Summary: Bacteria interact with fungi in a variety of ways to inhibit fungal growth, while the underlying mechanisms remain only partially characterized. The plant‐beneficial Bacillus and Pseudomonas species are well‐known antifungal biocontrol agents, whereas Lysobacter are far less studied. Members of Lysobacter are easy to grow in fermenters and are safe to humans, animals and plants. These environmentally ubiquitous bacteria use a diverse arsenal of weapons to prey on other microorganisms, including fungi and oomycetes. The small molecular toxins secreted by Lysobacter represent long‐range weapons effective against filamentous fungi. The secreted hydrolytic enzymes act as intermediate‐range weapons against non‐filamentous fungi. The contact‐dependent killing devices are proposed to work as short‐range weapons. We describe here the structure, biosynthetic pathway, action mode and applications of one of the best‐characterized long‐range weapons, the heat‐stable antifungal factor (HSAF) produced by Lysobacter enzymogenes . We discuss how the flagellar type III secretion system has evolved into an enzyme secretion machine for the intermediate‐range antifungal weapons. We highlight an intricate mechanism coordinating the production of the long‐range weapon, HSAF and the proposed contact‐dependent killing device, type VI secretion system. We also overview the regulatory mechanisms of HSAF production involving specific transcription factors and the bacterial second messenger c‐di‐GMP.

Enzymes controlling intracellular second messengers in bacteria, such as c-di-GMP, often affect some but not other targets. How such specificity is achieved is understood only partially. Here, we present a novel mechanism that enables specific c-di-GMP-dependent inhibition of the antifungal antibiotic production. Expression of the biosynthesis operon for Heat-Stable Antifungal Factor, HSAF, in Lysobacter enzymogenes occurs when the transcription activator Clp binds to two upstream sites. At high c-di-GMP levels, Clp binding to the lower-affinity site is compromised, which is sufficient to decrease gene expression. We identified a weak c-di-GMP phosphodiesterase, LchP, that plays a disproportionately high role in HSAF synthesis due to its ability to bind Clp. Further, Clp binding stimulates phosphodiesterase activity of LchP. An observation of a signaling complex formed by a c-di-GMP phosphodiesterase and a c-di-GMP-binding transcription factor lends support to the emerging paradigm that such signaling complexes are common in bacteria, and that bacteria and eukaryotes employ similar solutions to the specificity problem in second messenger-based signaling systems.

Lysobacter enzymogenes is a bacterial biological control agent emerging as a new source of antibiotic metabolites, such as heat-stable antifungal factor (HSAF) and the antibacterial factor WAP-8294A2. The regulatory mechanism(s) for antibiotic metabolite biosynthesis remains largely unknown in L. enzymogenes. Clp, a cyclic adenosine monophosphate (cAMP)-receptor-like protein, is shown to function as a global regulator in modulating biocontrol-associated traits in L. enzymogenes. However, the genetic basis of Clp signaling remains unclear. Here, we utilized transcriptome/microarray analysis to determine the Clp regulon in L. enzymogenes. We showed that Clp is a global regulator in gene expression, as the transcription of 775 genes belonging to 19 functional groups was differentially controlled by Clp signaling. Analysis of the Clp regulon detected previously characterized Clp-modulated functions as well as novel loci. These include novel loci involved in antibiotic metabolite biosynthesis and surface motility in L. enzymogenes. We further showed experimentally that Clp signaling played a positive role in regulating the biosynthesis of HSAF and WAP-8294A2, as well as surface motility which is a type-IV-pilus-dependent trait. The regulation by Clp signaling of antibiotic (HSAF and WAP-8294A2) biosynthesis and surface motility was found to be independent. Importantly, we identified a factor Lysobacter acetyltransferase (Lat), a homologue of histone acetyltransferase Hpa2, which was regulated by Clp and involved in HSAF biosynthesis, but not associated with WAP-8294A2 production and surface motility. Overall, our study provided new insights into the regulatory role and molecular mechanism of Clp signaling in L. enzymogenes.

Summary: Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell–cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact‐dependent, inter‐bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L . enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter‐bacterial killing system. Generation of knock‐out mutants revealed that L . enzymogenes OH11 uses T4SS as the main contact‐dependent weapon against other soilborne bacteria. The T4SS‐mediated killing behaviour of L . enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum . Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L . enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.

Vegetable oils are not only major components of human diet but also vital for industrial applications. WRINKLED1 (WRI1) is a pivotal transcription factor governing plant oil biosynthesis, but the underlying DNA-binding mechanism remains incompletely understood. Here, we resolved the structure of Arabidopsis WRI1 (AtWRI1) with its cognate double-stranded DNA (dsDNA), revealing two antiparallel β sheets in the tandem AP2 domains that intercalate into the adjacent major grooves of dsDNA to determine the sequence recognition specificity. We showed that AtWRI1 represented a previously unidentified structural fold and DNA-binding mode. Mutations of the key residues interacting with DNA element affected its binding affinity and oil biosynthesis when these variants were transiently expressed in tobacco leaves. Seed oil content was enhanced in stable transgenic wri1-1 expressing an AtWRI1 variant (W74R). Together, our findings offer a structural basis explaining WRI1 recognition and binding of DNA and suggest an alternative strategy to increase oil yield in crops through WRI1 bioengineering.