Heat-stable Antifungal Factor Production Research Articles

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

A Unique Role of the Pyrimidine de novo Synthesis Enzyme ODCase in Lysobacter enzymogenes1

Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential. Heat-Stable Antifungal Factor (HSAF) secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient antifungal activity. Studying the biosynthetic regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production. In this work, we demonstrate that Le0752, an orotidine-5’-phosphate decarboxylase enzyme (ODCase) catalyzing a pivotal step of the UMP de novo biosynthesis pathway, is vital for HSAF-mediated antimicrobial activities and growth of L. enzymogenes OH11, but not for twitching motility. This gene regulates the production of HSAF by affecting the expression of lafB, a key gene in the HSAF biosynthesis operon, through the transcription factor Clp. Interestingly, bioinformatics analysis revealed that Le0752 belongs to the Group III ODCases, whereas its homologs in the closely related genera Xanthomonas and Stenotrophomonas belong to Group I, which contains most ODCases from Gram-positive bacteria, Gram-negative bacteria and cyanobacteria. Moreover, the Group I ODCase PXO_3614 from the Xanthomonas oryzae pv. oryzae PXO99A strain complemented the Le0752 mutant in regulating HSAF-mediated antagonistic activity. Together, these results highlight the important requirement of de novo pyrimidine biosynthetic enzymes for antibiotic HSAF production in L. enzymogenes, which lays an important foundation for improving HSAF production via metabolic flow design and for dissecting the regulatory functions of bacterial ODCases.

Iron ions regulate antifungal HSAF biosynthesis in Lysobacter enzymogenes by manipulating the DNA-binding affinity of the ferric uptake regulator (Fur).

Heat-stable antifungal factor (HSAF), produced by Lysobacter enzymogenes OH11, is regarded as a potential biological pesticide due to its broad-spectrum antifungal activity and novel mode of action. However, the current production of HSAF is low and cannot meet the requirements for large-scale production. Herein, we discovered that iron ions greatly promoted HSAF production, and the ferric uptake regulator (Fur) was involved in this regulatory process. Fur was also found to participate in the regulation of iron homeostasis in OH11 via the classic inhibition mechanism of Holo-Fur. Furthermore, Fur was collectively observed to directly bind to the promoter of the HSAF biosynthesis gene, and its DNA-binding affinity was attenuated by the addition of iron ions in vitro and in vivo. Its regulatory mechanism followed the uncommon inhibition mechanism of Apo-Fur. In summary, Fur exhibited a bidirectional regulatory mechanism in OH11. This study reveals a novel regulatory mechanism whereby Fur upregulates the biosynthesis of secondary metabolites. These findings contribute to the improvement of HSAF production and may guide its development into biological pesticides. IMPORTANCE HSAF possesses potent and broad antifungal activity with a novel mode of action. The HSAF yield is critical for fermentation production. In this study, iron ions were found to increase HSAF production, and the specific mechanism was elaborated. These results provide theoretical support for genetic transformation to improve HSAF yield, supporting its development into biological pesticides.

The recombination regulator RecX negatively regulates heat-stable antifungal factor (HSAF) biosynthesis in Lysobacter enzymogenes

Bacteria often use multiple transcription factors to regulate specific biological processes. Biosynthesis of heat-stable antifungal factor (HSAF) is regulated by multiple factors in Lysobacter enzymogenes. However, the mechanism of HSAF biosynthesis regulation remains largely unknown. In this study, we screened a potential HSAF biosynthesis regulator, RecX, by a DNA pull-down assay. Deletion of recX resulted in a significant increase in the production of HSAF, and overexpression of recX significantly suppressed HSAF production. Importantly, our results showe that RecX directly binds to the promoter region of the lafB gene to inhibit its transcription and thus decreases HSAF production in L. enzymogenes. These findings reveal the novel mechanism of RecX regulation of antifungal antibiotic production in L. enzymogenes.

Antifungal weapons of Lysobacter, a mighty biocontrol agent.

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.

Outer Membrane Vesicle-Mediated Codelivery of the Antifungal HSAF Metabolites and Lytic Polysaccharide Monooxygenase in the Predatory Lysobacter enzymogenes.

Lysobacter are new biocontrol agents known for their prolific production of lytic enzymes and bioactive metabolites. L. enzymogenes is a predator of fungi and produces several structurally distinct antimicrobial compounds, such as the antifungal HSAF (heat stable antifungal factor) and analogs. The mechanism by which L. enzymogenes interacts with fungal prey is not well understood. Here, we found that the production of HSAF and analogs in L. enzymogenes OH11 was significantly induced in media supplemented with ground fungal mycelia or chitin. In the OH11 genome, we identified a gene (LeLPMO10A) that was annotated to encode a chitin-binding protein. The stimulation of HSAF and analogs by chitin was diminished when LeLPMO10A was deleted. We expressed the gene in E. coli and demonstrated that purified LeLPMO10A oxidatively cleaved chitin into oligomeric products, including 1,5 δ-lactones and aldonic acids. The results revealed that LeLPMO10A encodes a lytic polysaccharide monooxygenase, which has not been reported in Lysobacter. The metabolite analysis, antifungal assay, and proteomic analysis showed that the antifungal compounds and the chitin-cleaving LeLPMO10A are colocalized in outer membrane vesicles. The enzymatic products that resulted from in vitro LeLPMO10A-cleaved chitin also significantly induced HSAF and analogs in OH11. Scanning electron microscopic analysis indicated that spherical vesicles were formed outside of OH11 cells, and fewer OH11 cells were observed to attach to fungal hyphae when LeLPMO10A was deleted. Together, the study revealed a previously uncharacterized synergistic strategy utilized by the predatory Lysobacter during interaction with fungal prey.

Coordinated control of the type IV pili and c-di-GMP-dependent antifungal antibiotic production in Lysobacter by the response regulator PilR.

In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)‐mediated twitching motility as well as synthesis of the heat‐stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c‐di‐GMP; however, the mechanism remained unknown. Here, we describe how PilR, c‐di‐GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c‐di‐GMP synthases) and found that PilR binds to the promoter region of lchD and down‐regulates its transcription. The DNA‐binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c‐di‐GMP‐bound and c‐di‐GMP‐free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS–PilR two‐component system activates T4P‐motility while simultaneously decreasing c‐di‐GMP levels and promoting HSAF production via the highly specific LchD–c‐di‐GMP–Clp pathway. Coordinated increase in motility and secretion of the “long‐distance” antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili‐based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS–PilR system, and highlighted exquisite specificity in c‐di‐GMP‐mediated circuits.

An intrinsic mechanism for coordinated production of the contact-dependent and contact-independent weapon systems in a soil bacterium.

Soil bacteria possess multiple weapons to fend off microbial competitors. Currently, we poorly understand the factors guiding bacterial decisions about weapon systems deployment. In this study, we investigated how such decisions are made by the soil bacterium Lysobacter enzymogenes, used in antifungal plant protection. We found that weapons production is guided by environmental cues. In rich media, which likely mimic environments crowded with other microbes, L. enzymogenes produces a contact-dependent weapon, type six secretion system (T6SS). In nutrient-poor media, likely dominated by filamentous oomycetes and fungi, L. enzymogenes synthesizes and secretes a heat-stable antifungal factor (HSAF), a contact-independent weapon. Surprisingly, the T6SS inner tube protein Hcp is accumulated intracellularly even in nutrient-poor media, when the T6SS is not assembled. We found that Hcp interacts with the transcription factor Clp required for activating HSAF biosynthesis operon expression. Hcp protects Clp from binding to c-di-GMP, an intracellular second messenger inhibiting DNA binding. The increased concentration of c-di-GMP-free Clp thus leads to higher gene expression and HSAF production. Therefore, when the contact-dependent weapon, T6SS, is not in use, accumulation of one of its structural components, Hcp, serves as a signal to enhance production of the contact-independent weapon, HSAF. The uncovered environment-dependent and auto-regulatory mechanisms shed light on the processes governing deployment of various weapon systems in environmental bacteria.

A non-flagellated, predatory soil bacterium reprograms a chemosensory system to control antifungal antibiotic production via cyclic di-GMP signalling.

Lysobacter enzymogenes is a non-flagellated, soil proteobacterium that secretes a diffusible antibiotic known as heat-stable antifungal factor (HSAF) to kill nearby fungi for food. The genome of the model strain OH11 encodes a homologous Wsp system, which is generally deployed by flagellated bacteria to achieve flagella-dependent outputs via a c-di-GMP-FleQ complex, in which c-di-GMP is a ubiquitous dinucleotide second messenger and FleQ is a transcription factor (TF). Here, we show that the Wsp system in the non-flagellated OH11 participates in a unique c-di-GMP-dependent signalling pathway and forms a WspR-CdgL binary complex to alter HSAF production, in which WspR and CdgL act as a c-di-GMP diguanylate cyclase (DGC) and a non-TF binding protein respectively. We found that the phosphorylation of WspR activates its DGC activity and enhances c-di-GMP production while inhibiting HSAF biosynthesis. The phosphorylation of WspR also plays a key role in weakening WspR-CdgL binding and HSAF generation. Interestingly, c-di-GMP binding to CdgL did not seem to induce the disassociation of the WspR-CdgL complex. These observations, along with our earlier findings, lead us to propose a model in which L. enzymogenes re-programs the Wsp system via c-di-GMP signalling to regulate HSAF biosynthesis for the benefit of ecological adaptation.

LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in Lysobacter enzymogenes OH11

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf-orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13–14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent.

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Lysobacter enzymogenes Employs Diverse Genes for Inhibiting Hypha Growth and Spore Germination of Soybean Fungal Pathogens.

Lysobacter enzymogenes strain C3 (LeC3) is a potential biocontrol agent for plant diseases caused by fungi and oomycetes. Understanding the interaction between LeC3 and soybean pathogens at the molecular level could help improve its biocontrol efficacy. In this study, we obtained mutants with decreased abilities in inhibiting hypha growth of the white mold pathogen Sclerotinia sclerotiorum. Insertion sites for 50 mutants, which no longer inhibited S. sclerotiorum hypha growth in dual cultural assay, were determined and seven mutants were selected for further characterization. These seven mutants also completely lost their abilities in suppressing spore germination of Fusarium virguliforme, the causal agent of soybean sudden death syndrome. Furthermore, mutation of the seven genes, which encode diguanylate cyclase, transcriptional regulators from the TetR family, hemolysin III family channel protein, type IV secretion system VirB10 protein, phenol hydroxylase, and phosphoadenosine phosphosulfate reductase, respectively, led to reduced production or secretion of four extracellular enzymes and heat-stable antifungal factor (HSAF). These results suggest that these seven genes play important roles in L. enzymogenes in suppressing hypha growth and spore germination of fungal pathogens, probably by influencing production or secretion of extracellular enzymes and HSAF.

Knockout of Diguanylate Cyclase Genes in Lysobacter enzymogenes to Improve Production of Antifungal Factor and Increase Its Application in Seed Coating.

Heat-stable antifungal factor (HSAF) is a broad-spectrum antifungal antibiotic produced by the biological control agent, Lysobacter enzymogenes. In our earlier works, we have applied HSAF to effectively control wheat and pear fungal disease. However, a major bottleneck in its practical application is the low HSAF production level; therefore, boosting its production is essential for its wide application. In the past, we find that c-di-GMP, a universal bacterial second messenger, is inhibitory to HSAF production. In this work, we further identified eight active diguanylate cyclases (DGCs) responsible for c-di-GMP synthesis in Lysobacter enzymogenes via both bioinformatics and genetic analyses. We generated a strain lacking seven active DGC genes and found that this DGC-modified strain, OH11LC, produced a higher HSAF amount in a c-di-GMP concentration-dependent manner. Subsequently, by employing OH11LC as the host fermentation strain, we could even produce a much higher HSAF amount (> 200-fold). After improving the HSAF production, we further developed a technique of seed coating method with HSAF, which turned out to be effective in fighting against the maize seed-borne filamentous pathogen, Pythium gramineacola. Overall, via combining strain modification and fermentation optimization, we demonstrated a good example of translating fundamental knowledge of bacterial c-di-GMP signaling into biological control application in which we relieved the inhibitory effect of c-di-GMP on HSAF biosynthesis by deleting a bunch of potentially active L. enzymogenes DGC genes to improve HSAF yield and to expand its usage in antifungal seed coating.

Spermidine plays a significant role in stabilizing a master transcription factor Clp to promote antifungal activity in Lysobacter enzymogenes.

Spermidine is a common polyamine compound produced in bacteria, but its roles remain poorly understood. The bacterial SpeD encodes an S-adenosylmethionine decarboxylase that participates in spermidine synthesis. Lysobacter enzymogenes is an efficient environmental predator of crop fungal pathogens by secreting an antifungal antibiotic HSAF (heat-stable antifungal factor), while Clp is a master transcription factor essential for the antifungal activity of L. enzymogenes. In this work, we observed that speD was a close genomic neighbor of the clp gene. This genomic arrangement also seems to occur in many other bacteria, but the underlying reason remains unclear. By using L. enzymogenes OH11 as a working model, we showed that SpeD was involved in spermidine production that was essential for the L. enzymogenes antifungal activity. Spermidine altered the bacterial growth capability and HSAF production, both of which critically contributed to the L. enzymogenes antifungal activity. We further found that spermidine in L. enzymogenes was able to play a crucial, yet indirect role in maintaining the Clp level in vivo, at least partially accounting for its role in the antifungal activity. Thus, our findings suggested that spermidine probably plays an uncharacterized role in maintaining the levels of the master transcription regulator Clp to optimize its role in antifungal activity in an agriculturally beneficial bacterium.

Spermidine-Regulated Biosynthesis of Heat-Stable Antifungal Factor (HSAF) in Lysobacter enzymogenes OH11.

Heat-Stable Antifungal Factor (HSAF) and its analogs are antifungal natural products produced by the biocontrol agent Lysobacter enzymogenes. The production of HSAF is greatly influenced by environmental stimuli and nutrients, but the underlying molecular mechanism is mostly unclear. Here, we found that HSAF production in L. enzymogenes OH11 is strictly controlled by spermidine, which is the most prevalent triamine in bacteria. When added into OH11 cultures, spermidine regulated the production of HSAF and analogs in a concentration-dependent manner. To verify the role of spermidine, we deleted LeSDC and LeADC genes, encoding S-adenosylmethionine decarboxylase and arginine decarboxylase, respectively, that are the key enzymes for spermidine biosynthesis. Both deletion mutants produced barely detectable spermidine and HSAF including its analogs, whereas the antifungals production was restored by exogenous spermidine. The results showed that the OH11 cells must maintain a proper spermidine homeostasis for the antifungals production. Indeed, the expression level of the key HSAF biosynthetic genes was significantly impaired in LeSDC and LeADC mutants, and exogenous spermidine restored the gene expression level in the mutants. Ornithine is a key substrate for HSAF biosynthesis, and OH11 genome contains arg1 and arg2 genes, encoding arginases that convert arginine to ornithine. While the expression of arg1 and arg2 was affected slightly upon mutation of LeSDC and LeADC, exogenous spermidine significantly increased the arginase gene expression in LeSDC and LeADC mutants. Together, the data revealed a previously unrecognized mechanism, in which spermidine controls antibiotic production through controlling both the biosynthetic genes and the substrate-production genes.

Improving the production of a novel antifungal alteramide B in Lysobacter enzymogenes OH11 by strengthening metabolic flux and precursor supply

Lysobacter enzymogenes OH11 is currently considered to be a novel biocontrol agent for various plant fungi diseases. At present, only heat-stable antifungal factor (HSAF) has been isolated and identified in culture, although other active compounds also showed antifungal activity. In the present study, a novel active compound, alteramide B (ATB), which exhibits broad-spectrum antagonistic activity against phytopathogenic fungi and oomycetes, was isolated. The genes responsible for ATB biosynthesis were also determined. In addition, a strain producing ATB with minimal HSAF production was successfully generated by redirecting metabolic flux, namely L. enzymogenes OH57. Furthermore, ATB production increased to 893.32 ± 15.57 mg/L through medium optimization and precursor supply strategy, which was 24.36-fold higher than that of 10% tryptic soy broth (36.67 ± 1.63 mg/L). Taken together, this study indicates ATB has great development value as a biopesticide because of its bioactivity and high production.

Efficient production of heat-stable antifungal factor through integrating statistical optimization with a two-stage temperature control strategy in Lysobacter enzymogenes OH11

BackgroundHeat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has reported on the fermentation process involved, representing huge obstacles for large-scale industrial production.ResultsMedium capacity, culture temperature, and fermentation time were identified as the most significant factors affecting the production of HSAF and employed for further optimization through statistical methods. Based on the analysis of kinetic parameters at different temperatures, a novel two-stage temperature control strategy was developed to improve HSAF production, in which the temperature was increased to 32 °C during the first 12 h and then switched to 26 °C until the end of fermentation. Using this strategy, the maximum HSAF production reached 440.26 ± 16.14 mg L− 1, increased by 9.93% than that of the best results from single-temperature fermentation. Moreover, the fermentation time was shortened from 58 h to 54 h, resulting in the enhancement of HSAF productivity (17.95%) and yield (9.93%).ConclusionsThis study provides a simple and efficient method for producing HSAF that could be feasibly applied to the industrial-scale production of HSAF.

Enhanced heat stable antifungal factor production by Lysobacter enzymogenes OH11 with cheap feedstocks: medium optimization and quantitative determination.

Significance and Impact of the Study: Heat stable antifungal factor (HSAF) exhibits a potent and broad antifungal activity with a novel mode of action. Increased production and reduced cost of raw materials are particularly important for the future production of HSAF, however, no report was involved in these studies. This study aimed to improve the production of HSAF with cheap raw materials through the medium optimization, which would lay the foundation for the application of HSAF in biological control.

Heat-Stable Antifungal Factor (HSAF) Biosynthesis in Lysobacter enzymogenes Is Controlled by the Interplay of Two Transcription Factors and a Diffusible Molecule.

Lysobacter enzymogenes is a Gram-negative, environmentally ubiquitous bacterium that produces a secondary metabolite, called heat-stable antifungal factor (HSAF), as an antifungal factor against plant and animal fungal pathogens. 4-Hydroxybenzoic acid (4-HBA) is a newly identified diffusible factor that regulates HSAF synthesis via L. enzymogenes LysR (LysRLe), an LysR-type transcription factor (TF). Here, to identify additional TFs within the 4-HBA regulatory pathway that control HSAF production, we reanalyzed the LenB2-based transcriptomic data, in which LenB2 is the enzyme responsible for 4-HBA production. This survey led to identification of three TFs (Le4806, Le4969, and Le3904). Of them, LarR (Le4806), a member of the MarR family proteins, was identified as a new TF that participated in the 4-HBA-dependent regulation of HSAF production. Our data show the following: (i) that LarR is a downstream component of the 4-HBA regulatory pathway controlling the HSAF level, while LysRLe is the receptor of 4-HBA; (ii) that 4-HBA and LysRLe have opposite regulatory effects on larR transcription whereby larR transcript is negatively modulated by 4-HBA while LysRLe, in contrast, exerts positive transcriptional regulation by directly binding to the larR promoter without being affected by 4-HBA in vitro; (iii) that LarR, similar to LysRLe, can bind to the promoter of the HSAF biosynthetic gene operon, leading to positive regulation of HSAF production; and (iv) that LarR and LysRLe cannot interact and instead control HSAF biosynthesis independently. These results outline a previously uncharacterized mechanism by which biosynthesis of the antibiotic HSAF in L. enzymogenes is modulated by the interplay of 4-HBA, a diffusible molecule, and two different TFs.IMPORTANCE Bacteria use diverse chemical signaling molecules to regulate a wide range of physiological and cellular processes. 4-HBA is an "old" chemical molecule that is produced by diverse bacterial species, but its regulatory function and working mechanism remain largely unknown. We previously found that 4-HBA in L. enzymogenes could serve as a diffusible factor regulating HSAF synthesis via LysRLe Here, we further identified LarR, an MarR family protein, as a second TF that participates in the 4-HBA-dependent regulation of HSAF biosynthesis. Our results dissected how LarR acts as a protein linker to connect 4-HBA and HSAF synthesis, whereby LarR also has cross talk with LysRLe Thus, our findings not only provide fundamental insight regarding how a diffusible molecule (4-HBA) adopts two different types of TFs for coordinating HSAF biosynthesis but also show the use of applied microbiology to increase the yield of the antibiotic HSAF by modification of the 4-HBA regulatory pathway in L. enzymogenes.

Transcriptional and Antagonistic Responses of Biocontrol Strain Lysobacter enzymogenes OH11 to the Plant Pathogenic Oomycete Pythium aphanidermatum.

Lysobacter enzymogenes is a ubiquitous, beneficial, plant-associated bacterium emerging as a novel biological control agent. It has the potential to become a new source of antimicrobial secondary metabolites such as the Heat-Stable Antifungal Factor (HSAF), which is a broad-spectrum antimycotic with a novel mode of action. However, very little information about how L. enzymogenes detects and responds to fungi or oomycetes has been reported. An in vitro confrontation bioassay between the pathogenic oomycete Pythium aphanidermatum and the biocontrol bacterial strain L. enzymogenes OH11 was used to analyze the transcriptional changes in the bacteria that were induced by the oomycetes. Analysis was performed at three time points of the interaction, starting before inhibition zone formation until inhibition zone formation. A L. enzymogenes OH11 DNA microarray was constructed for the analysis. Microarray analysis indicated that a wide range of genes belonging to 14 diverse functions in L. enzymogenes were affected by P. aphanidermatum as critical antagonistic effects occurred. L. enzymogenes detected and responded to the presence of P. aphanidermatum early, but alteration of gene expression typically occurred after inhibition zone formation. The presence of P. aphanidermatum increased the twitching motility and HSAF production in L. enzymogenes. We also performed a contact interaction between L. enzymogenes and P. aphanidermatum, and found that HSAF played a critical role in the interaction. Our experiments demonstrated that L. enzymogenes displayed transcriptional and antagonistic responses to P. aphanidermatum in order to gain advantages in the competition with this oomycete. This study revealed new insights into the interactions between bacteria and oomycete.