Myxobacteria restrain Phytophthora invasion by scavenging thiamine in soybean rhizosphere via outer membrane vesicle-secreted thiaminase I.

Public metabolites such as vitamins play critical roles in maintaining the ecological functions of microbial community. However, the biochemical and physiological bases for fine-tuning of public metabolites in the microbiome remain poorly understood. Here, we examine the interactions between myxobacteria and Phytophthora sojae, an oomycete pathogen of soybean. We find that host plant and soil microbes complement P. sojae's auxotrophy for thiamine. Whereas, myxobacteria inhibits Phytophthora growth by a thiaminase I CcThi1 secreted into extracellular environment via outer membrane vesicles (OMVs). CcThi1 scavenges the required thiamine and thus arrests the thiamine sharing behavior of P. sojae from the supplier, which interferes with amino acid metabolism and expression of pathogenic effectors, probably leading to impairment of P. sojae growth and pathogenicity. Moreover, myxobacteria and CcThi1 are highly effective in regulating the thiamine levels in soil, which is correlated with the incidence of soybean Phytophthora root rot. Our findings unravel a novel ecological tactic employed by myxobacteria to maintain the interspecific equilibrium in soil microbial community.

Insights into the Antifungal Properties of Myxobacteria Outer Membrane ß-1,6-Glucanase.

Fungal cell wall decomposition enzymes exhibit great potential for the development of efficient antifungal agents. However, their practical application is restricted due to incomplete understanding of the action mode. In our previous study, we identified that a novel outer membrane (OM) ß-1,6-glucanase GluM is deployed by predatory myxobacteria to feed on fungi. In this work, we provide deep insights into the antifungal mechanism of ß-1,6-glucanase and its potential in improving plant disease resistance. The fungal cell wall decomposition ability of GluM resulted in irregular hyphae morphology, changed chitin distribution, increased membrane permeability, and leakage of cell constituents in Magnaporthe oryzae Guy11. Under the attack pattern, the cell wall integrity pathway was activated by strain Guy11 for self-protection. GluM exhibited a distinct endo-model toward fungal cell wall; the favorite substrate of GluM toward fungal ß-1,6-glucan may give reason for its efficient antifungal activity compared with Trichoderma ß-1,6-glucanase. Moreover, released glucans from GluM hydrolysis of fungal cell wall functioned as an elicitor and induced rice immunity by means of jasmonic acid pathway. Based on the dual roles of antifungal properties, gluM transgenic plants conferred enhanced resistance against fungal infection.

Predation of oomycetes by myxobacteria via a specialized CAZyme system arising from adaptive evolution.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

Myxobacterial Outer Membrane ß-1,6-Glucanase Induced the Cell Death of Fusarium oxysporum by Destroying the Cell Wall Integrity.

The ß-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and ß-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The ß-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 µg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of ß-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to ß-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and ß-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to ß-1,6-glucanase GluM treatment (0.08 µg/mL) than ß-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the ß-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the ß-1,3-glucan.

Preparation of chitooligosaccharides with a low degree of polymerization and anti-microbial properties using the novel chitosanase AqCsn1.

Chitosanases hydrolyze chitosan into chitooligosaccharides (COSs) with various biological activities, which are widely employed in many areas including plant disease management. In this study, the novel chitosanase AqCsn1 belonging to the glycoside hydrolase family 46 (GH46) was cloned from Aquabacterium sp. A7-Y and heterologously expressed in Escherichia coli BL21 (DE3). AqCsn1 displayed the highest hydrolytic activity towards chitosan with 95% degree of deacetylation at 40 °C and pH 5.0, with a specific activity of 13.18 U/mg. Product analysis showed that AqCsn1 hydrolyzed chitosan into (GlcN)2 and (GlcN)3 as the main products, demonstrating an endo-type cleavage pattern. Evaluation of antagonistic activity showed that the hydrolysis products of AqCsn1 suppress the mycelial growth of Magnaporthe oryzae and Phytophthora sojae in a concentration-dependent manner, and the inhibition rate of P. sojae reached 39.82% at a concentration of 8 g/L. Our study demonstrates that AqCsn1 and hydrolysis products with a low degree of polymerization might have potential applications in the biological control of agricultural diseases.

Glycogen Branching Enzyme with a Novel Chain Transfer Mode Derived from Corallococcus sp. Strain EGB and Its Potential Applications.

Dietary starch with an increased content of resistant starch (RS) has the potential to reduce the prevalence of diabetes, obesity, and cardiovascular diseases. Here, an efficient glycogen branching enzyme, CcGBE, from Corallococcus sp. strain EGB was identified, and its relevant properties, including potential application in the preparation of modified starch, were evaluated. The purified CcGBE exhibited a maximal specific activity of approximately 20,000 U/mg using cassava starch as the optimal substrate. The content of α-1,6-glucosidic bonds in CcGBE-modified cassava starch increased from 2.9 to 13.2%. Meanwhile, both the average chain length (CL) of CcGBE-modified starch and the blue value of the color complex formed by starch and iodine initially increased and then decreased, indicating that a new CL transfer mode was reported. Perforated small starch granules were released after CcGBE treatment, and a time-dependent decrease in the retrogradation enthalpy (ΔHr) of cassava starch indicated that CcGBE inhibited the long-term retrogradation of starch. Moreover, the RS content and cold water solubility (CWS) of CcGBE-modified starch increased from 3.3 to 12.8% and from 23.1 to 93.8%, respectively. These findings indicate the application potential of CcGBE for the preparation of modified starch with increased RS and CWS.

Yeast ß-1,3-glucan production by an outer membrane ß-1,6-glucanase: process optimization, structural characterization and immunomodulatory activity.

The ß-glucan from Saccharomyces cerevisiae is a potent adjuvant that exhibits a broad spectrum of biological activities and health benefits, and different processes have been established to prepare active ß-glucan from yeast. However, studies concerning the effect of ß-1,6-glucanase enzymolysis on the structure and immunomodulatory activity of yeast ß-1,3-glucan are scarce. In this study, we aim to develop a novel enzymatic process for the preparation of immunologically active ß-glucan (BYG) from baker's yeast using a ß-1,6-glucanase GluM. The ß-1,6-glucan in fungal cell wall was specifically hydrolyzed by GluM, and resulted in cell wall decomposition and ß-glucan release. Batch production of BYG was realized with 17.8% yield, 85.3% purity and 75.4% recovery rate. Structural characterization indicated that BYG exhibits rod-like structures with natural triplex and nanoparticle-like substructures compared with the commercial Glucan 300. BYG ameliorated inflammation in a DSS-induced mouse model of colitis through inhibiting oxidative stress (NO, MDA and MPO), inflammatory mediators (NLRP3, ASC, caspase-1, iNOS and COX-2), and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, IFN-γ), increasing the expression levels of tight junction proteins (ZO-1, occludin and claudin-1) and modulating the production of gut microbiota-synthesized SCFAs compared to the control. Our results showed that yeast ß-1,3-glucan prepared with ß-1,6-glucanase exhibits structural integrity that is responsible for its favorable immunomodulatory activity.

Phenylalanine 314 is essential for the activity of maltogenic amylase from Corallococcus sp. EGB.

Maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the hydrolysis and transglycosylation of maltooligosaccharides and soluble starch into maltose, the sole hydrolysate. This process yields pure maltose with potentially wide applications. Here, we identified and evaluated the role of phenylalanine 314 (F314), a key amino acid located near the active center, in the catalytic activities of the CoMA. Site-directed mutagenesis analysis showed that the activity of a F314L mutant on potato starch substrate decreased to 26% of that of wild-type protein. Compared with the wild-type, F314L exhibited similar substrate specificity, hydrolysis pattern, pH, and temperature requirements. Circular dichroism spectrum data showed that the F314L mutation did not affect the structure of the folded protein. In addition, kinetic analysis demonstrated that F314L exhibited an increased Km value with lower substrate affinity. Homology modeling showed that the benzene ring structure of F314L was involved in π-π conjugation, which might potentially affect the affinity of CoMA toward starch. Taken together, these data demonstrated that F314 is essential for the hydrolytic activity of the CoMA from Corallococcus sp. strain EGB.

Whole-Genome Sequencing of Corallococcus sp. Strain EGB Reveals the Genetic Determinants Linking Taxonomy and Predatory Behavior.

Corallococcus sp. strain EGB is a Gram-negative myxobacteria isolated from saline soil, and has considerable potential for the biocontrol of phytopathogenic fungi. However, the detailed mechanisms related to development and predatory behavior are unclear. To obtain a comprehensive overview of genetic features, the genome of strain EGB was sequenced, annotated, and compared with 10 other Corallococcus species. The strain EGB genome was assembled as a single circular chromosome of 9.4 Mb with 7916 coding genes. Phylogenomics analysis showed that strain EGB was most closely related to Corallococcus interemptor AB047A, and it was inferred to be a novel species within the Corallococcus genus. Comparative genomic analysis revealed that the pan-genome of Corallococcus genus was large and open. Only a small proportion of genes were specific to strain EGB, and most of them were annotated as hypothetical proteins. Subsequent analyses showed that strain EGB produced abundant extracellular enzymes such as chitinases and ß-(1,3)-glucanases, and proteases to degrade the cell-wall components of phytopathogenic fungi. In addition, 35 biosynthetic gene clusters potentially coding for antimicrobial compounds were identified in the strain EGB, and the majority of them were present in the dispensable pan-genome with unexplored metabolites. Other genes related to secretion and regulation were also explored for strain EGB. This study opens new perspectives in the greater understanding of the predatory behavior of strain EGB, and facilitates a potential application in the biocontrol of fungal plant diseases in the future.

Structural and digestion properties of potato starch modified using an efficient starch branching enzyme AqGBE.

Modified potato starch with slower digestion may aid the development of new starch derivatives with improved nutritional values, and strategies to increase nutritional fractions such as resistant starch (RS) are desired. In this study, a correspondence between starch structure and enzymatic resistance was provided based on the efficient branching enzyme AqGBE, and modified starches with different amylose content (Control, 100%; PS1, 90%; PS2, 72%; PS3, 32%; PS4, 18%) were prepared. Through SEM observation, NMR and X-ray diffraction analyses, we identified that an increased proportion of α-1,6-linked branches in potato starch changes its state of granule into large pieces with crystallinity. Molecular weight and chain-length distribution analysis showed a decrease of molecular weight (from 1.1 × 106 to 1.1 × 105 g/mol) without an obvious change of chain-length distribution in PS1, while PS2-4 exhibited an increased proportion of DP 6-12 with a stable molecular weight distribution, indicating a distinct model of structural modification by AqGBE. The enhancement of peak viscosity was related to increased hydrophobic interactions and pieces state of PS1, while the contents of SDS and RS in PS1 increased by 37.7 and 49.4%, respectively. Our result provides an alternative way to increase the RS content of potato starch by branching modification.

Preparation of Active Chitooligosaccharides with a Novel Chitosanase AqCoA and Their Application in Fungal Disease Protection.

Enzymes that degrade fungal cell walls and the resulting oligosaccharides are promising weapons to combat plant fungal disease. In this study, we identified a novel endo-chitosanase, AqCoA, from Aquabacterium sp. A7-Y. The enzyme showed a specific activity of 18 U/mg toward 95% deacetylated chitosan at pH 5.0 and 40 °C. AqCoA also showed activity toward sodium carboxymethylcellulose, indicating substrate promiscuity. AqCoA hydrolyzed chitosan into chitooligosaccharides (CoA-COSs) with degrees of polymerization (DPs) of 3-5 but showed no activity toward CoA-COSs with DPs <6, indicating an endo-type activity. At 2.5 µg/mL, AqCoA inhibited appressorium formation of Magnaporthe oryzae; the produced CoA-COSs also inhibited the growth of M. oryzae and Fusarium oxysporum. Furthermore, CoA-COSs acted as immune elicitors in rice by inducing the reactive oxygen species burst and the expression of defense genes. These results demonstrated that AqCoA and its resulting CoA-COSs might be effective tools for protecting plants against pathogenic fungi.

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.

Identification of an endo-chitinase from Corallococcus sp. EGB and evaluation of its antifungal properties.

As the main component of the fungal cell wall, chitin has been regarded as an optimal molecular target for the biocontrol of plant-pathogenic fungi. In this study, the chitin hydrolase CcCti1, which belongs to the glycoside hydrolase family 18 (GH 18) and exhibits potential antifungal activity, was identified from Corallococcus sp. EGB. CcCti1 lacks a fibronectin type-III (FN3) domain that is present in similar enzymes from most genera of myxobacteria, indicating that CcCti1 may have acquired chitinase activity due to the FN3 domain deletion during myxobacterial evolution. CcCti1 was expressed in Escherichia coli BL21 (DE3) with a specific activity of up to 10.5 U/µmol with colloidal chitin as the substrate. Product analysis showed that CcCti1 could hydrolyze chitin into N-acetylated chitohexaose (GlcNAc)6 as the major product, in addition to chitooligosaccharides. The analysis of biochemical properties indicated that the CBD and FN3 domains in CcCti1 determine the substrate affinity and pH stability. Otherwise, CcCti1 exhibited efficient biocontrol activity against the plant pathogen Magnaporthe oryzae in a dose-dependent manner, inhibiting the conidia germination and appressoria formation at a concentration of 0.08 mg/mL. Overall, the chitohexaose-producing chitinase CcCti1 with hydrolytic features may find potential application in chitin conversion and biocontrol of fungal plant diseases.