Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

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.

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 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.

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.

Discovery, Biosynthetic Origin, and Heterologous Production of Massinidine, an Antiplasmodial Alkaloid.

Bacteria of the genus Massilia represent an underexplored source of bioactive natural products. Here, we report the discovery of massinidine (1), a guanidine alkaloid with antiplasmodial activity, from these microbes. The unusual scaffold of massinidine is shown to originate from l-phenylalanine, acetate, and l-arginine. Massinidine biosynthesis genes were identified in the native producer and validated through heterologous expression in Myxococcus xanthus. Bioinformatic analyses indicate that the potential for massinidine biosynthesis is distributed in various proteobacteria.

Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism.

Encapsulins are bacterial organelle-like cages involved in various aspects of metabolism, especially protection from oxidative stress. They can serve as vehicles for a wide range of medical applications. Encapsulin shell proteins are structurally similar to HK97 bacteriophage capsid protein and their function depends on the encapsulated cargos. The Myxococcus xanthus encapsulin system comprises EncA and three cargos: EncB, EncC, and EncD. EncB and EncC are similar to bacterial ferritins that can oxidize Fe+2 to less toxic Fe+3. We analyzed EncA, EncB, and EncC by cryo-EM and X-ray crystallography. Cryo-EM shows that EncA cages can have T = 3 and T = 1 symmetry and that EncA T = 1 has a unique protomer arrangement. Also, we define EncB and EncC binding sites on EncA. X-ray crystallography of EncB and EncC reveals conformational changes at the ferroxidase center and additional metal binding sites, suggesting a mechanism for Fe oxidation and storage within the encapsulin shell.

Encapsulin Based Self-Assembling Iron-Containing Protein Nanoparticles for Stem Cells MRI Visualization.

Over the past decade, cell therapy has found many applications in the treatment of different diseases. Some of the cells already used in clinical practice include stem cells and CAR-T cells. Compared with traditional drugs, living cells are much more complicated systems that must be strictly controlled to avoid undesirable migration, differentiation, or proliferation. One of the approaches used to prevent such side effects involves monitoring cell distribution in the human body by any noninvasive technique, such as magnetic resonance imaging (MRI). Long-term tracking of stem cells with artificial magnetic labels, such as magnetic nanoparticles, is quite problematic because such labels can affect the metabolic process and cell viability. Additionally, the concentration of exogenous labels will decrease during cell division, leading to a corresponding decrease in signal intensity. In the current work, we present a new type of genetically encoded label based on encapsulin from Myxococcus xanthus bacteria, stably expressed in human mesenchymal stem cells (MSCs) and coexpressed with ferroxidase as a cargo protein for nanoparticles' synthesis inside encapsulin shells. mZip14 protein was expressed for the enhancement of iron transport into the cell. Together, these three proteins led to the synthesis of iron-containing nanoparticles in mesenchymal stem cells-without affecting cell viability-and increased contrast properties of MSCs in MRI.

In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis.

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a ß-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.

Genetically engineered magnetic nanocages for cancer magneto-catalytic theranostics.

The clinical applications of magnetic hyperthermia therapy (MHT) have been largely hindered by the poor magnetic-to-thermal conversion efficiency of MHT agents. Herein, we develop a facile and efficient strategy for engineering encapsulin-produced magnetic iron oxide nanocomposites (eMIONs) via a green biomineralization procedure. We demonstrate that eMIONs have excellent magnetic saturation and remnant magnetization properties, featuring superior magnetic-to-thermal conversion efficiency with an ultrahigh specific absorption rate of 2390 W/g to overcome the critical issues of MHT. We also show that eMIONs act as a nanozyme and have enhanced catalase-like activity in the presence of an alternative magnetic field, leading to tumor angiogenesis inhibition with a corresponding sharp decrease in the expression of HIF-1α. The inherent excellent magnetic-heat capability, coupled with catalysis-triggered tumor suppression, allows eMIONs to provide an MRI-guided magneto-catalytic combination therapy, which may open up a new avenue for bench-to-bed translational research of MHT.

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus.

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of the BacNOP bactofilins and PadC positions the essential ParB∙parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. We identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential, with Δsmc or ΔscpAB mutants being temperature sensitive. Inactivation of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold also caused chromosome segregation defects but this scaffold is not essential for viability. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems function together to enable chromosome segregation in M. xanthus. ParABS constitutes the basic and essential machinery. SMC and the BacNOP/PadC scaffold have different yet redundant roles in chromosome segregation with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly.

Biosynthesis of Cittilins, Unusual Ribosomally Synthesized and Post-translationally Modified Peptides from Myxococcus xanthus.

Cittilins are secondary metabolites from myxobacteria comprised of three l-tyrosines and one l-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in Streptomyces ssp. We also report first steps toward the biochemical characterization of the proposed biosynthetic pathway in vitro. An investigation of the cellular uptake properties of cittilin A connected it to a potential biological function as an inhibitor of the prokaryotic carbon storage regulator A (CsrA).

Adenosine Triphosphate and Carbon Efficient Route to Second Generation Biofuel Isopentanol.

Climate change necessitates the development of CO2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization.

Biosynthesis and Heterologous Production of Argyrins.

Argyrins represent a family of cyclic octapeptides exhibiting promising antimicrobial, antitumorigenic and immunosuppressant activities. They derive from a nonribosomal peptide synthetase pathway, which was identified and characterized in this study from the myxobacterial producer strain Cystobacter sp. SBCb004. Using the native biosynthetic gene cluster (BGC) sequence as template synthetic BGC versions were designed and assembled from gene synthesis fragments. A heterologous expression system was established after chromosomal deletion of a well-expressed lipopeptide pathway from the host strain Myxococcus xanthus DK1622. Different approaches were applied to engineer and improve heterologous argyrin production, which was finally increased to 160 mg/L, around 20-fold higher yields compared to the native producer. Heterologous production platform also led to identification of several novel argyrin derivatives (A2, F3, G3, I, J, K, and L). The optimized production system provides a versatile platform for future supply of argyrins and novel derivatives thereof.

Regulation of Pom cluster dynamics in Myxococcus xanthus.

Precise positioning of the cell division site is essential for the correct segregation of the genetic material into the two daughter cells. In the bacterium Myxococcus xanthus, the proteins PomX and PomY form a cluster on the chromosome that performs a biased random walk to midcell and positively regulates cell division there. PomZ, an ATPase, is necessary for tethering of the cluster to the nucleoid and regulates its movement towards midcell. It has remained unclear how the cluster dynamics change when the biochemical parameters, such as the attachment rates of PomZ dimers to the nucleoid and the cluster, the ATP hydrolysis rate of PomZ or the mobility of PomZ interacting with the nucleoid and cluster, are varied. To answer these questions, we investigate a one-dimensional model that includes the nucleoid, the Pom cluster and PomZ proteins. We find that a mechanism based on the diffusive PomZ fluxes on the nucleoid into the cluster can explain the latter's midnucleoid localization for a broad parameter range. Furthermore, there is an ATP hydrolysis rate that minimizes the time the cluster needs to reach midnucleoid. If the dynamics of PomZ on the nucleoid is slow relative to the cluster's velocity, we observe oscillatory cluster movements around midnucleoid. To understand midnucleoid localization, we developed a semi-analytical approach that dissects the net movement of the cluster into its components: the difference in PomZ fluxes into the cluster from either side, the force exerted by a single PomZ dimer on the cluster and the effective friction coefficient of the cluster. Importantly, we predict that the Pom cluster oscillates around midnucleoid if the diffusivity of PomZ on the nucleoid is reduced. A similar approach to that applied here may also prove useful for cargo localization in ParABS systems.

Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus.

Myxococcus xanthus generates diadenosine tetraphosphates (Ap4A) and diadenosine pentaphosphates (Ap5A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap4A from ATP, Ap5A from ATP and adenosine tetraphosphate (Ap4), and Ap4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap4A and Ap4 synthesis, 15 M. xanthus aminoacyl-tRNA synthetases (aaRSs), four acyl-CoA synthetases (Acys), three acetyl-CoA synthetases (Aces), phosphoglycerate kinase (Pgk), and adenylate kinase (Adk) were expressed in Escherichia coli and examined for Ap4A or Ap4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap4A synthetase activity. AlaRS, SerRS, and LeuRS1 showed high ADP synthetase activity with ATP as a substrate in the presence of pyrophosphatase, and also demonstrated the ability to produce Ap4 from ATP and triphosphate in the absence of pyrophosphatase. Ap4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap4A, suggesting that these enzymes prefer triphosphate over ATP as a substrate in the second reaction. Some of the recombinant M. xanthus Acys and Aces also synthesized Ap4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap4 from ATP and 3-phosphoglycerate in the presence of Mg2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap4 synthesis in M. xanthus.

Bactofilin-mediated organization of the ParABS chromosome segregation system in Myxococcus xanthus.

In bacteria, homologs of actin, tubulin, and intermediate filament proteins often act in concert with bacteria-specific scaffolding proteins to ensure the proper arrangement of cellular components. Among the bacteria-specific factors are the bactofilins, a widespread family of polymer-forming proteins whose biology is poorly investigated. Here, we study the three bactofilins BacNOP in the rod-shaped bacterium Myxococcus xanthus. We show that BacNOP co-assemble into elongated scaffolds that restrain the ParABS chromosome segregation machinery to the subpolar regions of the cell. The centromere (parS)-binding protein ParB associates with the pole-distal ends of these structures, whereas the DNA partitioning ATPase ParA binds along their entire length, using the newly identified protein PadC (MXAN_4634) as an adapter. The integrity of these complexes is critical for proper nucleoid morphology and chromosome segregation. BacNOP thus mediate a previously unknown mechanism of subcellular organization that recruits proteins to defined sites within the cytoplasm, far off the cell poles.

AibA/AibB Induces an Intramolecular Decarboxylation in Isovalerate Biosynthesis by Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals.

High concentrations of intracellular Ap4A and/or Ap5A in developing Myxococcus xanthus cells inhibit sporulation.

Diadenosine polyphosphates (ApnA) are thought to act as signalling molecules regulating stress responses and biofilm formation in prokaryotes. However, ApnA function in Myxococcus xanthus remains unknown. Here, we investigated the role of ApnA in M. xanthus, using the wild-type and ApnA hydrolase (apaH) mutant strains exposed to various stress conditions. In both wild-type and apaH mutant cells cultured on starvation medium (CF agar), the levels of intracellular diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) increased several fold during the first 16 h of development and decreased gradually thereafter. The levels of Ap4A and Ap5A in the apaH mutant were about 5- and 11-fold higher than those in the wild-type strain at 16 h, respectively. ApnA hydrolase activity of the wild-type strain increased 1.5-fold during the first 8 h of development, and it then gradually decreased. The apaH mutant formed spores 1-2 days after the wild-type strain did, and the yield of viable spores was 5.5 % of that in the wild-type strain 5 days after inoculation onto CF agar. These results suggest the possibility that high intracellular levels of Ap4A and/or Ap5A may inhibit M. xanthus sporulation at the early stage of development and that the bacteria reduce intracellular Ap4A and Ap5A accumulation through ApnA hydrolase activity.

In depth analysis of the mechanism of action of metal-dependent sigma factors: characterization of CorE2 from Myxococcus xanthus.

Extracytoplasmic function sigma factors represent the third pillar of signal-transduction mechanisms in bacteria. The variety of stimuli they recognize and mechanisms of action they use have allowed their classification into more than 50 groups. We have characterized CorE2 from Myxococcus xanthus, which belongs to group ECF44 and upregulates the expression of two genes when it is activated by cadmium and zinc. Sigma factors of this group contain a Cys-rich domain (CRD) at the C terminus which is essential for detecting metals. Point mutations at the six Cys residues of the CRD have revealed the contribution of each residue to CorE2 activity. Some of them are essential, while others are either dispensable or their mutations only slightly affect the activity of the protein. However, importantly, mutation of Cys174 completely shifts the specificity of CorE2 from cadmium to copper, indicating that the Cys arrangement of the CRD determines the metal specificity. Moreover, the conserved CxC motif located between the σ2 domain and the σ4.2 region has also been found to be essential for activity. The results presented here contribute to our understanding of the mechanism of action of metal-dependent sigma factors and help to define new common features of the members of this group of regulators.