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A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall.BioRxiv : the Preprint Server For... Apr 2024The Gram-negative bacterium glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into...
The Gram-negative bacterium glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here we show that AgmT, a highly abundant lytic PG transglycosylase homologous to MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of MltG restores the connection between PG and bFACs and thus rescues gliding motility in the cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall.
PubMed: 38617213
DOI: 10.1101/2024.04.04.588103 -
Protein Science : a Publication of the... May 2024Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV...
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 β-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.
Topics: Myxococcus xanthus; Adenosine Triphosphatases; Bacterial Proteins; Translesion DNA Synthesis; Escherichia coli; DNA; DNA Replication
PubMed: 38591662
DOI: 10.1002/pro.4981 -
Current Opinion in Microbiology Jun 2024Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require... (Review)
Review
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.
Topics: Fimbriae, Bacterial; Caulobacter crescentus; Bacteria; Bacterial Adhesion; Gene Transfer, Horizontal; Fimbriae Proteins; Bacterial Proteins; Myxococcus xanthus
PubMed: 38579360
DOI: 10.1016/j.mib.2024.102468 -
MSystems Apr 2024The chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes...
The chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes encode Hsp70 paralogs. Although the mechanisms of Hsp70 function have been clearly illuminated, the function and evolution of Hsp70 paralogs is not well studied. DnaK is a highly conserved bacterial Hsp70 family. Here, we show that is present in 98.9% of bacterial genomes, and 6.4% of them possess two or more DnaK paralogs. We found that the duplication of is positively correlated with an increase in proteomic complexity (proteome size, number of domains). We identified the interactomes of the two DnaK paralogs of DK1622 (MxDnaKs), which revealed that they are mostly nonoverlapping, although both prefer α and β domain proteins. Consistent with the entire proteome, MxDnaK substrates have both significantly more multi-domain proteins and a higher isoelectric point than that of , which encodes a single DnaK homolog. MxDnaK1 is transcriptionally upregulated in response to heat shock and prefers to bind cytosolic proteins, while MxDnaK2 is downregulated by heat shock and is more associated with membrane proteins. Using domain swapping, we show that the nucleotide-binding domain and the substrate-binding β domain are responsible for the significant differences in DnaK interactomes, and the nucleotide binding domain also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work suggests that bacterial DnaK has been duplicated in order to deal with a more complex proteome, and that this allows evolution of distinct domains to deal with different subsets of target proteins.IMPORTANCEAll eukaryotic and ~40% of prokaryotic species encode multiple 70 kDa heat shock protein (Hsp70) homologs with similar but diversified functions. Here, we show that duplication of canonical Hsp70 (DnaK in prokaryotes) correlates with increasing proteomic complexity and evolution of particular regions of the protein. Using the DnaK duplicates as a case, we found that their substrate spectrums are mostly nonoverlapping, and are both consistent to that of DnaK in structural and molecular characteristics, but show differential enrichment of membrane proteins. Domain/region swapping demonstrated that the nucleotide-binding domain and the β substrate-binding domain (SBDβ), but not the SBDα or disordered C-terminal tail region, are responsible for this functional divergence. This work provides the first direct evidence for regional evolution of DnaK paralogs.
Topics: Humans; Proteome; Escherichia coli Proteins; Proteomics; HSP70 Heat-Shock Proteins; Escherichia coli; Bacteria; Membrane Proteins; Nucleotides
PubMed: 38530057
DOI: 10.1128/msystems.01154-23 -
Genome Biology and Evolution May 2024Intrinsic rates of genetic mutation have diverged greatly across taxa and exhibit statistical associations with several other parameters and features. These include...
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 ∼400-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 ∼5.5 × 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 ∼107 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.
Topics: Myxococcus xanthus; Mutation Rate; Population Density; Genome, Bacterial
PubMed: 38526062
DOI: 10.1093/gbe/evae066 -
ACS Omega Mar 2024Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for...
Molecular Dynamics of Outer Membrane-Embedded Polysaccharide Secretion Porins Reveals Closed Resting-State Surface Gates Targetable by Virtual Fragment Screening for Drug Hotspot Identification.
Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and similarities to existing protein structures; the local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modeling, and characterization of 18-β-stranded outer-membrane (OM) WzpX, WzpS, and WzpB β-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium DZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains or on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using the WzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for a rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model structure validation, our molecular dynamics simulations revealed a minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each β-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the β-barrels and inhibiting polysaccharide secretion.
PubMed: 38524450
DOI: 10.1021/acsomega.3c09970 -
The Journal of Biological Chemistry Apr 2024Cell 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 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 (MglB or MglB linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB, but not MglB, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB and β of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB 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.
Topics: Bacterial Proteins; Enzyme Activation; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Myxococcus xanthus; Protein Multimerization; Models, Molecular; Protein Structure, Quaternary
PubMed: 38508314
DOI: 10.1016/j.jbc.2024.107197 -
BioRxiv : the Preprint Server For... Feb 2024Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry...
Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximises 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 novel symmetry-reduced structures. Starting with the encapsulin from , 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 cryo-EM, we determine the structures of a precedented 60-mer icosahedral assembly and an unprecedented 36-mer tetrahedron that features significant geometric rearrangements around a novel 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 the first example of tetrahedral geometry across all characterised 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 protein sequence.
PubMed: 38370832
DOI: 10.1101/2024.02.05.579038 -
Frontiers in Microbiology 2024Bacterial predators are widely distributed across a variety of natural environments. Understanding predatory interactions is of great importance since they play a... (Review)
Review
Bacterial predators are widely distributed across a variety of natural environments. Understanding predatory interactions is of great importance since they play a defining role in shaping microbial communities in habitats such as soils. is a soil-dwelling bacterial predator that can prey on Gram-positive and Gram-negative bacteria and even on eukaryotic microorganisms. This model organism has been studied for many decades for its unusual lifecycle, characterized by the formation of multicellular fruiting bodies filled with myxospores. However, less is known about its predatory behavior despite being an integral part of its lifecycle. Predation in is a multifactorial process that involves several mechanisms working synergistically, including motility systems to efficiently track and hunt prey, and a combination of short-range and contact-dependent mechanisms to achieve prey death and feed on them. In the short-range attack, is best known for the collective production of secondary metabolites and hydrolytic enzymes to kill prey and degrade cellular components. On the other hand, contact-dependent killing is a cell-to-cell process that relies on Tad-like and type III secretion systems. Furthermore, recent research has revealed that metals also play an important role during predation, either by inducing oxidative stress in the prey, or by competing for essential metals. In this paper, we review the current knowledge about predation, focusing on the different mechanisms used to hunt, kill, and feed on its prey, considering the most recent discoveries and the transcriptomic data available.
PubMed: 38328431
DOI: 10.3389/fmicb.2024.1339696 -
PLoS Biology Jan 2024Ecological variation influences the character of many biotic interactions, but examples of predator-prey reversal mediated by abiotic context are few. We show that the...
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.
Topics: Animals; Predatory Behavior; Antibiosis; Biological Evolution; Microbiota; Myxococcus xanthus
PubMed: 38261596
DOI: 10.1371/journal.pbio.3002454