RESUMO
Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.
Assuntos
Myxococcus xanthus , Polifosfatos , Esporos Bacterianos , Polifosfatos/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/crescimento & desenvolvimento , Myxococcus xanthus/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Meios de Cultura/químicaRESUMO
Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity is switched on during cytokinesis, resulting in a transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating phenotypically similar daughters with correct motility behaviours. Thus, our findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria can ensure the formation of dissimilar or similar daughter cells by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations.
Assuntos
Proteínas de Bactérias , GMP Cíclico , Citocinese , Myxococcus xanthus , Fenótipo , Fósforo-Oxigênio Liases , Citocinese/fisiologia , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fósforo-Oxigênio Liases/metabolismo , Fósforo-Oxigênio Liases/genética , Myxococcus xanthus/metabolismo , Myxococcus xanthus/citologia , Myxococcus xanthus/fisiologia , Myxococcus xanthus/genética , Divisão Celular , Regulação Bacteriana da Expressão Gênica , Proteínas de Escherichia coliRESUMO
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.
Assuntos
Adaptação Fisiológica , Myxococcus xanthus , Myxococcus xanthus/fisiologia , Myxococcus xanthus/metabolismo , Estresse Fisiológico , Interações Microbianas/fisiologiaRESUMO
Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.
Assuntos
Proteínas do Capsídeo , Capsídeo , Microscopia Crioeletrônica , Mutação Puntual , Capsídeo/metabolismo , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Modelos MolecularesRESUMO
We study traveling wave solutions for a reaction-diffusion model, introduced in the article Calvez et al. (Regime switching on the propagation speed of travelling waves of some size-structured myxobacteriapopulation models, 2023), describing the spread of the social bacterium Myxococcus xanthus. This model describes the spatial dynamics of two different cluster sizes: isolated bacteria and paired bacteria. Two isolated bacteria can coagulate to form a cluster of two bacteria and conversely, a pair of bacteria can fragment into two isolated bacteria. Coagulation and fragmentation are assumed to occur at a certain rate denoted by k. In this article we study theoretically the limit of fast coagulation fragmentation corresponding mathematically to the limit when the value of the parameter k tends to + ∞ . For this regime, we demonstrate the existence and uniqueness of a transition between pulled and pushed fronts for a certain critical ratio θ â between the diffusion coefficient of isolated bacteria and the diffusion coefficient of paired bacteria. When the ratio is below θ â , the critical front speed is constant and corresponds to the linear speed. Conversely, when the ratio is above the critical threshold, the critical spreading speed becomes strictly greater than the linear speed.
Assuntos
Conceitos Matemáticos , Modelos Biológicos , Myxococcus xanthus , Myxococcus xanthus/fisiologia , Simulação por Computador , DifusãoRESUMO
Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.
Assuntos
Proteínas de Bactérias , FMN Redutase , Myxococcus xanthus , Myxococcus xanthus/metabolismo , Myxococcus xanthus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , FMN Redutase/metabolismo , Cristalografia por Raios X , Mononucleotídeo de Flavina/metabolismo , Ferro/metabolismo , Modelos Moleculares , Microscopia CrioeletrônicaRESUMO
Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.
Assuntos
Células-Tronco Mesenquimais , Myxococcus xanthus , Transferrina , Células-Tronco Mesenquimais/metabolismo , Transferrina/metabolismo , Humanos , Myxococcus xanthus/metabolismo , Endocitose , Receptores da Transferrina/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/genéticaRESUMO
Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the ß-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.
Assuntos
Myxococcus xanthus , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Síntese de DNA Translesão , Escherichia coli/genética , Escherichia coli/metabolismo , DNA/genética , Replicação do DNARESUMO
Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.
Assuntos
Proteínas de Fímbrias , Myxococcus xanthus , Proteínas de Fímbrias/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Fímbrias Bacterianas/metabolismo , Estrutura Secundária de Proteína , VirulênciaRESUMO
Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts.
Assuntos
Fímbrias Bacterianas , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/fisiologia , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Bactérias/genética , Bactérias/metabolismo , Aderência Bacteriana/genética , Transferência Genética Horizontal , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Myxococcus xanthus/genética , Myxococcus xanthus/fisiologia , Myxococcus xanthus/metabolismoRESUMO
Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB1 or MglB2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB1, but not MglB2, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal ß-strand of MglB1 and ß0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.
Assuntos
Proteínas de Bactérias , Proteínas Ativadoras de GTPase , Myxococcus xanthus , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ativação Enzimática , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Myxococcus xanthus/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/enzimologia , Multimerização Proteica , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
Intrinsic rates of genetic mutation have diverged greatly across taxa and exhibit statistical associations with several other parameters and features. These include effective population size (Ne), genome size, and gametic multicellularity, with the latter being associated with both increased mutation rates and decreased effective population sizes. However, data sufficient to test for possible relationships between microbial multicellularity and mutation rate (µ) are lacking. Here, we report estimates of two key population-genetic parameters, Ne and µ, for Myxococcus xanthus, a bacterial model organism for the study of aggregative multicellular development, predation, and social swarming. To estimate µ, we conducted an â¼400-day mutation accumulation experiment with 46 lineages subjected to regular single colony bottlenecks prior to clonal regrowth. Upon conclusion, we sequenced one clonal-isolate genome per lineage. Given collective evolution for 85,323 generations across all lines, we calculate a per base-pair mutation rate of â¼5.5 × 10-10 per site per generation, one of the highest mutation rates among free-living eubacteria. Given our estimate of µ, we derived Ne at â¼107 from neutral diversity at four-fold degenerate sites across two dozen M. xanthus natural isolates. This estimate is below average for eubacteria and strengthens an already clear negative correlation between µ and Ne in prokaryotes. The higher and lower than average mutation rate and Ne for M. xanthus, respectively, amplify the question of whether any features of its multicellular life cycle-such as group-size reduction during fruiting-body development-or its highly structured spatial distribution have significantly influenced how these parameters have evolved.
Assuntos
Taxa de Mutação , Myxococcus xanthus , Myxococcus xanthus/genética , Densidade Demográfica , Genoma BacterianoRESUMO
Upon starvation, rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I, exoL-P, and nfsA-H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose produces spores suited to withstand starvation and environmental insults.
Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus , Óperon , Regiões Promotoras Genéticas , Esporos Bacterianos , Fatores de Transcrição , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Myxococcus xanthus/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Óperon/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Mutação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
Ecological variation influences the character of many biotic interactions, but examples of predator-prey reversal mediated by abiotic context are few. We show that the temperature at which prey grow before interacting with a bacterial predator can determine the very direction of predation, reversing predator and prey identities. While Pseudomonas fluorescens reared at 32°C was extensively killed by the generalist predator Myxococcus xanthus, P. fluorescens reared at 22°C became the predator, slaughtering M. xanthus to extinction and growing on its remains. Beyond M. xanthus, diffusible molecules in P. fluorescens supernatant also killed 2 other phylogenetically distant species among several examined. Our results suggest that the sign of lethal microbial antagonisms may often change across abiotic gradients in natural microbial communities, with important ecological and evolutionary implications. They also suggest that a larger proportion of microbial warfare results in predation-the killing and consumption of organisms-than is generally recognized.
Assuntos
Microbiota , Myxococcus xanthus , Animais , Comportamento Predatório , Antibiose , Evolução BiológicaRESUMO
The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new signals and molecules produced by one species that affects other species' behavior. In this work, we have studied the effects of the interaction of nine Streptomyces species (S. albidoflavus, S. ambofaciens, S. argillaceus, S. griseus, S. lividans, S. olivaceus, S. parvulus, S. peucetius, and S. rochei) with the predator bacteria Myxococcus xanthus, five of which (S. albidoflavus, S. griseus, S. lividans, S. olivaceus, and S. argillaceus) induce mound formation of M. xanthus on complex media (Casitone Yeast extract (CYE) and Casitone tris (CTT); media on which M. xanthus does not form these aggregates under normal culture conditions. An in-depth study on S. griseus-M. xanthus interactions (the Streptomyces strain producing the strongest effect) has allowed the identification of two siderophores produced by S. griseus, demethylenenocardamine and nocardamine, responsible for this grouping effect over M. xanthus. Experiments using pure commercial nocardamine and different concentrations of FeSO4 show that iron depletion is responsible for the behavior of M. xanthus. Additionally, it was found that molecules, smaller than 3 kDa, produced by S. peucetius can induce the production of DK-xanthenes by M. xanthus.
Assuntos
Myxococcus xanthus , Myxococcus , Streptomyces , Interações Microbianas , FerroRESUMO
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).1.
Assuntos
Biotina , Myxococcus xanthus , Biotina/metabolismo , Myxococcus xanthus/metabolismo , Proteômica/métodosRESUMO
IMPORTANCE: 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 Myxococcus xanthus 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.
Assuntos
Myxococcus xanthus , Esporos Bacterianos , Esporos Bacterianos/fisiologia , Biofilmes , Diferenciação CelularRESUMO
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 Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshEN, is conserved in the N-terminus of PilB (PilBN) in M. xanthus 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 M. xanthus 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 PilBN resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilBN 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 M. xanthus. 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 Myxococcus xanthus 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.
Assuntos
Myxococcus xanthus , Myxococcus xanthus/genética , GMP Cíclico , Adenosina Trifosfatases , BiofilmesRESUMO
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.
Assuntos
Myxococcus xanthus , Comportamento Predatório , Animais , Rastreamento de Células , Comportamento Cooperativo , Movimento (Física)RESUMO
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.