The predatory soil bacterium Myxococcus xanthus combines a Tad- and an atypical type 3-like protein secretion system to kill bacterial cells.

Predatory Myxobacteria employ a multilayered predation strategy to kill and lyse soil microorganisms. Aiming to dissect the mechanism of contact-dependent killing of bacteria, we analyze four protein secretion systems in Myxococcus xanthus and investigate the predation of mutant strains on different timescales. We find that a Tad-like and a type 3-like secretion system (Tad and T3SS∗) fulfill distinct functions during contact-dependent prey killing: the Tad-like system is necessary to induce prey cell death, while the needle-less T3SS∗ initiates prey lysis. Fluorescence microscopy reveals that components of both systems interdependently localize to the predator-prey contact site prior to killing. Swarm expansion assays show that both Tad and T3SS∗ are required to handle live prey and that nutrient extraction from prey bacteria is sufficient to power M. xanthus motility. In conclusion, our observations indicate the functional interplay of two types of secretion systems for killing and lysis of bacterial cells.

Exopolysaccharide microchannels direct bacterial motility and organize multicellular behavior.

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required for cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS- mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus.

Dual biochemical oscillators may control cellular reversals in Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium that glides on surfaces, reversing direction approximately once every 6 min. Motility in M. xanthus is governed by the Che-like Frz pathway and the Ras-like Mgl pathway, which together cause the cell to oscillate back and forth. Previously, Igoshin et al. (2004) suggested that the cellular oscillations are caused by cyclic changes in concentration of active Frz proteins that govern motility. In this study, we present a computational model that integrates both the Frz and Mgl pathways, and whose downstream components can be read as motor activity governing cellular reversals. This model faithfully reproduces wildtype and mutant behaviors by simulating individual protein knockouts. In addition, the model can be used to examine the impact of contact stimuli on cellular reversals. The basic model construction relies on the presence of two nested feedback circuits, which prompted us to reexamine the behavior of M. xanthus cells. We performed experiments to test the model, and this cell analysis challenges previous assumptions of 30 to 60 min reversal periods in frzCD, frzF, frzE, and frzZ mutants. We demonstrate that this average reversal period is an artifact of the method employed to record reversal data, and that in the absence of signal from the Frz pathway, Mgl components can occasionally reverse the cell near wildtype periodicity, but frz- cells are otherwise in a long nonoscillating state.

The lethal cargo of Myxococcus xanthus outer membrane vesicles.

Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 µm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

"Replica-extraction-transfer" nanostructure-initiator mass spectrometry imaging of acoustically printed bacteria.

Traditionally, microbes are studied under controlled laboratory conditions as isolates in planktonic culture. However, this is a vast extrapolation from their natural state; development of new techniques is required to decipher the largely unknown world of microbial chemical interactions in more realistic environments. The field of mass spectrometry imaging has made significant progress in localizing metabolites in and around bacterial colonies, primarily by using MALDI and ESI-based techniques that interrogate the top surface of the sample. Unfortunately, surface-based laser-desorption techniques, such as nanostructure-initiator mass spectrometry (NIMS), which has advantages in detection of small metabolite compounds and low background, has not been suitable for direct microbe imaging because desorption/ionization occurs on the bottom of the sample. Here, we describe a "replica-extraction-transfer" (REX) technique that overcomes this barrier by transferring biomolecules from agar cultures of spatially arrayed bacterial colonies onto NIMS surfaces; further, we demonstrate that acoustic printing of bacteria can be used to create complex colony geometries to probe microbial interactions with NIMS imaging. REX uses a solvent-laden semisolid (e.g., gel) to first extract metabolites from a microbial sample, such as a biofilm or agar culture; the metabolites are then replica "stamped" onto the NIMS surface. Using analytical standards we show that REX-NIMS effectively transfers and detects a range of small molecule compounds including amino acids and polyamines. This approach is then used to analyze the metabolite composition of streaked Shewanella oneidensis MR1 and Pseudomonas stutzeri RCH2 colonies and further resolve complex patterns produced by acoustic printing of liquid microbial cultures. Applying multivariate statistical analysis of the NIMS imaging data identified ions that were localized to different regions between and within colonies, as well as to the agar gel. Subsequent high-resolution tandem mass spectrometry was used to characterize two species-specific lipids that correlated with the spatial location of each microbial species and were found to be highly abundant in cell extracts. Overall, the use of acoustic printing of bacteria with REX-NIMS imaging will extend the range of analytical capabilities available for characterization of microbial interactions with mass spectrometry.

Chemosensory signaling controls motility and subcellular polarity in Myxococcus xanthus.

Myxococcus xanthus is a model system for the study of dynamic protein localization and cell polarity in bacteria. M. xanthus cells are motile on solid surfaces enabled by two forms of motility. Motility is controlled by the Che-like Frz pathway, which is essential for fruiting body formation and differentiation. The Frz signal is mediated by a GTPase/GAP protein pair that establishes cell polarity and directs the motility systems. Pilus driven motility at the leading pole of the cell requires dynamic localization of two ATPases and the coordinated production of EPS synthesis. Gliding motility requires dynamic movement of large protein complexes, but the mechanism by which this system generates propulsive force is still an active area of investigation.

FrzS regulates social motility in Myxococcus xanthus by controlling exopolysaccharide production.

Myxococcus xanthus Social (S) motility occurs at high cell densities and is powered by the extension and retraction of Type IV pili which bind ligands normally found in matrix exopolysaccharides (EPS). Previous studies showed that FrzS, a protein required for S-motility, is organized in polar clusters that show pole-to-pole translocation as cells reverse their direction of movement. Since the leading cell pole is the site of both the major FrzS cluster and type IV pilus extension/retraction, it was suggested that FrzS might regulate S-motility by activating pili at the leading cell pole. Here, we show that FrzS regulates EPS production, rather than type IV pilus function. We found that the frzS phenotype is distinct from that of Type IV pilus mutants such as pilA and pilT, but indistinguishable from EPS mutants, such as epsZ. Indeed, frzS mutants can be rescued by the addition of purified EPS, 1% methylcellulose, or co-culturing with wildtype cells. Our data also indicate that the cell density requirement in S-motility is likely a function of the ability of cells to construct functional multicellular clusters surrounding an EPS core.

Cyclic GMP controls Rhodospirillum centenum cyst development.

Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α-proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homologue of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homologue preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.

Deciphering the hunting strategy of a bacterial wolfpack.

Myxococcus xanthus is a common soil bacterium with an intricate multicellular lifestyle that continues to challenge the way in which we conceptualize the capabilities of prokaryotic organisms. Myxococcus xanthus is the preferred laboratory representative from the Myxobacteria, a family of organisms distinguished by their ability to form highly structured biofilms that include tentacle-like packs of surface-gliding cell groups, synchronized rippling waves of oscillating cells and massive spore-filled aggregates that protrude upwards from the substratum to form fruiting bodies. But most of the Myxobacteria are also predators that thrive on the degradation of macromolecules released through the lysis of other microbial cells. The aim of this review is to examine our understanding of the predatory life cycle of M. xanthus. We will examine the multicellular structures formed during contact with prey, and the molecular mechanisms utilized by M. xanthus to detect and destroy prey cells. We will also examine our understanding of microbial predator-prey relationships and the prospects for how bacterial predation mechanisms can be exploited to generate new antimicrobial technologies.

Predataxis behavior in Myxococcus xanthus.

Spatial organization of cells is important for both multicellular development and tactic responses to a changing environment. We find that the social bacterium, Myxococcus xanthus utilizes a chemotaxis (Che)-like pathway to regulate multicellular rippling during predation of other microbial species. Tracking of GFP-labeled cells indicates directed movement of M. xanthus cells during the formation of rippling wave structures. Quantitative analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell availability. Methylation of the receptor, FrzCD is required for this adaptation: a frzF methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in directing cell movement through prey colonies. These data indicate that the transition to an organized multicellular state during predation in M. xanthus relies on the tactic behavior of individual cells, mediated by a Che-like signal transduction pathway.

Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions.

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.

Rippling is a predatory behavior in Myxococcus xanthus.

Cells of Myxococcus xanthus will, at times, organize their movement such that macroscopic traveling waves, termed ripples, are formed as groups of cells glide together on a solid surface. The reason for this behavior has long been a mystery, but we demonstrate here that rippling is a feeding behavior which occurs when M. xanthus cells make direct contact with either prey or large macromolecules. Rippling has been observed during two fundamentally distinct environmental conditions: (i) starvation-induced fruiting body development and (ii) predation of other organisms. Our results indicate that case (i) does not occur in all wild-type strains and is dependent on the intrinsic level of autolysis. Analysis of predatory rippling indicates that rippling behavior is inducible during predation on proteobacteria, gram-positive bacteria, yeast (such as Saccharomyces cerevisiae), and phage. Predatory efficiency decreases under genetic and physiological conditions in which rippling is inhibited. Rippling will also occur in the presence of purified macromolecules such as peptidoglycan, protein, and nucleic acid but does not occur in the presence of the respective monomeric components and also does not occur when the macromolecules are physically separated from M. xanthus cells. We conclude that rippling behavior is a mechanism utilized to efficiently consume nondiffusing growth substrates and that developmental rippling is a result of scavenging lysed cell debris.

Involvement of a Che-like signal transduction cascade in regulating cyst cell development in Rhodospirillum centenum.

Homologues of the E. coli chemotaxis (Che) signal transduction pathway are present in nearly all motile bacteria. Although E. coli contains only one Che cascade, many other bacteria are known to possess multiple sets of che genes. The role of multiple che-like gene clusters could potentially code for parallel Che-like signal transduction pathways that have distinctly different input and output functions. In this study, we describe a che-like gene cluster in Rhodospirillum centenum that controls a developmental cycle. In-frame deletion mutants of homologues of CheW (DeltacheW(3a)and DeltacheW(3b)), CheR (DeltacheR(3)), CheA (DeltacheA(3)) and a methyl-accepting chemotaxis protein (Deltamcp(3)) are defective in starvation-induced formation of heat and desiccation resistant cyst cells. In contrast, mutants of homologues of CheY (DeltacheY(3)), CheB (DeltacheB(3)), and a second input kinase designated as CheS (DeltacheS(3)) result in cells that are derepressed in the formation of cysts. A model of signal transduction is presented in which there are three distinct Che-like signal transduction cascades; one that is involved in chemotaxis, one that is involved in flagella biosynthesis and the third that is involved in cyst development.

A che-like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum.

Rhodospirillum centenum is a photosynthetic bacterium capable of undergoing swim cell to swarm cell differentiation that allows this species to be motile on both liquid and solid media. Previous experiments have demonstrated that the che1 operon is required for the control of chemotactic and phototactic behaviour of both swim and swarm cells. In this report, we analyse the function of a second che-like gene cluster in R. centenum, the che2 gene cluster. In-frame deletion mutants of cheW2, cheB2, cheR2, cheY2, and of the entire che2 operon, exhibit defects in swim and swarm cell motility. Analysis of these strains demonstrates that they are non-motile, and that the non-motile phenotype is resulting from reduced polar and lateral flagella synthesis. Additionally, mutations in mcp2, ORF204, cheA2 and ORF74 remain chemotacticly and phototacticly competent at both high and low growth temperatures. Mutations in these che2 genes result in elevated levels of flagellin proteins giving rise to a hyperflagellate phenotype. We propose a model in which R. centenum utilizes a che-like signal transduction pathway (che2) for regulating flagellum synthesis in order to optimize swim cell-swarm cell differentiation in response to changing environmental conditions.

Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation.

Rhodospirillum centenum is a purple photosynthetic bacterium that forms resting cyst cells when starved for nutrients. In this study, we demonstrate that chalcone synthase gene (chsA) expression is developmentally regulated, with expression of chsA increasing up to 86-fold upon induction of the cyst developmental cycle. Screening for mini-Tn5-induced mutants that exhibit elevated chsA::lacZ expression has led to the isolation of a set of R. centenum mutants that display increased chsA gene expression concomitant with constitutive induction of the cyst developmental cycle. These "hypercyst" mutants have lost the ability to regulate cyst cell formation in response to nutrient availability. Sequence analysis indicates that the mini-Tn5-disrupted genes code for a variety of factors, including metabolic enzymes and a large set of potential regulatory factors, including four gene products with homology to histidine sensor kinases and three with homology to response regulators. Several of the disrupted genes also have sequence similarity to che-like signal transduction components.

Characterization of cyst cell formation in the purple photosynthetic bacterium Rhodospirillum centenum.

Rhodospirillum centenum is an anoxygenic photosynthetic bacterium that is capable of differentiating into several cell types. When grown phototrophically in liquid, cells exhibit a vibrioid shape and have a single polar flagellum. When grown on a solid surface, R. centenum will differentiate into rod-shaped swarm cells that display numerous lateral flagella. Upon starvation for nutrients, R. centenum also forms desiccation-resistant cysts. In this study, it was determined that R. centenum has heat- and desiccation-resistance properties similar to other cyst-forming species. In addition, microscopic analyses of the morphological changes that occur during cyst cell development were performed. It was observed that R. centenum typically forms multi-celled clusters of cysts that contain from four to more than 10 cells per cluster. It was also determined that cell density has a minor effect on the percentage of cyst cells formed, with cell densities of 10(5)-10(7) cells per 5 micro l spot yielding the highest percentage of cyst cells. The striking similarities between the life cycle of R. centenum and the life cycle exhibited by Azospirillum spp. are discussed.