A screen for antifungal compounds from Lysobacter enzymogenes strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.Heat-stable antifungal factor (HSAF) is a secondary metabolite produced by the bacterium Lysobacter enzymogenes strain C3 (originally called Stenotrophomonas maltophilia strain C3 [24]), a biological control agent originally isolated from grass foliage (7). Strain C3 was demonstrated in the field to reduce diseases caused by multiple fungal pathogens, including Bipolaris sorokiniana (28), Fusarium graminearum (26), Rhizoctonia solani (7), and Uromyces appendiculatus (27). It was also found to be effective in inhibiting the soilborne pathogens Pythium ultimum (12) and Magnaporthe poae (13) in greenhouse experiments. A search for antifungal factors in strain C3 led to the isolation of HSAF, which exhibits strong activity against a wide range of fungi (15).The mechanism by which HSAF inhibits the growth of Aspergillus nidulans has been studied (16). HSAF disrupts the polarized growth of the fungus. Genetic analysis of A. nidulans mutants suggests that HSAF targets the biosynthesis of sphingolipids (16), which are ubiquitous components of eukaryotic cell membranes and signaling molecules involved in numerous cellular processes. Interestingly, HSAF appears to target a distinct group of sphingolipids that are required for polarized growth of filamentous fungi and appears to be absent from mammals and plants. Th...

In filamentous fungi, the stabilization of a polarity axis is likely to be a pivotal event underlying the emergence of a germ tube from a germinating spore. Recent results implicate the polarisome in this process and also suggest that it requires localized membrane organization. Here, we employ a chemical genetic approach to demonstrate that ceramide synthesis is necessary for the formation of a stable polarity axis in the model fungus Aspergillus nidulans. We demonstrate that a novel compound (HSAF) produced by a bacterial biocontrol agent disrupts polarized growth and leads to loss of membrane organization and formin localization at hyphal tips. We show that BarA, a putative acyl-CoA-dependent ceramide synthase that is unique to filamentous fungi mediates the effects of HSAF. Moreover, A. nidulans possesses a second likely ceramide synthase that is essential and also regulates hyphal morphogenesis. Our results suggest that filamentous fungi possess distinct pools of ceramide that make independent contributions to polarized hyphal growth, perhaps through the formation of specialized lipid microdomains that regulate organization of the cytoskeleton.

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

Lysobacter enzymogenes strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and to evaluate their biocontrol efficacy. A single mini-Tn5-lacZ 1 -cat transposon mutant of L. enzymogenes strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a clp gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related N-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with clp. Chromosomal deletion mutants with mutations in clp and between clp and the acetyltransferase gene verified the 5E4 mutant phenotype. The clp gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain. clp mutant strains were significantly affected in biological control of pythium damping-off of sugar beet and bipolaris leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that clp is a global regulatory gene that controls biocontrol traits expressed by L. enzymogenes C3.

Lysobacter species are emerging as new sources of antibiotics. The regulation of these antibiotics is not well understood. Here, we identified a small molecule metabolite (LeDSF3) that regulates the biosynthesis of the antifungal antibiotic HSAF (heat-stable antifungal factor), a polycyclic tetramate macrolactam with a structure and mode of action distinct from the existing antifungal drugs. LeDSF3 was isolated from the culture broth of L. enzymogenes, and its chemical structure was established by NMR and MS. The purified compound induced green fluorescence in a reporter strain of Xanthomonas campestris, which contained gfp gene under the control of a DSF (diffusible signaling factor)-inducible promoter. Exogenous addition of LeDSF3 in L. enzymogenes cultures significantly increased the HSAF yield, the transcription of HSAF biosynthetic genes, and the antifungal activity of the organism. The LeDSF3-regulated HSAF production is dependent on the two-component regulatory system RpfC/RpfG. Moreover, LeDSF3 up-regulated the expression of the global regulator Clp (cAMP receptor-like protein). The disruption of clp led to no HSAF production. Together, the results show that LeDSF3 is a fatty acid-derived, diffusible signaling factor positively regulating HSAF biosynthesis and that the signaling is mediated by the RfpC/RpfG-Clp pathway. These findings may facilitate the antibiotic production through applied genetics and molecular biotechnology in Lysobacter, a group of ubiquitous yet underexplored microorganisms.