Bacillus Amyloliquefaciens C06 Employs Lipopeptides As Major Weapons To Control Monilinia Fructicola And Secrets γ-Polyglutamic Acid (γ-PGA) To Poromot Its Fruit Surface Colonization

In previous studies, Bacillus amyloliquefaciens C06 has been proved to be effective in controlling brown rot of stone fruit caused by Monilinia fructicola. When tested in vitro, culture filtrate of B. amyloliquefaciens C06 significantly inhibited mycelial growth and conidial germination of the fungal pathogen, indicating that the major antifungal compounds were in the culture filtrate. B. amyloliquefaciens C06 was also found to produce extra-celluLar mucilage and form mucoid colony on semi-solid surface. The present work consists of three parts:1, to characterize the antifungal compounds; 2, to characterize the mucilage produced by B. amyloliquefaciens C06 and make clear its role in promoting fruit surface colonization; 3, screen the mutant with enhanced productivity of the mucilage and its reguLatory mechanism. The resuLts obtained in this study were summarized as below:1. Sfp gene, encoding 4’-phosphopantetheinyl transferase, plays the key role in lipopeptides sysnthsis and once the gene was disrupted, Bacillus strains lost the ability of producing any lipopeptides. C06Δsfp, the site-directed muant of C06, was found unable to produce any lipopeptides and showed no biological control effects against mycelium growth and conidial germination of M. fructicola, indicating lipopeptides were the major weapons employed by C06 to control M. fructicola. To characterize the lipopeptides produced by C06 and the synthesizing genes, an approach combining DNA based suppression subtractive hybridization （SSH） method with MALDI-TOF-MS analysis was performed. MALDI-TOF-MS detected that B. amyliquefaciens C06 was a coproducer of bacillomycin D and fengycin, two lipopeptides belonging to iturin and fengycin family. In addition,43 DNA unique DNA fragments were obtained from B. amyloliquefaciens C06 with SSH method, by using digested B. amyloliquefaciens C06 chromosomal DNA as tester and digested B. subtilis chromosomal DNA as driver. Three SSH fragments bmyC, fenD and fenE were fonund to have high similarity with genes directing bacillomycin D synthetase and fengycin synthesizes respectively. Two mutants C06â–³bmyC and C06â–³fenD, unable to produce bacillomycin D and fengycin were constructed, which confirmed the roles of bmyC and fenD in lipopeptides synthesis. In vitro biological evaluation revealed that bacillomycin D and fengycin jointly contributed to inhibition of conidial germination of M. fructicola, and fengycin played a major role in suppressing mycelial growth of the fungal pathogen.2. Tranposon mutant library of B. amyloliquefaciens C06 was constructed for the purpose of charaterzaing genes directing the extra-cellualr mucilage synthesis in C06 by using pMarA, a shuttle vector harboring transposon TnYLB-1. The mucoid properties of 1000 mutants were evaluated and four mutants M105, M106, M107 and M108 unable to form sticky colony on semi-solid medium surface were selected. M106, M107 and M108, which harbored single copy of transposon insertion, were subjected to inverse PCR analysis, and the resuLt showed that two genes, ywsC whose translated product directedγ-polyglutamic acid （y-PGA） synthesis and comA a transcriptional reguLator with function to activate ywsC expression, were demonstrated to be responsible for the mucilage production in C06. The purified mucilage from C06 was characterized to be of high molecuLar weight, consisted of only glutamic acid and linked with non-peptide bond by hysical and chemical detection, and thus confirmed to be y-PGA. Compared with wild type B. amyloliquefaciens C06, its mutants deficient in producing y-PGA, e.g. M106 and C06ΔywsC showed less efficiency in biofilm formation, surface adhesion and swarming ability, which were considered as the key properties involved in bacteria colorzing fruit surface and paint root. In vivo evaluation showed that the efficiency of colonizing apple surface was impaired in strain C06 AywsC due to the disruption of ywsC gene.3. M306, a derivative strain of B. amyloliquefaciens C06 with defected ability of forming structured colony and enhanced ability of producing y-PGA, was obtained by screening the TnYLB-1 transposon mutant library of B. amyloliquefaciens C06. Southern blot analysis and inverse PCR demonstrated that M306 was inserted with single copy of transposon and insertion site was between two genes phrF and RBAM₀34450. qRT-PCR analysis showed that in M306 the expression level of RBAM₀34450 was obviously down-reguLated while the transcription of phrF showed no significant change compared to wild type C06, confirming that the disrupted gene responsible for the defective morphology in M306 was RBAM₀34450. Thus in the present study, RBAM₀34450 was designated wth the pbrA （PGA productivity and biofilm formation reguLation）. PbrA was a transcriptional reguLator and couLd be acitivated by the adverse environmental conditions. qRT-PCR analysis indicated in both M306 and C06ΔRBAM, the transcription levels of epsD and yqxM, the crucial genes involved in biofilm formation, were obviously down reguLated, while the expression of ywtB, the gene directing y-PGA synthesis, was non-affected. y-PGA productivity evaluation showed that the productivity of C06ΔepsA was enhaced to the same level as M306 and C06ARBAM, while no significant difference in y-PGA yield was observed between wild type C06 and C06ΔtasA. Both EPS production and y-PGA synthesis could utilize the intermediates of tricarboxylic acid cyle （TCA cycle） as the reaction substrates, and acoording to which, it was assumed that depressed production of EPS couLd promote the utilization efficiency of the reaction susbtrate and enhance the y-PGA productivity.