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.

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.

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.

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.

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.

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.

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.

Increasing creative self-efficacy: Developing the confidence of biochemistry undergraduates to innovate.

Biochemistry graduates need to be creative, however assessing creativity requires the production of novelty, judged by or against that of peers. A related phenomenon is 'creative self-efficacy' (CSE) - one's self-belief in producing creative outcomes. CSE is a contributor to creativity, but is more easily assessed, and thus more amenable for targeting pedagogically. To investigate interactions between student CSE and the learning environment, a biochemistry laboratory exercise was deployed within a 'creative' module, wherein students created their own experimental protocols. Students completed questionnaires at the beginning and end of the module. Compared to 'control' modules lacking overtly creative activities, the creative module significantly increased students' perceptions of their own creativity and whether their studies had increased their creativity. Students' confidence in meeting degree learning outcomes (for instance the ability to work productively in a laboratory), and motivation to study, were also significantly increased. Marks attained from the creative exercise correlated with students' CSE, but surprisingly, students' expected marks correlated negatively with their CSE, implying they had a poor understanding of the relationship between creativity and success. Our results suggest that the learning environment can positively affect students' CSE, promoting academic attainment of learning outcomes, motivation, and their confidence as biochemists.

Myxobacteria: Physiology and Regulation.

Myxobacteria are fascinating and important prokaryotes [...].

Myxobacterial Genomics and Post-Genomics: A Review of Genome Biology, Genome Sequences and Related 'Omics Studies.

Myxobacteria are fascinating and complex microbes. They prey upon other members of the soil microbiome by secreting antimicrobial proteins and metabolites, and will undergo multicellular development if starved. The genome sequence of the model myxobacterium Myxococcus xanthus DK1622 was published in 2006 and 15 years later, 163 myxobacterial genome sequences have now been made public. This explosion in genomic data has enabled comparative genomics analyses to be performed across the taxon, providing important insights into myxobacterial gene conservation and evolution. The availability of myxobacterial genome sequences has allowed system-wide functional genomic investigations into entire classes of genes. It has also enabled post-genomic technologies to be applied to myxobacteria, including transcriptome analyses (microarrays and RNA-seq), proteome studies (gel-based and gel-free), investigations into protein-DNA interactions (ChIP-seq) and metabolism. Here, we review myxobacterial genome sequencing, and summarise the insights into myxobacterial biology that have emerged as a result. We also outline the application of functional genomics and post-genomic approaches in myxobacterial research, highlighting important findings to emerge from seminal studies. The review also provides a comprehensive guide to the genomic datasets available in mid-2021 for myxobacteria (including 24 genomes that we have sequenced and which are described here for the first time).

Evolutionary history expands the range of signaling interactions in hybrid multikinase networks.

Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS-GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.

The Genetics of Prey Susceptibility to Myxobacterial Predation: A Review, Including an Investigation into Pseudomonas aeruginosa Mutations Affecting Predation by Myxococcus xanthus.

Bacterial predation is a ubiquitous and fundamental biological process, which influences the community composition of microbial ecosystems. Among the best characterised bacterial predators are the myxobacteria, which include the model organism Myxococcus xanthus. Predation by M. xanthus involves the secretion of antibiotic metabolites and hydrolytic enzymes, which results in the lysis of prey organisms and release of prey nutrients into the extracellular milieu. Due to the generalist nature of this predatory mechanism, M. xanthus has a broad prey range, being able to kill and consume Gram-negative/positive bacteria and fungi. Potential prey organisms have evolved a range of behaviours which protect themselves from attack by predators. In recent years, several investigations have studied the molecular responses of a broad variety of prey organisms to M. xanthus predation. It seems that the diverse mechanisms employed by prey belong to a much smaller number of general "predation resistance" strategies. In this mini-review, we present the current state of knowledge regarding M. xanthus predation, and how prey organisms resist predation. As previous molecular studies of prey susceptibility have focussed on individual genes/metabolites, we have also undertaken a genome-wide screen for genes of Pseudomonas aeruginosa which contribute to its ability to resist predation. P. aeruginosa is a World Health Organisation priority 1 antibiotic-resistant pathogen. It is metabolically versatile and has an array of pathogenic mechanisms, leading to its prevalence as an opportunistic pathogen. Using a library of nearly 5,500 defined transposon insertion mutants, we screened for "prey genes", which when mutated allowed increased predation by a fluorescent strain of M. xanthus. A set of candidate "prey proteins" were identified, which shared common functional roles and whose nature suggested that predation resistance by P. aeruginosa requires an effective metal/oxidative stress system, an intact motility system, and mechanisms for de-toxifying antimicrobial peptides.

A Genomic Survey of Signalling in the Myxococcaceae.

As prokaryotes diverge by evolution, essential 'core' genes required for conserved phenotypes are preferentially retained, while inessential 'accessory' genes are lost or diversify. We used the recently expanded number of myxobacterial genome sequences to investigate the conservation of their signalling proteins, focusing on two sister genera (Myxococcus and Corallococcus), and on a species within each genus (Myxococcus xanthus and Corallococcus exiguus). Four new C. exiguus genome sequences are also described here. Despite accessory genes accounting for substantial proportions of each myxobacterial genome, signalling proteins were found to be enriched in the core genome, with two-component system genes almost exclusively so. We also investigated the conservation of signalling proteins in three myxobacterial behaviours. The linear carotenogenesis pathway was entirely conserved, with no gene gain/loss observed. However, the modular fruiting body formation network was found to be evolutionarily plastic, with dispensable components in all modules (including components required for fruiting in the model myxobacterium M. xanthus DK1622). Quorum signalling (QS) is thought to be absent from most myxobacteria, however, they generally appear to be able to produce CAI-I (cholerae autoinducer-1), to sense other QS molecules, and to disrupt the QS of other organisms, potentially important abilities during predation of other prokaryotes.

Comparative Genomics and Pan-Genomics of the Myxococcaceae, including a Description of Five Novel Species: Myxococcus eversor sp. nov., Myxococcus llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogochensis sp. nov., Myxococcus vastator sp. nov., Pyxidicoccus caerfyrddinensis sp. nov., and Pyxidicoccus trucidator sp. nov.

Members of the predatory Myxococcales (myxobacteria) possess large genomes, undergo multicellular development, and produce diverse secondary metabolites, which are being actively prospected for novel drug discovery. To direct such efforts, it is important to understand the relationships between myxobacterial ecology, evolution, taxonomy, and genomic variation. This study investigated the genomes and pan-genomes of organisms within the Myxococcaceae, including the genera Myxococcus and Corallococcus, the most abundant myxobacteria isolated from soils. Previously, ten species of Corallococcus were known, whereas six species of Myxococcus phylogenetically surrounded a third genus (Pyxidicoccus) composed of a single species. Here, we describe draft genome sequences of five novel species within the Myxococcaceae (Myxococcus eversor, Myxococcus llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogochensis, Myxococcus vastator, Pyxidicoccus caerfyrddinensis, and Pyxidicoccus trucidator) and for the Pyxidicoccus type species strain, Pyxidicoccus fallax DSM 14698T. Genomic and physiological comparisons demonstrated clear differences between the five novel species and every other Myxococcus or Pyxidicoccus spp. type strain. Subsequent analyses of type strain genomes showed that both the Corallococcus pan-genome and the combined Myxococcus and Pyxidicoccus (Myxococcus/Pyxidicoccus) pan-genome are large and open, but with clear differences. Genomes of Corallococcus spp. are generally smaller than those of Myxococcus/Pyxidicoccus spp. but have core genomes three times larger. Myxococcus/Pyxidicoccus spp. genomes are more variable in size, with larger and more unique sets of accessory genes than those of Corallococcus species. In both genera, biosynthetic gene clusters are relatively enriched in the shell pan-genomes, implying they grant a greater evolutionary benefit than other shell genes, presumably by conferring selective advantages during predation.