Ann Microbiol (2016) 66:17–33 DOI 10.1007/s13213-015-1104-3

REVIEW ARTICLE

Current trends in myxobacteria research

Wioletta Wrótniak-Drzewiecka1 & Anna Joanna Brzezińska1 & Hanna Dahm1 & Avinash P. Ingle 2 & Mahendra Rai 2

Received: 5 March 2015 /Accepted: 19 May 2015 /Published online: 12 June 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract Myxobacteria are fascinating Gram-negative bacte- Keywords Myxobacteria . Ecology . Cytology . Enzymatic ria whose life cycle includes the formation of multicellular activity . Secondary metabolism . Social interactions fruiting bodies that contain about 100,000 cells differentiated as asexual spores for their long-term survival. They move by gliding on surfaces, an activity that helps them carry out their Introduction primitive kind of multicellular development. Myxobacteria have multiple traits that are clearly social in nature; they move Myxobacteria (slime bacteria) are rod-shaped Gram-negative and feed socially. These processes require specific intercellu- bacteria that move by gliding. They typically travel in swarms, lar signals, thereby exhibiting a sophisticated level of the inter- containing many cells kept together by intercellular molecular organismal communication. Myxobacteria are predators. signals. Bacterial gliding is a process whereby a bacterium can Predation is social not only with respect to searching for prey move under its own power. For many bacteria, the mechanism (motility) but also in the killing of prey. Swarming groups of of gliding is unknown or only partially known, and different cells secrete antibiotics and bacteriolytic compounds that kill bacteria (cyanobacteria, cytophaga-flavobacteria) have dis- and lyse their prey, and food is thereby released. Since the last tinct mechanisms of movement. When nutrients are scarce, three decades, myxobacteria are known as valuable producers cells of myxobacteria aggregate by chemotaxis, produce of secondary metabolites exhibiting various biological activi- fruiting bodies and avoid desiccation by forming resistant ties. Myxobacterial metabolites exhibit many unique structur- myxospores. al features as well as rare or novel modes of action, making Myxobacteria occur in at least two morphological types: them attractive lead structures for drug development. Both (1) slender flexible rods with more or less tapering ends, and genome sequencing and metabolic profiling of myxobacterial (2) cylindrical rods with rounded ends. Vegetative cells are strains suggest that the diversity of myxobacterial secondary relatively large, measuring 3–6 μm long and 0.7–1.0 μmwide metabolism is far greater than previously appreciated. The (Krzemieniewska and Krzemieniowski 1928). Different cell present review discusses the structure, cytology, physiology, types with other characters can be used to divide the and ecology of myxobacteria, as well as their secondary me- Myxobacteriales into three sub-orders, the Cytobacterineae, tabolite production and social interactions. Sorangiineae and Nannocystineae (Garcia et al. 2010). For a century, taxonomy and classification of myxobacteria were based on morphological traits such as shape of cell and * Wioletta Wrótniak-Drzewiecka fruiting bodies, size, colour and swarm patterns. However, [email protected] morphological similarity often does not represent genetic sim- ilarity, and morphology-based phylogenies may fail to model 1 Department of Microbiology, Faculty of Biology and Environmental relationships among species (Velicer and Hillesland 2008; Protection, Nicolaus Copernicus University, Lwowska 1, Garcia et al. 2010). 87-100 Torun, Poland The DNA sequence-based classification provides patterns 2 Department of Biotechnology, Sant Gadge Baba Amravati of ancestral relationship among myxobacterial species. In re- University, Amravat 444 602, Maharashtra, India cent years, 16S rRNA studies have shown that the group as a 18 Ann Microbiol (2016) 66:17–33 whole is phylogenetically coherent and belongs to the delta only M. xantus, but is also manifested by other bacteria. The group Proteobacteria, a large taxon of Gram-negative forms. mechanism whereby the bacterium senses the presence of a They are closely related to sulphate-reducing bacteria and physical or biological surface, specifically recognizes it and Bdellovibrio species, which are also predators of bacteria. associates with it is an area of microbiology that remains to be The sub-order Cystobacterinae includes the two most thor- clarified (Dawid et al. 1988). oughly studied species, Myxococcus xanthus and Stigmatella The present review is focused on different aspects of aurantiaca. The sub-order Soranginae includes the family myxobacteria, viz., ecology, cytology, social interactions, sig- Polyangiaceae with the genera Sorangium, an extensively nal transduction, predation, secondary metabolism and enzy- studied cellulose decomposer. The sub-order matic activity. Nannocystineae is a unique mixture of isolates grouped into two clusters: (1) the marine organisms Enhygromyxa and Plesiocystis, which are allied to the terrestrial nonhalophilic Ecology bacterium Nannocystis;and(2)theHaliangium-Kofleria clus- ter (Garcia et al. 2011). Jiang et al. (2007)proposedthatthe The myxobacteria seem to be a ubiquitous group of microor- myxobacteria exist in the environment in two forms — the ganisms that can inhabit very diverse habitats, including de- fruiting and the non-fruiting types. Most of the uncultured sert crust soils (Powell et al. 2015), the surface of the older myxobacteria may represent taxa that rarely form fruiting bod- leaves in grain crops, as well as the surface of wheat and ies, or may lack some or all of the developmental genes need- barley seeds (Leontievskaya and Dobrovol’skaya 2014), the ed for fruiting body formation. fruiting bodies and hyphosphere of several basidiomycetes Many phenomena and concepts originally described with (Zagriadskaia et al. 2014) and sewage sludge (Zhou et al. regard to social interactions among higher organisms have 2014a). Myxobacteria were dominant on the older leaves of equivalents in microorganisms. These include inter- grain crops and in grain of wheat and barley ears; species organismal communication, division of activity, self/non-self composition and structure of epiphytic bacterial communities recognition, kin selection vs. individual selection and social in the grain cereal crops changed in the course of vegetation conflict. Microorganisms offer the opportunity to study the (Leontievskaya and Dobrovol’skaya 2014). Nutrient-rich soils evolution of such social traits with a new level of rigor harbor more myxobacteria species, but these organisms can (Crespi 2001; Velicer and Stredwick 2002). Their rapid also live in rocky soils and pure sand (Dawid 2000). growth allows observations of evolutionary changes in labo- Myxobacteria are capable of bioreduction of water-soluble ratory populations. The social bacterium M. xanthus is a mod- uranium U(VI) to insoluble uranium U(IV). This phenomenon el system for the experimental study of microbial social evo- can be used for bioremediation of ground water contaminated lution. As a group, the myxobacteria produce a large variety of with uranium U(VI) and with other radionuclides (Newsome secondary metabolites, some of which may have medical uses et al. 2014). There is little contribution in the field of marine (Reichenbach and Höfle 1993; Gerth et al. 2003; Weissman myxobacteria (Felder et al. 2013). The marine myxobacteria, and Müller 2009;Diezetal.2012). The reasons why including Enhygromyxa, Plesiocystis, Pseudenhygromyxa, myxobacteria are multi-producers of secondary metabolites and Haliangium, are phylogenetically different from terrestrial are still not well understood. It has been argued that they myxobacteria. Most cultured species prefer mild temperatures confer a competitive advantage in soil environments, which (20–30 °C), neutral pH and high concentrations of organic maybeusedtomodulatecell–cell interactions and as weapons matter, but low ionic concentrations. Marine myxobacteria for predation. Answers to this question remain in the realm of occur in bottom sediments, and thus may have available high speculation. concentration of organic matter (Brinkhoff et al. 2012). Understanding the evolution of social phenotypes remains However, myxobacteria isolated from Antarctica grow at 4 ° a great challenge for the evolutionary biologist. This problem C (Dawid et al. 1988), and those from warm arid climates is daunting in higher organisms for many reasons, including have optimal growth temperatures of 42–44 °C (Gerth and limited knowledge of behavioral genetics and genotype–envi- Müller 2005). Only a few halophilic (Iizuka et al. 1998)or ronment interactions. Microbial social systems may help re- halotolerant (Li et al. 2002) strains of myxobacteria have been veal aspects of social evolution shared across all levels of isolated from coastal areas (Jiang et al. 2010). Many studies biological organization. It is now clear that cell–cell commu- suggest that our knowledge of the particular environments of nications and functional multicellularity among and between myxobacteria is limited by methods of isolation and cultiva- bacteria and eukaryotic cells are commonplace. These take tion. One of the commonly used myxobacteria isolation tech- place as a part of host–parasite interactions, biofilm formation, niques involves use of soil enrichment cultures (Fig. 1). multicellular development, and syntrophic interactions Myxobacteria are well known for three capabilities: (1) (Dworkin 1972). The physical connection between prokary- they move by gliding and their colonies are therefore thin, otic cells seems to give them an advantage. It concerns not film-like swarms; (2) they have sophisticated intercellular Ann Microbiol (2016) 66:17–33 19

Fig. 1 Isolation of myxobacteria—colonies of myxobacteria growing out of soil crust (f.b. fruiting bodies) communication systems and a highly developed social life; (3) Fig. 2 Young, vegetative cells of myxobacterium (Michałowska 2009) they show a remarkable morphogenetic potential. In coopera- (c.w. cell wall, c.m. cell membrane, n nucleoide, r ribosomes, i inclusions) tive starvation conditions, they may produce fruiting bodies. Within the fruiting body, a cellular morphogenesis takes place, those in other Gram-negative bacteria, the peptidoglycan of during which the vegetative cells convert into resistant M. xanthus had unique structural features. meso- myxospores (Reinchenbach 1999). In myxobacteria, diaminopimelic or ll-diaminopimelic acid was present in the swarming toward new nutrient sources is accomplished by stem peptides, and a new modification of N-acetylmuramic two genetically and physiologically distinct motility systems, acid was detected in a fraction of the muropeptides. adventure motility (A) (individual) and social (S) motility (Wu Peptidoglycan formed a continuous, bag-shaped sacculus in et al. 2007; Kaiser et al. 2010). The A-motility and S-motility vegetative cells. The sacculus was degraded during the tran- systems are synergistic, as colony spreading by wild-type cells sition from vegetative cells to glycerol-induced myxospores. + − − + is faster than the sum of those of individual A S and A S The spherical, bag-shaped coats isolated from glycerol- cells. In the absence of one or both motility systems, aggrega- induced spores contained no detectable muropeptides, but tion, fruiting body formation, and rippling are defective, indi- they contained small amounts of N-acetylmuramic acid and cating that motility is required for these social behaviors meso-diaminopimelic acid. (Mauriello et al. 2010). Recently, Nan et al. (2014)identified the helical protein track rotor mechanism of gliding motility in the myxobacterium. Periplasmic space

Cytology Little is known about myxobacteria periplasm specifically. The periplasmic space of M. xanthus contains, as in other Myxobacteria are typical Gram-negative bacteria with an out- er membrane. M. xanthus serves as a premier model bacterium for the study of cell envelope, cytological structures, social behavior, signals transduction and others. Myxobacteria are flexible cells and it has generally been assumed that their cell wall is fundamentally different from the wall of eubacteria, especially the layer of peptidoglycan (White et al. 1968). It would be expected that during differentiation, the M. xanthus cells would remodel their peptidoglycan as they change from rod-shaped vegetative cells to spherical spores (Yang et al. 2008). A comparison of peptidoglycan in vegetative cells and spherical spores of M. xanthus showed that although the overall composition of peptidoglycan of both cell types was similar, there appeared to be an increase in muropeptide cross- linking in the spherical spores (White et al. 1968)(Figs.2 and 3). Bui et al. (2009) observed that while the basic structural Fig. 3 Mature myxospore (Michałowska 2009)(c.w cell wall, c.m. cell elements of peptidoglycan in myxobacteria were identical to membrane, s.c. slime capsule, n nucleoide, r ribosomes, g granules) 20 Ann Microbiol (2016) 66:17–33

Gram-negative bacteria, many enzymes and structural and highly regulated process modulated by a phosphorylation- functional proteins. Among them, lipoproteins, hydrolytic en- dependent mechanism. Glucose from cellulose would, via zymes, proteins for nutrient acquisition, chaperons and senso- the pentose phosphate pathway, give rise to many other hex- ry proteins are important (Schlicker et al. 2004;Mogensenand oses and pentoses that are used to synthesize polysaccharides, Otzen 2005;Yangetal.2008). It was suggested that some including their capsule of slime and their lipopolysaccharide periplasmic structures could be a part of the gliding machinery (Kaiser et al. 2010). Biochemical analyses indicated that that may utilize membrane potential to power adventurous M. xanthus EPS contains five monosaccharides: galactose, gliding motility of myxobacteria. Freese et al. (1997)observed glucosamine, glucose, rhamnose and xylose (Merroun et al. the chain-like aggregates or strands in the periplasm of 2003;Yangetal.2008). Among the 41 proteins identified, 20 M. xanthus. The strands appear to be composed of ring-like are likely integral inner or outer membrane proteins or cyto- and centrally elongated elements and formed ribbon-like plasmic proteins and the remaining 21 have been suggested to structures. However, there is no direct evidence of linking be good ECM protein candidates (Konovalova et al. 2010). these structures to gliding (Yang et al. 2008). Only five of the 21 candidate ECM proteins have predicted functions. These functions include protease activity, amidohydrolase activity and coating of the myxospores. Outer membrane Inactivation of several of the genes encoding putative ECM proteins caused no defects in fruiting body formation, with the In Gram-negative bacteria, the outer layer of the cell envelope exception of the fibA gene, which encodes the FibA zinc is composed of lipopolysaccharide (LPS), which forms a se- metalloprotease (Kearns et al. 2002;Konovalovaetal.2010). lective barrier between the environment and the periplasmic space. In M. xanthus,the LPS is similar in structure to the LPS of other Gram-negative bacteria (Fink and Zissler 1989; Yang Pili et al. 2008). LPS molecule is composed of three parts: hydro- phobic lipid A, a covalently attached core oligosaccharide Pili (fimbrie) were first observed in myxobacteria by Mac-Rae region, and a distal repeating polysaccharide, termed O-anti- and Mc Curdy in 1976 (c.f. Rosenbluh and Eisenbach 1992), gen. The myxobacteria lipopolysaccharide O-antigen is simi- and were described as 6–8 nm diameter appendages, which lar in overall structure to that of lipopolysaccharide in other extend from one or both cell poles for up to a cell’slengthor Gram-negative bacteria, and the carbohydrate moiety consists further. M. xanthus pili belong to the class of Tfp (type IV of glucose, mannose, rhamnose, arabinose, xylose, galactos- pili), which are found in Gram-negative bacteria (Wu and amine, glucosamine, 2-keto-3-deoxyoctulosonic acid, 3-O- Kaiser 1995;Yangetal.2008). Pili have been studied in a methylpentose and 6-O-methylgalactosamine (Gill and variety of bacterial systems, and play an important role in Dworkin 1986;Yangetal.2008). Among the outer membrane cohesion, intercellular communication and host colonization proteins of M. xanthus, two lipoproteins, CgIB and Tgl, are by pathogens. In addition, the pili are thought to be involved important for motility. During the aggregation phase of in social motility (Merroun et al. 2003; Kaimer et al. 2012). fruiting body formation, lectin (MBHA) accumulates at a high level. It is suspected that MBHA plays an important role in myxobacteria (M. xanthus) cell adhesion, which is important Genome for fruiting body development (Yang and Kaplan 1997). Among the myxobacteria, M. xanthus is studied most geneti- cally, followed by Stigmatella aurantiaca. M. xanthus DK Extracellular matrix (ECM) 1622, apart from Anaeromyxobacter dehalogenans 2CP-C, was the first to have its genome sequenced. Further, different Cells of myxobacteria are covered by an extracellular matrix myxobacterial genomes have been completely sequenced: (ECM) comprised of protein and polysaccharide. Sorangium cellulosum So ce56 and Stigmatella aurantiaca Polysaccharide was previously referred to as fibrils on the cell DW 4/3-1, Haliangium ochraceum, Myxococcus fulvus, surface, observed by scanning microscopy. The structures ob- Corallococcus coralloides, Myxococcus stipitatus, served were most likely the results of dehydration of the poly- Cystobacter violaceus (Li et al. 2011; Huntley et al. 2012, saccharide during preparation. Now, the polysaccharide com- 2013; Muller et al. 2013; Stevens et al. 2014). Most ponent of ECM is referred to as exopolysaccharide (EPS) myxobacteria are composed of a single circular chromosome, (Behmlander and Dworkin 1994;Kaiseretal.2010). ECM and no extra-chromosomal plasmids were known until 1980, is required by these myxobacteria for integrity of cell groups, when the first endogenous plasmid was identified in which is important for cellular cohesion, social motility and Myxococcus fulvus. Endogenous plasmids have not been fruiting body morphogenesis. The production of EPS is a found in M. xanthus. However, this bacterium produces an Ann Microbiol (2016) 66:17–33 21 unusual satellite DNA known as multicopy single-stranded myxobacteria is coordinated by the regulatory (A) and (C) DNA (msDNA), which is highly conserved (Shimkets signal genes. The quorum sensing A-signal senses the ap- 1990). Myxobacteria possess giant chromosomes, belonging proach of starvation and induces cellular aggregation. In to the largest known in bacteria (Pradella et al. 2002;Kaiser M. xanthus, A-signal is controlled by five genes that may et al. 2010). Myxobacterial genomes are at the large end of the function together in response to the nutritional state of the cell eubacterial scale and approach the size of the lower eukaryotic (Kaiser 2004). Eight chemotaxis clusters have been identified genomes. A genome size ranging from 5690 to 12,727 kbp in M. xanthus. It is not clear that a large genome is the result of was noted (estimations were carried out on exponentially developmental complexity. It was detected that only about growing cells). Myxospores contain an average of 3.3 genome 8 % of the M. xanthus genome increases expression during equivalents per spore (Chen et al. 1990).Thegenomeof development and less than 1 % of the genome is essential for M. xanthus contains a much higher G+C proportion than does development. The function of the large genome remains Escherichia coli.TheM. xanthus (DK 1622) genome is com- unknown. Evidently, additional expansion within the posed of a single circular chromosome. Nearly half of the myxobacterial lineage led to Sorangium cellulosum, whose genes (48.9 %) have been assigned a putative function 13 Mbp genome encodes the largest number of serine/ (Goldman et al. 2006;RonningandNierman2008). threonine protein kinases of any organism (Perez et al. The unusually large size of the M. xanthus genome is re- 2008). portedly due to expansion by lineage-specific duplications of specific categories of genes, particularly those involved in cell–cell signaling, small molecule sensing and multi- Social interactions component transcriptional control of the complex molecular machinery required for development of a multicellular life- The conventional wisdom of microbiology has been that bac- style (Ronning and Nierman 2008). Genes involved in the teria are independent, unicellular organisms and that the prop- social and developmental behavior of this bacterium were erties of a population are the sum of the properties of the found in many regions of the chromosome. Thirty genes are individual cells (Dworkin 1999). It is now clear that cell–cell involved in the adventurous (individual) move: the type IV communications and functional multicellularity among bacte- pili genes and the sasA locus encoding the lipopolysaccharide ria are commonplace. Social cooperation is exhibited by many O-antigen biosynthesis genes (Wu and Kaiser 1995;Walletal. microbial species (Crespi 2001). Microbial sociality includes 1999; Lancero et al. 2002;Vlamakisetal.2004; Ronning and a quorum-sensing system, cooperative predation, suicidal al- Nierman 2008). Although the functions of most M. xanthus truism and the formation of complex developmental structures genes remain to be determined, bioinformatics analysis clearly such as biofilms and the fruiting structures of Streptomyces reveals complex signaling and regulatory networks. For in- and myxobacteria (Velicer and Stredwick 2002). stance, the M. xanthus genome encodes 53 s54 enhancer bind- Recently, it has been evaluated that during cell–cell inter- ing proteins, 38 extra-cytoplasmic function sigma factors, 97 action myxobacteria exchange their outer membrane (OM) serine threonine protein kinases and 272 two-component sig- proteins and lipids. The mechanism of transfer requires phys- nal transduction proteins (Goldman et al. 2006). Many of the- ical contact between aligned cells on hard surfaces. Transfer is se regulatory proteins are likely to function in pathways mediated by OM fusion, in which membrane contents lateral- governing interactions with other cells and their environment, ly diffuse and are exchanged bi-directionally between cells. and future work is needed to elucidate their roles. TraA and TraB are recently identified proteins that are re- Since the myxobacteria have a complex developmental cy- quired in donor and recipient cells for transfer to occur. OM cle, it is tempting to speculate that a large genome is necessary exchange results in phenotypic changes that can alter gliding for fruiting body development; however, only a small part of motility and development of myxobacteria. It is a novel mi- the genome is essential for fruiting body development (Chen crobial interaction coordinating multicellular activities et al. 1990). It has been estimated that secondary metabolite (Pathak et al. 2012a, b; Pathak and Wall 2012; Wall 2014a). synthetic genes constitute more than 3 % of some bacterial Recently, the traAB operon was discovered as a genetic deter- genomes (Sasse et al. 2000), e.g., 9 % of the total genetic minant for transfer. Because fluorescently labeled lipids are capacity in M. xanthus (Diez et al. 2012). Genes of secondary also exchanged by a TraAB-dependent mechanism, the model metabolism in myxobacteria have been intensively studied. for transfer invokes the transient fusion of the OM, resulting in Several secondary metabolic gene clusters have been analysed the exchange of content between cells (Pathak et al. 2012b). in detail (Beyer et al. 1999;Julienetal.2000; Silakowski et al. TraA functions as a cell surface adhesion and as a molecular 2001; Ligon et al. 2002;Pradellaetal.2002; Sandmann et al. recognition determinant that identifies other cells that express 2004; Carvalho et al. 2005;Fengetal.2005; Perlova et al. the same or similar traA alleles to coordinate social behaviors 2006). The myxobacteria have a large capacity for signal (Pathak et al. 2012b; Wall 2014a, b).Weietal.(2014)sug- transduction. Feeding and fruiting body development in the gested that traA functions as a fusogen to catalyse OM fusion 22 Ann Microbiol (2016) 66:17–33 between cells (Pathak and Wei 2012; Wall 2014a). The func- motility proteins, including AglT, AgmK, AgmX, AglW, tion of traB is less clear, although it contains an OmpA do- and CglB. These proteins likely form a large multiprotein main that is predicted to bind to the cell wall. Gram-negative complex that spans the membrane and periplasm of the cells. bacteria, including M. xanthus, secrete diffusible OM vesicles Swarming groups of cells secrete antibiotic and bacterio- that potentially can fuse with other cells, including eukaryotic lytic compounds that kill and lyse prey. The efficiency of this cells (Mashburn-Warren and Whiteley 2006; Kulp and Kuehn social predation appears to be density dependent (Velicer and 2010). Based on a bioinformatic analysis, TraAB orthologs Stredwick 2002). The M. xanthus process of fruiting body are restricted to myxobacteria, although other bacterial groups formation is a social behavior. Fruiting body development is may have functional analogs and may carry out similar behav- initiated by starvation for carbon or nitrogen or phosphorus. ior (Kudryashev et al. 2011). Using live imaging, Ducret et al. Some authors have shown that upon amino acids starvation, (2013) showed that transient contacts between two cells are swarms of about 105 organisms use movement together at sufficient to transfer OM materials, proteins and lipids, at high high-density aggregation loci, a process that has been com- efficiency. Transfer was associated with the formation of dy- pared to animal migration (Shimkets 1999). These aggregates namic OM tubes, strongly suggesting that transfer results from transform into fruiting bodies. Such process requires specific the local fusion of the OMs of two transferring cells. Last, intercellular signals, thereby exhibiting a sophisticated level of large amounts of OM materials were released in slime trails inter-organismal communication. A survey of literature re- deposited by gliding cells. Since cells tend to follow trails laid vealed that only a minority (ca. 10 %) of an initial aggregating by other cells, slime-driven OM material exchange may be an population differentiates into resistant spores, whereas most of important stigmergic regulation of Myxococcus social behav- the remaining cells autolyse (altruistic suicide) (Wiseman and iors. Similarly, during microscopic examination of cells, Wei Dworkin 1977;O’Connor and Zusman 1989)(Fig.4a–d). et al. (2014) discovered long tubular filaments emanating The ecological roles of fruiting bodies are unclear (Velicer from M. xanthus cells, which turned out to be OM tubes. and Stredwick 2002). One hypothesis holds that fruiting bod- M. xanthus and other species of myxobacteria have multi- ies may serve to improve the chance of spore dispersal to ple traits that are clearly social in nature. M. xanthus moves nutrient-rich habitats. Alternatively, they may help protect and feeds socially. Cells can move using one or both of two dormant spores from caustic soil compounds and facilitate genetically distinct motility systems. Social (S) motility is efficient germination. Individual spores may have higher fit- stimulated by cell–cell proximity and involves type IV pili ness during germination when clumped in packs than when in (McBride 2001). Adventurous (A) motility allows greater in- isolation. To carry out a program of morphological develop- dividual cell movement (Spormann 1999;Konovalovaetal. ment, the cells communicate with each other by emitting and 2010). Fibrils and type IV pili are the extracellular structures responding to extracellular chemicals signals, two of which that are associated with S-motility and physically link cells are A-factor and C-factor. Myxobacterial fruiting body devel- together. Fibril polysaccharides are frequently called opment requires extensive cell–cell signaling and interactions, exopolysaccharides (EPS). It should, however, be noted that as well as coordinated changes in gene expression and cell M. xanthus produces other forms of EPS that are not associ- movement (Kroos 2007;Leonardyetal.2008). The transition ated with fibrils (Ducret et al. 2012;Mulleretal.2012). Fibrils from vegetative growth to development in M. xanthus was play a key role in cell–cell adhesion (a.k.a. agglutination or previously shown to depend on the RelA-mediated stringent clumping), recognition, motility and development (Li et al. response (Singer and Kaiser 1995), but more recently, the 2003). Polarly localized type IV pili function as the motor that sRNA Pxr has also been implicated in governing this transi- powers S-motility; their retraction from the ‘front end’ of the tion (Yu et al. 2010). Starvation is recognized by M. xanthus rod-shaped cell pulls the cell forward (Skerker and Berg by a ribosome that lacks the charged tRNA specified by the 2001). As a recognition anchor for retraction, pili bind to codon positioned in the acceptor (A) site. Uncharged tRNA EPS deposited in the extracellular matrix (ECM) (Li et al. binds instead, and the highly phosphorylated guanosine nu- 2003). A-motility is associated with polar extracellular ‘slime’ cleotide (p)ppGpp is then synthesized from GTP and ATP. filaments, which are thought to be composed of an as-yet-to- Since formation of amino acyl tRNA depends on ATP, a be-characterized polysaccharide (Yu and Kaiser 2007; Ducret low-level energy or phosphate can be detected. Succinctly et al. 2012). The A-motility motor appears to be powered by and sensitively, (p)ppGpp directly assesses the cell’scapacity mobile cell surface adhesins. Thus, this mechanism of motility to synthesize protein (Kaiser 1998). In M. xanthus fruiting might be analogous to how tank or bulldozer tracks propel body development, ribosomal and tRNA synthesis are imme- those vehicles (Nan and Zusman 2011; Sun et al. 2011). A- diately inhibited in response to amino acid limitation. In ad- motility involves two proteins: AglZ, a cytoplasmic protein, dition, DNA, membrane and cell wall biosyntheses are and AgmU, a protein that localizes to both the cytoplasm and inhibited and proteolysis is increased. A response to amino periplasm (Mauriello et al. 2010). Recently, Nan et al. (2010) acid starvation is evident within 30 minutes as an elevation found that AgmU is also associated with many other A- of (p)ppGpp. (p)ppGpp is needed to induce production of the Ann Microbiol (2016) 66:17–33 23

Fig. 4 a Vegetative growth stage of myxobacterial cells (mature cells) (Michałowska 2009). b Early aggregation stage of vegetative cells (Michałowska 2009). c Differentiation stage of vegetative cells into myxospores (Michałowska 2009). d Mature myxospores (Michałowska 2009)

A-factor. The A signal appears to be a mixture of amino acids C–factor associated with the cell surface provides input to that are released from cells and function as relatively long- the Frz signal transduction cascade. Elements of this cascade range indicators of cell density. have sequence homology to bacterial chemotaxis systems and A second intercellular signal, C-factor is a 17 kDa protein are known to control the frequency of gliding reversal encodedbythecsgA gene. C-signaling controls three diverse (Kaimer et al. 2012). The extracellular C-signal is required process: cell aggregation, expression of variety of genes (in- for rippling, aggregation, sporulation and maximum induction cluding csgA) and sporulation. C-factor on the surface of one of developmentally regulated genes after 6 h during fruiting cell interacts with receptive cells and requires direct contact body morphogenesis in M. xanthus. C-signal is not only re- between the ends of two cells. C-factor bears sequence homol- quired for, but also induces these events (Kruse et al. 2001; ogy to a family of dehydrogenase enzymes, suggesting that Kaiser 2004, 2013). signaling involves a dehydrogenation (Kaiser 1998). Cell–cell interactions are often mediated by pili. The pili of M. xanthus have been shown to play an explicit role in social motility (Wu Signal transduction and Kaiser 1995; Mauriello et al. 2010;Berlemanetal.2011). However, an additional mechanism for mediating these con- The largest groups of prokaryotic signaling pathways are two tacts is that of extracellular appendages called fibrils. Fibrils component systems (TCSs). They are most abundant in the have been studied in detail in the myxobacteria (Behmlander myxobacteria (Whitworth and Cock 2007, 2008; Darnell et al. and Dworkin 1994;Kaiseretal.2010; Kaiser 2015). However, 2014). A typical TCS consists of a histidine protein kinase and a second extracellular appendage of fibrils has also been shown a response regulator protein. The genes for a histidine kinase to be necessary for social behavior. The fibrils are polysaccha- and response regulator pair are often found adjacent to one ride organelles containing a set of tightly adhering proteins. It another in the genome, and it is evident that they act together is proposed that cell–cell contact is perceived by the fibrils and to form a TCS. A large number of TCS proteins of is mediated by the action of a fibrillar ADP-ribosyl transferase myxobacteria have been identified and characterized (Perez (Dworkin 1999). Recently, Harvey et al. (2013)proposeda et al. 2008). continuum theory of clustering in self-propelled flexible rods Most of these proteins are known to regulate fruiting body with applications to collective dynamics of the common glid- development, motility and chemotaxis (Whitworth and Cock ing bacteria M. xanthus. Numerical simulations of this model 2007). The complex domain structures of myxobacterial TCS confirm the existence of stationary dense moving clusters and proteins suggest that the TCSs of myxobacteria operate in also elucidate the properties of their collisions. significantly different ways from those of most other bacteria 24 Ann Microbiol (2016) 66:17–33

(Whitworth and Cock 2008; Krell et al. 2009). One particu- larly unusual feature of myxobacteria is the conformation of a chemotactic signaling pathway to regulate gene expression (Kaiser et al. 2010). The low proportion of sensor kinases with transmembrane helices implies that myxobacteria are unusu- ally sensitive to changes in their internal state (Whitworth and Cock 2008) (Fig. 5). Myxobacteria also communicate with one another chemically by pheromones (Plaga et al. 1998) and bioactive secondary metabolites (Reinchenbach 2001), as well as mechanically by pili (Kaiser et al. 2010).

Fig. 6 Lysis of E. coli cells by Myxococcus virescens (E.c.–E. coli, M.v. M. virescens) Predation performance on individual prey species, thus reflecting some Populations of myxobacteria may significantly affect the den- degree of specialization. sity of a variety of organisms in the different environments. Myxobacteria are widespread in soils around the world. Many researchers reported predator prey interactions between This ubiquity, coupled with the ability to use a broad range myxobacteria and soil microorganisms. Myxobacteria prey of other microbial species (fungi, bacteria) as prey, suggest upon a diversity of Gram-negative as well as Gram-positive that myxobacteria strongly affect the populations of many species of bacteria. According to Guerrero et al. (1987), pre- microorganisms. Myxobacteria use gliding motility to search dation between prokaryotes is one of the most ancient forms the soil matrix for prey and produce a wide range of antibiotic of predation (Fig. 6). Myxobacteria employ a highly different and lytic compounds that kill and decompose prey cells. mode of predation than Bdellovibrio (Evans et al. 2008). They Myxobacterial predation is cooperative both in its searching use gliding motility and produce a wide range of antibiotics component and in its handling component (Morgan et al. and lytic enzymes and break down cell polymers for nutrients 2010). According to some research (McBride and Zusman (Morgan et al. 2010; Xiao et al. 2011). The Gram-positive 1996; Berleman et al. 2008), myxobacteria employ species are poorer prey for Myxococcus predators than are chemotaxis-like genes in their attack on prey, and predation Gram-negative ones. Such observations suggest a difference is stimulated by close contact with prey cells. M. xanthus cells in the susceptibility of Gram-positive and Gram-negative spe- cannot sense prey colonies until direct cell–cell contact is cies to predation by Myxococcus. It is suggested that the type made. There is no recognition of prey cells even at very short IV pili on Myxococcus cells play a key role in adhesion to prey distances, but when contact is made, the M. xanthus cells cells, and thereby facilitae predatory lysis (Morgan et al. began to alter their behavior. The Frz signal transduction sys- 2010). Some Myxococcus strains do vary in their prey specific tem is also responsible for keeping the M. xanthus cells in the

Fig. 5 Signaling pathways of two-component systems (TCS). (a) Sensor The MCP transducer is depicted as transmembrane and is coupled bx domain is covalently bound to histidine kinase domain. Phosphorylation CheW to the CheA kinase. Two methyl groups are shown to represent occurs on conserved histidine residue. The phosphoryl group is trans- methylation of the receptor bz CheR. Phosphorylated CheB can remove ferred to a conserved aspartate residue within the receiver domain (Rec) these methyl groups. Methylation is a hallmark feature of the chemotaxis in the response regulator. The prototypical RR output is a DNA-binding TCS systems. Phosphotransfer to the response regulator CheY influences domain capable of influencing gene expression. The vertical bar repre- its ability to bind the FliM switch component at the flagellar motor (ac- sents the cytoplasmic membrane. (b) TCS system controlling chemotaxis. cording to Kirby et al. 2008,modified) Ann Microbiol (2016) 66:17–33 25 vicinity of their prey after contact has been made and feeding compounds target the eukaryotic cytoskeleton; interference is underway. When cells start moving away from the source, with microtubule assembly plays a critical role in currently the Frz system senses this and induces a reverse in direction to available cancer chemotherapies, through the inhibition of cell keep them in contact with the prey colony (McBride and proliferation and the induction of apoptosis. Epothilones have Zusman 1996). been noted to have antineoplastic activity. This has led to the development of analogs that mimic its activity. One such an- alog, known as Ixabepilone, is a U.S. Food and Drug Production of secondary metabolites Administration-approved chemotherapy agent for the treat- ment of metastasis breast cancer. Several other metabolites Aside from actinomycetes and fungi, myxobacteria are one of are currently being evaluated in preclinical studies (Kim the important sources for natural microbial products. The main et al. 2013; Schmitz et al. 2013). From S. cellulosum (So ce producers of secondary metabolites are members of 12), five compound groups have been characterized. In addi- Actinomyces (ca. 8000 compounds characterized), genus tion to the highly active disorazole class of tubulin de-stabi- Bacillus (1400), as well as Pseudomonas (400). However, lizers, this strain produces sorangicins eubacterial RNA poly- over the last decade, the myxobacteria have emerged as a merase inhibitors, bactericidal sorangiolides, the antifungal promising alternative source of bioactive molecules (Huang chivosazoles and the sulfangolides (Irschik et al. 1987; et al. 2010; Weissman and Müller 2010; Altendorfer et al. Jansen et al. 1997; Wenzel and Müller 2007). At nanomolar 2012; Johnson et al. 2012; Kim et al. 2013; Schmitz et al. concentrations, Soraphen, a metabolite of S. cellulosum,in- 2013; Plaza and Müller 2014; Schäberle et al. 2014). hibits the biotin carboxylase domain of human, yeast and oth- Myxobacterial secondary metabolites present structural ele- er eukaryotic acetyl-coenzyme A carboxylases. The specific ments not commonly produced by other microbes, such as inhibitors of this enzyme show promise as a therapeutic agent unusual hybrids of polyketides and non-ribosomally made for cancer (Weissman and Müller 2010). Recently, Irschik peptides. In fact, around 40 % of the described myxobacterial et al. (2013) discovered a family of structurally related mac- compounds represent novel chemical structures. Furthermore, rocyclic lactones in the fermentation broth of the most small molecules from myxobacteria are not glycosylat- myxobacterium Sorangium cellulosum Soce1485. The most ed, as opposed to products derived from actinomycetes, and abundant member of this group was named maltepolide A, they target molecules that are often not targeted by metabolites since the bacterium was isolated from Malta Island. The novel from other microbes (Diez et al. 2012). Myxobacterial second- maltepolides demonstrated biological activity. It has been ary metabolites exhibit many unique structural features and shown that a metabolite derived from S. cellulosum, novel modes action, making them attractive and promising Ratjadone A, exhibits strong anti-HIV activity, but low selec- sources for drug development. Rare but notable properties tivity due to toxic effects. Although this limits its potential use include antibiotic, anti-malarial, immunosuppressive, antiviral as a therapeutic drug, further studies with derivatives of and insecticidal activities (Weissman and Müller 2010). ratjadones might help to overcome these difficulties in the The many different compounds from myxobacteria show future (Fleta-Soriano et al. 2014). quite different mechanisms of action. There are inhibitors of Corallopyronin A is a myxobacterial compound with po- prokaryotic (myxovalargin) and eukaryotic (gephyronic acid) tent antibacterial activity, isolated from the Corallococcus protein synthesis, compounds that stimulate potassium export coralloides. Corallopyronin A is a novel inhibitor of bacterial from Gram-positive bacteria (tartrolon) and compounds that RNA polymerases that is being developed as an antifilarial bind to DNA (saframycin). In some cases, the mechanism of drug, targeting lymphatic filariasis and onchocerciasis, which action has not yet been elucidated, e.g., for the highly cyto- are tropical diseases caused by parasitic nematodes. The toxic vioprolids or for the antifungal leupyrrin. antifilarial activity of corallopyronin is due to its bactericidal The genus Sorangium produces almost half of the second- effect on the Wolbachia endosymbionts, leading to a block of ary metabolites isolated from myxobacteria. Different strains oogenesis, embryo-genesis and development of larvae, and of Sorangium produce several novel antimicrobial macrolides, eventually to death of the adult worms (Schäberle et al. 2014). the leupyrrins (Kopp et al. 2011), the thuggacins (Irschik et al. The majority of myxobacterial secondary metabolites rep- 2007; Buntin et al. 2008), sorangicin (Irschik et al. 2013), resent polyketides, non-ribosomally made peptides or hybrids phoxalone (Guo et al. 2008) and other components, like a of the two structural types (Wenzel and Müller 2007). The new sesquiterpene, sorangiodenosine (Ahn et al. 2008), a free corresponding genes of multi-enzymes are organized in clus- radial scavenger, soraphinol C (Li et al. 2008), as well as a ters that are located on genomic regions 20–200 kilobases (kb) novel class of antineoplastic agents, the epothilones and their in size (Bode and Müller 2005). Polyketide synthase and non- analogs (Mulzer 2009). Epothilones and their analogues have ribosomal peptide synthetase are composed of highly con- demonstrated antitumor activity towards multidrug resistant served regions. Analysis of the chromosome of Stigmatella tumor cells (Mulzer 2009;Gongetal.2014). These aurantiaca revealed the presence of 8656 putative genes, a 26 Ann Microbiol (2016) 66:17–33 significant number of which are involved in secondary metab- blocks the maturation of key lipoproteins required for murein olism (Wenzel and Müller 2007). The strain DW 4/3–1of biosynthesis, and thus indirectly blocks cell wall biosynthesis. S. aurantiaca synthesizes five different compound families: For a number of reasons, LspA is an attractive target for anti- myxochromides S, aurafurones, myxothiazoles, DK xan- biotic drug discovery. First, this signal peptidase is universally thenes and dawenol. Aurachins are a family of secondary found in bacteria and is broadly essential in Gram-negative metabolites produced by the myxobacterium Stigmatella bacteria. In Gram-positive organisms, LspA appears to be aurantiaca strain Sg a15, and possess numerous bioactivities, conditionally essential or nonessential and plays a key role such as anti-bacterial, anti-fungal and anti-plasmodial proper- in pathogenesis, as many virulence factors are lipoproteins ties. Furthermore, these isoprenoid quinoline alkaloids are po- or require lipoprotein function (Hutchings et al. 2009). tent inhibitors of mitochondrial respiration by targeting the Second, LspA is absent in eukaryotic cells, which eliminates cytochrome b6/f-complex, as well as the complexes I and III any concerns about target-based toxicity in animals. Third, in the respiratory chain (Dejon and Speicher 2013). from a clinical perspective, LspA represents a novel target. Some time ago, using high performance liquid chromatog- Interestingly, some of the secondary metabolites isolated raphy and high resolution mass spectrometry, 37 novel natural thus far play a significant role in cellular development or in products were identified from 98M. xanthus isolates (Krug the predatory lifestyle of M.xanthus. DKxanthenes have been et al. 2008). These results suggest that M. xanthus and reported to be essential for the formation of viable spores myxobacteria in general are promising sources for natural sec- within fruiting bodies, and myxovirescin plays a major role ondary metabolites. Scanning of the M. xanthus DK 1622 in the predatory lifestyle of M.xanthus (Xiao et al. 2011; Volz genome sequence for the presence of polyketide synthase et al. 2012). and non-ribosomal peptide synthetase-encoding genes re- An exciting discovery was that about 10 % of myxobacterial vealed the presence of 18 biosynthetic gene clusters, account- compounds interact specifically with the cytoskeleton of eu- ing for around 9 % of the genome (Diez et al. 2012), but until karyotic cells. Such compounds might become useful drugs 2005, no substance could be identified in the screening pro- for the control of cancer (Reinchenbach 2001). Other cesses. However postgenomic examination of extracts by myxobacterial compounds bind to DNA (saframycin), alter HPLC-MS, combined with mutagenesis, led to the identifica- the osmoregulation of fungi (ambruticin) and inhibit eukaryotic tion of five compound families: myxovirescines, (gephyronic acid) and prokaryotic (myxovalargin) protein syn- myxalamides, myxochelines, myxochromides, A and thesis, as well as viral nucleic acid polymerases (etnangien). DKxanthenes (Simunowic et al. 2006; Bode et al. 2007; Etnangien also targets eubacterial RNA polymerases. One of Volz et al. 2012). These compounds show different activities, the classes of compounds used clinically is rifampicin. e.g., electron transport inhibition, anticancer activity, antibac- Myxobacterial compounds ripostatin and corallopyronin show terial, antifungal or cytotoxic activity. Myxovirescin (TA an- no significant cross-resistance with rifampicin, and are there- tibiotics), is a promising compound. TA is a rapid bactericidal fore likely to act by a different mechanism. This observation agent and has activity against many Gram-negative and some suggests the potential utility of the metabolites in overcoming Gram-positive bacteria. Antibacterial activity is specific, as rifampicin-resistant bacteria (O’Neilletal.2000). TA shows no toxicity toward fungi, protozoa, eukaryotic cells, It is generally assumed that microbial secondary metabo- rodents, or even humans (Xiao et al. 2012). TA also exhibits lites are preferentially synthesized during the late logarithmic unusually high adhesive properties toward biological and abi- and stationary growth phases, the so-called idiophase, when otic materials. For these reasons, TA has been proposed for the metabolism is no longer fully occupied with growth. treatment or prevention of biofilm infections, such as peri- However, with myxobacteria this is not the usual case. odontal diseases or infections derived from indwelling medi- Many substances are synthesized from the beginning of cal devices (Schierholz and Beuth 2001). The bactericidal growth, or shortly after (Reinchenbach 2001). The new anti- activity of myxovirescin requires de novo protein synthesis, biotic scaffold elansolid exhibiting potent anti-MRSA activity suggesting that synthesis of new proteins may be required for (Steinmetz et al. 2011, 2012)wasfoundinChitinophaga killing. Genetic and biochemical results show that sanctii, and the novel genus Aetherobacter was identified as myxovirescin targets type II signal peptidase (LspA) encoded a producer of a novel scaffold, the aetheramides, showing by the lspA gene. Killing likely occurs by two mechanisms. activity against HIV (Plaza et al. 2012). Cystobacter One mechanism involves the aforementioned mislocalization ferrugineus was found to produce roimatacenes representing and toxic buildup of lipoprotein (Lpp). The second mecha- a novel class of antibiotics (Zander et al. 2011), hyalidione nism likely prevents essential lipoproteins from being proper- was identified from Hyalangium minutum, and icumazoles ly localized to the outer membrane. Previous metabolic label- (Barbier et al. 2012) were described as a new class of antifun- ing studies found that TA causes a delayed inhibition in cell gals from Sorangium cellulosum. The latter genus was also wall biosynthesis (Zafriri et al. 1981). Xiao et al. (2012) found to produce carolactone, a novel cyclic lactone that spe- reinterpreted those results; namely, the inhibition of LspA cifically inhibits growth of streptococci (Jansen et al. 2010). Ann Microbiol (2016) 66:17–33 27

However, to develop a metabolite hit into an applicable enzymes in Sorangium are arranged into a complex of 1000– pharmaceutical compound is not an easy task, especially given 2000 kDa, which at least contains cellulase and xylanase ac- the complexity of their natural product chemistry, side effects tivities (Hou et al. 2006). In the studies on the communities of and poor bioavailability. Therefore, to make better use of na- epiphytic bacteria in grain crops, Leontievskaya and ture’s pharmaceutical factories, new technologies, such as en- Dobrovol’skaya (2014)definedaso-calledBhydrolytic block^ gineering of microorganisms to synthesize complex molecular of bacteria, which comprised myxobacteria and bacilli. structures, in silico tools to predict the target profile and an- Occurrence (percentage) of this group of bacteria on the cereal ticipate potential side effects of those metabolites, and targeted plant leaves (wheat and barley) grew up along with duration of delivery strategies, for example via nanoparticles, are under the vegetation period; the authors stressed the group's cellulo- the spotlight and will play an increasing role in the future lytic capabilities. According to Zhou et al. (2014b), (Villaverde 2010;Diezetal.2012) myxobacteria in sewage sludge (e.g., members of the genera: Recently, we reported extracellular synthesis of silver Nannocystis, Chondromyces, Haliangium and Stigmatella) nanoparticles by Myxococcus virescens. The silver nanoparti- are responsible for hydrolysis of some macromolecules cles obtained demonstrated antibacterial activity against hu- (among others—hemicellulose); they can also prey on true man pathogenic bacteria (Wrótniak-Drzewiecka et al. 2013, eubacteria. 2014). Lipids play a pivotal role in the Myxococcus life cycle during predation and development. Lipids containing the fatty acids c16:1ω5c are among the most abundant lipids in Enzymatic activity M. xanthus, but are rare in other bacteria (Moraleda-Muñoz and Shimkets 2007). They are chemo-attractants and may play Myxobacteria are considered micropredators. Antibiotics and an important role in development. Lipids containing the fatty enzymes produced by myxobacteria kill microorganisms and acids c18:1ω9c are not found in M. xanthus and appear to lyse cells. Cell wall degrading, lipases, nucleases, serve as chemo-attractants for detecting prey bacteria (Curtis polysaccharidases and proteases appear to be involved in the et al. 2006). Fatty acids are utilized for carbon and energy lysis of prey microbes as well as in autolysis or programmed during growth, along with protein that is locked in the prey cell death, which is simultaneous with myxospore develop- cytoplasm. M. xanthus remove the membrane barrier with ment. Despite numerous studies on myxobacteria, their enzy- lipolytic enzymes that not only release fatty acids, but also matic activity has received little attention (Alvarez-Sieiro et al. empty the cytoplasmic contents of the prey (Moraleda- 2014;Dahmetal.2015; Leontievskaya and Dobrovol’skaya Muñoz and Shimkets 2007). The lipids have a prominent role 2014; Mori and Kimura 2014). Myxobacteria are character- in the life cycle of M. xanthus. This bacterium has a large ized by their efficient degradation abilities of number of putative lipase genes in three main families— biomacromolecules (Dahm et al. 2015; Leontievskaya and patatin lipases, α/β hydrolases and GDSL lipases. An extra- Dobrovol’skaya 2014; Saraf et al. 2014;Zhouetal.2014b). cellular alkaline protease was purified from M. xanthus culture Based on their activity in degradation of biomacromolecules, supernatant. This enzyme was specific for hydrophobic or myxobacteria are divided into two groups. One is bacteriolyt- aromatic residues. In our experiments, myxobacteria produced ic, lysing living cells of other microorganisms, and the other is extracellular alkaline as well as acid proteases (Brzezińska cellulolytic, decomposing cellulose (Reichenbach and 2012). Proteolytic enzymes are produced both by the cellulo- Dworkin 1992). In general, the cellulolytic enzymes are orga- lytic myxobacteria (e.g., members of the genus Sorangium), nized in two strategies, extracellular free enzymes and cell- as well as by the predatory ones (e.g., belonging to the genus bound complex enzymes. Cellulolytic activity in Myxococcus)(Kimetal.2009). myxobacteria Sorangium sp. is rather low compared to that Three possible functions for myxobacterial extracellular of the extracellular free cellulases in aerobic cellulolytic fungi proteases are suggested: (1) proteases may supply amino acids such as Penicillium, Aspergillus and Trichoderma. However, to the myxobacteria by hydrolysing soil proteins derived from the degradation of cellulose is complete (e.g., lyse of cell wall plant, animal and soil microorganisms; (2) after activity of cell lower fungi), which does not correspond to the assayed activ- wall lytic enzymes, a protease may disrupt the cell membrane ities (Hou et al. 2006;Brzezińska 2012). These authors found of the eubacterium, releasing its intracellular content accessi- the protuberant structure on the surface of Sorangium that is ble to the surrounding environment, (3) the ultimate lysis of responsible for cellulose degradation, and cellulose materials the prey is likely to involve proteases. Gnosspelius (1978) destroyed were limited to the region of cellular swarm. purified three proteases produced by Myxococcus virescens. Beyond the contact with cells, the cellulose (filter paper) The purified enzyme hydrolysed casein and hemoglobin and remained intact. The authors suggested that the cellulases in was specific for peptide bonds involving amino acids with Sorangium exist on cellular surfaces and are organized as a nonpolar side-chains. Its optimal pH was between 7 and 10 complex, which might be cellular protuberances. Cellulolytic and it was inhibited by diisopropylphosphorofluoridate. These 28 Ann Microbiol (2016) 66:17–33 properties and its substrate specificity suggest that the enzyme and Kimura 2014). Saraf et al. (2014) point out that was a serine protease, even though it was inhibited by metal myxobacteria that act as a biocontrol agent (e.g., towards plant chelating agents. pathogenic fungi) produce large amounts of lytic enzymes. Several microorganisms secret slime that is more or less firmly bound to the cell. The slime is generally considered to be an external coat preventing drying of the cell and Conclusion protecting the cell wall against attack by various antimicrobial agents. The myxobacterial slime is involved in gliding motil- Myxobacteria are unique microorganisms known for broad ity (Gnosspelius 1978) and the matrix of the fruiting body is range of social adaptations. Myxobacteria from different hab- presumably polysaccharide (Dworkin 1972). Slime might also itats are not well explored, and hence there is a pressing need be involved in the nutrition of myxobacteria and serves as a to search for the myxobacteria from different environments in substrate for extracellular enzymes. During exponential general and in extreme environments like acidic/alkaline soils, growth, M. virescens secrets bacteriolytic enzymes followed marine environments, etc. Furthermore, a thorough screening by proteolytic enzymes and slime. This secretion order might of myxobacteria isolated from various habitats (including ex- reflect the fact that the natural substrates for the proteolytic treme environments) is needed in order to obtain potential enzymes are native cell components, released from eubacterial pharmaceuticals (e.g., for fighting against multi-drug resis- cells after their lysis by bacteriolytic enzymes (Gnosspelius tance problems and also to provide a better solution for new 1978). Myxobacteria (mainly members of the genera and emerging/ re-emerging diseases like AIDS and cancer). It Corallococcus and Myxococcus)isolatedfromsoilundervar- is believed that myxobacteria may open up new avenues in the ious trees (Scots pine, birch, black alder, oak) did not have any field of pharmaceuticals. cellulolytic and/or chitinolytic activity, but they produced ex- tracellular acidic and neutral proteinases (Dahm et al. 2015). Acknowledgments This work has been supported by Grant from the Studies on the M. virescens show that an extracellular protein Polish Ministry of Science and Higher Education (Grant No. 2 PO4 C 040 29). Dr Mahendra Rai is thankful to Nicolaus Copernicus University polysaccharide-lipid complex exhibited proteolytic activity (Torun, Poland) for fellowships to Visiting Professors within the project against gelatin and totally inactivated lysozyme. In the super- BEnhancing Educational Potential of Nicolaus Copernicus University in natant fluid of cultures of some myxobacteria the Disciplines of Mathematical and Natural Sciences^ conducted under – (Chondrococcus corraloides, Myxococcus xanthus, Sub-measure 4.1.1 Human Capital Operational Programme Task 7 (Project No.POKL.04.01.01-00-081/10)^ M. virescens, Sorangium sp.), an enzyme was detected that hydrolyses the β-1,4-bond between muramic acid and glucos- amine, thus having the substrate specificity of muraminidase References with an activity similar but not identical to that of lysozyme (Rosenberg and Varon 1984). Ahn JW, Jang KH, Chung SC, Oh KB, Shin J (2008) Sorangiadenosine, a Studies on M. virescens show that prolyl endopeptidases new sesquiterpene adenoside from the myxobacterium Sorangium (PEP) (EC 3.4.21.26), a family of serine proteases with the cellulosum. Org Lett 10:1167–1169 ability to hydrolyse the peptide bond on the carboxyl side of Altendorfer M, Irschik H, Menche D (2012) Design, synthesis and bio- an internal proline residue, isolated from this strain are able to logical evaluation of simplified side chains of the macrolide antibi- otic etnangien. Bioorg Med Chem Lett 22:5731–5734 degrade immunotoxic peptides responsible for celiac disease, Alvarez-Sieiro P, Martin MC, Redruello B, Rio B, Ladero V, Palanski such as a 33-residue gluten peptide (33-mer). The gene of this BA, Khosla C, Fernandez M, Alvarez MA (2014) Generation of proteinase — prolyl endopeptidase (produced by Myxococcus food-grade recombinant Lactobacillus casei delivering xanthus) was cloned into a food-grade recombinant Myxococcus xanthus prolyl endopeptidase. Appl Microbiol – Lactobacillus casei strain, thus obtaining a new tool to cure Biotechnol 98(15):6689 6700 Barbier J, Jansen R, Irschik H, Benson S, Gerth K, Böhlendorf B, Höfle celiac disease (Alvarez-Sieiro et al. 2014). Sasaki et al. (2014) G, Reichenbach H, Wegner J, Zeilinger C, Kirschning A, Müller R characterised the activities of the Myxococcus xanthus ApaH- (2012) Isolation and total synthesis of icumazoles and like phosphatases PrpA and ApaH, which share homologies noricumazoles antifungal antibiotics and cation-channel blockers with both phosphoprotein phosphatases and diadenosine from Sorangium cellulosum. Angew Chem Int Ed 44(42):6828– 6846 tetraphosphate (Ap A) hydrolases. The above authors stated 4 Behmlander RM, Dworkin M (1994) Biochemical and structural analyses that PrpA and ApaH may function mainly as a tyrosine protein of the extracellular matrix fibrils of Myxococcus xanthus.J phosphatase and an Ap4A hydrolase, respectively (Sasaki Bacteriology 176:6295–6303 et al. 2014). According to Mori and Kimura (2014), an en- Berleman JE, Scott J, Chumley T, Kirby JR (2008) Predataxis behavior in – zyme produced by Myxococcus xanthus, named ArsA, exhib- Myxococcus xanthus. Proc Natl Acad Sci U S A 105:17127 17132 ited weak phosphatase activity toward p-nitrophenyl phos- Berleman JE, Vicente JJ, Davis AE, Jiang SY, Seo Y-E et al (2011) FrzS regulates social motility in Myxococcus xanthus by controlling phate, and high arsenate reductase activity, which indicates exopolysaccharide production. PLoS ONE 6(8):e23920. doi:10. that it can reduce arsenates under natural conditions (Mori 1371/journal.pone.0023920 Ann Microbiol (2016) 66:17–33 29

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