REVIEWARTICLE

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-ria whose life cycle includes the formation of multicellularfruiting bodies that contain about 100,000 cells differentiatedas asexual spores for their long-term survival. They move bygliding on surfaces, an activity that helps them carry out theirprimitive kind of multicellular development. Myxobacteriahave multiple traits that are clearly social in nature; they moveand feed socially. These processes require specific intercellu-lar signals, thereby exhibiting a sophisticated level of the inter-organismal communication. Myxobacteria are predators.Predation is social not only with respect to searching for prey(motility) but also in the killing of prey. Swarming groups ofcells secrete antibiotics and bacteriolytic compounds that killand lyse their prey, and food is thereby released. Since the lastthree decades, myxobacteria are known as valuable producersof secondary metabolites exhibiting various biological activi-ties. Myxobacterial metabolites exhibit many unique structur-al features as well as rare or novel modes of action, makingthem attractive lead structures for drug development. Bothgenome sequencing and metabolic profiling of myxobacterialstrains suggest that the diversity of myxobacterial secondarymetabolism is far greater than previously appreciated. Thepresent review discusses the structure, cytology, physiology,and ecology of myxobacteria, as well as their secondary me-tabolite production and social interactions.

Keywords Myxobacteria . Ecology . Cytology . Enzymaticactivity . Secondary metabolism . Social interactions

Introduction

Myxobacteria (slime bacteria) are rod-shaped Gram-negativebacteria that move by gliding. They typically travel in swarms,containing many cells kept together by intercellular molecularsignals. Bacterial gliding is a process whereby a bacterium canmove under its own power. For many bacteria, the mechanismof gliding is unknown or only partially known, and differentbacteria (cyanobacteria, cytophaga-flavobacteria) have dis-tinct mechanisms of movement. When nutrients are scarce,cells of myxobacteria aggregate by chemotaxis, producefruiting bodies and avoid desiccation by forming resistantmyxospores.

Myxobacteria occur in at least two morphological types:(1) slender flexible rods with more or less tapering ends, and(2) cylindrical rods with rounded ends. Vegetative cells arerelatively large, measuring 3–6 μm long and 0.7–1.0 μmwide(Krzemieniewska and Krzemieniowski 1928). Different celltypes with other characters can be used to divide theMyxobacteriales into three sub-orders, the Cytobacterineae,Sorangiineae and Nannocystineae (Garcia et al. 2010). For acentury, taxonomy and classification of myxobacteria werebased on morphological traits such as shape of cell andfruiting bodies, size, colour and swarm patterns. However,morphological similarity often does not represent genetic sim-ilarity, and morphology-based phylogenies may fail to modelrelationships among species (Velicer and Hillesland 2008;Garcia et al. 2010).

The DNA sequence-based classification provides patternsof ancestral relationship among myxobacterial species. In re-cent years, 16S rRNA studies have shown that the group as a

* Wioletta Wrótniak-Drzewieckawiolawr@umk.pl

1 Department of Microbiology, Faculty of Biology and EnvironmentalProtection, Nicolaus Copernicus University, Lwowska 1,87-100 Torun, Poland

2 Department of Biotechnology, Sant Gadge Baba AmravatiUniversity, Amravat 444 602, Maharashtra, India

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

Current trends in myxobacteria research

Wioletta Wrótniak-Drzewiecka1 &

Anna Joanna Brzezińska1 & Hanna Dahm1&

Avinash P. Ingle2 & Mahendra Rai2

whole is phylogenetically coherent and belongs to the deltagroup Proteobacteria, a large taxon of Gram-negative forms.They are closely related to sulphate-reducing bacteria andBdellovibrio species, which are also predators of bacteria.The sub-order Cystobacterinae includes the two most thor-oughly studied species, Myxococcus xanthus and Stigmatellaaurantiaca. The sub-order Soranginae includes the familyPolyangiaceae with the genera Sorangium, an extensivelys t ud i ed c e l l u l o s e de compos e r. The sub - o r d e rNannocystineae is a unique mixture of isolates grouped intotwo clusters: (1) the marine organisms Enhygromyxa andPlesiocystis, which are allied to the terrestrial nonhalophilicbacteriumNannocystis; and (2) theHaliangium-Kofleria clus-ter (Garcia et al. 2011). Jiang et al. (2007) proposed that themyxobacteria exist in the environment in two forms — thefruiting and the non-fruiting types. Most of the unculturedmyxobacteria may represent taxa that rarely form fruiting bod-ies, or may lack some or all of the developmental genes need-ed for fruiting body formation.

Many phenomena and concepts originally described withregard to social interactions among higher organisms haveequivalents in microorganisms. These include inter-organismal communication, division of activity, self/non-selfrecognition, kin selection vs. individual selection and socialconflict. Microorganisms offer the opportunity to study theevolution of such social traits with a new level of rigor(Crespi 2001; Velicer and Stredwick 2002). Their rapidgrowth allows observations of evolutionary changes in labo-ratory populations. The social bacteriumM. xanthus is a mod-el system for the experimental study of microbial social evo-lution. As a group, the myxobacteria produce a large variety ofsecondary metabolites, some of which may have medical uses(Reichenbach and Höfle 1993; Gerth et al. 2003; Weissmanand Müller 2009; Diez et al. 2012). The reasons whymyxobacteria are multi-producers of secondary metabolitesare still not well understood. It has been argued that theyconfer a competitive advantage in soil environments, whichmay be used tomodulate cell–cell interactions and as weaponsfor predation. Answers to this question remain in the realm ofspeculation.

Understanding the evolution of social phenotypes remainsa great challenge for the evolutionary biologist. This problemis daunting in higher organisms for many reasons, includinglimited knowledge of behavioral genetics and genotype–envi-ronment interactions. Microbial social systems may help re-veal aspects of social evolution shared across all levels ofbiological organization. It is now clear that cell–cell commu-nications and functional multicellularity among and betweenbacteria and eukaryotic cells are commonplace. These takeplace as a part of host–parasite interactions, biofilm formation,multicellular development, and syntrophic interactions(Dworkin 1972). The physical connection between prokary-otic cells seems to give them an advantage. It concerns not

only M. xantus, but is also manifested by other bacteria. Themechanism whereby the bacterium senses the presence of aphysical or biological surface, specifically recognizes it andassociates with it is an area of microbiology that remains to beclarified (Dawid et al. 1988).

The present review is focused on different aspects ofmyxobacteria, viz., ecology, cytology, social interactions, sig-nal transduction, predation, secondary metabolism and enzy-matic activity.

Ecology

The myxobacteria seem to be a ubiquitous group of microor-ganisms that can inhabit very diverse habitats, including de-sert crust soils (Powell et al. 2015), the surface of the olderleaves in grain crops, as well as the surface of wheat andbarley seeds (Leontievskaya and Dobrovol’skaya 2014), thefruiting bodies and hyphosphere of several basidiomycetes(Zagriadskaia et al. 2014) and sewage sludge (Zhou et al.2014a). Myxobacteria were dominant on the older leaves ofgrain crops and in grain of wheat and barley ears; speciescomposition and structure of epiphytic bacterial communitiesin the grain cereal crops changed in the course of vegetation(Leontievskaya and Dobrovol’skaya 2014). Nutrient-rich soilsharbor more myxobacteria species, but these organisms canalso live in rocky soils and pure sand (Dawid 2000).Myxobacteria are capable of bioreduction of water-solubleuraniumU(VI) to insoluble uraniumU(IV). This phenomenoncan be used for bioremediation of ground water contaminatedwith uranium U(VI) and with other radionuclides (Newsomeet al. 2014). There is little contribution in the field of marinemyxobacteria (Felder et al. 2013). The marine myxobacteria,including Enhygromyxa, Plesiocystis, Pseudenhygromyxa,andHaliangium, are phylogenetically different from terrestrialmyxobacteria. Most cultured species prefer mild temperatures(20–30 °C), neutral pH and high concentrations of organicmatter, but low ionic concentrations. Marine myxobacteriaoccur in bottom sediments, and thus may have available highconcentration of organic matter (Brinkhoff et al. 2012).However, myxobacteria isolated from Antarctica grow at 4 °C (Dawid et al. 1988), and those from warm arid climateshave optimal growth temperatures of 42–44 °C (Gerth andMüller 2005). Only a few halophilic (Iizuka et al. 1998) orhalotolerant (Li et al. 2002) strains of myxobacteria have beenisolated from coastal areas (Jiang et al. 2010). Many studiessuggest that our knowledge of the particular environments ofmyxobacteria is limited by methods of isolation and cultiva-tion. One of the commonly used myxobacteria isolation tech-niques involves use of soil enrichment cultures (Fig. 1).

Myxobacteria are well known for three capabilities: (1)they move by gliding and their colonies are therefore thin,film-like swarms; (2) they have sophisticated intercellular

18 Ann Microbiol (2016) 66:17–33

communication systems and a highly developed social life; (3)they show a remarkable morphogenetic potential. In coopera-tive starvation conditions, they may produce fruiting bodies.Within the fruiting body, a cellular morphogenesis takes place,during which the vegetative cells convert into resistantmyxospores (Reinchenbach 1999). In myxobacteria,swarming toward new nutrient sources is accomplished bytwo genetically and physiologically distinct motility systems,adventure motility (A) (individual) and social (S) motility (Wuet al. 2007; Kaiser et al. 2010). The A-motility and S-motilitysystems are synergistic, as colony spreading bywild-type cellsis faster than the sum of those of individual A+S− and A−S+

cells. In the absence of one or both motility systems, aggrega-tion, fruiting body formation, and rippling are defective, indi-cating that motility is required for these social behaviors(Mauriello et al. 2010). Recently, Nan et al. (2014) identifiedthe helical protein track rotor mechanism of gliding motility inthe myxobacterium.

Cytology

Myxobacteria are typical Gram-negative bacteria with an out-er membrane.M. xanthus serves as a premier model bacteriumfor the study of cell envelope, cytological structures, socialbehavior, signals transduction and others. Myxobacteria areflexible cells and it has generally been assumed that their cellwall is fundamentally different from the wall of eubacteria,especially the layer of peptidoglycan (White et al. 1968). Itwould be expected that during differentiation, the M. xanthuscells would remodel their peptidoglycan as they change fromrod-shaped vegetative cells to spherical spores (Yang et al.2008). A comparison of peptidoglycan in vegetative cellsand spherical spores of M. xanthus showed that although theoverall composition of peptidoglycan of both cell types wassimilar, there appeared to be an increase in muropeptide cross-linking in the spherical spores (White et al. 1968) (Figs. 2 and3). Bui et al. (2009) observed that while the basic structuralelements of peptidoglycan in myxobacteria were identical to

those in other Gram-negative bacteria, the peptidoglycan ofM. xanthus had unique structural features. meso-diaminopimelic or ll-diaminopimelic acid was present in thestem peptides, and a new modification of N-acetylmuramicacid was detected in a fraction of the muropeptides.Peptidoglycan formed a continuous, bag-shaped sacculus invegetative cells. The sacculus was degraded during the tran-sition from vegetative cells to glycerol-induced myxospores.The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, butthey contained small amounts of N-acetylmuramic acid andmeso-diaminopimelic acid.

Periplasmic space

Little is known about myxobacteria periplasm specifically.The periplasmic space of M. xanthus contains, as in other

Fig. 1 Isolation of myxobacteria—colonies of myxobacteria growingout of soil crust (f.b. fruiting bodies)

Fig. 2 Young, vegetative cells of myxobacterium (Michałowska 2009)(c.w. cell wall, c.m. cell membrane, n nucleoide, r ribosomes, i inclusions)

Fig. 3 Mature myxospore (Michałowska 2009) (c.w cell wall, c.m. cellmembrane, s.c. slime capsule, n nucleoide, r ribosomes, g granules)

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Gram-negative bacteria, many enzymes and structural andfunctional proteins. Among them, lipoproteins, hydrolytic en-zymes, proteins for nutrient acquisition, chaperons and senso-ry proteins are important (Schlicker et al. 2004;Mogensen andOtzen 2005; Yang et al. 2008). It was suggested that someperiplasmic structures could be a part of the glidingmachinerythat may utilize membrane potential to power adventurousglidingmotility of myxobacteria. Freese et al. (1997) observedthe chain-like aggregates or strands in the periplasm ofM. xanthus. The strands appear to be composed of ring-likeand centrally elongated elements and formed ribbon-likestructures. However, there is no direct evidence of linkingthese structures to gliding (Yang et al. 2008).

Outer membrane

In Gram-negative bacteria, the outer layer of the cell envelopeis composed of lipopolysaccharide (LPS), which forms a se-lective barrier between the environment and the periplasmicspace. InM. xanthus,the LPS is similar in structure to the LPSof other Gram-negative bacteria (Fink and Zissler 1989; Yanget al. 2008). LPS molecule is composed of three parts: hydro-phobic lipid A, a covalently attached core oligosaccharideregion, and a distal repeating polysaccharide, termed O-anti-gen. The myxobacteria lipopolysaccharide O-antigen is simi-lar in overall structure to that of lipopolysaccharide in otherGram-negative bacteria, and the carbohydrate moiety consistsof glucose, mannose, rhamnose, arabinose, xylose, galactos-amine, glucosamine, 2-keto-3-deoxyoctulosonic acid, 3-O-methylpentose and 6-O-methylgalactosamine (Gill andDworkin 1986; Yang et al. 2008). Among the outer membraneproteins of M. xanthus, two lipoproteins, CgIB and Tgl, areimportant for motility. During the aggregation phase offruiting body formation, lectin (MBHA) accumulates at a highlevel. It is suspected that MBHA plays an important role inmyxobacteria (M. xanthus) cell adhesion, which is importantfor fruiting body development (Yang and Kaplan 1997).

Extracellular matrix (ECM)

Cells of myxobacteria are covered by an extracellular matrix(ECM) comprised of protein and polysaccharide.Polysaccharide was previously referred to as fibrils on the cellsurface, observed by scanning microscopy. The structures ob-served were most likely the results of dehydration of the poly-saccharide during preparation. Now, the polysaccharide com-ponent of ECM is referred to as exopolysaccharide (EPS)(Behmlander and Dworkin 1994; Kaiser et al. 2010). ECMis required by these myxobacteria for integrity of cell groups,which is important for cellular cohesion, social motility andfruiting body morphogenesis. The production of EPS is a

highly regulated process modulated by a phosphorylation-dependent mechanism. Glucose from cellulose would, viathe pentose phosphate pathway, give rise to many other hex-oses and pentoses that are used to synthesize polysaccharides,including their capsule of slime and their lipopolysaccharide(Kaiser et al. 2010). Biochemical analyses indicated thatM. xanthus EPS contains five monosaccharides: galactose,glucosamine, glucose, rhamnose and xylose (Merroun et al.2003; Yang et al. 2008). Among the 41 proteins identified, 20are likely integral inner or outer membrane proteins or cyto-plasmic proteins and the remaining 21 have been suggested tobe good ECM protein candidates (Konovalova et al. 2010).Only five of the 21 candidate ECM proteins have predictedfunctions. These functions include protease activity,amidohydrolase activity and coating of the myxospores.Inactivation of several of the genes encoding putative ECMproteins caused no defects in fruiting body formation, with theexception of the fibA gene, which encodes the FibA zincmetalloprotease (Kearns et al. 2002; Konovalova et al. 2010).

Pili

Pili (fimbrie) were first observed in myxobacteria byMac-Raeand Mc Curdy in 1976 (c.f. Rosenbluh and Eisenbach 1992),and were described as 6–8 nm diameter appendages, whichextend from one or both cell poles for up to a cell’s length orfurther. M. xanthus pili belong to the class of Tfp (type IVpili), which are found in Gram-negative bacteria (Wu andKaiser 1995; Yang et al. 2008). Pili have been studied in avariety of bacterial systems, and play an important role incohesion, intercellular communication and host colonizationby pathogens. In addition, the pili are thought to be involvedin social motility (Merroun et al. 2003; Kaimer et al. 2012).

Genome

Among the myxobacteria, M. xanthus is studied most geneti-cally, followed by Stigmatella aurantiaca. M. xanthus DK1622, apart from Anaeromyxobacter dehalogenans 2CP-C,was the first to have its genome sequenced. Further, differentmyxobacterial genomes have been completely sequenced:Sorangium cellulosum So ce56 and Stigmatella aurantiacaDW 4/3-1, Haliangium ochraceum, Myxococcus fulvus,Corallococcus coralloides, Myxococcus stipitatus ,Cystobacter violaceus (Li et al. 2011; Huntley et al. 2012,2013; Muller et al. 2013; Stevens et al. 2014). Mostmyxobacteria are composed of a single circular chromosome,and no extra-chromosomal plasmids were known until 1980,when the first endogenous plasmid was identified inMyxococcus fulvus. Endogenous plasmids have not beenfound in M. xanthus. However, this bacterium produces an

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unusual satellite DNA known as multicopy single-strandedDNA (msDNA), which is highly conserved (Shimkets1990). Myxobacteria possess giant chromosomes, belongingto the largest known in bacteria (Pradella et al. 2002; Kaiseret al. 2010). Myxobacterial genomes are at the large end of theeubacterial scale and approach the size of the lower eukaryoticgenomes. A genome size ranging from 5690 to 12,727 kbpwas noted (estimations were carried out on exponentiallygrowing cells). Myxospores contain an average of 3.3 genomeequivalents per spore (Chen et al. 1990). The genome ofM. xanthus contains a much higher G+C proportion than doesEscherichia coli. TheM. xanthus (DK 1622) genome is com-posed of a single circular chromosome. Nearly half of thegenes (48.9 %) have been assigned a putative function(Goldman et al. 2006; Ronning and Nierman 2008).

The unusually large size of the M. xanthus genome is re-portedly due to expansion by lineage-specific duplications ofspecific categories of genes, particularly those involved incell–cell signaling, small molecule sensing and multi-component transcriptional control of the complex molecularmachinery required for development of a multicellular life-style (Ronning and Nierman 2008). Genes involved in thesocial and developmental behavior of this bacterium werefound in many regions of the chromosome. Thirty genes areinvolved in the adventurous (individual) move: the type IVpili genes and the sasA locus encoding the lipopolysaccharideO-antigen biosynthesis genes (Wu and Kaiser 1995;Wall et al.1999; Lancero et al. 2002; Vlamakis et al. 2004; Ronning andNierman 2008). Although the functions of most M. xanthusgenes remain to be determined, bioinformatics analysis clearlyreveals complex signaling and regulatory networks. For in-stance, theM. xanthus genome encodes 53 s54 enhancer bind-ing proteins, 38 extra-cytoplasmic function sigma factors, 97serine threonine protein kinases and 272 two-component sig-nal transduction proteins (Goldman et al. 2006). Many of the-se regulatory proteins are likely to function in pathwaysgoverning interactions with other cells and their environment,and future work is needed to elucidate their roles.

Since the myxobacteria have a complex developmental cy-cle, it is tempting to speculate that a large genome is necessaryfor fruiting body development; however, only a small part ofthe genome is essential for fruiting body development (Chenet al. 1990). It has been estimated that secondary metabolitesynthetic genes constitute more than 3 % of some bacterialgenomes (Sasse et al. 2000), e.g., 9 % of the total geneticcapacity inM. xanthus (Diez et al. 2012). Genes of secondarymetabolism in myxobacteria have been intensively studied.Several secondary metabolic gene clusters have been analysedin detail (Beyer et al. 1999; Julien et al. 2000; Silakowski et al.2001; Ligon et al. 2002; Pradella et al. 2002; Sandmann et al.2004; Carvalho et al. 2005; Feng et al. 2005; Perlova et al.2006). The myxobacteria have a large capacity for signaltransduction. Feeding and fruiting body development in the

myxobacteria is coordinated by the regulatory (A) and (C)signal genes. The quorum sensing A-signal senses the ap-proach of starvation and induces cellular aggregation. InM. xanthus, A-signal is controlled by five genes that mayfunction together in response to the nutritional state of the cell(Kaiser 2004). Eight chemotaxis clusters have been identifiedinM. xanthus. It is not clear that a large genome is the result ofdevelopmental complexity. It was detected that only about8 % of the M. xanthus genome increases expression duringdevelopment and less than 1 % of the genome is essential fordevelopment. The function of the large genome remainsunknown. Evidently, additional expansion within themyxobacterial lineage led to Sorangium cellulosum, whose13 Mbp genome encodes the largest number of serine/threonine protein kinases of any organism (Perez et al.2008).

Social interactions

The conventional wisdom of microbiology has been that bac-teria are independent, unicellular organisms and that the prop-erties of a population are the sum of the properties of theindividual cells (Dworkin 1999). It is now clear that cell–cellcommunications and functional multicellularity among bacte-ria are commonplace. Social cooperation is exhibited bymanymicrobial species (Crespi 2001). Microbial sociality includesa quorum-sensing system, cooperative predation, suicidal al-truism and the formation of complex developmental structuressuch as biofilms and the fruiting structures of Streptomycesand myxobacteria (Velicer and Stredwick 2002).

Recently, it has been evaluated that during cell–cell inter-action myxobacteria exchange their outer membrane (OM)proteins and lipids. The mechanism of transfer requires phys-ical contact between aligned cells on hard surfaces. Transfer ismediated by OM fusion, in which membrane contents lateral-ly diffuse and are exchanged bi-directionally between cells.TraA and TraB are recently identified proteins that are re-quired in donor and recipient cells for transfer to occur. OMexchange results in phenotypic changes that can alter glidingmotility and development of myxobacteria. It is a novel mi-crobial interaction coordinating multicellular activities(Pathak et al. 2012a, b; Pathak and Wall 2012; Wall 2014a).Recently, the traAB operon was discovered as a genetic deter-minant for transfer. Because fluorescently labeled lipids arealso exchanged by a TraAB-dependent mechanism, the modelfor transfer invokes the transient fusion of the OM, resulting inthe exchange of content between cells (Pathak et al. 2012b).TraA functions as a cell surface adhesion and as a molecularrecognition determinant that identifies other cells that expressthe same or similar traA alleles to coordinate social behaviors(Pathak et al. 2012b; Wall 2014a, b). Wei et al. (2014) sug-gested that traA functions as a fusogen to catalyse OM fusion

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between cells (Pathak and Wei 2012; Wall 2014a). The func-tion of traB is less clear, although it contains an OmpA do-main that is predicted to bind to the cell wall. Gram-negativebacteria, includingM. xanthus, secrete diffusible OM vesiclesthat potentially can fuse with other cells, including eukaryoticcells (Mashburn-Warren andWhiteley 2006; Kulp and Kuehn2010). Based on a bioinformatic analysis, TraAB orthologsare restricted to myxobacteria, although other bacterial groupsmay have functional analogs and may carry out similar behav-ior (Kudryashev et al. 2011). Using live imaging, Ducret et al.(2013) showed that transient contacts between two cells aresufficient to transfer OMmaterials, proteins and lipids, at highefficiency. Transfer was associated with the formation of dy-namic OM tubes, strongly suggesting that transfer results fromthe local fusion of the OMs of two transferring cells. Last,large amounts of OM materials were released in slime trailsdeposited by gliding cells. Since cells tend to follow trails laidby other cells, slime-driven OMmaterial exchange may be animportant stigmergic regulation of Myxococcus social behav-iors. Similarly, during microscopic examination of cells, Weiet al. (2014) discovered long tubular filaments emanatingfrom M. xanthus cells, which turned out to be OM tubes.

M. xanthus and other species of myxobacteria have multi-ple traits that are clearly social in nature. M. xanthus movesand feeds socially. Cells can move using one or both of twogenetically distinct motility systems. Social (S) motility isstimulated by cell–cell proximity and involves type IV pili(McBride 2001). Adventurous (A) motility allows greater in-dividual cell movement (Spormann 1999; Konovalova et al.2010). Fibrils and type IV pili are the extracellular structuresthat are associated with S-motility and physically link cellstogether. Fibril polysaccharides are frequently calledexopolysaccharides (EPS). It should, however, be noted thatM. xanthus produces other forms of EPS that are not associ-ated with fibrils (Ducret et al. 2012; Muller et al. 2012). Fibrilsplay a key role in cell–cell adhesion (a.k.a. agglutination orclumping), recognition, motility and development (Li et al.2003). Polarly localized type IV pili function as the motor thatpowers S-motility; their retraction from the ‘front end’ of therod-shaped cell pulls the cell forward (Skerker and Berg2001). As a recognition anchor for retraction, pili bind toEPS deposited in the extracellular matrix (ECM) (Li et al.2003). A-motility is associated with polar extracellular ‘slime’filaments, which are thought to be composed of an as-yet-to-be-characterized polysaccharide (Yu and Kaiser 2007; Ducretet al. 2012). The A-motility motor appears to be powered bymobile cell surface adhesins. Thus, this mechanism ofmotilitymight be analogous to how tank or bulldozer tracks propelthose vehicles (Nan and Zusman 2011; Sun et al. 2011). A-motility involves two proteins: AglZ, a cytoplasmic protein,and AgmU, a protein that localizes to both the cytoplasm andperiplasm (Mauriello et al. 2010). Recently, Nan et al. (2010)found that AgmU is also associated with many other A-

motility proteins, including AglT, AgmK, AgmX, AglW,and CglB. These proteins likely form a large multiproteincomplex that spans the membrane and periplasm of the cells.

Swarming groups of cells secrete antibiotic and bacterio-lytic compounds that kill and lyse prey. The efficiency of thissocial predation appears to be density dependent (Velicer andStredwick 2002). The M. xanthus process of fruiting bodyformation is a social behavior. Fruiting body development isinitiated by starvation for carbon or nitrogen or phosphorus.Some authors have shown that upon amino acids starvation,swarms of about 105 organisms use movement together athigh-density aggregation loci, a process that has been com-pared to animal migration (Shimkets 1999). These aggregatestransform into fruiting bodies. Such process requires specificintercellular signals, thereby exhibiting a sophisticated level ofinter-organismal communication. A survey of literature re-vealed that only a minority (ca. 10 %) of an initial aggregatingpopulation differentiates into resistant spores, whereas most ofthe remaining cells autolyse (altruistic suicide) (Wiseman andDworkin 1977; O’Connor and Zusman 1989) (Fig. 4a–d).

The ecological roles of fruiting bodies are unclear (Velicerand Stredwick 2002). One hypothesis holds that fruiting bod-ies may serve to improve the chance of spore dispersal tonutrient-rich habitats. Alternatively, they may help protectdormant spores from caustic soil compounds and facilitateefficient germination. Individual spores may have higher fit-ness during germination when clumped in packs than when inisolation. To carry out a program of morphological develop-ment, the cells communicate with each other by emitting andresponding to extracellular chemicals signals, two of whichare A-factor and C-factor. Myxobacterial fruiting body devel-opment requires extensive cell–cell signaling and interactions,as well as coordinated changes in gene expression and cellmovement (Kroos 2007; Leonardy et al. 2008). The transitionfrom vegetative growth to development in M. xanthus waspreviously shown to depend on the RelA-mediated stringentresponse (Singer and Kaiser 1995), but more recently, thesRNA Pxr has also been implicated in governing this transi-tion (Yu et al. 2010). Starvation is recognized by M. xanthusby a ribosome that lacks the charged tRNA specified by thecodon positioned in the acceptor (A) site. Uncharged tRNAbinds instead, and the highly phosphorylated guanosine nu-cleotide (p)ppGpp is then synthesized from GTP and ATP.Since formation of amino acyl tRNA depends on ATP, alow-level energy or phosphate can be detected. Succinctlyand sensitively, (p)ppGpp directly assesses the cell’s capacityto synthesize protein (Kaiser 1998). In M. xanthus fruitingbody development, ribosomal and tRNA synthesis are imme-diately inhibited in response to amino acid limitation. In ad-dition, DNA, membrane and cell wall biosyntheses areinhibited and proteolysis is increased. A response to aminoacid starvation is evident within 30 minutes as an elevationof (p)ppGpp. (p)ppGpp is needed to induce production of the

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A-factor. The A signal appears to be a mixture of amino acidsthat are released from cells and function as relatively long-range indicators of cell density.

A second intercellular signal, C-factor is a 17 kDa proteinencoded by the csgA gene. C-signaling controls three diverseprocess: cell aggregation, expression of variety of genes (in-cluding csgA) and sporulation. C-factor on the surface of onecell interacts with receptive cells and requires direct contactbetween the ends of two cells. C-factor bears sequence homol-ogy to a family of dehydrogenase enzymes, suggesting thatsignaling involves a dehydrogenation (Kaiser 1998). Cell–cellinteractions are often mediated by pili. The pili of M. xanthushave been shown to play an explicit role in social motility (Wuand Kaiser 1995; Mauriello et al. 2010; Berleman et al. 2011).However, an additional mechanism for mediating these con-tacts is that of extracellular appendages called fibrils. Fibrilshave been studied in detail in the myxobacteria (BehmlanderandDworkin 1994; Kaiser et al. 2010; Kaiser 2015). However,a second extracellular appendage of fibrils has also been shownto be necessary for social behavior. The fibrils are polysaccha-ride organelles containing a set of tightly adhering proteins. Itis proposed that cell–cell contact is perceived by the fibrils andis mediated by the action of a fibrillar ADP-ribosyl transferase(Dworkin 1999). Recently, Harvey et al. (2013) proposed acontinuum theory of clustering in self-propelled flexible rodswith applications to collective dynamics of the common glid-ing bacteria M. xanthus. Numerical simulations of this modelconfirm the existence of stationary dense moving clusters andalso elucidate the properties of their collisions.

C–factor associated with the cell surface provides input tothe Frz signal transduction cascade. Elements of this cascadehave sequence homology to bacterial chemotaxis systems andare known to control the frequency of gliding reversal(Kaimer et al. 2012). The extracellular C-signal is requiredfor rippling, aggregation, sporulation and maximum inductionof developmentally regulated genes after 6 h during fruitingbody morphogenesis in M. xanthus. C-signal is not only re-quired for, but also induces these events (Kruse et al. 2001;Kaiser 2004, 2013).

Signal transduction

The largest groups of prokaryotic signaling pathways are twocomponent systems (TCSs). They are most abundant in themyxobacteria (Whitworth and Cock 2007, 2008; Darnell et al.2014). A typical TCS consists of a histidine protein kinase anda response regulator protein. The genes for a histidine kinaseand response regulator pair are often found adjacent to oneanother in the genome, and it is evident that they act togetherto form a TCS. A large number of TCS proteins ofmyxobacteria have been identified and characterized (Perezet al. 2008).

Most of these proteins are known to regulate fruiting bodydevelopment, motility and chemotaxis (Whitworth and Cock2007). The complex domain structures of myxobacterial TCSproteins suggest that the TCSs of myxobacteria operate insignificantly different ways from those of most other bacteria

Fig. 4 aVegetative growth stageof myxobacterial cells (maturecells) (Michałowska 2009). bEarly aggregation stage ofvegetative cells (Michałowska2009). c Differentiation stage ofvegetative cells into myxospores(Michałowska 2009). d Maturemyxospores (Michałowska 2009)

Ann Microbiol (2016) 66:17–33 23

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

Predation

Populations of myxobacteria may significantly affect the den-sity of a variety of organisms in the different environments.Many researchers reported predator prey interactions betweenmyxobacteria and soil microorganisms. Myxobacteria preyupon a diversity of Gram-negative as well as Gram-positivespecies of bacteria. According to Guerrero et al. (1987), pre-dation between prokaryotes is one of the most ancient formsof predation (Fig. 6). Myxobacteria employ a highly differentmode of predation than Bdellovibrio (Evans et al. 2008). Theyuse gliding motility and produce a wide range of antibioticsand lytic enzymes and break down cell polymers for nutrients(Morgan et al. 2010; Xiao et al. 2011). The Gram-positivespecies are poorer prey for Myxococcus predators than areGram-negative ones. Such observations suggest a differencein the susceptibility of Gram-positive and Gram-negative spe-cies to predation by Myxococcus. It is suggested that the typeIV pili onMyxococcus cells play a key role in adhesion to preycells, and thereby facilitae predatory lysis (Morgan et al.2010). SomeMyxococcus strains do vary in their prey specific

performance on individual prey species, thus reflecting somedegree of specialization.

Myxobacteria are widespread in soils around the world.This ubiquity, coupled with the ability to use a broad rangeof other microbial species (fungi, bacteria) as prey, suggestthat myxobacteria strongly affect the populations of manymicroorganisms. Myxobacteria use gliding motility to searchthe soil matrix for prey and produce a wide range of antibioticand lytic compounds that kill and decompose prey cells.Myxobacterial predation is cooperative both in its searchingcomponent and in its handling component (Morgan et al.2010). According to some research (McBride and Zusman1996; Berleman et al. 2008), myxobacteria employchemotaxis-like genes in their attack on prey, and predationis stimulated by close contact with prey cells.M. xanthus cellscannot sense prey colonies until direct cell–cell contact ismade. There is no recognition of prey cells even at very shortdistances, but when contact is made, the M. xanthus cellsbegan to alter their behavior. The Frz signal transduction sys-tem is also responsible for keeping theM. xanthus cells in the

Fig. 5 Signaling pathways of two-component systems (TCS). (a) Sensordomain is covalently bound to histidine kinase domain. Phosphorylationoccurs on conserved histidine residue. The phosphoryl group is trans-ferred to a conserved aspartate residue within the receiver domain (Rec)in the response regulator. The prototypical RR output is a DNA-bindingdomain capable of influencing gene expression. The vertical bar repre-sents the cytoplasmicmembrane. (b) TCS system controlling chemotaxis.

The MCP transducer is depicted as transmembrane and is coupled bxCheW to the CheA kinase. Two methyl groups are shown to representmethylation of the receptor bz CheR. Phosphorylated CheB can removethese methyl groups. Methylation is a hallmark feature of the chemotaxisTCS systems. Phosphotransfer to the response regulator CheY influencesits ability to bind the FliM switch component at the flagellar motor (ac-cording to Kirby et al. 2008, modified)

Fig. 6 Lysis of E. coli cells byMyxococcus virescens (E.c.–E. coli,M.v.M. virescens)

24 Ann Microbiol (2016) 66:17–33

vicinity of their prey after contact has been made and feedingis underway. When cells start moving away from the source,the Frz system senses this and induces a reverse in direction tokeep them in contact with the prey colony (McBride andZusman 1996).

Production of secondary metabolites

Aside from actinomycetes and fungi, myxobacteria are one ofthe important sources for natural microbial products. Themainproducers of secondary metabolites are members ofActinomyces (ca. 8000 compounds characterized), genusBacillus (1400), as well as Pseudomonas (400). However,over the last decade, the myxobacteria have emerged as apromising alternative source of bioactive molecules (Huanget al. 2010; Weissman and Müller 2010; Altendorfer et al.2012; Johnson et al. 2012; Kim et al. 2013; Schmitz et al.2013; Plaza and Müller 2014; Schäberle et al. 2014).Myxobacterial secondary metabolites present structural ele-ments not commonly produced by other microbes, such asunusual hybrids of polyketides and non-ribosomally madepeptides. In fact, around 40 % of the described myxobacterialcompounds represent novel chemical structures. Furthermore,most small molecules from myxobacteria are not glycosylat-ed, as opposed to products derived from actinomycetes, andthey target molecules that are often not targeted bymetabolitesfrom other microbes (Diez et al. 2012).Myxobacterial second-ary metabolites exhibit many unique structural features andnovel modes action, making them attractive and promisingsources for drug development. Rare but notable propertiesinclude antibiotic, anti-malarial, immunosuppressive, antiviraland insecticidal activities (Weissman and Müller 2010).

The many different compounds from myxobacteria showquite different mechanisms of action. There are inhibitors ofprokaryotic (myxovalargin) and eukaryotic (gephyronic acid)protein synthesis, compounds that stimulate potassium exportfrom Gram-positive bacteria (tartrolon) and compounds thatbind to DNA (saframycin). In some cases, the mechanism ofaction has not yet been elucidated, e.g., for the highly cyto-toxic vioprolids or for the antifungal leupyrrin.

The genus Sorangium produces almost half of the second-ary metabolites isolated from myxobacteria. Different strainsof Sorangium produce several novel antimicrobial macrolides,the leupyrrins (Kopp et al. 2011), the thuggacins (Irschik et al.2007; Buntin et al. 2008), sorangicin (Irschik et al. 2013),phoxalone (Guo et al. 2008) and other components, like anew sesquiterpene, sorangiodenosine (Ahn et al. 2008), a freeradial scavenger, soraphinol C (Li et al. 2008), as well as anovel class of antineoplastic agents, the epothilones and theiranalogs (Mulzer 2009). Epothilones and their analogues havedemonstrated antitumor activity towards multidrug resistanttumor cells (Mulzer 2009; Gong et al. 2014). These

compounds target the eukaryotic cytoskeleton; interferencewith microtubule assembly plays a critical role in currentlyavailable cancer chemotherapies, through the inhibition of cellproliferation and the induction of apoptosis. Epothilones havebeen noted to have antineoplastic activity. This has led to thedevelopment of analogs that mimic its activity. One such an-alog, known as Ixabepilone, is a U.S. Food and DrugAdministration-approved chemotherapy agent for the treat-ment of metastasis breast cancer. Several other metabolitesare currently being evaluated in preclinical studies (Kimet al. 2013; Schmitz et al. 2013). From S. cellulosum (So ce12), five compound groups have been characterized. In addi-tion to the highly active disorazole class of tubulin de-stabi-lizers, this strain produces sorangicins eubacterial RNA poly-merase inhibitors, bactericidal sorangiolides, the antifungalchivosazoles and the sulfangolides (Irschik et al. 1987;Jansen et al. 1997; Wenzel and Müller 2007). At nanomolarconcentrations, Soraphen, a metabolite of S. cellulosum, in-hibits the biotin carboxylase domain of human, yeast and oth-er eukaryotic acetyl-coenzyme A carboxylases. The specificinhibitors of this enzyme show promise as a therapeutic agentfor cancer (Weissman and Müller 2010). Recently, Irschiket al. (2013) discovered a family of structurally related mac-rocyclic lactones in the fermentation broth of themyxobacterium Sorangium cellulosum Soce1485. The mostabundant member of this group was named maltepolide A,since the bacterium was isolated fromMalta Island. The novelmaltepolides demonstrated biological activity. It has beenshown that a metabolite derived from S. cellulosum,Ratjadone A, exhibits strong anti-HIVactivity, but low selec-tivity due to toxic effects. Although this limits its potential useas a therapeutic drug, further studies with derivatives ofratjadones might help to overcome these difficulties in thefuture (Fleta-Soriano et al. 2014).

Corallopyronin A is a myxobacterial compound with po-tent antibacterial activity, isolated from the Corallococcuscoralloides. Corallopyronin A is a novel inhibitor of bacterialRNA polymerases that is being developed as an antifilarialdrug, targeting lymphatic filariasis and onchocerciasis, whichare tropical diseases caused by parasitic nematodes. Theantifilarial activity of corallopyronin is due to its bactericidaleffect on theWolbachia endosymbionts, leading to a block ofoogenesis, embryo-genesis and development of larvae, andeventually to death of the adult worms (Schäberle et al. 2014).

The majority of myxobacterial secondary metabolites rep-resent polyketides, non-ribosomally made peptides or hybridsof the two structural types (Wenzel and Müller 2007). Thecorresponding genes of multi-enzymes are organized in clus-ters that are located on genomic regions 20–200 kilobases (kb)in size (Bode and Müller 2005). Polyketide synthase and non-ribosomal peptide synthetase are composed of highly con-served regions. Analysis of the chromosome of Stigmatellaaurantiaca revealed the presence of 8656 putative genes, a

Ann Microbiol (2016) 66:17–33 25

significant number of which are involved in secondary metab-olism (Wenzel and Müller 2007). The strain DW 4/3–1 ofS. aurantiaca synthesizes five different compound families:myxochromides S, aurafurones, myxothiazoles, DK xan-thenes and dawenol. Aurachins are a family of secondarymetabolites produced by the myxobacterium Stigmatellaaurantiaca strain Sg a15, and possess numerous bioactivities,such as anti-bacterial, anti-fungal and anti-plasmodial proper-ties. Furthermore, these isoprenoid quinoline alkaloids are po-tent inhibitors of mitochondrial respiration by targeting thecytochrome b6/f-complex, as well as the complexes I and IIIin the respiratory chain (Dejon and Speicher 2013).

Some time ago, using high performance liquid chromatog-raphy and high resolution mass spectrometry, 37 novel naturalproducts were identified from 98M. xanthus isolates (Kruget al. 2008). These results suggest that M. xanthus andmyxobacteria in general are promising sources for natural sec-ondary metabolites. Scanning of the M. xanthus DK 1622genome sequence for the presence of polyketide synthaseand non-ribosomal peptide synthetase-encoding genes re-vealed the presence of 18 biosynthetic gene clusters, account-ing for around 9 % of the genome (Diez et al. 2012), but until2005, no substance could be identified in the screening pro-cesses. However postgenomic examination of extracts byHPLC-MS, combined with mutagenesis, led to the identifica-tion of five compound families: myxovirescines,myxalamides, myxochelines, myxochromides, A andDKxanthenes (Simunowic et al. 2006; Bode et al. 2007;Volz et al. 2012). These compounds show different activities,e.g., electron transport inhibition, anticancer activity, antibac-terial, antifungal or cytotoxic activity. Myxovirescin (TA an-tibiotics), is a promising compound. TA is a rapid bactericidalagent and has activity against many Gram-negative and someGram-positive bacteria. Antibacterial activity is specific, asTA shows no toxicity toward fungi, protozoa, eukaryotic cells,rodents, or even humans (Xiao et al. 2012). TA also exhibitsunusually high adhesive properties toward biological and abi-otic materials. For these reasons, TA has been proposed for thetreatment or prevention of biofilm infections, such as peri-odontal diseases or infections derived from indwelling medi-cal devices (Schierholz and Beuth 2001). The bactericidalactivity of myxovirescin requires de novo protein synthesis,suggesting that synthesis of new proteins may be required forkilling. Genetic and biochemical results show thatmyxovirescin targets type II signal peptidase (LspA) encodedby the lspA gene. Killing likely occurs by two mechanisms.One mechanism involves the aforementioned mislocalizationand toxic buildup of lipoprotein (Lpp). The second mecha-nism likely prevents essential lipoproteins from being proper-ly localized to the outer membrane. Previous metabolic label-ing studies found that TA causes a delayed inhibition in cellwall biosynthesis (Zafriri et al. 1981). Xiao et al. (2012)reinterpreted those results; namely, the inhibition of LspA

blocks the maturation of key lipoproteins required for mureinbiosynthesis, and thus indirectly blocks cell wall biosynthesis.For a number of reasons, LspA is an attractive target for anti-biotic drug discovery. First, this signal peptidase is universallyfound in bacteria and is broadly essential in Gram-negativebacteria. In Gram-positive organisms, LspA appears to beconditionally essential or nonessential and plays a key rolein pathogenesis, as many virulence factors are lipoproteinsor require lipoprotein function (Hutchings et al. 2009).Second, LspA is absent in eukaryotic cells, which eliminatesany concerns about target-based toxicity in animals. Third,from a clinical perspective, LspA represents a novel target.

Interestingly, some of the secondary metabolites isolatedthus far play a significant role in cellular development or inthe predatory lifestyle ofM.xanthus. DKxanthenes have beenreported to be essential for the formation of viable sporeswithin fruiting bodies, and myxovirescin plays a major rolein the predatory lifestyle ofM.xanthus (Xiao et al. 2011; Volzet al. 2012).

An exciting discovery was that about 10% of myxobacterialcompounds interact specifically with the cytoskeleton of eu-karyotic cells. Such compounds might become useful drugsfor the control of cancer (Reinchenbach 2001). Othermyxobacterial compounds bind to DNA (saframycin), alterthe osmoregulation of fungi (ambruticin) and inhibit eukaryotic(gephyronic acid) and prokaryotic (myxovalargin) protein syn-thesis, as well as viral nucleic acid polymerases (etnangien).Etnangien also targets eubacterial RNA polymerases. One ofthe classes of compounds used clinically is rifampicin.Myxobacterial compounds ripostatin and corallopyronin showno significant cross-resistance with rifampicin, and are there-fore likely to act by a different mechanism. This observationsuggests the potential utility of the metabolites in overcomingrifampicin-resistant bacteria (O’Neill et al. 2000).

It is generally assumed that microbial secondary metabo-lites are preferentially synthesized during the late logarithmicand stationary growth phases, the so-called idiophase, whenmetabolism is no longer fully occupied with growth.However, with myxobacteria this is not the usual case.Many substances are synthesized from the beginning ofgrowth, or shortly after (Reinchenbach 2001). The new anti-biotic scaffold elansolid exhibiting potent anti-MRSA activity(Steinmetz et al. 2011, 2012) was found in Chitinophagasanctii, and the novel genus Aetherobacter was identified asa producer of a novel scaffold, the aetheramides, showingactivity against HIV (Plaza et al. 2012). Cystobacterferrugineus was found to produce roimatacenes representinga novel class of antibiotics (Zander et al. 2011), hyalidionewas identified from Hyalangium minutum, and icumazoles(Barbier et al. 2012) were described as a new class of antifun-gals from Sorangium cellulosum. The latter genus was alsofound to produce carolactone, a novel cyclic lactone that spe-cifically inhibits growth of streptococci (Jansen et al. 2010).

26 Ann Microbiol (2016) 66:17–33

However, to develop a metabolite hit into an applicablepharmaceutical compound is not an easy task, especially giventhe complexity of their natural product chemistry, side effectsand poor bioavailability. Therefore, to make better use of na-ture’s pharmaceutical factories, new technologies, such as en-gineering of microorganisms to synthesize complex molecularstructures, in silico tools to predict the target profile and an-ticipate potential side effects of those metabolites, and targeteddelivery strategies, for example via nanoparticles, are underthe spotlight and will play an increasing role in the future(Villaverde 2010; Diez et al. 2012)

Recently, we reported extracellular synthesis of silvernanoparticles byMyxococcus virescens. The silver nanoparti-cles obtained demonstrated antibacterial activity against hu-man pathogenic bacteria (Wrótniak-Drzewiecka et al. 2013,2014).

Enzymatic activity

Myxobacteria are considered micropredators. Antibiotics andenzymes produced by myxobacteria kill microorganisms andlyse cells. Cell wall degrading, lipases, nucleases,polysaccharidases and proteases appear to be involved in thelysis of prey microbes as well as in autolysis or programmedcell death, which is simultaneous with myxospore develop-ment. Despite numerous studies on myxobacteria, their enzy-matic activity has received little attention (Alvarez-Sieiro et al.2014; Dahm et al. 2015; Leontievskaya and Dobrovol’skaya2014; Mori and Kimura 2014). Myxobacteria are character-i zed by the i r e ff i c ien t degrada t ion ab i l i t i e s o fbiomacromolecules (Dahm et al. 2015; Leontievskaya andDobrovol’skaya 2014; Saraf et al. 2014; Zhou et al. 2014b).Based on their activity in degradation of biomacromolecules,myxobacteria are divided into two groups. One is bacteriolyt-ic, lysing living cells of other microorganisms, and the other iscellulolytic, decomposing cellulose (Reichenbach andDworkin 1992). In general, the cellulolytic enzymes are orga-nized in two strategies, extracellular free enzymes and cell-bound complex enzymes. Cellulolytic activity inmyxobacteria Sorangium sp. is rather low compared to thatof the extracellular free cellulases in aerobic cellulolytic fungisuch as Penicillium, Aspergillus and Trichoderma. However,the degradation of cellulose is complete (e.g., lyse of cell walllower fungi), which does not correspond to the assayed activ-ities (Hou et al. 2006; Brzezińska 2012). These authors foundthe protuberant structure on the surface of Sorangium that isresponsible for cellulose degradation, and cellulose materialsdestroyed were limited to the region of cellular swarm.Beyond the contact with cells, the cellulose (filter paper)remained intact. The authors suggested that the cellulases inSorangium exist on cellular surfaces and are organized as acomplex, which might be cellular protuberances. Cellulolytic

enzymes in Sorangium are arranged into a complex of 1000–2000 kDa, which at least contains cellulase and xylanase ac-tivities (Hou et al. 2006). In the studies on the communities ofepiphytic bacteria in grain crops, Leontievskaya andDobrovol’skaya (2014) defined a so-called Bhydrolytic block^of bacteria, which comprised myxobacteria and bacilli.Occurrence (percentage) of this group of bacteria on the cerealplant leaves (wheat and barley) grew up along with duration ofthe vegetation period; the authors stressed the group's cellulo-lytic capabilities. According to Zhou et al. (2014b),myxobacteria in sewage sludge (e.g., members of the genera:Nannocystis, Chondromyces, Haliangium and Stigmatella)are responsible for hydrolysis of some macromolecules(among others—hemicellulose); they can also prey on trueeubacteria.

Lipids play a pivotal role in the Myxococcus life cycleduring predation and development. Lipids containing the fattyacids c16:1ω5c are among the most abundant lipids inM. xanthus, but are rare in other bacteria (Moraleda-Muñozand Shimkets 2007). They are chemo-attractants and may playan important role in development. Lipids containing the fattyacids c18:1ω9c are not found in M. xanthus and appear toserve as chemo-attractants for detecting prey bacteria (Curtiset al. 2006). Fatty acids are utilized for carbon and energyduring growth, along with protein that is locked in the preycytoplasm. M. xanthus remove the membrane barrier withlipolytic enzymes that not only release fatty acids, but alsoempty the cytoplasmic contents of the prey (Moraleda-Muñoz and Shimkets 2007). The lipids have a prominent rolein the life cycle of M. xanthus. This bacterium has a largenumber of putative lipase genes in three main families—patatin lipases, α/β hydrolases and GDSL lipases. An extra-cellular alkaline protease was purified fromM. xanthus culturesupernatant. This enzyme was specific for hydrophobic oraromatic residues. In our experiments, myxobacteria producedextracellular alkaline as well as acid proteases (Brzezińska2012). Proteolytic enzymes are produced both by the cellulo-lytic myxobacteria (e.g., members of the genus Sorangium),as well as by the predatory ones (e.g., belonging to the genusMyxococcus) (Kim et al. 2009).

Three possible functions for myxobacterial extracellularproteases are suggested: (1) proteases may supply amino acidsto the myxobacteria by hydrolysing soil proteins derived fromplant, animal and soil microorganisms; (2) after activity of cellwall lytic enzymes, a protease may disrupt the cell membraneof the eubacterium, releasing its intracellular content accessi-ble to the surrounding environment, (3) the ultimate lysis ofthe prey is likely to involve proteases. Gnosspelius (1978)purified three proteases produced by Myxococcus virescens.The purified enzyme hydrolysed casein and hemoglobin andwas specific for peptide bonds involving amino acids withnonpolar side-chains. Its optimal pH was between 7 and 10and it was inhibited by diisopropylphosphorofluoridate. These

Ann Microbiol (2016) 66:17–33 27

properties and its substrate specificity suggest that the enzymewas a serine protease, even though it was inhibited by metalchelating agents.

Several microorganisms secret slime that is more or lessfirmly bound to the cell. The slime is generally consideredto be an external coat preventing drying of the cell andprotecting the cell wall against attack by various antimicrobialagents. The myxobacterial slime is involved in gliding motil-ity (Gnosspelius 1978) and the matrix of the fruiting body ispresumably polysaccharide (Dworkin 1972). Slimemight alsobe involved in the nutrition of myxobacteria and serves as asubstrate for extracellular enzymes. During exponentialgrowth, M. virescens secrets bacteriolytic enzymes followedby proteolytic enzymes and slime. This secretion order mightreflect the fact that the natural substrates for the proteolyticenzymes are native cell components, released from eubacterialcells after their lysis by bacteriolytic enzymes (Gnosspelius1978). Myxobacteria (mainly members of the generaCorallococcus andMyxococcus) isolated from soil under var-ious trees (Scots pine, birch, black alder, oak) did not have anycellulolytic and/or chitinolytic activity, but they produced ex-tracellular acidic and neutral proteinases (Dahm et al. 2015).Studies on theM. virescens show that an extracellular proteinpolysaccharide-lipid complex exhibited proteolytic activityagainst gelatin and totally inactivated lysozyme. In the super-na tan t f lu id of cu l tu res of some myxobac te r i a(Chondrococcus corraloides, Myxococcus xanthus,M. virescens, Sorangium sp.), an enzyme was detected thathydrolyses the β-1,4-bond between muramic acid and glucos-amine, thus having the substrate specificity of muraminidasewith an activity similar but not identical to that of lysozyme(Rosenberg and Varon 1984).

Studies on M. virescens show that prolyl endopeptidases(PEP) (EC 3.4.21.26), a family of serine proteases with theability to hydrolyse the peptide bond on the carboxyl side ofan internal proline residue, isolated from this strain are able todegrade immunotoxic peptides responsible for celiac disease,such as a 33-residue gluten peptide (33-mer). The gene of thisproteinase— prolyl endopeptidase (produced byMyxococcusxanthus) was cloned into a food-grade recombinantLactobacillus casei strain, thus obtaining a new tool to cureceliac disease (Alvarez-Sieiro et al. 2014). Sasaki et al. (2014)characterised the activities of theMyxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologieswith both phosphoprotein phosphatases and diadenosinetetraphosphate (Ap4A) hydrolases. The above authors statedthat PrpA and ApaHmay functionmainly as a tyrosine proteinphosphatase and an Ap4A hydrolase, respectively (Sasakiet al. 2014). According to Mori and Kimura (2014), an en-zyme produced byMyxococcus xanthus, named ArsA, exhib-ited weak phosphatase activity toward p-nitrophenyl phos-phate, and high arsenate reductase activity, which indicatesthat it can reduce arsenates under natural conditions (Mori

and Kimura 2014). Saraf et al. (2014) point out thatmyxobacteria that act as a biocontrol agent (e.g., towards plantpathogenic fungi) produce large amounts of lytic enzymes.

Conclusion

Myxobacteria are unique microorganisms known for broadrange of social adaptations. Myxobacteria from different hab-itats are not well explored, and hence there is a pressing needto search for the myxobacteria from different environments ingeneral and in extreme environments like acidic/alkaline soils,marine environments, etc. Furthermore, a thorough screeningof myxobacteria isolated from various habitats (including ex-treme environments) is needed in order to obtain potentialpharmaceuticals (e.g., for fighting against multi-drug resis-tance problems and also to provide a better solution for newand emerging/ re-emerging diseases like AIDS and cancer). Itis believed that myxobacteria may open up new avenues in thefield of pharmaceuticals.

Acknowledgments This work has been supported by Grant from thePolishMinistry of Science andHigher Education (Grant No. 2 PO4C 04029). Dr Mahendra Rai is thankful to Nicolaus Copernicus University(Torun, Poland) for fellowships to Visiting Professors within the projectBEnhancing Educational Potential of Nicolaus Copernicus University inthe Disciplines of Mathematical and Natural Sciences^ conducted underSub-measure 4.1.1 Human Capital Operational Programme – Task 7(Project No.POKL.04.01.01-00-081/10)^

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