centrifuge microfilter sections with diffusible membranes (DM), inoculated with different types of media within the chamber (AC) and the plastic holder (H) for fixing within the sponge

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Most of the isolation media contained fungicidal agents like cycloheximide and nystatin to reduce fungal contamination as well as rifampicin and nalidixic acid to dispatch fast-growing Gram-negative bacteria. All plates were incubated between 2 and 6 weeks between room temperature and 28 °C.

1.5 Bioactive Substances Produced by Marine Actinobacteria

While we concentrate in this review on the bioactive secondary metabolites like antibiotics, cytotoxic agents and fungicides, marine Actinobacteria also produce melanins, enzymes, enzyme inhibitors, single cell proteins as well as probiotics which can be used for example in aquaculture (Manivasagan et al. 2013).

Novel metabolites isolated from marine Actinobacteria include the antitumor agents chromomycins B, A2 and A3 from Streptomyces coelicolor (Lu et al. 2012), the antibacterial frigocyclinone from Streptomyces griseus (Bruntner et al. 2005), the antifungal and anticancer agents, daryamides from Streptomyces sp. (Sivakumar et al. 2007) and further ones as listed in Table 1. However, it becomes apparent that most of the producer strains were classified as Streptomyces species, which are common terrestrial bacteria. To date, “real new structures” were only isolated from the two genera of genuine marine Actinobacteria Salinispora and Marinospora (Manivasagan et al. 2013). Salinosporamides A and B (Fig. 3) are both produced by Salinispora tropica. These compounds are β-lactone-γ-lactams produced by a mixed PKS/NRPS biosynthesis. Salinosporamide A is an orally active proteasome inhibitor that induced apoptosis in multiple myeloma cells with mechanisms distinct from the commercial proteasome inhibitor anticancer drug Bortezomib (Chauhan et al. 2005). As NPI-0052, salinosporamide A entered as the first compound isolated out of an obligate marine organism, the clinical studies in multiple phase I trials for solid tumors, lymphoma, and multiple myeloma (http://www.nereuspharm.com/NPI-0052.shtml). The second compound, salinisporamide B, differs only in a lack of chorine and the subsequently less activity by a factor of 500 (Manivasagan et al. 2013). However, the terrestrial Streptomyces strain JS360 is the producer strain of the cinnabaramides A-G which are structurally close related to salinosporamide A (Stadler et al. 2007). These substances are also described as strong proteasome inhibitors. However, Rachid et al. (2011) detected a significantly weaker cytotoxic effect then caused by salinosporamide A. Other unprecedented substances produced by Salinispora tropica are sporolide A, arenicolide A, cyanosporaside A, and salinipyrone A (Fig. 3). Even though, the salinipyrones A and B (Fig. 3) were first isolated out of the obligate marine Actinobacterium Salinispora pacifica (Oh et al. 2008). Both substance did not show antibiotic activity against drug resistance human pathogens, however a moderate cytotoxic activity was detected (Jensen et al. 2007; Manivasagan et al. 2013).

Table 1 Examples of secondary metabolites produced by marine actinobacteria
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Fig. 3
figure 3

Examples of new bioactive compounds and their corresponding structures produced by novel genera of marine actinobacteria

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Marinomycin A (Fig. 3), produced by a Marinophilus strain (Kwon et al. 2006), is a new polyene macrolide with a high toxicity to tumor cells and antibiotic effect against vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus.

In conclusion, the discovery of the new marine Actinobacteria genera Salinispora and Marinophilus could be directly correlated to the new and unprecedented compounds with new structures and partially new modes of action. Like described before, molecular studies indicate a great potential for the isolation of novel genera of marine Actinobacteria. However, new isolation techniques will have to be established and unexplored environments have to be sampled to avoid re-isolations of bacteria out of known genera producing common compounds.

2 Myxobacteria—The Underestimated Bacterial Resource

2.1 History of Myxobacteria

The first myxobacterium was discovered in 1809 by the German botanist H.F. Link and named Polyangium vitellinum, but erroneously it was characterized as fungus because of the characteristic fungi-like life cycle (Link 1809). It took many years, until 1892, until Roland Thaxter identified these organisms as bacteria (Thaxter 1892). Actually, the order of Myxococcales consists of 55 species including 28 genera (Fig. 4) and differentiates from other Gram-negative prokaryotes by the mutuality to have a special life cycle.

Fig. 4
figure 4

Current taxonomy of the order Myxococcales

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Myxobacteria belong to the δ-subgroup of proteobacteria and therefore are Gram-negative. The vegetative cells are rod-shaped with the ability to glide over solid surfaces and form some kind of multicellular, species-specific “fruiting-bodies” under starvation conditions without access to a sufficient nutrient storage (Reichenbach et al. 1988) (Fig. 5). Those fruiting bodies can comprise up to 105 individuals and show a wide range of differences between the genera and species referring their height, shape and color, which mostly varies from yellow, orange or red until brown or even black (Reichenbach 1983; Garcia and Müller 2014a, b, c, d, e).

Fig. 5
figure 5

a Fruiting bodies of Stigmatella aurantiaca (bar = 1000 µm); b Cells of Myxococcus virescens (bar = 50 µm); c Swarming of Corallococcus coralloides (bar = 5000 µm)

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Within the fruiting bodies, the vegetative cells transform to short, so called myxospores, often enclosed in sporangioles. Because of desiccation resistance, myxospores are able to survive in unfavorable environmental conditions for many years (Reichenbach et al. 2006a, b). Myxobacteria can be found in large populations on many substrates in nature all over the world, e.g., soil, rotting wood, and other habitats. Due to their nutritional requirements, myxobacteria can be divided into two ecological groups that are also in agreement with their phylogeny. Predators use other bacteria or yeasts as food source, and cellulose decomposers, belonging to the genera Sorangium and Byssovorax, decompose organic materials by producing different types of lytic exoenzymes (Reichenbach et al. 1988; Reichenbach et al. 2006a, b).

Another important and interesting feature of myxobacteria is their ability to produce a broad range of structurally diverse secondary metabolites, many of them with bioactivity. The working group Microbial Strain Collection at the Helmholtz-Center for Infection Research (HZI) in Braunschweig, has the largest collection of myxobacteria worldwide with more than 9000 strains, including all validly described type strains (Fig. 6).

Fig. 6
figure 6

Number of strains at the HZI

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The major part of the collection is represented by species of the frequently occurring genera Corallococcus, Myxococcus, Nannocystis, and Sorangium (Gerth et al. 2003). Chondromyces, Myxococcus, and Sorangium are producers of most of the interesting natural products, consequently they became enriched in the collection (Fig. 7).

Fig. 7
figure 7

Percentage of genera in the producers of our novel secondary metabolites from myxobacteria (modified according to Gerth et al. 2003)

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2.2 Isolation Methods

To find new bioactive substances, it seems reasonable to exploit new producer organisms and to insert these novel species and genera in the latest taxonomic classification (Müller and Wink 2014). It is necessary to use basic routine methods (Dawid 2000; Shimkets et al. 2006) as well as new approaches (Garcia et al. 2009; Mohr et al. 2015) for the isolation and cultivation of potential and interesting new producer strains.

In general, myxobacteria are characterized as mesophilic soil organisms which prefer a temperature of 30° and a neutral pH. Nevertheless, also acidophilic (Brockman and Boyd 1963), alkaliphilic (Hook 1977) and psychrophilic (Dawid et al. 1988) species were found in the last decades, demonstrating the enormous adaptability of myxobacteria even to extreme uncomfortable habitats. Myxobacteria can also be found in marine, saline habitats, for example Haliangium ochraceum and Haliangium tepidum (Iizuka et al. 1998). To isolate new myxobacterial strains from different biotopes, environmental samples (e.g., soil, sand, dead wood, bark, leaves, compost) were treated by a standardized procedure. For soil samples, an amount of 50–150 g of sample is collected in 1–5 cm depth below the surface in sterile vials. Before the enrichment on different plates, the sample has to be air dried and characterized concerning different parameters like color, size, grain, content, and acidity (Dawid 2000). Commonly, different types of plates are used for the enrichment of a wide range especially of myxobacteria species referring to the methods of Reichenbach et al. (2006). Living E. coli cells on water agar are suitable as the feed organism for predatory myxobacteria (Shimkets et al. 2006). Many soil bacteria are able to degrade dead, but not living, microorganisms. Furthermore, most soil bacteria grow faster than myxobacteria. Using living bait organisms, myxobacteria have an advantage in comparison to other, not or less swarming, competitors (Garcia et al. 2009). To enrich and isolate specifically cellulose degrading myxobacteria, mineral salt agar Stan 21 is used. Sterile filter paper is placed on the top of the agar and serves as food source (Shimkets et al. 2006). The plates are normally incubated at 30° over a period of several weeks, until the visually striking myxobacteria can be seen with the naked eye. The following isolation, purification, and identification of new strains are carried out on VY/2 plates, containing yeast as nutrient source (Dawid 2000). It is supposed, that the preparation of the medium, combining the important characteristics like in the natural habitats of myxobacteria, leads to high advantage finding novel strains. For example, novel strains were isolated by growing at room temperature (23°) as well as under white light or sun light exposure and on acidic or saline agar plates (Mohr et al. 2015; Garcia et al. 2009). The identification of the facultative anaerobic genus Anaeromyxobacter (Sanford et al. 2002) leads to the assumption, that different air conditions tried within the isolation procedures can reveal facultative anaerobic and microaerophilic species (Garcia et al. 2009). Culture-independent methods, like clone bank analyzes, revealed that there is a high number of new potential producer species (Mohr et al. 2015). The development of new cultivation methods is important to find the best growth conditions for all these different types of uncultivated myxobacteria.

The procedure of the standardized screening method, developed at the HZI, starts with the cultivation of a new isolated strain in different liquid complete media (each 100 ml) with different C- and N-sources. The bacteria are cultivated at 30 °C and shaking (180 rpm) 7–14 days. Myxobacteria secrete the secondary metabolites out of the cells into their environment, in this case into the medium. To bind the metabolites and thereby preventing a feedback inhibition or a degradation of the metabolite by a producer strain, XAD16 adsorber resin is added to the culture. After sieving, the resin, and the adsorbed compounds are extracted with acetone, evaporated and finally eluted with methanol, resulting in a raw extract of a 1:100 concentration which is used for further analyzes (Reichenbach and Höfle 1993). However, it is necessary to find the best growth conditions and to optimize the specific production rate for each producer strain, as well as an economic justifiable fermentation process has to be established.

To investigate the effect of a crude extract from a new potential myxobacterial producer strain against different pathogens with clinical importance and to find the active principle, the working group Microbial Strain Collection at the HZI use a dereplication system, which is a combination of a biological activity assay and a chemical screening with liquid chromatography-high resolution mass spectrometry (LC/HRMS). First, the minimum inhibitory concentration (MIC) of the extract is determined by the use of a microtiter plate based serial dilution assay containing different Gram-positive and Gram-negative bacteria as well as fungi to assess selectivity. After semipreparative high performance liquid chromatography (HPLC), the inhibitory effect of the fractionated extract is examined against the affected organisms and further analyzed by ultra-high resolution-time of flight mass spectrometry (UHR-TOF-MS) regarding molecular masses and molecular formulae (Fig. 8). These obtained molecular features are matched against known myxobacterial compounds from the in-house database (Myxobase) or Dictionary of Natural Products (Taylor and Francis Group 2016). The strategy consisting of the use of the different methods above gives possibilities to find and identify new bioactive substances from myxobacteria.

Fig. 8
figure 8

Combination of activity assay and HPLC-MS

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2.3 Genetical Potential

The genomes of myxobacteria belong to the largest ever found in bacteria. The genome of the Sorangium strain So ce56, for example, consists of more than 13,000 base pairs and includes seventeen secondary metabolite gene clusters (Schneiker et al. 2007) and the genome of M. xanthus comprises at least around 9000 base pairs (Goldman et al. 2006). It is suggested that this large genome size is connected to the extraordinary and complex life cycle of myxobacteria. In contrast to other bacteria, they need a much more complex genetic constitution to demonstrate their special social behavior including swarming and culminating fruiting bodies. Within these enormously large genomes, there probably exists a high capacity concerning to the unlimited number of unknown and promising secondary metabolites (Wenzel and Müller 2009).

It is hardly possible to figure out the potential of a strain to produce bioactive substances only by phenotypic and biochemic analyzes, because some metabolites cannot be detected due to the applied extraction and detection methods (Wenzel and Müller 2009). Therefore, our research is focusing on the discovery of biosynthetic pathways and the connected gene clusters of novel, potentially anti-infective natural compounds as well as the full genome sequences of scientific interesting bacteria strains. With this information, it might become easier to find possibilities for enhancing the production of known metabolites as well as activating unused “silent” genes for novel substances (Müller and Wink 2014). The genome mining approach is based on the genome sequences, which are used to predict synthesized compounds with the help of bioinformatics analyzes. The recovered genetic information can lead to specific inducing of the supposed biosynthetic gene clusters that encode for new bioactive products (Müller and Wink 2014).

2.4 Pharmaceutically Important Secondary Metabolites

Myxobacteria are a rich source of novel and unique secondary metabolites, mainly polyketides and nonribosomal polypetides. Many of these metabolites show antibiotic activities and are urgently needed as new drugs for a broad range of applications. These skills probably evolved because of the natural competition between different kinds of organisms in varying habitats. For example, the cellulose degrading members of the genus Sorangium have to combat other cellulose degraders sharing the same habitat like fungi which also use wood (cellulose) as nutrient source, whereas proteolytic myxobacteria need to stand up to other degraders of decaying organic material (Gerth et al. 2003). The existence of a link between the production of biological active secondary metabolites and microbial predation is supposed, because about 20 % of the known myxobacterial compounds show antibiotic effects. The importance of secondary metabolites derived from natural producers should not be underestimated, because they are the source of almost 50 % of most important medications for humans (Demain 1999).

The scientific interest for myxobacteria increased already in the year 1947, when it was shown that a strain of Myxococcus virescens has a significant inhibitory effect on the growth of Staphylococcus aureus (Oxford 1947). In the following years many research groups all over the world tried to find the active compounds by optimizing the growth conditions. It was believed for a long time that it is very difficult to cultivate myxobacteria in liquid medium and that these organisms are somewhat problematic with regard to their axenic growth in general (Reichenbach and Höfle 1993). The breakthrough in discovering antibiotic substances from myxobacteria happened in 1977 by elucidation of the complete chemical structure of the potent antifungal secondary metabolite ambruticin, produced by Sorangium cellulosum (Connor et al. 1977; Ringel et al. 1977). Next to the use as antibiotic or antifungal drug, biologically active secondary metabolites can also be used as antiparasitic, antiviral and antitumor drugs in human and veterinary medicine and also as insecticides, acaricides, and herbicides. They can act, e.g., as inhibitors of carboxylases, polymerases, or mitochondrial respiration as well as inhibitors of eukaryotic protein synthesis (Weissmann and Müller 2009). Furthermore, also antidiabetic, antimalarial, antihypertensive, antihypercholesterolemic, insuline-sensitizing, and immunoregulatory characteristics can be attributed to microbial products (Grabley and Thiericke 1999; Schreurs et al. 2009; Berod et al. 2014). A molecule with biological effects can also work as a model for a synthetic production to get a higher output under better economic conditions (Reichenbach and Höfle 1993). More than 100 new and important myxobacterial core structures have been discovered (Garcia et al. 2009), some of them are summarized in Table 2. Another important ability of myxobacteria is the production of polyunsaturated fatty acids (PUFAs) like eicosaoentaenoic acid (EPA) and docosahexaenoic acid (DHA), which play an important role in food industry and for pharmaceutical applications (Garcia et al. 2011; Gemperlein et al. 2016).

Table 2 Important compounds and their biological activity found in myxobacteria
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Furthermore, the natural compounds can also work as a basic structure for chemical modifications which can lead to an exploitation in the pharmaceutical area. Epothilones A and B, for example, were originally found in a Sorangium cellulosum strain and are distinguished by antifungal and cytotoxic activity (Gerth et al. 1996). Meanwhile, there are modified versions of these molecules already used in the active treatment against different types of cancer or are present in clinical trials. Until epothilones were discovered, the common medication for advanced and early-stage breast cancer was taxanes, anthracyclines, and capecetabine, but these agents are often subjected to a multidrug-resistance, which has a natural origin in the patient. It is suggested that there is an overexpression of efflux pumps and other proteins serving as efflux pumps, so that the anticancer agents can be removed very easy out of the targeted cancer cell (Egerton 2008). In fact, this resistance limits dramatically the chances of success within a therapy (Burger et al. 2003). As an effective alternative to the chemotherapies with taxanes and anthracyclines, the semi-synthetic epothilone derivative ixabepilone (Ixempra®) was developed in October 2007 for monotherapie of different stages of breast cancer (Fig. 9) (Reichenbach and Höfle 2008). In contrast to the other available pharmaceuticals referring to this disease, ixabepilone is just low affected by multidrug-resistant mechanisms and consequently offers a chance to eliminate the tumor cells effectively (Pivot et al. 2007).

Fig. 9
figure 9

Chemical structures of epothilon B and ixabepilon

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