*5.4. Marine/Saline Environments*

Covering more than roughly 78% of the earth's surface, water is the most prevalent natural substance, of which approximately 97.5% is salt water in the world's oceans [96]. The salt tolerance of myxobacteria is low in general. It was assumed for a long time that myxobacteria exclusively live in terrestrial habitats. Indeed, even in 1963, Brockman observed fruiting myxobacteria in sand samples from an ocean beach in South Carolina [97]. Species of the already known terrestrial genera *Archangium*, *Chondrococcus* (*Corallococcus*), *Chondromyces*, *Myxococcus*, and *Polyangium*, could be cultivated. As late as 2002 with *Haliangium ochraceum* and *H. tepidum*, the first myxobacterial genus was isolated and described from coastal salt marshes. The strains differ from known terrestrial myxobacteria with regard to salt requirements (2–3% NaCl) and the presence of anteiso-branched fatty acids [4]. Other genera, exclusively detected in marine habitats like *Plesiocystis* [5], *Enhygromyxa* [6], and *Pseudenhygromyxa* [7] (all Nannocystineae-suborder) followed (Figure 9).

**Figure 9.** Marine myxobacterial type strains on agar plates. (**a**) *Haliangium tepidum* (DSM 14436T) on VY/2SWS, (**b**) *Enhygromyxa salina* (DSM 15217T) on VY/4SWS, (**c**) *Pseudenhygromyxa salsuginis* (DSM 21377T) on 1102, (**d**) *Plesiocystis pacifica* (DSM 14875T) on VY/2SWS.

In 2010, Jiang et al. investigated the diversity of marine myxobacteria in comparison to terrestrial soil myxobacteria [82]. Therefore, they established myxobacteria enriched libraries of 16S rRNA gene sequences from four deep-sea sediments and a hydrothermal vent and identified 68 different myxobacteria related sequences from randomly sequenced clones of these libraries. The authors concluded that the myxobacterial sequences were diverse but phylogenetically similar at different locations and depths. However, they separate from terrestrial myxobacteria at high levels of classification.

In 2012, the study of Brinkhoff and co-workers gave an impressive insight to the marine myxobacterial community [83]. They detected a cluster of exclusively marine myxobacteria (marine myxobacteria cluster, MMC) in sediments of the North Sea, but not in the limnetic section of the Weser estuary and other freshwater habitats. In a quantitative real-time PCR approach, the authors found out that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In addition, in a global survey including sediments from the Mediterranean Sea, the Atlantic, Pacific, and Indian Oceans, and various climatic regions, the MMC appears in most samples and to a water depth of 4300 m, but there was no synteny to other myxobacterial genomes. The study of Brinkhoff et al. showed that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities. Figure 10 shows some representative clones from different marine habitats mentioned in the Brinkhoff study. The 17 clones show 95.1–100% similarity to each other and 88–91% to the next type strain *Sandaracinus amylolyticus.* This implies that members of the MMC cluster do at least belong to new genera if not even families, but presumably belong to the Sorangiineae suborder. However, the genus *Sandaracinus* was published shortly after the Brinkhoff study, so this relative was not mentioned there.

In 2013, Zhang et al. isolated fifty-eight terrestrial and salt-tolerant myxobacteria from the saline-alkaline soils collected from Xinjiang, China [95]. Based on morphology and 16S rRNA gene sequences, the authors identified species of *Myxococcus*, *Cystobacter*, *Corallococcus*, *Sorangium*, *Nannocystis*, and *Polyangium*. They reported that all the strains grew better with 1% NaCl than without salt; some *Myxococcus* strains even grow with 2% NaCl.

Li et al. (2014) chose a cultivation-independent approach to analyze the diversity of myxobacteria from saline-alkaline soils of Xinjiang, China, too. A semi-nested PCR-denaturing gradient gel electrophoresis (DGGE) based on the taxon-specific gene mglA (a key gene involved in gliding motility) was used [98]. In accordance to previous studies, Li et al. also suggested that there are still many viable, but under standard laboratory conditions uncultured myxobacterial strains in the investigated saline-alkaline habitat. Natural product classes discovered from marine Myxococcales strains include polyketides, hybrid polyketide-nonribosomal peptides, degraded sterols, diterpenes, cyclic depsipeptides, and alkylidenebutenolides [99]. Four genera of marine/saline origin are known so far (Figure 10) and from two, *Haliangium* and *Enhygromyxa*, numerous (bioactive) secondary metabolites could be isolated [100]: Haliangicin [101], salimabromide [102], salimyxins, enhygrolides [103], and haliamide [104]. The above-mentioned data reveal that marine/saline environments as oceans harbor

an enormous potential of new myxobacteria. These organisms are an unexplored resource of novel antibiotics of novel chemical scaffolds, as mentioned by Albataineh and Stevens, who highlighted the need for continued discovery and exploration of marine myxobacteria as producers of novel natural products [104].

**Figure 10.** Neighbour joining tree of myxobacterial type strains (16S rRNA-genes), some representative clones from the MMC-cluster (blue) and the next cultivated relative *Sandaracinus amylolyticus*. Genera isolated from marine environment are in green blue. Suborders of the order Myxococcales, origin of clones, and accession numbers are shown. Bar, 0.1 substitutions per nucleotide position.

#### *5.5. Facultative or Strictly Anaerobic Myxobacteria*

All known myxobacteria live aerobically, with one exception: The facultative-anaerobic genus *Anaeromyxobacter* comprises one species, *A. dehalogenans.* The type strain was isolated from stream sediment and grows with acetate as electron donor and 2-chlorophenol (2-CPh) as electron acceptor [11]. Since 2002, several strains of *Anaeromyxobacter* were isolated from various habitats. Flooded rice field soil [105], uranium contaminated surface environment [106], corrosion material of drinking water pipelines [107], arsenic-contaminated soils [108], or chemically and electro-chemically enriched sodic-saline soil (unpublished) served as sources for the cultivation of *Anaeromyxobacter*-strains. A total of 23 sequences designated as *Anaeromyxobacter* (sequence lengths > 1000 bp) are available from the NCBI database (FJ90053–FJ90062, FJ90048, FJ90049, FJ90051, EF067314, AJ504438, KF952446, AF382397, AF382399, AF382400, FJ939131, KF952441, KF952438, KC921178) and were added to a phylogenetic tree of myxobacterial type strains. A similarity matrix calculated with arb (www.arb-home.de) revealed 98.4–100% similarity (on basis of 16S rRNA gene) for 20 of these strains to the type strain of *A. dehalogenans*. Assuming that the standard value for the definition of a new species is 98.65% [109] and 94% for a new genus, respectively, the above-mentioned 20 cultures probably do belong to *A. dehalogenans*. However, strains OnlyC-B2 (KF952441) and SSS-B8 (KF952438) show only 96.3% and 96.0% similarity to the corresponding type strain (but 99.4% to each other) and putatively represent a new species. One culture, isolated from ginger foundation soil, and also designated as *A. dehalogenans* (KC921178), shows only 86% similarity to the next cultivated (myxobacterial) type strain *Vulgatibacter incomptus*. This culture definitely represents at least a new family if not even a new suborder of myxobacteria (unpublished).

In summary, there are currently two cultivated species of *Anaeromyxobacter,* but only one is validly described. But what about further facultative or even strictly anaerobic myxobacteria?

The NCBI search for 16S rRNA gene sequences of "uncultured *Anaeromyxobacter*" revealed more than 1200 hits. Nevertheless, not all sequences designated as "Uncultured *Anaeromyxobacter*" are close relatives of *Anaeromyxobacter*, as a revision of randomly selected sequences revealed. For example: the

sequence GU271851, mentioned as "Uncultured *Anaeromyxobacter*", shows 91% similarity to the next type strain *Haliangium tepidum,* but only 87% to the type strain of *A. dehalogenans* [110]. Clone GU271788, also mentioned as *A. dehalogenans*, shows 92% to the next type strain, *Sorangium cellulosum*, but only 86% to *Anaeromyxobacter* [111]. On the other hand, there are probably numerous sequences deposited at NCBI which are close relatives of *Anaeromyxobacter*. However, these sequences are just mentioned as "uncultivated Myxococcales", "uncultivated (delta) proteobacteria" or "uncultured bacterium clone" such as clone EUB\_19 (FJ189540), which shows 98.7% [112] or clone A\_Ac-2\_16 (EU307085), which shows 97.8% similarity to the next type strain: *A. dehalogenans* [113]. In 2009, Thomas et al. analysed the diversity and distribution of *Anaeromyxobacter* strains in a uranium-contaminated environment by mainly cultivation-independent methods. Phylogenetic analyses of the clone and culture sequences revealed that there are at least three distinct *Anaeromyxobacter* clusters at the IFC (Integrated Field-Scale Subsurface Research Challenge) site near Oak Ridge, whereby two sides are exclusively represented by clones. As mentioned above, quantitative PCR assay and pyrosequencing analysis of 16S rRNA genes also revealed *A. dehalogenans* as a part of the microbial community in the sediment of Poyang Lake, the largest freshwater lake in China [95].

To get an impression about the diversity of uncultured *Anaeromyxobacter*, I added about 80 clone sequences from NCBI with corresponding designation to a phylogenetic tree of myxobacterial type strains (data not shown). The clones revealed 90.0–100% similarity on the basis of 16S rRNA gene to the type strain of *A. dehalogenans* (AF382396). Figure 11 shows the affiliation of some representative clones. These clones represent at least several new genera, if not even families of myxobacteria, which are probably also facultative or strictly anaerobic and which could not be cultivated so far.

Although the 16S affiliation of clones does not give any information about metabolism of the corresponding organism, high similarities to aerobic or anaerobic cultures indicate similar metabolic capabilities. However, no bioactive secondary metabolites have been described so far from *Anaeromyxobacter* strains, which is certainly because anaerobic isolation, cultivation, and large scale fermentation requires special efforts regarding equipment and microbiological skills and experience.
