| Lysobacter enzymogenes OH11, isolated from rhizosphere soil of pepper, is a novel biocontrol bacterium. It was categorized into Xanthomonadaceae, Lysobacter, and exhibited gliding motility and high GC content. OH11 has a bright future in biocontrol application, as it could produce diverse extracellular enzymes, and exhibited strong antifungal activity against many phytopathogens. The objective of this paper was to detect the regulatory mechanism on secondary metabolites and surface motility involved in biocontrol activity in OH 11.Function of chitinase and β-1,3-glucanase has been reported in recent years. In this paper, we addressed the function of other two important extracellular enzymes:a-lytic protease and cellulase. Mutation of them did not impair the antifungal activity, indicated these two enzymes were not essential for the antifungal activity of L. enzymogenes OH 11 against pathogens.HSAF (heat-stable antifungal factor) was a kind of secondary metabolites reported recently. Based on the genome sequence of OH11, we constructed pks-nrps deletion mutant Apks with L. enzymogenes OH11. Compared to wild type of OH11, Apks lost the ability to inhibit the growth of pathogens, and exhibited significant reduction of HSAF production. Additionally, the extraction of HSAF from wild type of OH11 could inhibit the growth of pathogens. Importantly, pks-nrps deletion did not impair the activities of several important extracellular emzymes. These results indicated HSAF was a crucial factor in biocontrol process of OH11, and pks-nrps was a crucial gene in HSAF biosynthesis.In order to screen the genes regulating HSAF biosynthesis, we constructed engineering strain OH11-GP112 with LacZ inserted into pks-nrps of L. enzymogenes OH11. OH11-GP112 exhibited slight-blue color on medium containing X-gal, and showed similar antifungal activity and HSAF production to OH11. We constructed transposon library with Tn5 transposon and OH11-GP112. Attractively, the variety of the colony color was consistent with the HSAF production in OH11-GP112 harboring Tn5. Based on these results, with utilization of the variety of the colony color, we could filtrate the transformants and screened the candidates with reduced HSAF production. Then, we tested the candidates with antifungal ability and got several strains with reduced or disappeared antifungal activity. Finally, the insertion sites of Tn5 in the strains were identified with subclone, and we explored an important transformant with dp (encoding for cAMP-receptor like protein) inserted by Tn5.Based on gene-deletion strategy, we constructed c/p-deletion mutant Ac/p. Compared to wild type of OH11, Δclp lost the antifungal activity against phytopathogens. HPLC revealed HSAF production had been dramatically reduced inAc/p. Additionally, dp deletion impaired the activities of several extracellular enzymes in Lysobacter enzymogenes OH11. Based on these results, we analyzed the dp regulon with transcriptomic analysis. According to transcriptomic analysis, the number of the genes modulated by Clp was up to 775, and could be categorized into 19 groups based on COG function classification. Further analysis indicated Clp could modulate 20 putative gene clusters. Among these gene clusters, we observed a gene cluster responsible for WAP-8294A2 biosynthesis. This means Clp could modulate the biosynthesis of WAP-8294A2. Finally, we confirmed this possibility with experiments.Among the gene clusters modulated by Clp, there were 4 gene clusters related to cell motility, including 3 gene clusters involved in type IV pili (T4P) and one gene cluster involved in flagella biosynthesis. Based on the gene deletion strategy, we revealed Clp could modulate surface motility via T4P and pilA was a crucial gene. Finally, we presented Clp could independently regulate biosynthesis of HSAF, WAP-8294A2 and surface motility in L. enzymogenes.In Xanthomonas. campestris pv. campestris, Clp could via FhrR and Zur to modulate a set of genes expression. However, we did not detect the FhrR homo log in OH11 according to the genome sequence. Attractively, Clp did not via Zur to modulate HSAF and WAP-8294A2 biosynthesis and surface motility. These results indicated Clp signaling in OH11 was much different to that in Xcc. Among the regulatory factors downstream of Clp, we detected two novel factors Lat and Lys1456. Both of them were modulated by Clp, and Clp could bind to the predicted promoter of Iys1456. Lat and Lys1456 were involved in the biosynthesis of HSAF and WAP-8294A2, respectively. Further more, we revealed over-expression of Clp could bypass Lat to modulate HSAF biosynthesis. Based on these results, we unveiled a schematic model of Clp signaling in L. enzymogenes OH11. |
