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Science (New York, N.Y.) Mar 2016Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used...
Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, four interconnected ring structures in the periplasm and cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. By systematically imaging mutants lacking defined T4PM proteins or with individual proteins fused to tags, we mapped the locations of all 10 T4PM core components and the minor pilins, thereby providing insights into pilus assembly, structure, and function.
Topics: Bacterial Adhesion; Cryoelectron Microscopy; Fimbriae, Bacterial; Microscopy, Electron, Transmission; Models, Molecular; Mutation; Myxococcus xanthus
PubMed: 26965631
DOI: 10.1126/science.aad2001 -
Applied and Environmental Microbiology Aug 2021Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase...
Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Here, we investigated the transcription of , , and in UV-C-irradiated Myxococcus xanthus and found that the induction of occurred significantly earlier than that of the other two genes. Mutant analysis showed that unlike that of or , the deletion of significantly delayed bacterial regrowth and slightly reduced the bacterial mutation frequency and UV resistance. Transcriptomic analysis revealed that the absence of ImuA released the expression of some known SOS genes, including , , , and . Yeast two-hybrid and pulldown analyses proved that ImuA interacts physically with RecA1 besides ImuB. Protein activity analysis indicated that ImuA had no DNA-binding activity but inhibited the DNA-binding and recombinase activity of RecA1. These findings indicate the new role of ImuA in SOS mutagenesis; that is, ImuA inhibits the recombinase activity of RecA1, thereby facilitating SOS mutagenesis in M. xanthus. DnaE2 is responsible for bacterial SOS mutagenesis in nearly one-third of sequenced bacterial strains. However, its mechanism, especially the function of one of its accessory proteins, ImuA, is still unclear. Here, we report that M. xanthus ImuA could affect SOS mutagenesis by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the selection of the two restart pathways to repair the stalled replication fork.
Topics: Bacterial Proteins; DNA; Mutagenesis; Myxococcus xanthus; Rec A Recombinases; SOS Response, Genetics; Two-Hybrid System Techniques
PubMed: 34190612
DOI: 10.1128/AEM.00919-21 -
PLoS Genetics Sep 2022The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates...
The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.
Topics: Bacterial Proteins; Cell Polarity; GTP Phosphohydrolases; Guanosine Triphosphate; Myxococcus xanthus
PubMed: 36067225
DOI: 10.1371/journal.pgen.1010384 -
Journal of Bacteriology Feb 2016When starved for nutrients, Myxococcus xanthus produces a biofilm that contains a mat of rod-shaped cells, known as peripheral rods, and aerial structures called... (Review)
Review
When starved for nutrients, Myxococcus xanthus produces a biofilm that contains a mat of rod-shaped cells, known as peripheral rods, and aerial structures called fruiting bodies, which house thousands of dormant and stress-resistant spherical spores. Because rod-shaped cells differentiate into spherical, stress-resistant spores and spore differentiation occurs only in nascent fruiting bodies, many genes and multiple levels of regulation are required. Over the past 2 decades, many regulators of the temporal and spatial expression of M. xanthus sporulation genes have been uncovered. Of these sporulation gene regulators, two-component signal transduction circuits, which typically contain a histidine kinase sensor protein and a transcriptional regulator known as response regulator, are among the best characterized. In this review, we discuss prototypical two-component systems (Nla6S/Nla6 and Nla28S/Nla28) that regulate an early, preaggregation phase of sporulation gene expression during fruiting body development. We also discuss orphan response regulators (ActB and FruA) that regulate a later phase of sporulation gene expression, which begins during the aggregation stage of fruiting body development. In addition, we summarize the research on a complex two-component system (Esp) that is important for the spatial regulation of sporulation.
Topics: Gene Expression Regulation, Bacterial; Myxococcus xanthus; Signal Transduction; Spores, Bacterial; Time Factors
PubMed: 26369581
DOI: 10.1128/JB.00474-15 -
Angewandte Chemie (International Ed. in... Dec 2022During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with...
During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non-cyclized thiamyxin D fused to a glycerol unit at the C-terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety by a GCN5-related N-acetyltransferase-like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.
Topics: Humans; Myxococcales; RNA; Polyketide Synthases; Multigene Family; Polyketides; Zika Virus; Zika Virus Infection
PubMed: 36208117
DOI: 10.1002/anie.202212946 -
MBio Feb 2019Self-recognition underlies sociality in many group-living organisms. In bacteria, cells use various strategies to recognize kin to form social groups and, in some cases,...
Self-recognition underlies sociality in many group-living organisms. In bacteria, cells use various strategies to recognize kin to form social groups and, in some cases, to transition into multicellular life. One strategy relies on a single genetic locus that encodes a variable phenotypic tag ("greenbeard") for recognizing other tag bearers. Previously, we discovered a polymorphic cell surface receptor called TraA that directs self-identification through homotypic interactions in the social bacterium Recognition by TraA leads to cellular resource sharing in a process called outer membrane exchange (OME). A second gene in the operon, , is also required for OME but is not involved in recognition. Our prior studies of TraA identified only six recognition groups among closely related isolates. Here we hypothesize that the number of polymorphisms and, consequently, the diversity of recognition in wild isolates are much greater. To test this hypothesis, we expand the scope of TraA characterization to the order From genomic sequences within the three suborders of , we identified 90 orthologs. Sequence analyses and functional characterization of loci suggest that OME is well maintained among diverse myxobacterial taxonomic groups. Importantly, TraA orthologs are highly polymorphic within their variable domain, the region that confers selectivity in self-recognition. We experimentally defined 10 distinct recognition groups and, based on phylogenetic and experimental analyses, predicted >60 recognition groups among the 90 alleles. Taken together, our findings revealed a widespread greenbeard locus that mediates the diversity of self-recognition across the order Many biological species distinguish self from nonself by using different mechanisms. Higher animals recognize close kin via complex processes that often involve the five senses, cognition, and learning, whereas some microbes achieve self-recognition simply through the activity of a single genetic locus. Here we describe a single locus, , in myxobacteria that governs cell-cell recognition within natural populations. We found that is widespread across the order TraA is highly polymorphic among diverse myxobacterial isolates, and such polymorphisms determine selectivity in self-recognition. Through bioinformatic and experimental analyses, we showed that governs many distinct recognition groups within This report provides an example in which a single locus influences social recognition across a wide phylogenetic range of natural populations.
Topics: Bacterial Outer Membrane Proteins; Computational Biology; Data Mining; Genome, Bacterial; Microbial Interactions; Myxococcales; Polymorphism, Genetic; Sequence Homology
PubMed: 30755513
DOI: 10.1128/mBio.02751-18 -
Applied and Environmental Microbiology Nov 2018Myxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores, along...
Myxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores, along with a new myxochelin derivative named pseudochelin A, was reported for the marine bacterium S2040. The latter derivative features a characteristic imidazoline moiety, which was proposed to originate from an intramolecular condensation reaction of the β-aminoethyl amide group in myxochelin B. To identify the enzyme catalyzing this conversion, we compared the myxochelin regulons of two myxobacterial strains that produce solely myxochelin A and B with those of S2040. This approach revealed a gene exclusive to the myxochelin regulon in S2040, coding for an enzyme of the amidohydrolase superfamily. To prove that this enzyme is indeed responsible for the postulated conversion, the reaction was reconstituted using a hexahistidine-tagged recombinant protein made in , with myxochelin B as the substrate. To test the production of pseudochelin A under conditions, the amidohydrolase gene was cloned into the myxobacterial plasmid pZJY156 and placed under the control of a copper-inducible promoter. The resulting vector was introduced into the myxobacterium DSM 16526, a native producer of myxochelin A and B. Following induction with copper, the myxobacterial expression strain was found to synthesize small quantities of pseudochelin A. Replacement of the copper-inducible promoter with the constitutive promoter led to increased production levels in , which facilitated the isolation and subsequent structural verification of the heterologously produced compound. In this study, an enzyme for imidazoline formation in pseudochelin biosynthesis was identified. Evidence for the involvement of this enzyme in the postulated reaction was obtained after reconstitution. Furthermore, the function of this enzyme was demonstrated by transferring the corresponding gene into the bacterium , which thereby became a producer of pseudochelin A. In addition to clarifying the molecular basis of imidazoline formation in siderophore biosynthesis, we describe the heterologous expression of a gene in a myxobacterium without chromosomal integration. Due to its metabolic proficiency, represents an interesting alternative to established host systems for the reconstitution and manipulation of biosynthetic pathways. Since the plasmid used in this study is easily adaptable for the expression of other enzymes as well, we expand the conventional expression strategy for myxobacteria, which is based on the integration of biosynthetic genes into the host genome.
Topics: Amidohydrolases; Bacterial Proteins; Biosynthetic Pathways; Catechols; Gene Expression Regulation, Bacterial; Genetic Engineering; Lysine; Myxococcus xanthus; Plasmids; Promoter Regions, Genetic; Regulon
PubMed: 30217842
DOI: 10.1128/AEM.01789-18 -
ACS Chemical Biology Apr 2023In this study, an unprecedented myxobacterial siderophore termed sorangibactin was discovered by heterologous expression of a coelibactin-like nonribosomal peptide...
In this study, an unprecedented myxobacterial siderophore termed sorangibactin was discovered by heterologous expression of a coelibactin-like nonribosomal peptide synthetase (NRPS) gene cluster from the strain MSr11367 in the host DK1622. De novo structure elucidation uncovered a linear polycyclic structure consisting of an N-terminal phenol group, an oxazole, tandem -methyl-thiazolidines, and an unusual C-terminal γ-thiolactone moiety. Except for the unprecedented oxazoline dehydrogenation to form an oxazole, which we show to be catalyzed by a cytochrome P450-dependent enzyme, other tailoring steps were found necessary for efficient downstream processing. The unusual thioesterase (TE) domain is proposed to select homocysteine or methionine for offloading involving an intramolecular γ-thiolactone formation. Its active site comprises a rare cysteine, which was found essential for product formation by point mutation to alanine or serine, which both abolished its activity. This unusual release mechanism and the resulting rare thiolactone structure can serve as a starting point for detailed biochemical investigations.
Topics: Myxococcales; Myxococcus xanthus; Phenols; Oxazoles
PubMed: 37014749
DOI: 10.1021/acschembio.3c00063 -
The ISME Journal Aug 2021Extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) extend from plant roots into the soil environment and interact with soil microbial communities. Evidence...
Extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) extend from plant roots into the soil environment and interact with soil microbial communities. Evidence of positive and negative interactions between AMF and soil bacteria point to functionally important ERH-associated communities. To characterize communities associated with ERH and test controls on their establishment and composition, we utilized an in-growth core system containing a live soil-sand mixture that allowed manual extraction of ERH for 16S rRNA gene amplicon profiling. Across experiments and soils, consistent enrichment of members of the Betaproteobacteriales, Myxococcales, Fibrobacterales, Cytophagales, Chloroflexales, and Cellvibrionales was observed on ERH samples, while variation among samples from different soils was observed primarily at lower taxonomic ranks. The ERH-associated community was conserved between two fungal species assayed, Glomus versiforme and Rhizophagus irregularis, though R. irregularis exerted a stronger selection and showed greater enrichment for taxa in the Alphaproteobacteria and Gammaproteobacteria. A distinct community established within 14 days of hyphal access to the soil, while temporal patterns of establishment and turnover varied between taxonomic groups. Identification of a conserved ERH-associated community is consistent with the concept of an AMF microbiome and can aid the characterization of facilitative and antagonistic interactions influencing the plant-fungal symbiosis.
Topics: Bacteria; Fungi; Hyphae; Mycorrhizae; Plant Roots; RNA, Ribosomal, 16S; Soil; Soil Microbiology
PubMed: 33649552
DOI: 10.1038/s41396-021-00920-2 -
MSphere May 2021Hsp70 proteins are among the most ubiquitous chaperones and play important roles in maintaining proteostasis and resisting environmental stress. Multiple copies of...
Hsp70 proteins are among the most ubiquitous chaperones and play important roles in maintaining proteostasis and resisting environmental stress. Multiple copies of Hsp70s are widely present in eukaryotic cells with redundant and divergent functions, but they have been less well investigated in prokaryotes. DK1622 is annotated as having many genes. In this study, we performed a bioinformatic analysis of Hsp70 proteins and investigated the functions of six genes in DK1622, including two genes that encode proteins with the conserved PRK00290 domain (MXAN_3192 and MXAN_6671) and four genes that encode proteins with the cl35085 or cd10170 domain. We found that only MXAN_3192 is essential for cell survival and heat shock induction. MXAN_3192, compared with the other genes, has a high transcriptional level, far exceeding that of any other gene, which, however, is not the reason for its essentiality. Deletion of MXAN_6671 () led to multiple deficiencies in development, social motility, and oxidative resistance, while deletion of each of the other four genes decreased sporulation and oxidative resistance. MXAN_3192 or , but not the other genes, restored the growth deficiency of the mutant. Our results demonstrated that the PRK00290 proteins play a central role in the complex cellular functions of , while the other diverse Hsp70 superfamily homologues probably evolved as helpers with some unknown specific functions. Hsp70 proteins are highly conserved chaperones that occur in all kingdoms of life. Multiple copies of Hsp70s are often present in genome-sequenced prokaryotes, especially taxa with complex life cycles, such as myxobacteria. We investigated the functions of six genes in DK1622 and demonstrated that the two Hsp70 proteins with the PRK00290 domain play a central role in complex cellular functions in , while other Hsp70 proteins probably evolved as helpers with some unknown specific functions.
Topics: Bacterial Proteins; Computational Biology; HSP72 Heat-Shock Proteins; Myxococcus xanthus; Phylogeny; Stress, Physiological; Transcription, Genetic
PubMed: 34011688
DOI: 10.1128/mSphere.00305-21