The Role Of Regulatory Gene GacA In The Biocontrol Activity Of Pseudomonas Fluorescens 2P24

Pseudomonas fluorescens 2P24, a biocontrol agent isolated from wheat take-all decline soil, could protect plants against several soil-borne diseases. This strain produces several antifungal secondary metabolites, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide (HCN), siderophore and proteases. The main aims of the present study are to understand 1) the colonization pattern of strain 2P24 on rhizosphere by using GFP as a visual marker; 2) the role of regulatory gene gacA in the antifungal productions and biocontrol activity.Pseudomonas fluorescens 2P24 was tagged by a plasmid-borne gfp marker and by a chromosome-integrated Tn5::gfp, resulted in the labeled strains PM-21 and PM-g1, respectively. No significant difference was found in the growth rate, biofilm formation and antibiosis ability between the gfp-tagged strains and their parent. Tn5::gfp in PM-g1 showed a relatively high stability compared with plasmid pSMC21 in PM-21. The fluorescence intensity of PM-21 is 3～4-fold stronger than that of PM-g1. The fluorescence microscope observation revealed that most of the labeled bacteria aggregated at the grooves of the root, the junctions of the epidermal cells and the wounds of the root, rather than distributed on the smooth surface of the epidermis. In the direction from root base to tip, the bacterial distribution showed a clear decreasing tendency in wheat root.The two-component regulatory system, typically composed of a membrane-bound sensor kinase (GacS) and a cytoplasmic response protein (GacA), distribute widely in bacteria. A number of publications demonstrated that the GacS/GacA system globally regulated the production of the extracellular compounds and exoenzymes and played an important role in the biocontrol activity of the Pseudomonas spp. A mutant of P. fluorescens 2P24 named PM3390 was generated through Tn5 mutagenesis. PM3390 had multiple changes resemble to gacA- phenotypes in other Pseudomonas spp., including lost of the production of 2,4-DAPG, HCN, protease and overproduction of siderosphore. PM3390 also lost the ability of biofilm formation. A cosmid containing gacA gene was obtained from a genomic library of 2P24 by using PCR-mediated screening method. The positive cosmid was introduced into PM3390, and recovered the lost ability of gacA- mutant, indicating that the cosmid contained the functional gacA fragment. The gacA gene was subcloned and sequenced. An in-frame deletion of the gacA ORF was generated by two homologous recombination. The mutant named PM395 showed the same phenotypes with PM3390. The shuttle vector pRK12 containing a 1.2 kb gacA fragment was introduced into PM395, and recovered the lost ability of PM395. In greenhouse, the gacA–negative mutant showed decreased ability to suppress soil-borne diseases compared with that of wild type 2P24. The results reveal that gacA is a global regulator in the 2P24, which plays an important role in biocontrol of soil-borne plant diseases. By using PCR, the native RBS (GAGG) and the translation initiation codon (TTG) of gacA gene were changed into GGAGG and ATG, and a promoterlessed lacZ gene was inserted at the downstream of gacA(gacA' ) to obtain a gacA (gacA' )-lacZ transcriptional fusion in shuttle vector pRK415. 2P24-A and 2P24-A' , the derivates of strain 2P24 containing gacA-lacZ and gacA'-lacZ, respectively, produced 2～4 fold of 2,4-DAPG compared to the wild type strain 2P24. These results showed that genetic modifications of the gacA gene might increase the antibiotic 2,4-DAPG biosynthesis. Subsequently, the gacA'-lacZ gene was integrated into the chromosome of 2P24 by the Tn5-mediated mutagenisis. Ten modified 2P24 strains were selected through in vitro antibiosis test and competitive colonization assay on the tomato root with the wild type. A positive correlation between gacA expression and the antibiotic 2,4-DAPG production was demonstrated by comparing the activity of (-galactosidase and production of 2,4-DAPG.The modified strains described above were tested for biocontrol activities in a tomato...