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STAR Protocols Dec 2023Protein-protein interactions are foundational for many cellular processes. Such interactions are especially challenging to identify if they are transient or depend on...
Protein-protein interactions are foundational for many cellular processes. Such interactions are especially challenging to identify if they are transient or depend on environmental conditions. This protocol details steps to identify stable and transient protein interactomes in the bacterium Myxococcus xanthus using biotin ligase miniTurbo-based proximity labeling. We include instructions for optimizing the expression of control proteins, in vivo biotin labeling of bacteria grown on a surface or in suspension culture, enrichment of biotinylated proteins, and sample processing for proteomic analysis. For complete details on the use and execution of this protocol, please refer to Branon et al. (2018)..
Topics: Biotin; Myxococcus xanthus; Proteomics
PubMed: 37883223
DOI: 10.1016/j.xpro.2023.102657 -
Frontiers in Microbiology 2023Myxobacteria are widely distributed in various habitats of soil and oceanic sediment. However, it is unclear whether soil-dwelling myxobacteria tolerate a saline...
Myxobacteria are widely distributed in various habitats of soil and oceanic sediment. However, it is unclear whether soil-dwelling myxobacteria tolerate a saline environment. In this study, a salt-tolerant myxobacterium sp. strain MxC21 was isolated from forest soil with NaCl tolerance >2% concentration. Under 1% salt-contained condition, strain MxC21 could kill and consume bacteria prey and exhibited complex social behaviors such as S-motility, biofilm, and fruiting body formation but adopted an asocial living pattern with the presence of 1.5% NaCl. To investigate the genomic basis of stress tolerance, the complete genome of MxC21 was sequenced and analyzed. Strain MxC21 consists of a circular chromosome with a total length of 9.13 Mbp and a circular plasmid of 64.3 kb. Comparative genomic analysis revealed that the genomes of strain MxC21 and DK1622 share high genome synteny, while no endogenous plasmid was found in DK1622. Further analysis showed that approximately 21% of its coding genes from the genome of strain MxC21 are predominantly associated with signal transduction, transcriptional regulation, and protein folding involved in diverse niche adaptation such as salt tolerance, which enables social behavior such as gliding motility, sporulation, and predation. Meantime, a high number of genes are also found to be involved in defense against oxidative stress and production of antimicrobial compounds. All of these functional genes may be responsible for the potential salt-toleration. Otherwise, strain MxC21 is the second reported myxobacteria containing indigenous plasmid, while only a small proportion of genes was specific to the circular plasmid of strain MxC21, and most of them were annotated as hypothetical proteins, which may have a direct relationship with the habitat adaptation of strain MxC21 under saline environment. This study provides an inspiration of the adaptive evolution of salt-tolerant myxobacterium and facilitates a potential application in the improvement of saline soil in future.
PubMed: 37789850
DOI: 10.3389/fmicb.2023.1250602 -
MBio Oct 2023Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different...
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 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.
Topics: Fimbriae Proteins; Myxococcus xanthus; Fimbriae, Bacterial
PubMed: 37754549
DOI: 10.1128/mbio.01593-23 -
MSystems Oct 2023Understanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these...
Understanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacterium forms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.
Topics: Spores, Bacterial; Myxococcus xanthus; Biofilms; Cell Differentiation
PubMed: 37747885
DOI: 10.1128/msystems.00425-23 -
Nature Communications Sep 2023Many species, such as fish schools or bird flocks, rely on collective motion to forage, prey, or escape predators. Likewise, Myxococcus xanthus forages and moves...
Many species, such as fish schools or bird flocks, rely on collective motion to forage, prey, or escape predators. Likewise, Myxococcus xanthus forages and moves collectively to prey and feed on other bacterial species. These activities require two distinct motility machines enabling adventurous (A) and social (S) gliding, however when and how these mechanisms are used has remained elusive. Here, we address this long-standing question by applying multiscale semantic cell tracking during predation. We show that: (1) foragers and swarms can comprise A- and S-motile cells, with single cells exchanging frequently between these groups; (2) A-motility is critical to ensure the directional movement of both foragers and swarms; (3) the combined action of A- and S-motile cells within swarms leads to increased predation efficiencies. These results challenge the notion that A- and S-motilities are exclusive to foragers and swarms, and show that these machines act synergistically to enhance predation efficiency.
Topics: Animals; Predatory Behavior; Cell Tracking; Cooperative Behavior; Motion; Myxococcus xanthus
PubMed: 37696789
DOI: 10.1038/s41467-023-41193-x -
Journal of Bacteriology Sep 2023The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger...
The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in . Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshE, is conserved in the N-terminus of PilB (PilB) in and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilB resulted in mutants with distinct and differential phenotypes in piliation and biofilm levels in . This suggests that the PilB domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in . IMPORTANCE The regulation of motility and biofilm by cyclic-di-GMP in flagellated bacteria has been extensively investigated. However, our knowledge regarding this regulation in motile bacteria without flagella remains limited. Here, we aimed to address this gap by investigating a non-flagellated bacterium with motility powered by bacterial type-IV pilus (T4P). Previous studies hinted at the possibility of PilB, the T4P assembly ATPase, serving as a cyclic-di-GMP effector involved in regulating both motility and biofilm. Our findings strongly support the hypothesis that PilB directly interacts with cyclic-di-GMP to act as a potential switch to promote biofilm formation or T4P-dependent motility. These results shed light on the bifurcation of PilB functions and its pivotal role in coordinating biofilm formation and T4P-mediated motility.
Topics: Myxococcus xanthus; Cyclic GMP; Adenosine Triphosphatases; Biofilms
PubMed: 37695853
DOI: 10.1128/jb.00221-23 -
Nature Communications Sep 2023Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that...
Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.
Topics: Peptidoglycan; Bambermycins; Nitric Oxide Synthase; Peptide Hydrolases; Cell Wall; Myxococcus xanthus; Penicillin-Binding Proteins
PubMed: 37660104
DOI: 10.1038/s41467-023-41082-3 -
Frontiers in Microbiology 2023Natural products discovered from bacteria provide critically needed therapeutic leads for drug discovery, and myxobacteria are an established source for metabolites with...
INTRODUCTION
Natural products discovered from bacteria provide critically needed therapeutic leads for drug discovery, and myxobacteria are an established source for metabolites with unique chemical scaffolds and biological activities. Myxobacterial genomes accommodate an exceptional number and variety of biosynthetic gene clusters (BGCs) which encode for features involved in specialized metabolism.
METHODS
In this study, we describe the collection, sequencing, and genome mining of 20 myxobacteria isolated from rhizospheric soil samples collected in North America.
RESULTS
Nine isolates were determined to be novel species of myxobacteria including representatives from the genera , and . Growth profiles, biochemical assays, and descriptions were provided for all proposed novel species. We assess the BGC content of all isolates and observe differences between Myxococcia and Polyangiia clusters.
DISCUSSION
Continued discovery and sequencing of novel myxobacteria from the environment provide BGCs for the genome mining pipeline. Utilizing complete or near-complete genome sequences, we compare the chromosomal organization of BGCs of related myxobacteria from various genera and suggest that the spatial proximity of hybrid, modular clusters contributes to the metabolic adaptability of myxobacteria.
PubMed: 37601375
DOI: 10.3389/fmicb.2023.1227206 -
ArXiv Aug 2023Colonies of the social bacterium go through a morphological transition from a thin colony of cells to three-dimensional droplet-like fruiting bodies as a strategy to...
Colonies of the social bacterium go through a morphological transition from a thin colony of cells to three-dimensional droplet-like fruiting bodies as a strategy to survive starvation. The biological pathways that control the decision to form a fruiting body have been studied extensively. However, the mechanical events that trigger the creation of multiple cell layers and give rise to droplet formation remain poorly understood. By measuring cell orientation, velocity, polarity, and force with cell-scale resolution, we reveal a stochastic local polar order in addition to the more obvious nematic order. Average cell velocity and active force at topological defects agree with predictions from active nematic theory, but their fluctuations are anomalously large due to polar active forces generated by the self-propelled rod-shaped cells. We find that M. xanthus cells adjust their reversal frequency to tune the magnitude of this local polar order, which in turn controls the mechanical stresses and triggers layer formation in the colonies.
PubMed: 37576128
DOI: No ID Found -
BioRxiv : the Preprint Server For... Jul 2023Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and...
Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.
PubMed: 37546724
DOI: 10.1101/2023.07.26.550694