| Currently food safety and environment have been seriously threatened due to the overuse of chemical pesticides and fertilizer to control plant diseases or promote plant growth. The plant disease management through developing and applying microorganisms with biological control potential has been recognized as an environmentally friendly alternative to chemicals to keep ecosystem in dynamic equilibrium and improve food safety for sustainable agriculture. However, the biological control microorganisms have some disadvantages such as narrow antimicrobiol spectrum and non-stablityetc, thereby screening of novel target genes to construct more efficient and stable engineered biocontrol strains is of paramount importance. GGDEF/EAL domain proteins are ubiquitous in bacteria, and numerous studies have established that they are involved in regulation of a variey of biological processes including motility, biofilm formation, virulence, cell differentiation and proliferation etc., yet little is known about the GGDEF/EAL domain protein in biocontrol strains of plant-associated bacteria.Serratia plymuthica G3 isolated from wheat (Triticum aestivum L.) is capable to produce an array of antifungal factors such as proteases, chitinases, and the antibiotic pyrrolnitrin etc., as well as plant hormone IAA and acetoin. Previous studies have indicated that several global regulators including QS system, Rsm system and regulators Hfq and RpoS are involved in the regulation of biocontrol activities of strain G3. A gene encoding GGDEF/EAL domain protein, designated as pigX, was identified in the S. plymuthica G3 genome, but its function remains unknown.In this study, S. plymuthica G3 was used as a model organism to investigate the biological function of the gene pigX. Firstly, an insertion mutant pigX::Gm was constructed, followed by mutational analysis to characterize the biocontrol-related traits regulated by PigX in strain G3. Secondly, using lux-or lacZ-based transcriptional and translational fusions, the molecular basis involved in regulation of cell motility and PRN biosynthesis by PigX was examined; the interactions betweenPigX and the QS system, the Rsm system, and Hfq and RpoS regulators, as well as autoregulation of PigX were analyzed. Finaly, comparative proteomics strategy was used to determine the global impact of pigX inactivation on the expression profiles of the cellular proteins during stationary phase. Unravelling the biological function of PigX can not only gain more insights into the regulatory network in S. plymuthica, but also lay a foundation to improve biocontrol efficacy and stability of strain G3. The major results achieved in this study were shown as follows:(1) Mutational analysis showed that PigX positively regulates proteolytic and chitinolytic activities, cell motility and antifiingal activity. However, PigX negatively controls biofilm formation. Detection of exoproducts in spent cultures suggested that PigX up-regulates PRN production, but inhibits IAA and acetoin biosynthesis. These findings indicated that PigX as a pleiotropic regulator plays an important role in the beneficial interactions between the endophyte S. plymuthica G3 and host plants.(2) lux or/ocZ-based transcriptional and translational fusion analysis was performed in order to reveal the molecular basis of the PigX regulation of cell motility and PRN biosynthesis. The results showed that PigX up-regulates the transcription and translation of both the flagella master regulator flhDC and the prnABCD gene cluster which are responsible for the synthesis of the antibiotic PRN suggesting that PigX may modulate motility via FlhDC, as well as PigX activation of PRN production due to up-regulating the expression of the prnABCD operon at both transcriptional and translational levels.(3) Assay of the lux-based transcriptional fusions and lacZ-based translational fusions demonstrated the existence of the interactions between PigX and other global regulators/systems. The results showed that PigX positively regulates the transcription of spll and spsI encoding AHLs synthetases indicating that PigX might have an impact on QS system. Additionally, PigX represses the rsmB transcription, but stimulates rsmA transcription during the stationary phase; Inversely, RsmB and RsmC positively control the transcription and translation of pigX. PigX is also positively auto-regulated, apart from up-regulation of the flhDC operon encoding the flagellar master regulator. PigX positively controls the transcription of hfq, reciprocally Hfq up-regulates pigX at both transcriptional and translational levels; RpoS negatively regulates the transcription and translation of pigX.(4) Comparative proteomics was performed to identify the differential protein expression profiles in cellular proteomes of the wild type G3 and the mutant pigX::Gm after incubation for 24 h. The results showed that there were significant differences between the cellular protein expression profiles of G3 and the mutant pigX::Gm.133 protein spots more than two-fold change in abundance were identified by MALDI-TOF/TOF MS representing 128 unique proteins. The abundance of 79 protein spots was increased and that of 54 protein spots was decreased in the pigX::Gm mutant relative to the wild type. Functional analyses by GO and COG demonstrated that these proteins were involved in a variety of biological processes including energy production and conversion, metabolism and transport of amino acid, carbohydrate, lipid, nucleotide and inorganic ion, protein folding, oxygen stress response, cell membrane/envelope biogenesis, signal transduction, etc.. Among the identified proteins,15 are involved in metabolism and transport of carbohydrate,17 are related to the stress response and six are outer membrane porin proteins. KEGG pathway analyses showed that many proteins are involved into metabolism pathways such as tricarboxylic acid (TCA) cycle, glycolysis and gluconeogenesis.Especially, mutation in pigX caused membrane structure’s changes due to the decreased expression of outer membrane protein OmpA, OmpC and TolC, which may affect bacterial secretion transport. PigX has an impact on ecological fitness of strain G3. For example, the expression of DNA starvation/stationary phase protection protein Dps, superoxide dismutase (SOD), catalase and universal stress protein UspE was decreased in the mutant pigX::Gm. The activities of SOD and catalase were measured exhibiting the same trend as the proteomics data presented above, confirmed that PigX positively regulate the expression of SOD and catalase encoding genes, and hence enhance tolerance to oxidation stress. Flagellin was only detected in protein expression profile of the wild type G3, which was verified by flagellin sheering and purification, SDS-PAGE and mass spectrometry characterization, in agreement with the phenotypic analysis above showing PigX is required for flagellar-dependent swimming motility. Additionally, the transcription repressor LrhA of the flagellar master regulator FlhDC was up-regulated in the mutant pigX::Gm relative to the wild type G3, which was verified using lacZ-based translational fusion assay. These findings suggested that PigX at least partially modulates flagellar-dependent motility via FlhDC and its transcriptional repressor LrhA to up-regulate flagellin production in strain G3.In conclusion, PigX plays a global role in both transcriptional and post-transcriptional regulation of a wide range of physiological processes and behaviors through the interactions with the QS and Rsm systems, and other global regulators such as Hfq and RpoS integrated into the complex regulatory networks including the production of PRN, IAA and acetoin, stress response, cell motility and biofilm formationto modulate biocontrol and plant-growth promoting potential, as well as bacterial ecological fitness. |
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