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Microbiology Spectrum Aug 2023Myxobacteria serve as a treasure trove of secondary metabolites. During our ongoing search for bioactive natural products, a novel subclass of disorazoles termed...
Myxobacteria serve as a treasure trove of secondary metabolites. During our ongoing search for bioactive natural products, a novel subclass of disorazoles termed disorazole Z was discovered. Ten disorazole Z family members were purified from a large-scale fermentation of the myxobacterium Sorangium cellulosum So ce1875 and characterized by electrospray ionization-high-resolution mass spectrometry (ESI-HRMS), X-ray, nuclear magnetic resonance (NMR), and Mosher ester analysis. Disorazole Z compounds are characterized by the lack of one polyketide extension cycle, resulting in a shortened monomer in comparison to disorazole A, which finally forms a dimer in the bis-lactone core structure. In addition, an unprecedented modification of a geminal dimethyl group takes place to form a carboxylic acid methyl ester. The main component disorazole Z1 shows comparable activity in effectively killing cancer cells to disorazole A1 via binding to tubulin, which we show induces microtubule depolymerization, endoplasmic reticulum delocalization, and eventually apoptosis. The disorazole Z biosynthetic gene cluster (BGC) was identified and characterized from the alternative producer So ce427 and compared to the known disorazole A BGC, followed by heterologous expression in the host Myxococcus xanthus DK1622. Pathway engineering by promoter substitution and gene deletion paves the way for detailed biosynthesis studies and efficient heterologous production of disorazole Z congeners. Microbial secondary metabolites are a prolific reservoir for the discovery of bioactive compounds, which prove to be privileged scaffolds for the development of new drugs such as antibacterial and small-molecule anticancer drugs. Consequently, the continuous discovery of novel bioactive natural products is of great importance for pharmaceutical research. Myxobacteria, especially spp., which are known for their large genomes with yet-underexploited biosynthetic potential, are proficient producers of such secondary metabolites. From the fermentation broth of Sorangium cellulosum strain So ce1875, we isolated and characterized a family of natural products named disorazole Z, which showed potent anticancer activity. Further, we report on the biosynthesis and heterologous production of disorazole Z. These results can be stepping stones toward pharmaceutical development of the disorazole family of anticancer natural products for (pre)clinical studies.
Topics: Biological Products; Antineoplastic Agents; Lactones; Myxococcales
PubMed: 37318329
DOI: 10.1128/spectrum.00730-23 -
Synthetic and Systems Biotechnology Sep 2024The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new...
The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new hosts, but has been less explored rationally. The bacterium harbors a large circular high-GC genome, and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products. In this study, we conducted transposon insertion at TA sites on the genome, and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites. The enrichment sites are characteristically distributed along the genome, and the dilution sites are overlapped well with the horizontal transfer genes. We experimentally demonstrated the enrichment sites as high expression integration sites (HEISs), and the dilution sites unsuitable for gene integration expression. This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.
PubMed: 38680947
DOI: 10.1016/j.synbio.2024.04.007 -
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 -
Frontiers in Microbiology 2023MrpC, a member of the CRP/Fnr transcription factor superfamily, is necessary to induce and control the multicellular developmental program of the bacterium, . During...
INTRODUCTION
MrpC, a member of the CRP/Fnr transcription factor superfamily, is necessary to induce and control the multicellular developmental program of the bacterium, . During development, certain cells in the population first swarm into haystack-shaped aggregates and then differentiate into environmentally resistant spores to form mature fruiting bodies (a specialized biofilm). transcriptional regulation is controlled by negative autoregulation (NAR).
METHODS
Wild type and mutant promoter regions were fused to a fluorescent reporter to examine effects on expression in the population and in single cells . Phenotypic consequences of the mutant promoter were assayed by deep convolution neural network analysis of developmental movies, sporulation efficiency assays, and anti-MrpC immunoblot. In situ analysis of single cell MrpC levels in distinct populations were assayed with an MrpC-mNeonGreen reporter.
RESULTS
Disruption of MrpC binding sites within the promoter region led to increased and broadened distribution of expression levels between individual cells in the population. Expression of from the mutant promoter led to a striking phenotype in which cells lose synchronized transition from aggregation to sporulation. Instead, some cells abruptly exit aggregation centers and remain locked in a cohesive swarming state we termed developmental swarms, while the remaining cells transition to spores inside residual fruiting bodies. examination of a fluorescent reporter for MrpC levels in developmental subpopulations demonstrated cells locked in the developmental swarms contained MrpC levels that do not reach the levels observed in fruiting bodies.
DISCUSSION
Increased cell-to-cell variation in expression upon disruption of MrpC binding sites within its promoter is consistent with NAR motifs functioning to reducing noise. Noise reduction may be key to synchronized transition of cells in the aggregation state to the sporulation state. We hypothesize a novel subpopulation of cells trapped as developmental swarms arise from intermediate levels of MrpC that are sufficient to promote aggregation but insufficient to trigger sporulation. Failure to transition to higher levels of MrpC necessary to induce sporulation may indicate cells in developmental swarms lack an additional positive feedback signal required to boost MrpC levels.
PubMed: 38075919
DOI: 10.3389/fmicb.2023.1293966 -
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 -
Frontiers in Microbiology 2023The soil-dwelling delta-proteobacterium is a model organism to study predation and competition. preys on a broad range of bacteria mediated by lytic enzymes,...
The soil-dwelling delta-proteobacterium is a model organism to study predation and competition. preys on a broad range of bacteria mediated by lytic enzymes, exopolysaccharides, Type-IV pilus-based motility, and specialized metabolites. Competition between and prey bacterial strains with various specialized metabolite profiles indicates a range of fitness, suggesting that specialized metabolites contribute to prey survival. To expand our understanding of how specialized metabolites affect predator-prey dynamics, we assessed interspecies interactions between and two strains of . While strain ATCC 14579 resisted predation, strain T was found to be highly sensitive to predation. The interaction between ATCC 14579 and appears to be competitive, resulting in population loss for both predator and prey. Genome analysis revealed that ATCC 14579 belongs to a clade that possesses the biosynthetic gene cluster for production of thiocillins, whereas strain T lacks those genes. Further, purified thiocillin protects strains unable to produce this specialized metabolite, strengthening the finding that thiocillin protects against predation and contributes to the ecological fitness of ATCC 14579. Lastly, strains that produce thiocillin appear to confer some level of protection to their own antibiotic by encoding an additional copy of the L11 ribosomal protein, a known target for thiopeptides. This work highlights the importance of specialized metabolites affecting predator-prey dynamics in soil microenvironments.
PubMed: 38075900
DOI: 10.3389/fmicb.2023.1295262 -
Journal of Bacteriology Jan 2024The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small...
The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small C-terminal sorting domains. Sortase A cleaves LPXTG, exosortases (XrtA and XrtB) cleave the PEP-CTERM sorting signal, archaeosortase A cleaves PGF-CTERM, and rhombosortase cleaves GlyGly-CTERM domains. Four sorting signal domains without previously known processing proteases are the MYXO-CTERM, JDVT-CTERM, Synerg-CTERM, and CGP-CTERM domains. These exhibit the standard tripartite architecture of a short signature motif, a hydrophobic transmembrane segment, and an Arg-rich cluster. Each has an invariant cysteine in its signature motif. Computational evidence strongly suggests that each of these four Cys-containing sorting signals is processed, at least in part, by a cognate family of glutamic-type intramembrane endopeptidases related to the eukaryotic type II CAAX-processing protease Rce1. For the MYXO-CTERM sorting signals of different lineages, their sorting enzymes, called myxosortases, include MrtX (MXAN_2755 in ), MrtC, and MrtP, all with radically different N-terminal domains but with a conserved core. Related predicted sorting enzymes were also identified for JDVT-CTERM (MrtJ), Synerg-CTERM (MrtS), and CGP-CTERM (MrtA). This work establishes a major new family of protein-sorting housekeeping endopeptidases contributing to the surface attachment of proteins in prokaryotes. IMPORTANCE Homologs of the eukaryotic type II CAAX-box protease Rce1, a membrane-embedded endopeptidase found in yeast and human ER and involved in sorting proteins to their proper cellular locations, are abundant in prokaryotes but not well understood there. This bioinformatics paper identifies several subgroups of the family as cognate endopeptidases for four protein-sorting signals processed by previously unknown machinery. Sorting signals with newly identified processing enzymes include three novel ones, but also MYXO-CTERM, which had been the focus of previous experimental work in the model fruiting and gliding bacterium . The new findings will substantially improve our understanding of Cys-containing C-terminal protein-sorting signals and of protein trafficking generally in bacteria and archaea.
Topics: Humans; Cysteine; Protein Transport; Peptide Hydrolases; Membrane Proteins; Bacteria; Saccharomyces cerevisiae
PubMed: 38084967
DOI: 10.1128/jb.00173-23 -
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 -
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 -
Proceedings of the National Academy of... Apr 2024Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are...
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 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 T4aP (T4aP) at a resolution of 3.0 Å using cryo-EM. The T4aP 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 T4aP is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aP 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.
Topics: Fimbriae Proteins; Myxococcus xanthus; Fimbriae, Bacterial; Protein Structure, Secondary; Virulence
PubMed: 38625941
DOI: 10.1073/pnas.2321989121