Functional investigation of the two ClpPs and three ClpXs in Myxococcus xanthus DK1622.

ClpXP is a protease complex that plays important roles in protein quality control and cell cycle regulation, but the functions of multiple ClpXs and multiple ClpPs in M. xanthus remain unknown. The genome of Myxococcus xanthus DK1622 contains two clpPs and three clpXs. The clpP1 and clpX1 genes are cotranscribed and are both essential, while the other copies are isolated in the genome and are deletable. The deletion of clpX2 caused the mutant to be deficient in fruiting body development, while the clpX3 gene is involved in resistance to thermal stress. Both ClpPs possess catalytic active sites, but only ClpP1 shows in vitro peptidase activity on the typical substrate Suc-LY-AMC. All of these clpP and clpX genes exhibit strong transcriptional upregulation in the stationary phase, and the transcription of the three clpX genes appears to be coordinated. Our results demonstrated that multiple ClpPs and multiple ClpXs are functionally divergent and may assist in the environmental adaptation and functional diversification of M. xanthus.IMPORTANCEClpXP is an important protease complex of bacteria and is involved in various physiological processes. Myxococcus xanthus DK1622 possesses two ClpPs and three ClpXs with unclear functions. We investigated the functions of these genes and demonstrated the essential roles of clpP1 and clpX1. Only ClpP1 has in vitro peptidase activity on Suc-LY-AMC, and the isolated clpX copies participate in distinct cellular processes. All of these genes exhibited significant transcriptional upregulation in the stationary phase. Divergent functions appear in multiple ClpPs and multiple ClpXs in M. xanthus DK1622.

Enzymatic characterization of five thioredoxins and a thioredoxin reductase from Myxococcus xanthus.

Thioredoxin (Trx) is a disulfide-containing redox protein that functions as a disulfide oxidoreductase. Myxococcus xanthus contains five Trxs (Trx1-Trx5) and one Trx reductase (TrxR). Trxs typically have a CGPC active-site motif; however, M. xanthus Trxs have slightly different active-site sequences, with the exception of Trx4. The five Trxs of M. xanthus exhibited reduced activities against insulin, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, glutathione disulfide (GSSG), S-nitrosoglutathione (GSNO), and H2O2 in the presence of TrxR. Myxococcus xanthus adenylate kinase and serine/threonine phosphatase activities, which were increased by the addition of dithiothreitol, were activated by the addition of Trxs and TrxR. Among these, Trx1, which has a CAPC sequence in its active site, exhibited the highest reducing activity with the exception of GSNO. Myxococcus xanthus TrxR showed weak reducing activity towards DTNB, GSSG, GSNO, and H2O2, suggesting that it has broad substrate specificity, unlike previously reported low-molecular-weight TrxRs. TrxR reduced oxidized Trx1 as the best substrate, with a kcat/Km value of 0.253 min-1 µM-1, which was 10-28-fold higher than that of the other Trxs. These results suggest that all Trxs possess reducing activity and that Trx1 may be the most functional in M. xanthus because TrxR most efficiently reduces oxidized Trx1.

Cell-cell transfer of adaptation traits benefits kin and actor in a cooperative microbe.

Microbes face many physical, chemical, and biological insults from their environments. In response, cells adapt, but whether they do so cooperatively is poorly understood. Here, we use a model social bacterium, Myxococcus xanthus, to ask whether adapted traits are transferable to naïve kin. To do so we isolated cells adapted to detergent stresses and tested for trait transfer. In some cases, strain-mixing experiments increased sibling fitness by transferring adaptation traits. This cooperative behavior depended on a kin recognition system called outer membrane exchange (OME) because mutants defective in OME could not transfer adaptation traits. Strikingly, in mixed stressed populations, the transferred trait also benefited the adapted (actor) cells. This apparently occurred by alleviating a detergent-induced stress response in kin that otherwise killed actor cells. Additionally, this adaptation trait when transferred also conferred resistance against a lipoprotein toxin delivered to targeted kin. Based on these and other findings, we propose a model for stress adaptation and how OME in myxobacteria promotes cellular cooperation in response to environmental stresses.

Siderophores and competition for iron govern myxobacterial predation dynamics.

Bacterial predators are decisive organisms that shape microbial ecosystems. In this study, we investigated the role of iron and siderophores during the predatory interaction between two rhizosphere bacteria: Myxococcus xanthus, an epibiotic predator, and Sinorhizobium meliloti, a bacterium that establishes nitrogen-fixing symbiosis with legumes. The results show that iron enhances the motility of the predator and facilitates its predatory capability, and that intoxication by iron is not used by the predator to prey, although oxidative stress increases in both bacteria during predation. However, competition for iron plays an important role in the outcome of predatory interactions. Using combinations of predator and prey mutants (nonproducers and overproducers of siderophores), we have investigated the importance of competition for iron in predation. The results demonstrate that the competitor that, via the production of siderophores, obtains sufficient iron for growth and depletes metal availability for the opponent will prevail in the interaction. Consequently, iron fluctuations in soils may modify the composition of microbial communities by altering the activity of myxobacterial predators. In addition, siderophore overproduction during predation can alter soil properties, affecting the productivity and sustainability of agricultural operations.

Characteristics and immune functions of the endogenous CRISPR-Cas systems in myxobacteria.

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery. IMPORTANCE: Serving as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.

Bacterial cell differentiation enables population level survival strategies.

Clonal reproduction of unicellular organisms ensures the stable inheritance of genetic information. However, this means of reproduction lacks an intrinsic basis for genetic variation, other than spontaneous mutation and horizontal gene transfer. To make up for this lack of genetic variation, many unicellular organisms undergo the process of cell differentiation to achieve phenotypic heterogeneity within isogenic populations. Cell differentiation is either an inducible or obligate program. Induced cell differentiation can occur as a response to a stimulus, such as starvation or host cell invasion, or it can be a stochastic process. In contrast, obligate cell differentiation is hardwired into the organism's life cycle. Whether induced or obligate, bacterial cell differentiation requires the activation of a signal transduction pathway that initiates a global change in gene expression and ultimately results in a morphological change. While cell differentiation is considered a hallmark in the development of multicellular organisms, many unicellular bacteria utilize this process to implement survival strategies. In this review, we describe well-characterized cell differentiation programs to highlight three main survival strategies used by bacteria capable of differentiation: (i) environmental adaptation, (ii) division of labor, and (iii) bet-hedging.

Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells.

The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new hosts, but has been less explored rationally. The bacterium Myxococcus xanthus harbors a large circular high-GC genome, and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products. In this study, we conducted transposon insertion at TA sites on the M. xanthus genome, and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites. The enrichment sites are characteristically distributed along the genome, and the dilution sites are overlapped well with the horizontal transfer genes. We experimentally demonstrated the enrichment sites as high expression integration sites (HEISs), and the dilution sites unsuitable for gene integration expression. This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.

DnaK duplication and specialization in bacteria correlates with increased proteome complexity.

The chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes encode Hsp70 paralogs. Although the mechanisms of Hsp70 function have been clearly illuminated, the function and evolution of Hsp70 paralogs is not well studied. DnaK is a highly conserved bacterial Hsp70 family. Here, we show that dnaK is present in 98.9% of bacterial genomes, and 6.4% of them possess two or more DnaK paralogs. We found that the duplication of dnaK is positively correlated with an increase in proteomic complexity (proteome size, number of domains). We identified the interactomes of the two DnaK paralogs of Myxococcus xanthus DK1622 (MxDnaKs), which revealed that they are mostly nonoverlapping, although both prefer α and ß domain proteins. Consistent with the entire M. xanthus proteome, MxDnaK substrates have both significantly more multi-domain proteins and a higher isoelectric point than that of Escherichia coli, which encodes a single DnaK homolog. MxDnaK1 is transcriptionally upregulated in response to heat shock and prefers to bind cytosolic proteins, while MxDnaK2 is downregulated by heat shock and is more associated with membrane proteins. Using domain swapping, we show that the nucleotide-binding domain and the substrate-binding ß domain are responsible for the significant differences in DnaK interactomes, and the nucleotide binding domain also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work suggests that bacterial DnaK has been duplicated in order to deal with a more complex proteome, and that this allows evolution of distinct domains to deal with different subsets of target proteins.IMPORTANCEAll eukaryotic and ~40% of prokaryotic species encode multiple 70 kDa heat shock protein (Hsp70) homologs with similar but diversified functions. Here, we show that duplication of canonical Hsp70 (DnaK in prokaryotes) correlates with increasing proteomic complexity and evolution of particular regions of the protein. Using the Myxococcus xanthus DnaK duplicates as a case, we found that their substrate spectrums are mostly nonoverlapping, and are both consistent to that of Escherichia coli DnaK in structural and molecular characteristics, but show differential enrichment of membrane proteins. Domain/region swapping demonstrated that the nucleotide-binding domain and the ß substrate-binding domain (SBDß), but not the SBDα or disordered C-terminal tail region, are responsible for this functional divergence. This work provides the first direct evidence for regional evolution of DnaK paralogs.

Mutation of self-binding sites in the promoter of the MrpC transcriptional regulator leads to asynchronous Myxococcus xanthus development.

Introduction: MrpC, a member of the CRP/Fnr transcription factor superfamily, is necessary to induce and control the multicellular developmental program of the bacterium, Myxococcus xanthus. During development, certain cells in the population first swarm into haystack-shaped aggregates and then differentiate into environmentally resistant spores to form mature fruiting bodies (a specialized biofilm). mrpC transcriptional regulation is controlled by negative autoregulation (NAR). Methods: Wild type and mutant mrpC promoter regions were fused to a fluorescent reporter to examine effects on mrpC expression in the population and in single cells in situ. Phenotypic consequences of the mutant mrpC promoter were assayed by deep convolution neural network analysis of developmental movies, sporulation efficiency assays, and anti-MrpC immunoblot. In situ analysis of single cell MrpC levels in distinct populations were assayed with an MrpC-mNeonGreen reporter. Results: Disruption of MrpC binding sites within the mrpC promoter region led to increased and broadened distribution of mrpC expression levels between individual cells in the population. Expression of mrpC from the mutant promoter led to a striking phenotype in which cells lose synchronized transition from aggregation to sporulation. Instead, some cells abruptly exit aggregation centers and remain locked in a cohesive swarming state we termed developmental swarms, while the remaining cells transition to spores inside residual fruiting bodies. In situ examination of a fluorescent reporter for MrpC levels in developmental subpopulations demonstrated cells locked in the developmental swarms contained MrpC levels that do not reach the levels observed in fruiting bodies. Discussion: Increased cell-to-cell variation in mrpC expression upon disruption of MrpC binding sites within its promoter is consistent with NAR motifs functioning to reducing noise. Noise reduction may be key to synchronized transition of cells in the aggregation state to the sporulation state. We hypothesize a novel subpopulation of cells trapped as developmental swarms arise from intermediate levels of MrpC that are sufficient to promote aggregation but insufficient to trigger sporulation. Failure to transition to higher levels of MrpC necessary to induce sporulation may indicate cells in developmental swarms lack an additional positive feedback signal required to boost MrpC levels.

Genetic components of Escherichia coli involved in its complex prey-predator interaction with Myxococcus xanthus.

Myxococcus xanthus and Escherichia coli represent a well-studied microbial predator-prey pair frequently examined in laboratory settings. While significant progress has been made in comprehending the mechanisms governing M. xanthus predation, various aspects of the response and defensive mechanisms of E. coli as prey remain elusive. In this study, the E. coli MG1655 large-scale chromosome deletion library was screened, and a mutant designated as ME5012 was identified to possess significantly reduced susceptibility to predation by M. xanthus. Within the deleted region of ME5012 encompassing seven genes, the significance of dusB and fis genes in driving the observed phenotype became apparent. Specifically, the deletion of fis resulted in a notable reduction in flagellum production in E. coli, contributing to a certain level of resistance against predation by M. xanthus. Meanwhile, the removal of dusB in E. coli led to diminished inducibility of myxovirescin A production by M. xanthus, accompanied by a slight decrease in susceptibility to myxovirescin A. These findings shed light on the molecular mechanisms underlying the complex interaction between M. xanthus and E. coli in a predatory context.

Genome sequences of Mx1, the first Myxococcus phage isolated, and Mx4, a generalized transducing myxophage.

Myxococcus xanthus is the best-studied member of the phylum Myxococcota, but the bacteriophages infecting it and their characterization remain limited. Here, we present complete genomes of Mx1, the first Myxococcus phage isolated, and of an Mx4 derivative widely used for generalized transduction, both unclassified Caudoviricetes with long, contractile tails.

The mechanism for polar localization of the type IVa pilus machine in Myxococcus xanthus.

IMPORTANCE: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

Microbially induced calcium carbonate precipitation in fossil consolidation treatments: Preliminary results inducing exogenous Myxococcus xanthus bacteria in a miocene Cheirogaster richardi specimen.

This research paper proposes Microbially Induced Calcium Carbonate Precipitation (MICP) as an innovative approach for palaeontological heritage conservation, specifically on deteriorated carbonate fossils. Due to its efficiency in bioconsolidation of carbonate ornamental rocks, Myxococcus xanthus inoculation on carbonate fossils was studied in this research. Treatment was tested on nine fossil samples from decontextualized fragments of Cheirogaster richardi specimens (Can Mata site, Hostalets de Pierola, Catalonia, Spain). The main objective was to evaluate whether treatment with Myxococcus xanthus improved fossil surface cohesion and hardness and mechanical strength without significant physicochemical and aesthetic changes to the surface. Chemical compatibility of the treatment, penetration capacity and absence of noticeable changes in substrate porosity were considered as important issues to be evaluated. Samples were analysed, before and after treatment, by scanning electron microscopy, weight control, spectrophotometry, X-ray diffraction analysis, water absorption analysis, pH and conductivity control, Vickers microindentation and tape test. Results show that hardness increases by a factor of almost two. Cohesion also increases and surface disaggregated particles are bonded together by a calcium carbonate micrometric layer with no noticeable changes in surface roughness. Colour and gloss variations are negligible, and pH, conductivity and weight hardly change. Slight changes in porosity were observed but without total pore clogging. To sum up, results indicate that Myxococcus xanthus biomineralisation is an effective consolidation treatment for carbonate fossils and highly compatible with carbonate substrates. Furthermore, bacterial precipitation of calcium carbonate is a safe and eco-friendly consolidation treatment.

Two reasons to kill: predation and kin discrimination in myxobacteria.

Myxobacteria are social microbial predators that use cell-cell contacts to identify bacterial or fungal prey and to differentiate kin relatives to initiate cellular responses. For prey killing, they assemble Tad-like and type III-like secretion systems at contact sites. For kin discrimination (KD), they assemble outer membrane exchange complexes composed of the TraA and TraB receptors at contacts sites. A type VI secretion system and Rhs proteins also mediate KD. Following cellular recognition, these systems deliver appropriate effectors into target cells. For prey, this leads to cell death and lysis for nutrient consumption by myxobacteria. In KD, a panel of effectors are delivered, and if adjacent cells are clonal cells, resistance ensues because they express a cognate panel of immunity factors; while nonkin lack complete immunity and are intoxicated. This review compares and contrasts recent findings from these systems in myxobacteria.

Ribonuclease D Processes a Small RNA Regulator of Multicellular Development in Myxobacteria.

By targeting mRNA transcripts, non-coding small RNAs (sRNAs) regulate the expression of genes governing a wide range of bacterial functions. In the social myxobacterium Myxococcus xanthus, the sRNA Pxr serves as a gatekeeper of the regulatory pathway controlling the life-cycle transition from vegetative growth to multicellular fruiting body development. When nutrients are abundant, Pxr prevents the initiation of the developmental program, but Pxr-mediated inhibition is alleviated when cells starve. To identify genes essential for Pxr function, a developmentally defective strain in which Pxr-mediated blockage of development is constitutively active (strain "OC") was transposon-mutagenized to identify suppressor mutations that inactivate or bypass Pxr inhibition and thereby restore development. One of the four loci in which a transposon insertion restored development is rnd, encoding the Ribonuclease D protein (RNase D). RNase D is an exonuclease important for tRNA maturation. Here, we show that disruption of rnd abolishes the accumulation of Pxr-S, the product of Pxr processing from a longer precursor form (Pxr-L) and the active inhibitor of development. Additionally, the decrease in Pxr-S caused by rnd disruption was associated with increased accumulation primarily of a longer novel Pxr-specific transcript (Pxr-XL) rather than of Pxr-L. The introduction of a plasmid expressing rnd reverted cells back to OC-like phenotypes in development and Pxr accumulation, indicating that a lack of RNase D alone suppresses the developmental defect of OC. Moreover, an in vitro Pxr-processing assay demonstrated that RNase D processes Pxr-XL into Pxr-L; this implies that overall, Pxr sRNA maturation requires a sequential two-step processing. Collectively, our results indicate that a housekeeping ribonuclease plays a central role in a model form of microbial aggregative development. To our knowledge, this is the first evidence implicating RNase D in sRNA processing.

Synthesis and degradation of polyphosphate in Myxococcus xanthus.

Polyphosphate kinase 1 (Ppk1) generates polyphosphates (polyPs) by catalyzing phosphate transfer from ATP. In the presence of ATP, Myxococcus xanthus Ppk1 showed the highest activity with polyP60-70 but also showed high activity with orthophosphate and pyrophosphate. Ppk1 synthesizes long-chain polyPs with >1 000 phosphate residues from orthophosphate or pyrophosphate present in high concentrations, suggesting that in M. xanthus, Ppk1 uses intracellular ortho/pyrophosphate as an initial primer for polyP production. During M. xanthus starvation-induced development, the specific activity of Ppk1 peaked at 12 h (300-800 nmol/min/mg) and then gradually decreased. The polyP concentration was highest during mound formation (45 nmol phosphate/mg protein); then, the level of long-chain polyPs decreased and that of short-chain polyPs increased during fruiting body and spore formation. Myxococcus xanthus expresses two exopolyphosphatases, Ppx1 and Ppx2, which mainly degrade short- and long-chain polyPs, respectively, both of which were highest in vegetative cells and were detected during starvation, which may account for the degradation of polyPs. Thus, polyPs synthesized by Ppk1 early in starvation-induced development could be degraded by exopolyphosphatases and may also be used as substrates by polyP:AMP phosphotransferases and polyphosphate/ATP-NAD kinases to generate ADP and NADP+, respectively.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

FrzS acts as a polar beacon to recruit SgmX, a central activator of type IV pili during Myxococcus xanthus motility.

In rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polar-binding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium.

Myxococcus xanthus as Host for the Production of Benzoxazoles.

Benzoxazoles are important structural motifs in pharmaceutical drugs. Here, we present the heterologous production of 3-hydroxyanthranilate-derived benzoxazoles in the host bacterium Myxococcus xanthus following the expression of two genes from the nataxazole biosynthetic gene cluster of Streptomyces sp. Tü 6176. The M. xanthus expression strain achieved a benzoxazole titer of 114.6±7.4 mg L-1 upon precursor supplementation, which is superior to other bacterial production systems. Crosstalk between the heterologously expressed benzoxazole pathway and the endogenous myxochelin pathway led to the combinatorial biosynthesis of benzoxazoles featuring a 2,3-dihydroxybenzoic acid (2,3-DHBA) building block. Subsequent in vitro studies confirmed that this crosstalk is not only due to the availability of 2,3-DHBA in M. xanthus, rather, it is promoted by the adenylating enzyme MxcE from the myxochelin pathway, which contributes to the activation of aryl carboxylic acids and delivers them to benzoxazole biosynthesis.

Unorthodox regulation of the MglA Ras-like GTPase controlling polarity in Myxococcus xanthus.

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns.