Unlocking the bacterial contact-dependent antibacterial activity to engineer a biocontrol alliance of two species from natural incompatibility to artificial compatibility

Plant growth-promoting rhizobacteria (PGPR) contain various biocontrol bacteria with broad-spectrum antimicrobial activity, and their single species has been extensively applied to control crop diseases. The development of complex biocontrol community by mixing two or more PGPR members together is a promising strategy to enlarge the efficacy and scope of biocontrol. However, an effective method to assess the natural compatibility of PGPR members has not yet been established to date. Here, we developed such a tool by using the bacterial contact-dependent antibacterial activity (CDAA) as a probe. We showed that the CDAA events are common in two-species interactions in the four selected representative PGPRs, represented by the incompatible interaction of Lysobacter enzymogenes strain OH11 (OH11) and Lysobacter antibioticus strain OH13 (OH13). We further showed that the CDAA between OH11 and OH13 is jointly controlled by a contact-dependent killing device, called the type IV secretion system (T4SS). By deleting the respective T4SS synthesis genes, the T4SS in both strains was co-inactivated and this step unlocked their natural CDAA, resulting in an engineered, compatible mutant alliance that co-displayed antibacterial and antifungal activity. Therefore, this study reveals that releasing bacterial CDAA is effective to rationally engineer the biocontrol community.

Diguanylate cyclase and phosphodiesterase interact to maintain the specificity of c-di-GMP signaling in the regulation of antibiotic synthesis in Lysobacter enzymogenes

Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs) and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components deploy various methods to avoid undesired crosstalk to maintain signaling specificity. Synthesis of the antibiotic HSAF ( H eat S table A ntifungal F actor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE LchP and a c-di-GMP effector Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, which participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP dispaly local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. Importance Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. Signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases. DGCs), c-di-GMP degrading enzymes (phosphodiesterases, PDEs), and c-di-GMP effectors. These components deploy various methods to avoid undesired crosstalk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes . We provide direct evidence to show that the DGC and PDE form a complex, and also indirect evidence to argue that they may balance a local c-di-GMP pool to control the antibiotic production. The results represent an important finding regarding the mechanism of a pair of DGC and PDE to control the expression of specific c-di-GMP signaling pathways.

Antimicrobial Compounds Biosynthesis and Biocontrol Mechanisms of Lysobacter enzymogenes

Biocontrol of plant pathogens is considered an environmentally friendly strategy and it is preferred over the use of chemicals which cause environmental pollution. Lysobacter enzymogenes is a bacterium that has been identified as an agriculturally important biocontrol agent. L. enzymogenes possess antagonistic activity against numerous phytopathogens such as fungi, oomycetes, bacteria and nematodes. Its antagonistic activity is conferred by its ability to produce bioactive secondary metabolites such as the Heat-Stable Anti-Fungal Factor (HSAF), Heat-Stable Degrading Metabolite (HSDM) and WAP-8294A2. It can also produce abundant lytic enzymes such as; chitinases, proteases, glucanases and cellulases that can degrade fungal cell walls and therefore inhibit their growth. To design effective biocontrol strategies employing L. enzymogenes, it is important to understand its antagonistic mechanisms. This review crystalizes information on the biosynthesis mechanisms and biocontrol mechanisms of various antimicrobial compounds produced by L. enzymogenes, this information is essential in designing biocontrol strategies against phytopathogens. Further, this review highlights the uncharacterized HSDM and proposes the need for its future characterization, determination of its biosynthetic gene cluster and characterization of its antagonistic mechanisms against various phytopathogens. Also, the mechanism of clp regulation of lytic enzymes biosynthesis needs to be further studied.

Separation of Heat-Stable Antifungal Factor From Lysobacter enzymogenes Fermentation Broth via Photodegradation and Macroporous Resin Adsorption

Heat-stable antifungal factor (HSAF) is produced by the fermentation of Lysobacter enzymogenes, which is known for its broad-spectrum antifungal activity and novel mode of action. However, studies on the separation of HSAF have rarely been reported. Herein, alteramide B (the main byproduct) was removed firstly from the fermentation broth by photodegradation to improve the purity of HSAF. Then, the separation of HSAF via adsorption by macroporous adsorption resins (MARs) was evaluated and NKA resin showed highest static adsorption and desorption performances. After optimizing the static and dynamic adsorption characteristics, the content of HSAF in the purified product increased from 8.67 ± 0.32% (ethyl acetate extraction) to 31.07 ± 1.12% by 3.58-fold. These results suggest that the developed strategy via photodegradation and macroporous resin adsorption is an effective process for the separation of HSAF, and it is also a promising method for the large-scale preparation of HSAF for agricultural applications.

Antifungal polycyclic tetramate macrolactam HSAF is a novel oxidative stress modulator in Lysobacter enzymogenes

Polycyclic tetramate macrolactams (PoTeM) are a fast-growing family of antibiotic natural products found in phylogenetically diverse microorganisms. Surprisingly, none of the PoTeM had been investigated for potential physiological functions in their producers. Here, we used HSAF (heat-stable antifungal factor), an antifungal PoTeM from Lysobacter enzymogenes, as a model to show that PoTeM forms complexes with iron ion, with a Ka of 2.71*106. The in vivo and in vitro data showed formation of 2:1 and 3:1 complexes between HSAF and iron ions, which were confirmed by molecular mechanical and quantum mechanical calculations. HSAF protected DNA from degradation in high concentrations of iron and H2O2 or under UV radiation. HSAF mutants of L. enzymogenes barely survived under oxidative stresses and markedly increased the production of reactive oxygen species (ROS). Exogenous addition of HSAF into the mutants significantly prevented ROS production and rescued the mutants to normal growth under the oxidative stresses. The results reveal that the function of HSAF is to protect the producer microorganism from oxidative damages, rather than as an iron-acquisition siderophore. The characteristic structure of PoTeM, 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. Together, the study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damages to the cells. Importance Polycyclic tetramate macrolactams (PoTeM) are a family of structurally distinct metabolites that have been found in a large number of bacteria. Although PoTeM exhibit diverse therapeutic properties, the physiological function of PoTeM in the producer microorganisms had not been investigated. HSAF from Lysobacter enzymogenes is an antifungal PoTeM that has been subjected to extensive studies for mechanism of biosynthesis, regulation and the antifungal activity. Using HSAF as a model system, we here showed that the characteristic structure of PoTeM, 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. In L. enzymogenes, HSAF functions as a small molecule modulator for oxidative damages caused by iron, H2O2 and UV light. Together, the study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damages to the cells. HSAF represents the first member of the fast growing PoTeM family of microbial metabolites whose potential biological function has been studied.