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.

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.

Unorthodox regulation of the MglA Ras-like GTPase controlling polarity in Myxococcus xanthus.

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns.

The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2.

Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP3 and polyP4), whereas mxPpx2 preferred long-chain polyP60-70 and polyP700-1000. mxPpx2 was activated by 25-50 mM KCl, but mxPpx1 did not significantly depend on K+. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K+, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K+. The exopolyphosphatase activity of mxPpx1 toward polyP3 was inhibited by polyP700-1000 and that of mxPpx2 toward polyP60-70 and polyP700-1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2∆C). Compared to wild-type mxPpx2, mxPpx2∆C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP3 and polyP4 was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30-75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2∆C lost the requirement for K+ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K+ for activation.

Catalytic activity profile of polyP:AMP phosphotransferase from Myxococcus xanthus.

Myxococcus xanthus generates polyphosphates (polyPs) during starvation and forms fruiting bodies through the activity of polyphosphate kinase (Ppk). M. xanthus polyP:AMP phosphotransferase (Pap), a class II Ppk2, catalyzes the transfer of the terminal phosphate from polyP to AMP to yield ADP, but its enzymatic properties have not been investigated in detail. In this study, we found that Pap was activated by Mn2+ or Mg2+ and required higher concentrations of these ions in reactions with longer polyPs to demonstrate maximum activity. The Km of Pap for polyP700-1000 was significantly lower than that for shorter polyPs, but the highest catalytic constant (kcat) was observed for polyP60-70. When Pap was incubated with polyP60-70 and AMP for 3 h, it first generated ADP and then gradually produced ATP, suggesting that M. xanthus Pap also has polyP:ADP phosphotransferase activity similar to that of class III Ppk2 enzymes. During starvation, the specific activity of Pap in M. xanthus was increased by 2.3-2.4-fold at days 1 and 2 of incubation. In addition, recombinant Pap in combination with M. xanthus recombinant enzymes Ppk1 or adenylate kinase (AdkA) could generate ATP from AMP and polyP60-70. These results suggest a functional role of Pap during M. xanthus starvation, when it might act in cooperation with Ppk1 and/or AdkA to produce ATP from AMP, ADP, and polyP.

Mechanism of 3-Methylglutaconyl CoA Decarboxylase AibA/AibB: Pericyclic Reaction versus Direct Decarboxylation.

The enzyme 3-methylglutaconyl coenzyme A (CoA) decarboxylase (called AibA/AibB) catalyzes the decarboxylation of 3-methylglutaconyl CoA to generate 3,3-dimethylacrylyl-CoA, representing an important step in the biosynthesis of isovaleryl-coenzyme A in Myxococcus xanthus when the regular pathway is blocked. A novel mechanism involving a pericyclic transition state has previously been proposed for this enzyme, making AibA/AibB unique among decarboxylases. Herein, density functional calculations are used to examine the energetic feasibility of this mechanism. It is shown that the intramolecular pericyclic reaction is associated with a very high energy barrier that is similar to the barrier of the same reaction in the absence of the enzyme. Instead, the calculations show that a direct decarboxylation mechanism has feasible energy barriers that are in line with the experimental observations.

Bioconversion of arachidonic acid into human 14,15-hepoxilin B3 and 13,14,15-trioxilin B3 by recombinant cells expressing microbial 15-lipoxygenase without and with epoxide hydrolase.

OBJECTIVE: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B3, 14,15-HXB3) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B3, 13,14,15-TrXB3) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively. RESULTS: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB3 and 13,14,15-TrXB3, respectively. Under the optimal conditions of 30 g cells L-1, 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB3 and 100 mM 13,14,15-TrXB3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h-1, respectively. CONCLUSION: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

Identification of the Wzx flippase, Wzy polymerase and sugar-modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus.

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.

Enzymatic characteristics of Nudix hydrolase 2 (Nud2), an 8-oxo-dGTP hydrolase from Myxococcus xanthus.

Myxococcus xanthus Nudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8-oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn2+ was the most effective co-factor for stimulating oxidized deoxynucleotide hydrolase activity. The Km of Nud2 with 8-oxo-dGTP for Mn2+ was 19-fold lower than that for Mg2+, and was 2-fold lower than that with dGTP for Mn2+. The specificity constant (kcat/Km) for 8-oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif (84AX590GX7REX2EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.

Second messengers and divergent HD-GYP phosphodiesterases regulate 3',3'-cGAMP signaling.

3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.

Enzymatic Characteristics of a Polyphosphate/ATP-NAD Kinase, PanK, from Myxococcus xanthus.

NAD kinase is a crucial enzyme for production of NADP+. Myxococcus xanthus is a gram-negative soil bacterium that forms fruiting bodies and spores under starvation, and it accumulates polyphosphate (poly(P)) during early development. We found that M. xanthus NAD kinase (PanK) utilized both ATP and poly(P) as phosphoryl donors; therefore, PanK was designated as a poly(P)/ATP-NAD kinase. Unlike other poly(P)/ATP-NAD kinases, PanK hardly exhibited NADH kinase activity. The NAD kinase activity of PanK was inhibited by NADPH, but not NADH. Replacement of Thr-90 in the GGDGT motif of PanK with Asn decreased both ATP- and poly(P)-dependent NAD kinase activities; however, poly(P)-dependent NAD kinase activity was further decreased by approximately 6- to 10-fold compared with ATP-dependent NAD kinase activity, suggesting that Thr-90 in the GGDGT motif of PanK may be important for poly(P) utilization. PanK preferred ATP and short-chain poly(P) as phosphoryl donors. The Km of PanK for ATP, poly(P)4, and poly(P)10-15 was 0.66 mM, 0.08 mM, and 0.71 mM, respectively, and the catalytic efficiency (kcat/Km) for poly(P)4 was 2.4-fold higher than that for ATP, suggesting that M. xanthus under starvation conditions may be able to efficiently generate NADP+ using PanK, ATP, and poly(P).

A bacterial light response reveals an orphan desaturase for human plasmalogen synthesis.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond. These lipids are found in animals and some bacteria and have proposed membrane organization, signaling, and antioxidant roles. We discovered the plasmanylethanolamine desaturase activity that is essential for vinyl ether bond formation in a bacterial enzyme, CarF, which is a homolog of the human enzyme TMEM189. CarF mediates light-induced carotenogenesis in Myxococcus xanthus, and plasmalogens participate in sensing photooxidative stress through singlet oxygen. TMEM189 and other animal homologs could functionally replace CarF in M. xanthus, and knockout of TMEM189 in a human cell line eliminated plasmalogens. Discovery of the human plasmanylethanolamine desaturase will spur further study of plasmalogen biogenesis, functions, and roles in disease.

Allosteric regulation of a prokaryotic small Ras-like GTPase contributes to cell polarity oscillations in bacterial motility.

Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases.

A novel outer membrane ß-1,6-glucanase is deployed in the predation of fungi by myxobacteria.

Myxobacterial predation on bacteria has been investigated for several decades. However, their predation on fungi has received less attention. Here, we show that a novel outer membrane ß-1,6-glucanase GluM from Corallococcus sp. strain EGB is essential for initial sensing and efficient decomposition of fungi during predation. GluM belongs to an unstudied family of outer membrane ß-barrel proteins with potent specific activity up to 24,000 U/mg, whose homologs extensively exist in myxobacteria. GluM was able to digest fungal cell walls efficiently and restrict Magnaporthe oryzae infection of rice plants. Genetic complementation with gluM restored the fungal predation ability of Myxococcus xanthus CL1001, which was abolished by the disruption of gluM homolog oar. The inability to prey on fungi with cell walls that lack ß-1,6-glucans indicates that ß-1,6-glucans are targeted by GluM. Our results demonstrate that GluM confers myxobacteria with the ability to feed on fungi, and provide new insights for understanding predator-prey interactions. Considering the attack mode of GluM, we suggest that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents.

Microbial Synthesis of Linoleate 9 S-Lipoxygenase Derived Plant C18 Oxylipins from C18 Polyunsaturated Fatty Acids.

Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9 S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy- cis, cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy- cis, cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy- cis, cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.

Enzymatic characteristics of two adenylate kinases, AdkA and AdkB, from Myxococcus xanthus.

Adenylate kinase (Adk) plays a critical role in energy metabolism and adaptation of bacteria to environmental stresses. We have previously shown that Myxococcus xanthus expresses polyphosphate kinase 1 (Ppk1) that also has Adk activity in the absence of polyphosphates. In this study, we investigated the Adk activity of the other two M. xanthus enzymes, AdkA and AdkB. The activity of AdkA was increased by dithiothreitol (DTT), which also enhanced enzyme stability. Site-directed mutagenesis of three cysteine residues (C130, C150, and C153) present in the LID domain of AdkA revealed that the Adk activity and stability of C150S and C153S mutants were not affected by DTT addition, suggesting formation of a disulfide bond between C150 and C153 in AdkA. The Km of AdkA for AMP was 8 and 17 times lower than that for ADP and ATP, respectively. AdkB is a polyphosphate kinase 2 (Ppk2) homolog lacking the Ppk2 middle region and, consequently, Ppk activity. According to our analysis, AdkB also had Adk activity and its affinity for substrates was higher than that of AdkA. Thus, M. xanthus expresses three enzymes, AdkA, AdkB, and Ppk1, with Adk activity, which may function to support energy metabolism of the bacteria in different environmental conditions.

Stabilization and improved activity of arachidonate 11S-lipoxygenase from proteobacterium Myxococcus xanthus.

Lipoxygenases (LOXs) catalyze the dioxygenation of PUFAs to produce regio- and stereospecific oxygenated fatty acids. The identification of regio- and stereospecific LOXs is important because their specific products are involved in different physiological activities in various organisms. Bacterial LOXs are found only in some proteobacteria and cyanobacteria, and they are not stable in vitro. Here, we used C20 and C22 PUFAs such as arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid to identify an 11S-specific LOX from the proteobacterium Myxococcus xanthus and explore its in vitro stability and activity. The activity and stability of M. xanthus ARA 11S-LOX as well as the production of 11S-hydroxyeicosatetraenoic acid from ARA were significantly increased by the addition of phosphatidylcholine, Ca2+, and coactosin-like protein (newly identified in the yeast Rhodosporidium toluroides) as stimulatory factors; in fact, LOX activity in the presence of all three factors increased approximately 3-fold. Our results indicate that these stimulatory factors can be used to increase the activity and stability of bacterial LOX and the production of bioactive hydroxy fatty acids, which can contribute to new academic research.

An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622.

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs.IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.

A nuclease-toxin and immunity system for kin discrimination in Myxococcus xanthus.

The use of toxin to attack neighbours and immunity proteins to protect against toxin has been observed in bacterial conflicts, including kin discrimination. Here, we report a novel nuclease-toxin and its immunity protein function in the colony-merger incompatibility, a kind of bacterial kin discrimination, in Myxococcus xanthus DK1622. The MXAN_0049 gene was determined to be a genetic determinant for colony-merger incompatibility, and the incompatibility could be eliminated by deletion of the upstream co-transcribed MXAN_0050 gene. We demonstrated that the MXAN_0050 protein was a nuclease, and MXAN_0049 protein was able to bind to MXAN_0050 to block nuclease activity in vitro. Expression of MXAN_0050 in Escherichia coli inhibited cellular growth, and the inhibition effect could be recovered by co-expression of MXAN_0049. We found that deletion of the PAAR-encoding gene (MXAN_0044) or the type VI secretion system led to the colony-merger and co-existence with the ΔMXAN_0049 mutant, suggesting that they were associated with colony-merger incompatibility. Homologues of the nuclease-toxin and cognate immunity pair are widely distributed in bacteria. We propose a simplified model to explain the kin discrimination mechanism mediated by the nuclease-toxin and immunity protein.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.