Characterization Of Lsp Proteins And NRPS/PKS Gene Clusters In The Lysobacter Enzymogenes

Lysobacter enzymogenes belongs to the Lysobacter genus of the Xanthomodaceae family. Lysobacter enzymogenes strain OH 11 is emerging as a novel biocontrol agent, which was isolated and identified by our laboratory independently. It was reported that HSAF, a novel structure and functional way, is the natural product of broad-spectrum antifungal and anti-oomycetous. WAP-8294A2, a novel product to antibacterial activity, is also found from Lysobacter enzymogenes strain OH11. Ax21, a novel small molecule secreted protein, participated in the pathogenicity of pathogenic bacteria of the Xanthomodaceae family. The gene that encoding the protein of pathogenic bacteria has only one copy.Using Stenotrophomonas maltophilia (Sm) in Ax21sm (Smlt0387) protein as a blast sequence in the first chapter, we found three homologous Ax21sm proteins by bioinformatics analysis in the whole genome of Lysobacter enzymogenes strain OH11, which were named as Lsp1, Lsp2 and Lsp3 (Lysobacter Small Protein). These three proteins were compared with Ax21Sm having 44%(Lspl),26%(Lsp2) and 26%(Lsp3) homology at the amino acid level. At the same time, there are 28%(Lsp1 and Lsp2),43% (Lsp2 and Lsp3) and 34%(Lsp1 and Lsp3) of the amino acid sequence homology to each other. In addition, the three proteins were predicted with conserved secretory signal peptide in the N terminal, which is similar with Ax21Sm.Any one of three lsp mutation significanty impaired the production of HSAF and WAP-8294A2 biosynthetic synthesis. This result showed that three lsp genes are all involved in the biosynthetic regulation of the two antimicrobial substances. We obtained three pairs of double-gene deletion mutants and a three-gene deletion mutant by combined mutant. Then, we found that the production of HSAF and WAP-8294A2 decreased significantly. However, it is no significant difference between combined mutants of three lsp genes and single gene deletion mutants. It reveals that three lsp genes do not the accumulative effect in the biosynthetic regulation of HSAF and WAP-8294A2. Besides, we also found that the lsp3 gene can compensate the reduce of HSAF in the lspl and lspl mutation, but the other two genes do not have the same function.Furthermore, the growth of wild-type strain OH 11 was influenced because of lsp genes deletion. But the production of three extracellular enzymes (cellulase, protease and chitinase) did not change.In this study, we reported, for the first time, that three homologous Ax21sm were cloned in Lysobacter enzymogens of the Xanthomonas, which played important roles in the biosynthetic regulation of secondary metabolite. In addition, we testified that lsp3 gene plays a key role in the the recovery of HSAF production of lspl and lspl genes.In the second chapter, we knew that polyketides and non-ribosomal peptides represented two large families of natural products (NPs) with diverse structure and important functions. They are synthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modular biosynthetic enzymes, respectively. Lysobacter enzymogenes is emerging as a novel biocontrol agent against pathogens of crop plants and a new source of bioactive NPs, such as antibacterial antibiotic WAP-8294A2 and antifungal antibiotic HSAF. Genome survey of strain OH11, a Chinese L. enzymogenes isolate, detected four novel PKS, NRPS or hybrid gene clusters, designed as Cluster A to D. Here, we individually mutated a total of five genes (PKS or NRPS) located in these four gene clusters, and showed that both a PKS (located in Cluster A) and NRPS gene (distributed in Cluster D) were involved in the antibacterial activity via a WAP-8294A2 dependent way. We also showed that these two genes were involved in the regulation of colony morphology via a nutrient-dependent, but WAP-8294A2 independent way. However, all five genes are not associated with antifungal activity and the regulation of HSAF biosynthesis. The results demonstrated for the first time that the regulatory roles of uninvestigated PKS and NRPS gene systems in L. enzymogenes.