Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

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.

Internalization of transferrin-tagged Myxococcus xanthus encapsulins into mesenchymal stem cells.

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.

Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity.

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.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

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.

Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA.

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.

Regulation of late-acting operons by three transcription factors and a CRISPR-Cas component during Myxococcus xanthus development.

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.

Mechanism of GTPase activation of a prokaryotic small Ras-like GTPase MglA by an asymmetrically interacting MglB dimer.

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.

A miniTurbo-based proximity labeling protocol to identify conditional protein interactomes in vivo in Myxococcus xanthus.

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.

The mechanism for polar localization of the type IVa pilus machine in Myxococcus xanthus.

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.

Molecular basis and design principles of switchable front-rear polarity and directional migration in Myxococcus xanthus.

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that breaks the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.

Two reasons to kill: predation and kin discrimination in myxobacteria.

Myxobacteria are social microbial predators that use cell-cell contacts to identify bacterial or fungal prey and to differentiate kin relatives to initiate cellular responses. For prey killing, they assemble Tad-like and type III-like secretion systems at contact sites. For kin discrimination (KD), they assemble outer membrane exchange complexes composed of the TraA and TraB receptors at contacts sites. A type VI secretion system and Rhs proteins also mediate KD. Following cellular recognition, these systems deliver appropriate effectors into target cells. For prey, this leads to cell death and lysis for nutrient consumption by myxobacteria. In KD, a panel of effectors are delivered, and if adjacent cells are clonal cells, resistance ensues because they express a cognate panel of immunity factors; while nonkin lack complete immunity and are intoxicated. This review compares and contrasts recent findings from these systems in myxobacteria.

Genome-Guided Discovery of the Myxobacterial Thiolactone-Containing Sorangibactins.

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 Sorangiineae strain MSr11367 in the host Myxococcus xanthus DK1622. De novo structure elucidation uncovered a linear polycyclic structure consisting of an N-terminal phenol group, an oxazole, tandem N-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.

Synthesis and degradation of polyphosphate in Myxococcus xanthus.

Polyphosphate kinase 1 (Ppk1) generates polyphosphates (polyPs) by catalyzing phosphate transfer from ATP. In the presence of ATP, Myxococcus xanthus Ppk1 showed the highest activity with polyP60-70 but also showed high activity with orthophosphate and pyrophosphate. Ppk1 synthesizes long-chain polyPs with >1 000 phosphate residues from orthophosphate or pyrophosphate present in high concentrations, suggesting that in M. xanthus, Ppk1 uses intracellular ortho/pyrophosphate as an initial primer for polyP production. During M. xanthus starvation-induced development, the specific activity of Ppk1 peaked at 12 h (300-800 nmol/min/mg) and then gradually decreased. The polyP concentration was highest during mound formation (45 nmol phosphate/mg protein); then, the level of long-chain polyPs decreased and that of short-chain polyPs increased during fruiting body and spore formation. Myxococcus xanthus expresses two exopolyphosphatases, Ppx1 and Ppx2, which mainly degrade short- and long-chain polyPs, respectively, both of which were highest in vegetative cells and were detected during starvation, which may account for the degradation of polyPs. Thus, polyPs synthesized by Ppk1 early in starvation-induced development could be degraded by exopolyphosphatases and may also be used as substrates by polyP:AMP phosphotransferases and polyphosphate/ATP-NAD kinases to generate ADP and NADP+, respectively.

Whole-genome sequencing of a biocontrol Myxococcus xanthus R31 isolate and comparative genomic analysis.

Tomato bacterial wilt (TBW) caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases. Myxococcus xanthus R31, isolated from healthy tomato rhizosphere soil using the R. solanacearum baiting method, exhibiting good biocontrol efficacy against TBW. However, the genomic information and evolutionary features of R31 are largely unclear. Here, the high-quality genome assembly of R31 was presented. Using Nanopore sequencing technology, we assembled the 9.25 Mb complete genome of R31 and identified several extracellular enzyme proteins, including carbohydrate-active enzymes (CAZymes) and peptidases. We also performed a comparative genome analysis of R31 and 17 other strains of M. xanthus with genome sequences in the NCBI database to gain insights into myxobacteria predation and genome size expansion. Average nucleotide identity and digital DNA-DNA hybridization calculation and phylogenetic analysis indicated that R31 was closely related to the species M. xanthus. Further comparative genomics analysis suggested that, in addition to characteristics of predatory microorganisms, R31 contains many strain-specific genes, which may provide a genetic basis for its proficient predatory ability. This study provides new insights into R31 and other closely related species and facilitates studies using genetic approaches to further elucidate the predation mechanism of myxobacteria.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

Myxococcus xanthus as Host for the Production of Benzoxazoles.

Benzoxazoles are important structural motifs in pharmaceutical drugs. Here, we present the heterologous production of 3-hydroxyanthranilate-derived benzoxazoles in the host bacterium Myxococcus xanthus following the expression of two genes from the nataxazole biosynthetic gene cluster of Streptomyces sp. Tü 6176. The M. xanthus expression strain achieved a benzoxazole titer of 114.6±7.4 mg L-1 upon precursor supplementation, which is superior to other bacterial production systems. Crosstalk between the heterologously expressed benzoxazole pathway and the endogenous myxochelin pathway led to the combinatorial biosynthesis of benzoxazoles featuring a 2,3-dihydroxybenzoic acid (2,3-DHBA) building block. Subsequent in vitro studies confirmed that this crosstalk is not only due to the availability of 2,3-DHBA in M. xanthus, rather, it is promoted by the adenylating enzyme MxcE from the myxochelin pathway, which contributes to the activation of aryl carboxylic acids and delivers them to benzoxazole biosynthesis.

FrzS acts as a polar beacon to recruit SgmX, a central activator of type IV pili during Myxococcus xanthus motility.

In rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polar-binding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium.

Myxococcus xanthus Encapsulin as a Promising Platform for Intracellular Protein Delivery.

Introducing a new genetically encoded material containing a photoactivatable label as a model cargo protein, based on Myxococcus xanthus (Mx) encapsulin system stably expressed in human 293T cells. Encapsulin from Mx is known to be a protein-based container for a ferritin-like cargo in its shell which could be replaced with an exogenous cargo protein, resulting in a modified encapsulin system. We replaced Mx natural cargo with a foreign photoactivatable mCherry (PAmCherry) fluorescent protein and isolated encapsulins, containing PAmCherry, from 293T cells. Isolated Mx encapsulin shells containing photoactivatable label can be internalized by macrophages, wherein the PAmCherry fluorescent signal remains clearly visible. We believe that a genetically encoded nanocarrier system obtained in this study, can be used as a platform for controllable delivery of protein/peptide therapeutics in vitro.

Total synthesis of Myxoprincomide, a secondary metabolite from Myxococcus xanthus.

Myxoprincomide, a secondary metabolite of the myxobacterium Myxococcus xanthus DK 1622, is synthesised for the first time. The central, unusual α-ketoamide is generated at the end of the synthesis to avoid side reactions during the synthesis of this rather reactive subunit. Nevertheless, the synthetic natural product is obtained as an isomeric mixture. Detailed analytical investigations show that the identical isomeric mixture is found in the isolated natural product.

Physiological roles of catalases Cat1 and Cat2 in Myxococcus xanthus.

Catalases are key antioxidant enzymes in aerobic organisms. Myxococcus xanthus expresses two monofunctional catalases, small-subunit Cat1 and large-subunit Cat2. The Km of H2O2 for recombinant Cat1 and Cat2 were 14.0 and 9.0 mM, respectively, and the catalytic efficiency of Cat2 (kcat/Km = 500 sec-1 mM-1) was 4-fold higher than that of Cat1. The activity ratio of Cat1 to Cat2 in the exponential growth phase of M. xanthus was 1 to 3-4. A Cat1-deficient strain was constructed, whereas a Cat2-deficient strain could not be produced In H2O2-supplemented medium, the cat1 mutant exhibited marked growth retardation and a longer generation time than the wild-type (wt) strain. After 2 h of incubation in 0.5 mM H2O2-supplemented medium, the catalase activity of the wt strain significantly increased (by 64-fold), but that of the cat1 mutant strain did not. Under starvation-induced developmental conditions, catalase activity was induced by approximately 200-fold in both wt and cat1 strains, although in the mutant the activity increase as well as spore formation occurred one day later, indicating that the induction of catalase activity during starvation was due to Cat2. In wt starved cells, catalase activity was not induced by H2O2. These results suggest that Cat2 is the primary housekeeping catalase during M. xanthus growth and starvation-induced development, whereas Cat1 may have a complementary role, being responsible for the rapid degradation of H2O2 in proliferating vegetative cells subjected to oxidative stress.