Role of Gluconic Acid Production in the Regulation of Biocontrol Traits of Pseudomonas fluorescens CHA0

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Δgcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.Plant growth-promoting rhizobacteria (PGPR) (36) are root-colonizing bacteria that enhance the performance of crop plants by several mechanisms. First, they antagonize plant-pathogenic fungi, mainly by the production of antimicrobial metabolites, but also by competition for iron or rhizosphere niches (9, 23, 24, 59). The biocontrol activity of many disease-suppressive microorganisms is also attributed to stimulation of host defense (induced systemic resistance). Other mechanisms by which these rhizobacteria directly promote plant growth are the production of phytohormones and the increase of nutrient, in particular phosphate, availability to plants (18, 37). Certain rhizobacteria are able to solubilize insoluble or poorly soluble mineral phosphates by producing acid phosphatases and organic acids, mainly gluconic acid (2, 34, 60). Some PGPR combine these different plant-beneficial activities and are able to suppress soilborne plant diseases, as well as to increase phosphate availability for plants (72).In fluorescent pseudomonads, gluconic acid production is catalyzed by periplasmic oxidation of glucose by membrane-bound glucose dehydrogenase (Gcd) (Fig. ​(Fig.1A)1A) (16, 43). In many gram-negative bacteria, the synthesis of gluconic acid has been shown to be dependent on pyrroloquinoline quinone (PQQ) as an enzymatic cofactor of the Gcd (1, 14). A consecutive oxidation reaction is mediated by gluconate dehydrogenase (Gad), which converts gluconic acid to 2-ketogluconate (Fig. ​(Fig.1A)1A) (11, 12, 44, 50). These enzymes should be of predominant importance for biocontrol soil pseudomonads, as major carbon sources provided by plant root exudates in the rhizosphere are made up of glucose (29, 69, 70). The two enzymes involved in glucose metabolism may have a substantial influence on general nutrient availability in the rhizosphere. First, Gcd and Gad affect glucose levels, and second, they may modulate the availability of soluble phosphates by controlling the amount of gluconic acid released into the rhizosphere. Furthermore, the production of gluconic acid might substantially change the rhizosphere pH. Therefore, Gcd and Gad enzymes produced by fluorescent pseudomonads are likely to be important for soil fertility and to impact the activities of other organisms living in the rhizosphere, e.g., fungal pathogens attacking the roots. Indeed, gluconic acid metabolism has already been linked to antifungal activity. Recently, Kaur et al. (30) proposed that gluconic acid produced by a nonfluorescent Pseudomonas isolate may be important for the biological control of take-all disease.Open in a separate windowFIG. 1.(A) Periplasmic and intracellular glucose catabolism in pseudomonads based on studies with P. aeruginosa (10), P. putida (11, 12), and P. fluorescens (17, 28). Shown are membrane-bound enzymes involved in periplasmic glucose metabolism, Gcd (glucose dehydrogenase) and Gad (gluconate dehydrogenase), and enzymes involved in cytoplasmic glucose metabolism, Glk (glucokinase), Zwf (glucose-6-phosphate 1-dehydrogenase), GnuK (gluconokinase), KguK (2-ketogluconate kinase), and KguD (2-ketogluconate 6-phosphate reductase) (the names of the enzymes are derived from the nomenclature for P. putida KT2440 [12, 54]). (B and C) Physical locations of the gcd (B) and gad (C) genes in the genome of P. fluorescens strain CHA0. The shaded arrows show the sequenced or partly sequenced genes. The representation is based on the sequence data for strain CHA0 obtained by sequencing the chromosomal fragments inserted in the indicated vectors. The designations of the ORFs flanking the gcd and gad genes are based on the corresponding locus tags in the complete annotated sequence of the closely related P. fluorescens strain Pf-5 (56). Δ, region deleted in strains CHA1196 and CHA1197 and in plasmids pME3087::F34 and pME3087::F12. The bars designate the fragments cloned into the vector pME3087 to give pME3087::F34 and pME3087::F12 and into pColdI to give pColdI::gcd and pColdI::gad. Artificial restriction sites on the cloned fragments are marked with asterisks.Pseudomonas fluorescens CHA0 is a bacterial strain known to be able to suppress various soilborne plant diseases (24). Its biocontrol ability has been linked to the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) (31, 33) and pyoluteorin (PLT) (46, 47). The strain is also able to solubilize mineral phosphate and to improve plant growth under phosphate-limiting conditions (A. von Felten, personal communication). Gluconic acid is supposed to play a predominant role in the phosphate solubilization activity of P. fluorescens CHA0, and we hypothesize that the metabolite also has an impact on the biocontrol activity of this PGPR strain.The aim of this study was to elucidate the role of gluconic acid production in P. fluorescens CHA0 with respect to its phosphate-solubilizing ability, antifungal metabolite production, and ability to suppress fungal root diseases. To this end, mutants of strain CHA0 carrying deletions in the gcd gene, encoding Gcd, and the gad gene, encoding Gad (Fig. ​(Fig.1),1), were created. The three in-frame deletion mutants, CHA1196 (Δgcd), CHA1197 (Δgad), and CHA1198 (Δgcd Δgad), were compared with their parental strain for the ability to produce organic acids, to solubilize inorganic phosphate, to produce the antifungal metabolites DAPG and PLT, to inhibit the growth of fungal pathogens, and to suppress different soilborne diseases. We provide evidence that in fact, gluconic acid production by P. fluorescens CHA0 is involved not only in the solubilization of phosphate, but also in the regulation of antifungal compound production and, as a consequence, can influence the level of plant protection provided by the strain.