| Lysobacter enzymogenes strain oH11, isolated from cayenne-root soil, is reported for the first time as a bacterial biological control agent in China. It is characterizaed with a high G+C content and gliding motility, and has been shown to produce extracellular lytic enzymes, including chitinase, protease, cellulase andβ-1,3-glucanas. Furhtermore, it displays in vitro activity against fungal and oomycetous plant pathogens. The objectives of this work are to:(i) establish the genetic manipulation system in strain OH 11 and construct a mutant library by a mariner transposon mutagenesis; (ii) to identify the pigment and exopolysacchide formation related genes, and to investigate the function of these cloned genes in colonization, antimicrobial activity and biocontrol efficiency of strain OH11; (ii) to construct an engineering strain with AHL degradation capacity to biocontrol bacterial plant diseases, broadening the biocontrol spectrum of L. enzymogenes.Transposon mutagenesis was first performed in Lysobacter enzymogenes strain OH 11 by a mariner transposon and a large-scale mutant library was obtained. Four chitinase-defective mutants, which could not produce hydrolytic zones around colonies in chitine selection medium, were selected from over 2,000 chloramphenicol-resistant (Cm1) mutants. By using arbitrary PCR techonology, the transposon insertion in 4 chitinase-defective mutants were identified as regulatory gene clp, chitinase encoding gene chiA, carboxyl-terminal protease encoding gene cptA and hyprothetical protein encoding gene. Subsequenctly, three different suicide vectors (pMW91CM, pJQ200SK, pEX18GM) were evaluated for the possibility of performing gene mutagenesis in strain OH 11 using the chiA gene (accession number:DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied for disruption and in-frame deletions in the chiA gene in strain OH11. The chiA-deletion mutant OH11-3 did not have the ability to produce chitinase on chitine selection medium, whereas the corresponding complemented strain OP1 (chiA gene was expressed under an Escherichia coli constitutive promoter Plpp in strain OH11-3) was partially restored in chitinase production in the same medium. Interestingly, the chiA-deletion mutants displayed wild-type antimicrobial activity against Phytophthora capsici, Rhizoctonia solani, Sclerotinia sclerotiorum and Fusarium graminearum. Our data suggest that chitinase might not be a unique lytic enzyme in controlling R. solani, S. sclerotiorum and F. graminearum. Also, the established genetic manipulation system might be explored as a potential tool for functional gene deletion and complementation, and alien gene expression in L. enzymogenes, which will facilitate the molecular study of mechanisms of biological control in L. enzymogenes.According to the phenotype changes, two pigment changing mutants, named strain OH11H and OH11B were isolated from the mutant library constructed by mariner transposon mutagenesis. In LA agar plates, wild-type OH11 exhibited a yellow colony, while strain OH11H produced dark brown pigment around yellow colonies, and strain OH11B was defective in yellow pigment production, resulting in a white colony. The transposon insertion in strain OH11H and OH11B was identified as hmgA and acp by using arbitrary PCR and sub-cloning technology. Based on the results of suicide-vector selections in a previous work, in this study, the hmgA gene was further used as a new reporter to confirm the results of suicide-vector selections, ultimately confirming that only pEX18GM is an available suicide vector for performing gene deletions in L. enzymogenes OH11. After that, the hmgA and acp disruption mutants were constructed by homologue recombination technology, respectively. Compared to wild-type OH11, the hmgA-disruption mutants changed the cell shape, enhanced the ability of biofilm formation, colonization in rice leaves and bicontrol efficiency against rice sheath blight. However, compared to wild-type OH11, the acp mutants showed a reduction of biofilm formation, colonization in rice leaves and biocontrol efficiency against rice sheat blight. Meanwhile, interestingly, on one hand, the hmgA and acp mutants exhibited wild-type tolerance to ultraviolet light (UV); on the other hand, the hmgA and acp mutation did not affect 4 type lytic enzyme production (chitinase, a-lytic protease, cellulase andβ-1,3-glucanase), and extropolysaccharide formation.According to the phenotype changes,5 exopolysacchide (EPS)-defective mutants, named MEPS1-MESP5 were isolated from the mutant library constructed by mariner transposon mutagenesis. In LAS agar plates, wild-type OH11 displayed a plump and smooth colony, wherease the EPS-defective mutants exhibited dry, crimple and yellow colonies. By using arbitrary PCR, the transposon insertion in MEPS1-MESP5 was identified as tonB, permease encoding gene, pilin encoding gene and hypothetical protein encoding genes, respectively. After that, the whole tonB gene in strain MESP1 was obtained by using sub-cloning technology. Subsequently, the tonB deletion mutants were constructed by homologue technology. Compared to wild-type OH11, the tonB mutation in strain OH11 decreased gliding motility, tolerance to UV, the ability of biofilm formation, colonization in rice leaves and bicontrol efficiency against rice sheath blight, and changed the bacterial colony. However, the tonB mutants exhibited wild-type cell shape and ability of 4 type lytic enzyme production and antimicrobial activity against R. solani and Phytophthora capsici.An N-acyl homoserine lactonase gene aiiA, transcribed by a strong and constitutive Escherichia coli promoter Plpp (accession no. EU723847), was transformed into strain OH11, creating strain OH11A. The AHL-degradation assay showed that transformant OH11A acquired the ability to degrade AHL molecules produced by Agrobacterium tumefaciens, Pectobacterium carotovorum, Pseudomonas syringae. pv. tomato strain DC3000 and Acidovorax avenae subsp. citrulli. Pathogenicity tests showed that while the parental strain OH11 did not reduce P. carotovorum infection, the transformant OH11A caused a strong reduction of Pectobacterium virulence on Chinese cabbages and cactus, whereas strain OH11A did not seem to interfere with the normal growth of this pathogen in cabbages. In antimicrobial activity assays, strain OH11A and OH11 showed similar antimicrobial activity against Phytophthora capsici and Sclerotinia sclerotiorum. In the following work, a promoter-trapping vector was constructed using lacZ as a reporter, and a strong and constitutive promoter, named PB500, was isolated from the promoter library of strain OH11 by using this promoter-trapping vector. Subsequently, promoter PB500 and aiiA was spliced by overlap PCR amplification, and the spliced gene PB500-aii4 was integrated into hmgA by homologue recombination technology, creating unmarked strain OHl1PA. Similar with strain OH11 A, strain OH11PA exhibited strong degradation ability to AHL signals (AHLA) produced by Agrobacterium tumefaciens, and was able to control soft rot of Chinese cabbages in vitro. This work provided a new strategy for developing genetically engineered multi-functional L. enzymogenes strains that possessed the ability to biologically control fungal pathogens and reduce bacterial pathogenicity. |
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