Comparative Proteomic Analysis Of The Role Of GacA In Pseudomonas Chlororaphis G5

GacS/GacA two-component signal transduction system is widespread in Gram negative bacteria.Gac system globally regulates bacterial physiology and behaviours via activating the transcription of small RNAs in RsmB family which can sequester the RNA-bingding protein RsmA/CsrA to release their translational repression.These phenotypes regulated by the Gac/Rsm system including cell motility, biofilmformation, production of exoenzymes, antibiotics and indole-3-acetic acid （IAA） are closely related to biocontrol activity or pathogenicity, drug resistance, plant growth promotion. Thereby, the GacS/GacA system has been extensively studied in bacteria related to food safety, environment, medicine, pharmaceutical industry and agricultural production. GacS/GacA is comprised of a sensor kinase GacS and a response regulator GacA. GacA as a global regulator is highly conserved among Gram negative bacteria, but functions differentially depending on strains or their habitats. Endophytic Psuedomonas.chlororaphis G5 was isolated from the stems of Chinese parsley（Coriandrum sativum L.）, previous studies displayed that G5 can produce a variety of secondary metabolites such as hydrogen cyanide （HCN）, phenazine and its derivatives, indole-3-acetic acid （IAA）, as well as siderophores and protease, which contribute to biocontrol of many plant diseases and plant growth promotion in strain G5. However, the specific role of GacA playes in G5 remains to be investigated.In the study, the endophytic starin G5 was used as a bacterial model to determine the global influence of gacA inactivation during stationary phase on the expression profiles of the cellular and extracellular proteins, respectively through comparative proteomics strategy. The identified proteins differentially expressed were annotated by bioinformatics technique for functional analysis. Furthermore, some key protein spots differentially expressed were confirmed by phenotypic analysis with a view to get more insights into the biological function of GacA in G5, which will pave the way for developing environmentally friendly biopesticides/biofertilizer to conserve ecological environment and realize sustainable agriculture, especially for food security and human health.The results obtained in this experiment were shown as follows:1)Wild type G5 and the gacA mutant were grown for 24 hours entry into stationary phase, comparative proteomic was performed to identify the differential protein expression profiles in cellular and extracellular proteomes, respectively. The total of 244 protein spots more than two-fold change in abundance were identified by MALDI-TOF mass spectrometry representing two hundreds and ten of unique proteins. Comparison with wild type G5, the expression of 38,91 protein spots were up-regulated and 89,26 protein spots were down-regulated in cellular and extracellular components by gacA mutation, respectively.2) Function analysis by GO annotation showed these proteins were involved in a wide range of biological processes including biosynthesis and metabolism of amino acid, polysaccharide, lipid, fatty acid, nucleotide and inorganic substrate, transport, secretion, protein folding and degradation, defense response, signal transduction, oxidation-reduction stress etc.. Among identified proteins,39,33,29 and 26 spots are related to stress response, substrate transport, amino acid biosynthesis and metabolism, carbohydrate metabolism and energy conversion, respectively. KEGG analysis indicated many proteins are involved into metabolism pathway such as tricarboxylic acid cycle （TCA）, glycolysis and gluconeogenesis.3) gacA mutation had a profound influence on biocontrol activity of strain G5. The expressions of identified proteins of PhzB, PhzF, PhzD and PhzO were abolished in the gacA mutant, which are required for biosynthesis of the orange pigment phenazine. In addition, the expression of alkaline metalloprotease AprA, chitinase ChiC₁, ChiC₂ and serine protease PspA, PspB were dramatically decreased. Further protease activity assay verified that gacA inactivation resulted in the loss of proteolytic activity.4) GacA also positively regulated type Ⅵ secretion system. gacA inactivation reduced the abundance of TssC2, effector Hcp and Vgr family proteins.It suggested that gacA mutation had an influence on the interaction mediated by type VI secretion system between strain G5 and plant hosts.5) GacA is closely related to ecology fitness in strain G5. The expression of stringent starvation protein SspA and superoxide dismutase SodB was increased （> 10-fold in abundance） in the gacA mutant. The SOD and catalase activities were measured, in agreement with the data from proteomics showing GacA negatively regulate the expression of sodB gene, hence enhanced tolerance to oxidation stress.6) Four proteins of FliC,F1gK,F1gL and FliI were identified by MALDI-TOF/MS and significantly enhanced in abundance in gacA mutant, which are required for flagellar biosynthesis and assembly, Flagellin shearing was done, followed by SDS-PAGE and MS analysis. The results verified that GacA negatively regulated the biosynthesis of flagellin FliC. The relation between the increase of flagellar biosynthesis and the decrease of motility ability suggested that flagellin is not a limitation factor for motility in G5.7) gacA mutation altered the structure of cell envelope. The expression of protein Ddl required for peptidoglycan biosynthesis of cell wall and LpxC responsible for lipid A biosynthesis were induced.Tricine SDS-PAGE analysis revealed that gacA mutation resulted in enhanced rough-type LPS production.8) Unlike in P. chlororaphis 06, gacA inactivation in strain G5 inhibited the expression of anthranilate synthase TrpE responsible for IAA biosynthesis. HPLC analysis verified that IAA production was significantly lower in the gacA mutant than the wild type G5 implying that the GacA regulation of IAA biosynthesis shows niche specificity.Taken together, the results presented here revealed that GacA is involved in global regulation of a diversity of biological processes including primary and secondary metabolism, and plays a central role in biological control, plant growth promotion, stress resistance, competitive fitness and the interactions between bacteria and plant hosts.