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 β-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 β-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 β-1,6-glucans indicates that β-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 β-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents.

Background: Myxobacteria are micropredators in the soil ecosystem with the capacity to move and feed cooperatively. Some myxobacterial strains have been used to control soil-borne fungal phytopathogens. However, interactions among myxobacteria, plant pathogens, and the soil microbiome are largely unexplored. In this study, we aimed to investigate the behaviors of the myxobacterium Corallococcus sp. strain EGB in the soil and its effect on the soil microbiome after inoculation for controlling cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC). Results: A greenhouse and a 2-year field experiment demonstrated that the solid-state fermented strain EGB significantly reduced the cucumber Fusarium wilt by 79.6% (greenhouse), 66.0% (2015, field), and 53.9% (2016, field). Strain EGB adapted to the soil environment well and decreased the abundance of soil-borne FOC efficiently. Spatiotemporal analysis of the soil microbial community showed that strain EGB migrated towards the roots and root exudates of the cucumber plants via chemotaxis. Cooccurrence network analysis of the soil microbiome indicated a decreased modularity and community number but an increased connection number per node after the application of strain EGB. Several predatory bacteria, such as Lysobacter, Microvirga, and Cupriavidus, appearing as hubs or indicators, showed intensive connections with other bacteria. Conclusion: The predatory myxobacterium Corallococcus sp. strain EGB controlled cucumber Fusarium wilt by migrating to the plant root and regulating the soil microbial community. This strain has the potential to be developed as a novel biological control agent of soil-borne Fusarium wilt.

A novel ␣-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaoseforming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The K m and V max of recombinant AmyM for soluble starch were 6.61 mg ml ؊1 and 44,301.5 mol min ؊1 mg ؊1 , respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.

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The gene encoding the novel amylolytic enzyme designated CoMA was cloned from sp. strain EGB. The deduced amino acid sequence contained a predicted lipoprotein signal peptide (residues 1 to 18) and a conserved glycoside hydrolase family 13 (GH13) module. The amino acid sequence of CoMA exhibits low sequence identity (10 to 19%) with cyclodextrin-hydrolyzing enzymes (GH13_20) and is assigned to GH13_36. The most outstanding feature of CoMA is its ability to catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. Moreover, it can hydrolyze γ-cyclodextrin and starch to maltose and hydrolyze pullulan exclusively to panose with relative activities of 0.2, 1, and 0.14, respectively. CoMA showed both hydrolysis and transglycosylation activities toward α-1,4-glycosidic bonds but not to α-1,6-linkages. Moreover, glucosyl transfer was postulated to be the major transglycosidation reaction for producing a high level of maltose without the attendant production of glucose. These results indicated that CoMA possesses some unusual properties that distinguish it from maltogenic amylases and typical α-amylases. Its physicochemical properties suggested that it has potential for commercial development. The α-amylase from sp. EGB, which was classified to the GH13_36 subfamily, can catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. An action mechanism for producing a high level of maltose without the attendant production of glucose has been proposed. Moreover, it also can hydrolyze γ-cyclodextrin and pullulan. Its biochemical characterization suggested that CoMA may be involved the accumulation of maltose in media.

c Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a widely applied herbicide with potential carcinogenic properties. N-Deethoxymethylation is the key step in acetochlor biodegradation. N-Deethoxymethylase is a multicomponent enzyme that catalyzes the conversion of acetochlor to 2=-methyl-6=-ethyl-2-chloroacetanilide (CMEPA). Fast detection of CMEPA by a two-enzyme (N-deethoxymethylase-amide hydrolase) system was established in this research. Based on the fast detection method, a three-component enzyme was purified from Rhodococcus sp. strain T3-1 using ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular masses of the components of the purified enzyme were estimated to be 45, 43, and 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of peptide mass fingerprint analysis, acetochlor N-deethoxymethylase was identified as a cytochrome P450 system, composed of a cytochrome P450 oxygenase (43-kDa component; EthB), a ferredoxin (45 kDa; EthA), and a reductase (11 kDa; EthD), that is involved in the degradation of methyl tert-butyl ether. The gene cluster ethABCD was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). Resting cells of a recombinant E. coli strain showed deethoxymethylation activity against acetochlor. Subcloning of ethABCD showed that ethABD expressed in E. coli BL21(DE3) has the activity of acetochlor N-deethoxymethylase and is capable of converting acetochlor to CMEPA. Chloroacetanilide herbicides are a class of important herbicides used worldwide. The chloroacetanilide herbicide acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a selective preemergent herbicide that has been used to effectively control broadleaf weeds and annual grasses in corn fields for almost 40 years (1). The excessive and frequent application of acetochlor may result in high levels of acetochlor residues, which have been detected in ground and surface waters and negatively impact both the environment and agricultural ecosystems (2). The U.S. Environmental Protection Agency (EPA) has classified acetochlor as a B-2 carcinogen and a probable human carcinogen (3). Studies of different soil types treated with acetochlor have demonstrated that 2=-methyl-6=-ethyl-2-chloroacetanilide (CMEPA), a compound derived from acetochlor N-deethoxymethylation, is a major product of the microbial degradation process (1, 3-5).We previously isolated an efficient acetochlor N-deethoxymethylation strain, T3-1, from a microbial consortium that could mineralize acetochlor completely. The strain was identified as a Rhodococcus sp. (1). The biochemical pathway of acetochlor degradation by the three bacteria in the consortium was proposed to be as follows: acetochlor to CMEPA by Rhodococcus sp. strain T3-1, CMEPA to 2-methyl-6-ethyl aniline (MEA) by Delftia sp. strain T3-6, and MEA by Sphingobium sp. strain MEA3-1, based on the identified degradation intermediates (1). In this study, a three-...

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