Corallococcus senghenyddensis sp. nov., a myxobacterium with potent antimicrobial activity.

AIM: Corallococcus species are diverse in the natural environment with 10 new Corallococcus species having been characterized in just the last 5 years. As well as being an abundant myxobacterial genus, they produce several secondary metabolites, including Corallopyronin, Corramycin, Coralmycin, and Corallorazine. We isolated a novel strain Corallococcus spp RDP092CA from soil in South Wales, UK, using Candida albicans as prey bait and characterized its predatory activities against pathogenic bacteria and yeast. METHODS AND RESULTS: The size of the RDP092CA genome was 8.5 Mb with a G + C content of 71.4%. Phylogenetically, RDP092CA is closely related to Corallococcus interemptor, C. coralloides, and C. exiguus. However, genome average nucleotide identity and digital DNA-DNA hybridization values are lower than 95% and 70% when compared to those type strains, implying that it belongs to a novel species. The RDP092CA genome harbours seven types of biosynthetic gene clusters (BGCs) and 152 predicted antimicrobial peptides. In predation assays, RDP092CA showed good predatory activity against Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, and Staphylococcus aureus but not against Enterococcus faecalis. It also showed good antibiofilm activity against all five bacteria in biofilm assays. Antifungal activity against eight Candida spp. was variable, with particularly good activity against Meyerozyma guillermondii DSM 6381. Antimicrobial peptide RDP092CA_120 exhibited potent antibiofilm activity with >50% inhibition and >60% dispersion of biofilms at concentrations down to 1 µg/ml. CONCLUSIONS: We propose that strain RDP092CA represents a novel species with promising antimicrobial activities, Corallococcus senghenyddensis sp. nov. (=NBRC 116490T =CCOS 2109T), based on morphological, biochemical, and genomic features.

Is "Wolf-Pack" Predation by Antimicrobial Bacteria Cooperative? Cell Behaviour and Predatory Mechanisms Indicate Profound Selfishness, Even when Working Alongside Kin.

For decades, myxobacteria have been spotlighted as exemplars of social "wolf-pack" predation, communally secreting antimicrobial substances into the shared public milieu. This behavior has been described as cooperative, becoming more efficient if performed by more cells. However, laboratory evidence for cooperativity is limited and of little relevance to predation in a natural setting. In contrast, there is accumulating evidence for predatory mechanisms promoting "selfish" behavior during predation, which together with conflicting definitions of cooperativity, casts doubt on whether microbial "wolf-pack" predation really is cooperative. Here, it is hypothesized that public-goods-mediated predation is not cooperative, and it is argued that a holistic model of microbial predation is needed, accounting for predator and prey relatedness, social phenotypes, spatial organization, activity/specificity/transport of secreted toxins, and prey resistance mechanisms. Filling such gaps in our knowledge is vital if the evolutionary benefits of potentially costly microbial behaviors mediated by public goods are to be properly understood.

Predatory Organisms with Untapped Biosynthetic Potential: Descriptions of Novel Corallococcus Species C. aberystwythensis sp. nov., C. carmarthensis sp. nov., C. exercitus sp. nov., C. interemptor sp. nov., C. llansteffanensis sp. nov., C. praedator sp. nov., C. sicarius sp. nov., and C. terminator sp. nov.

Corallococcus spp. are common soil-dwelling organisms which kill and consume prey microbes through the secretion of antimicrobial substances. Two species of Corallococcus have been described previously (Corallococcus coralloides and Corallococcus exiguus). A polyphasic approach, including biochemical analysis of fatty acid methyl esters, substrate utilization, and sugar assimilation assays, was taken to characterize eight Corallococcus species strains and the two type strains. The genomes of all strains, including that of C. exiguus DSM 14696T (newly reported here), shared an average nucleotide identity below 95% and digital DNA-DNA hybridization scores of less than 70%, indicating that they belong to distinct species. In addition, we characterized the prey range and antibiotic resistance profile of each strain, illustrating the diversity of antimicrobial activity and, thus, the potential for drug discovery within the Corallococcus genus. Each strain gave a distinct profile of properties, which together with their genomic differences supports the proposal of the eight candidate strains as novel species. The eight candidates are as follows: Corallococcus exercitus sp. nov. (AB043AT= DSM 108849T = NBRC 113887T), Corallococcus interemptor sp. nov. (AB047AT= DSM 108843T = NBRC 113888T), Corallococcus aberystwythensis sp. nov. (AB050AT = DSM 108846T = NBRC 114019T), Corallococcus praedator sp. nov. (CA031BT= DSM 108841T = NBRC 113889T), Corallococcus sicarius sp. nov. (CA040BT= DSM 108850T = NBRC 113890T), Corallococcus carmarthensis sp. nov. (CA043DT= DSM 108842T = NBRC 113891T), Corallococcus llansteffanensis sp. nov. (CA051BT= DSM 108844T = NBRC 114100T), and Corallococcus terminator sp. nov. (CA054AT= DSM 108848T = NBRC 113892T).IMPORTANCECorallococcus is a genus of predators with broad prey ranges, whose genomes contain large numbers of gene clusters for secondary metabolite biosynthesis. The physiology and evolutionary heritage of eight Corallococcus species strains were characterized using a range of analyses and assays. Multiple metrics confirmed that each strain belonged to a novel species within the Corallococcus genus. The strains exhibited distinct patterns of drug resistance and predatory activity, which mirrored their possession of diverse sets of biosynthetic genes. The breadth of antimicrobial activities observed within the Corallococcus genus highlights their potential for drug discovery and suggests a previous underestimation of both their taxonomic diversity and biotechnological potential. Taxonomic assignment of environmental isolates to novel species allows us to begin to characterize the diversity and evolution of members of this bacterial genus with potential biotechnological importance, guiding future bioprospecting efforts for novel biologically active metabolites and antimicrobials.

Genome Analysis, Metabolic Potential, and Predatory Capabilities of Herpetosiphon llansteffanense sp. nov.

Herpetosiphon spp. are ubiquitous, chemoheterotrophic, filamentous gliding bacteria with the ability to prey on other microbes through a "wolf pack" mechanism. The genus currently comprises four known species (H. aurantiacus, H. geysericola, H. giganteus, and H. gulosus), which produce antimicrobial secondary metabolites such as siphonazole. As part of a study isolating myxobacterial wolf pack predators, we serendipitously isolated a novel environmental strain (CA052B) from the edge of a stream at Llansteffan, United Kingdom, which was identified as a member of the Herpetosiphon genus. A lawn culture method was utilized to analyze the predatory activity of CA052B against 10 prey organisms of clinical relevance. CA052B was found to prey on Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus faecalis, Bacillus subtilis, and Candida albicans Purified CA052B outer membrane vesicles also exhibited killing activity against the prey organisms when tested by flow cytometry. 16S rRNA sequencing of CA052B showed 98 to 99% identity with other Herpetosiphon species members. Comparing the genome of CA052B with the publicly available genomes of H. aurantiacus and H. geysericola revealed average nucleotide identities of only 84% and 91%, respectively, whereas the genome-to-genome distance calculation showed sequence identities of 28.2% and 46.6%, respectively. Biochemical characterization also revealed distinctions between CA052B and both H. gulosus and H. giganteus Thus, strain CA052BT (= DSM 107618T = NBRC 113495T) is proposed to be the type strain of a novel species, Herpetosiphon llansteffanense sp. nov. The genome sequence of CA052B also revealed diverse secondary metabolite biosynthetic clusters, encouraging further exploration of its antibiotic production potential.IMPORTANCE Predatory bacteria are able to kill and consume other microbes and are therefore of interest as potential sources of new antimicrobial substances for applications in the clinic. "Wolf pack" predators kill prey by secreting antimicrobial substances into their surroundings, and those substances can kill prey organisms independently of the predatory cells. The genus Herpetosiphon exhibits wolf pack predation, yet its members are poorly described compared to other wolf pack predators, such as the myxobacteria. By providing a thorough characterization of a novel Herpetosiphon species, including its predatory, biochemical, and genomic features, this study increases our understanding of genomic variation within the Herpetosiphon genus and how that variation affects predatory activity. This will facilitate future rational exploitation of genus members (and other wolf pack predators) as sources of novel antimicrobials.

MetaPred2CS: a sequence-based meta-predictor for protein-protein interactions of prokaryotic two-component system proteins.

MOTIVATION: Two-component systems (TCS) are the main signalling pathways of prokaryotes, and control a wide range of biological phenomena. Their functioning depends on interactions between TCS proteins, the specificity of which is poorly understood. RESULTS: The MetaPred2CS web-server interfaces a sequence-based meta-predictor specifically designed to predict pairing of the histidine kinase and response-regulator proteins forming TCSs. MetaPred2CS integrates six sequence-based methods using a support vector machine classifier and has been intensively tested under different benchmarking conditions: (i) species specific gene sets; (ii) neighbouring versus orphan pairs; and (iii) k-fold cross validation on experimentally validated datasets. AVAILABILITY AND IMPLEMENTATION: Web server at: http://metapred2cs.ibers.aber.ac.uk/, Source code: https://github.com/martinjvickers/MetaPred2CS or implemented as Virtual Machine at: http://metapred2cs.ibers.aber.ac.uk/download CONTACT: naf4@aber.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

P2CS: updates of the prokaryotic two-component systems database.

The P2CS database (http://www.p2cs.org/) is a comprehensive resource for the analysis of Prokaryotic Two-Component Systems (TCSs). TCSs are comprised of a receptor histidine kinase (HK) and a partner response regulator (RR) and control important prokaryotic behaviors. The latest incarnation of P2CS includes 164,651 TCS proteins, from 2758 sequenced prokaryotic genomes. Several important new features have been added to P2CS since it was last described. Users can search P2CS via BLAST, adding hits to their cart, and homologous proteins can be aligned using MUSCLE and viewed using Jalview within P2CS. P2CS also provides phylogenetic trees based on the conserved signaling domains of the RRs and HKs from entire genomes. HK and RR trees are annotated with gene organization and domain architecture, providing insights into the evolutionary origin of the contemporary gene set. The majority of TCSs are encoded by adjacent HK and RR genes, however, 'orphan' unpaired TCS genes are also abundant and identifying their partner proteins is challenging. P2CS now provides paired HK and RR trees with proteins from the same genetic locus indicated. This allows the appraisal of evolutionary relationships across entire TCSs and in some cases the identification of candidate partners for orphan TCS proteins.

Genome-wide prediction of prokaryotic two-component system networks using a sequence-based meta-predictor.

BACKGROUND: Two component systems (TCS) are signalling complexes manifested by a histidine kinase (receptor) and a response regulator (effector). They are the most abundant signalling pathways in prokaryotes and control a wide range of biological processes. The pairing of these two components is highly specific, often requiring costly and time-consuming experimental characterisation. Therefore, there is considerable interest in developing accurate prediction tools to lessen the burden of experimental work and cope with the ever-increasing amount of genomic information. RESULTS: We present a novel meta-predictor, MetaPred2CS, which is based on a support vector machine. MetaPred2CS integrates six sequence-based prediction methods: in-silico two-hybrid, mirror-tree, gene fusion, phylogenetic profiling, gene neighbourhood, and gene operon. To benchmark MetaPred2CS, we also compiled a novel high-quality training dataset of experimentally deduced TCS protein pairs for k-fold cross validation, to act as a gold standard for TCS partnership predictions. Combining individual predictions using MetaPred2CS improved performance when compared to the individual methods and in comparison with a current state-of-the-art meta-predictor. CONCLUSION: We have developed MetaPred2CS, a support vector machine-based metapredictor for prokaryotic TCS protein pairings. Central to the success of MetaPred2CS is a strategy of integrating individual predictors that improves the overall prediction accuracy, with the in-silico two-hybrid method contributing most to performance. MetaPred2CS outperformed other available systems in our benchmark tests, and is available online at http://metapred2cs.ibers.aber.ac.uk, along with our gold standard dataset of TCS interaction pairs.

Genome-wide analysis of myxobacterial two-component systems: genome relatedness and evolutionary changes.

BACKGROUND: Two-component systems (TCSs) are abundant prokaryotic signaling pathways, whose evolution is of particular importance because of their role in bacterial pathogenicity. Comparative genomics can provide important insights into the evolution of these genes, but inferences are dependent on the relatedness of the compared genomes. This study investigated the relationship between evolutionary distance and TCS evolution in myxobacterial genomes, of which there are several sequenced examples, of varying relatedness, and which encode large numbers of TCSs. METHODS: Myxobacterial TCS gene sets were compared, orthologues defined, and changes in TCS properties such as gene organisation, domain architecture and size identified. RESULTS: Genome relatedness/evolutionary distance was found to have a large effect on the apparent frequency of evolutionary events affecting TCS genes, but not on the relative dominance of different types of mutations. Large (≥1 gene) indels were the most common changes, often giving rise to gene organisation changes. Smaller indels were also common, sometimes changing domain architecture, and/or leading to pseudogene formation. Individuality of myxobacterial TCS gene sets seems primarily due to lineage specific gene loss. However, there is also evidence of extensive acquisition of genes by lateral transfer, with gene duplication also creating new TCS genes. CONCLUSIONS: This study provides catalogues of myxobacterial TCS gene sets and their orthology relationships, benchmarked against genome relatedness. It also provides insights into the relationship between evolutionary distance and the inference of TCS estudies of TCS evolution beyond the myxobacteriavolution, which may be important for studies of TCS evolutiThe online version of this articleon beyond the myxobacteria.

Composition of distinct sub-proteomes in Myxococcus xanthus: metabolic cost and amino acid availability.

Subsets of proteins involved in distinct functional processes are subject to different selective pressures. We investigated whether there is an amino acid composition bias (AACB) inherent in discrete subsets of proteins, and whether we could identify changing patterns of AACB during the life cycle of the social bacterium Myxococcus xanthus. We quantitatively characterised the cellular, soluble secreted, and outer membrane vesicle (OMV) sub-proteomes of M. xanthus, identifying 315 proteins. The AACB of the cellular proteome differed only slightly from that deduced from the genome, suggesting that genome-inferred proteomes can accurately reflect the AACB of their host. Inferred AA deficiencies arising from prey consumption were exacerbated by the requirements of the 68%GC genome, whose character thus seems to be selected for directly rather than via the proteome. In our analysis, distinct subsets of the proteome (whether segregated spatially or temporally) exhibited distinct AACB, presumably tailored according to the needs of the organism's lifestyle and nutrient availability. Secreted AAs tend to be of lower cost than those retained in the cell, except for the early developmental A-signal, which is a particularly costly sub-proteome. We propose a model of AA reallocation during the M. xanthus life cycle, involving ribophagy during early starvation and sequestration of limiting AAs within cells during development.

The genetic basis of predation by myxobacteria.

Myxobacteria (phylum Myxococcota) are abundant and virtually ubiquitous microbial predators. Facultatively multicellular organisms, they are able to form multicellular fruiting bodies and swarm across surfaces, cooperatively hunting for prey. Myxobacterial communities are able to kill a wide range of prey microbes, assimilating their biomass to fuel population growth. Their mechanism of predation is exobiotic - hydrolytic enzymes and toxic metabolites are secreted into the extracellular environment, killing and digesting prey cells from without. However, recent observations of single-cell predation and contact-dependent prey killing challenge the dogma of myxobacterial predation being obligately cooperative. Regardless of their predatory mechanisms, myxobacteria have a broad prey range, which includes Gram-negative bacteria, Gram-positive bacteria and fungi. Pangenome analyses have shown that their extremely large genomes are mainly composed of accessory genes, which are not shared by all members of their species. It seems that the diversity of accessory genes in different strains provides the breadth of activity required to prey upon such a smorgasbord of microbes, and also explains the considerable strain-to-strain variation in predatory efficiency against specific prey. After providing a short introduction to general features of myxobacterial biology which are relevant to predation, this review brings together a rapidly growing body of work into the molecular mechanisms and genetic basis of predation, presenting a summary of current knowledge, highlighting trends in research and suggesting strategies by which we can potentially exploit myxobacterial predation in the future.

The RNA cargo of Myxococcus outer membrane vesicles.

The outer membrane vesicles (OMVs) secreted by some Gram-negative bacteria contain RNA cargo, which can be introduced into target cells, affecting their cellular processes. To test whether the antimicrobial OMVs secreted by predatory myxobacteria might contain cargo RNA with a role in prey killing, we purified OMVs and cells from four different strains of Myxococcus spp. for RNA-seq transcriptome sequencing. Myxobacterial OMVs contained distinct sets of RNA molecules. The abundance of major cellular transcripts correlated strongly with their abundance in OMVs, suggesting non-specific packaging into OMVs. However, many major cellular transcripts were absent entirely from OMVs and some transcripts were found exclusively in OMVs, suggesting OMV RNA cargo loading is not simply a consequence of sampling the cellular transcriptome. Despite considerable variation in OMV RNA cargo between biological replicates, a small number of transcripts were found consistently in replicate OMV preparations. These 'core' OMV transcripts were often found in the OMVs from multiple strains, and sometimes enriched relative to their abundance in cellular transcriptomes. In addition to providing the first transcriptomes for myxobacterial OMVs, and the first cellular transcriptomes for three strains of Myxococcus spp., we highlight five transcripts for further study. These transcripts are 'core' for at least two of the three strains of M. xanthus studied, and encode two alkyl hydroperoxidase proteins (AhpC and AhpD), two ribosome-associated inhibitors (RaiA-like) and a DO-family protease. It will be interesting to test whether the transcripts serve a biological function within OMVs, potentially being transported into prey cells for translation into toxic proteins.

MyxoPortal: a database of myxobacterial genomic features.

Myxobacteria are predatory bacteria with antimicrobial activity, utilizing complex mechanisms to kill their prey and assimilate their macromolecules. Having large genomes encoding hundreds of secondary metabolites, hydrolytic enzymes and antimicrobial peptides, these organisms are widely studied for their antibiotic potential. MyxoPortal is a comprehensive genomic database hosting 262 genomes of myxobacterial strains. Datasets included provide genome annotations with gene locations, functions, amino acids and nucleotide sequences, allowing analysis of evolutionary and taxonomical relationships between strains and genes. Biosynthetic gene clusters are identified by AntiSMASH, and dbAMP-generated antimicrobial peptide sequences are included as a resource for novel antimicrobial discoveries, while curated datasets of CRISPR/Cas genes, regulatory protein sequences, and phage associated genes give useful insights into each strain's biological properties. MyxoPortal is an intuitive open-source database that brings together application-oriented genomic features that can be used in taxonomy, evolution, predation and antimicrobial research. MyxoPortal can be accessed at http://dicsoft1.physics.iisc.ac.in/MyxoPortal/. Database URL:  http://dicsoft1.physics.iisc.ac.in/MyxoPortal/. Graphical Abstract.

P2RP: a Web-based framework for the identification and analysis of regulatory proteins in prokaryotic genomes.

BACKGROUND: Regulatory proteins (RPs) such as transcription factors (TFs) and two-component system (TCS) proteins control how prokaryotic cells respond to changes in their external and/or internal state. Identification and annotation of TFs and TCSs is non-trivial, and between-genome comparisons are often confounded by different standards in annotation. There is a need for user-friendly, fast and convenient tools to allow researchers to overcome the inherent variability in annotation between genome sequences. RESULTS: We have developed the web-server P2RP (Predicted Prokaryotic Regulatory Proteins), which enables users to identify and annotate TFs and TCS proteins within their sequences of interest. Users can input amino acid or genomic DNA sequences, and predicted proteins therein are scanned for the possession of DNA-binding domains and/or TCS domains. RPs identified in this manner are categorised into families, unambiguously annotated, and a detailed description of their features generated, using an integrated software pipeline. P2RP results can then be outputted in user-specified formats. CONCLUSION: Biologists have an increasing need for fast and intuitively usable tools, which is why P2RP has been developed as an interactive system. As well as assisting experimental biologists to interrogate novel sequence data, it is hoped that P2RP will be built into genome annotation pipelines and re-annotation processes, to increase the consistency of RP annotation in public genomic sequences. P2RP is the first publicly available tool for predicting and analysing RP proteins in users' sequences. The server is freely available and can be accessed along with documentation at http://www.p2rp.org.

P2CS: a database of prokaryotic two-component systems.

P2CS (http://www.p2cs.org) is a specialized database for prokaryotic two-component systems (TCSs), virtually ubiquitous signalling proteins which regulate a wide range of physiological processes. The primary aim of the database is to annotate and classify TCS proteins from completely sequenced prokaryotic genomes and metagenomes. Information within P2CS can be accessed through a variety of routes-TCS complements can be browsed by metagenome, replicon or sequence cluster (and these genesets are available for download by users). Alternatively a variety of database-wide or taxon-specific searches are supported. Each TCS protein is fully annotated with sequence-feature information including replicon context, while properties of the predicted proteins can be queried against several external prediction servers to suggest homologues, interaction networks, sub-cellular localization and domain complements. Another unique feature of P2CS is the analysis of ORFeomes to identify TCS genes missed during genome annotation. Recent innovations for P2CS include a CGView representation of the distribution of TCS genes around a replicon, categorization of TCS genes based on gene organization, an expanded domain-based classification scheme, a P2CS 'gene cart' and categorization on the basis of sequence clusters.

Myxobacterial Predation: A Standardised Lawn Predation Assay Highlights Strains with Unusually Efficient Predatory Activity.

Myxobacteria prey upon a broad range of microorganisms. Lawn assays are commonly used to quantify myxobacterial predation-myxobacterial suspensions are spotted onto prey lawns, and monitored via spot expansion. The diversity in motility behaviours of myxobacterial strains and differing assay protocols in myxobacteriology laboratories led us to develop a highly-specified assay, which was applied to 28 myxobacterial strains preying on seven phytopathogenic prey species. Generally, prey organisms showed no qualitative differences in their susceptibility/resistance to myxobacterial predation. For most myxobacteria, prey did not stimulate, and in ~50% of cases actively hindered colony expansion. Only ~25% of predator/prey strain combinations exhibited greater colony expansion than in the absence of nutrients. The activity of predatory strains against different prey correlated, implying effective predators may have relatively non-specific predation mechanisms (e.g., broad specificity proteases/lipases), but no correlation was observed between predatory activity and phylogeny. Predation on dead (but intact) or lysed prey cells gave greater colony expansion than on live prey. Occasional strains grew substantially faster on dead compared to lysed cells, or vice-versa. Such differences in accessing nutrients from live, dead and lysed cells indicates there are strain-specific differences in the efficiencies/machineries of prey killing and nutrient acquisition, which has important implications for the ecology of myxobacterial predators and their prey.

Identification of secondary metabolites containing a diketopiperazine core in extracts from myxobacterial strains with growth inhibition activity against a range of prey species.

Myxobacteria produce a variety of bioactive secondary metabolites, and with a wealth of under-researched species they hold vast potential for undiscovered compounds. With the ever-increasing need for new antibiotics, the development of novel therapeutics is vitally important. Therefore, this study aimed to extract and elucidate antimicrobial metabolites from the following myxobacteria: Myxococcus xanthus CA010 and AB022; Corallococcus exiguus DSM 14696T; Myxococcus stipitatus DSM 14675T; and Corallococcus aberystwythensis AB050AT. Metabolite mixtures were extracted in acetone from XAD-16 resin incubated in liquid cultures and analysed using GC-MS. Bioactivity was identified using a growth inhibition assay against a panel of clinically relevant prey species including Gram-positive and Gram-negative bacteria and a fungus. Growth of Klebsiella pneumoniae and Enterococcus faecalis was most affected by the metabolite mixtures and the mixtures from AB022 and AB050AT were effective against the most prey. GC-MS analysis revealed metabolites with roles in the synthesis and degradation of amino acids and fatty acids, but also identified compounds A and B with a diketopiperazine (DKP) core. With previously confirmed bioactivity of compound A, it is suggested that these DKP compounds are contributing to the antimicrobial activity observed. Furthermore, many compounds could not be identified and so these unknowns present further potential for novel bioactive compounds.

Predatory Strategies of Myxococcus xanthus: Prey Susceptibility to OMVs and Moonlighting Enzymes.

Predatory outer membrane vesicles (OMVs) secreted by myxobacteria fuse readily with the outer membranes of Gram-negative bacteria, introducing toxic cargo into their prey. Here we used a strain of the myxobacterium Myxococcus xanthus that produces fluorescent OMVs to assay the uptake of OMVs by a panel of Gram-negative bacteria. M. xanthus strains took up significantly less OMV material than the tested prey strains, suggesting that re-fusion of OMVs with producing organisms is somehow inhibited. The OMV killing activity against different prey correlated strongly with the predatory activity of myxobacterial cells, however, there was no correlation between OMV killing activity and their propensity to fuse with different prey. It has previously been proposed that M. xanthus GAPDH stimulates the predatory activity of OMVs by enhancing OMV fusion with prey cells. Therefore, we expressed and purified active fusion proteins of M. xanthus glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (GAPDH and PGK; moonlighting enzymes with additional activities beyond their roles in glycolysis/gluconeogenesis) to investigate any involvement in OMV-mediated predation. Neither GAPDH nor PGK caused lysis of prey cells or enhanced OMV-mediated lysis of prey cells. However, both enzymes were found to inhibit the growth of Escherichia coli, even in the absence of OMVs. Our results suggest that fusion efficiency is not a determinant of prey killing, but instead resistance to the cargo of OMVs and co-secreted enzymes dictates whether organisms can be preyed upon by myxobacteria.

In Silico and In Vitro Analyses Reveal Promising Antimicrobial Peptides from Myxobacteria.

Antimicrobial resistance (AMR) is a global concern, and as soon as new antibiotics are introduced, resistance to those agents emerges. Therefore, there is an increased appetite for alternative antimicrobial agents to traditional antibiotics. Here, we used in silico methods to investigate potential antimicrobial peptides (AMPs) from predatory myxobacteria. Six hundred seventy-two potential AMP sequences were extracted from eight complete myxobacterial genomes. Most putative AMPs were predicted to be active against Klebsiella pneumoniae with least activity being predicted against Staphylococcus aureus. One hundred seventeen AMPs (defined here as 'potent putative AMPs') were predicted to have very good activity against more than two bacterial pathogens, and these were characterized further in silico. All potent putative AMPs were predicted to have anti-inflammatory and antifungal properties, but none was predicted to be active against viruses. Twenty six (22%) of them were predicted to be hemolytic to human erythrocytes, five were predicted to have anticancer properties, and 56 (47%) were predicted to be biofilm active. In vitro assays using four synthesized AMPs showed high MIC values (e.g. So_ce_56_913 250 µg/ml and Coral_AMP411 125 µg/ml against E. coli). However, antibiofilm assays showed a substantial reduction in numbers (e.g. Coral_AMP411 and Myxo_mac104 showed a 69% and 73% reduction, respectively, at the lowest concentration against E. coli) compared to traditional antibiotics. Fourteen putative AMPs had high sequence similarity to proteins which were functionally associated with proteins of known function. The myxobacterial genomes also possessed a variety of biosynthetic gene clusters (BGCs) that can encode antimicrobial secondary metabolites, but their numbers did not correlate with those of the AMPs. We suggest that AMPs from myxobacteria are a promising source of novel antimicrobial agents with a plethora of biological properties.

A Synthetic Polymicrobial Community Biofilm Model Demonstrates Spatial Partitioning, Tolerance to Antimicrobial Treatment, Reduced Metabolism, and Small Colony Variants Typical of Chronic Wound Biofilms.

Understanding chronic wound infection is key for successful treatment and requires accurate laboratory models. We describe a modified biofilm flow device that effectively mimics the chronic wound environment, including simulated wound fluid, a collagen-based 3D biofilm matrix, and a five-species mixture of clinically relevant bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Citrobacter freundii). Mixed biofilms were cultured for between 3 and 14 days with consistent numbers of bacteria that exhibited reduced metabolic activity, which increased with a high dose of glucose. S. aureus was recovered from biofilms as a small colony variant, but as a normal colony variant if P. aeruginosa was excluded from the system. Bacteria within the biofilm did not co-aggregate but formed discrete, species-specific clusters. Biofilms demonstrated differential tolerance to the topical antimicrobials Neosporin and HOCl, consistent with protection due to the biofilm lifestyle. The characteristics exhibited within this model match those of real-world wound biofilms, reflecting the clinical scenario and yielding a powerful in vitro tool that is versatile, inexpensive, and pivotal for understanding chronic wound infection.