Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440

Abstract

Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator. IMPORTANCEPlant growth promotion can be enhanced by soil-and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.

Regulation of carbohydrate degradation pathways in Pseudomonas involves a versatile set of transcriptional regulators

“…However, there is also evidence for global regulation and interconnection with other processes, although the precise mechanisms have not been elucidated yet. Available information suggests the ketogluconate branch plays a role in the control virulence; and An and Moe () showed that Gcd levels varied significantly with the carbon source used by Pseudomonas , showing that expression in glucose was higher than in glycerol, LB and citrate, which was consistent with the requirements in glucose dehydrogenase activity. These authors also showed that gcd expression was downregulated by inorganic phosphate, a demonstration of interconnection among metabolism of different nutrients.…”

Contrasting P acquisition strategies of the bacterial communities associated with legume and grass in subtropical orchard soil

“…Conversely, immobilized P can be remobilized and thus release Pi for plant uptake via biochemical processes mediated by soil microorganisms (Richardson and Simpson, 2011). For example, P solubilizing bacteria can produce low molecular-weight organic acids (OAs) to solubilize sparingly soluble Pi (Oteino et al, 2015;An and Moe, 2016), and P mineralizing bacteria and arbuscular mycorrhizal fungi are capable of hydrolyzing Po via secreting phosphatase (Tan et al, 2013;Cui et al, 2015;Ragot et al, 2016). Microbial biomass P also contributes to the soil P pool and is normally in the form of watersoluble Po after microbial cell lysis (Turner and Haygarth, 2001).…”

“…For the mobilization of sparingly soluble Pi, P solubilizing bacteria adopt the nonenzymatic mechanisms, mainly via exuding OAs and H 1 (Zaidi et al, 2009;Jones and Oburger, 2011;Hsu et al, 2015). Gluconic acid and 2-ketogluconic acid are the principle soil microbial-derived OAs involved, whose biosynthesis requires the redox cofactor pyrroloquinoline quinone (PQQ) (An and Moe, 2016). Recently, the genes (gcd, gad, pqq) encoding the related enzymes have been investigated (de Werra et al, 2009;Hsu et al, 2015;An and Moe, 2016).…”

“…Gluconic acid and 2-ketogluconic acid are the principle soil microbial-derived OAs involved, whose biosynthesis requires the redox cofactor pyrroloquinoline quinone (PQQ) (An and Moe, 2016). Recently, the genes (gcd, gad, pqq) encoding the related enzymes have been investigated (de Werra et al, 2009;Hsu et al, 2015;An and Moe, 2016). In addition to OA biosynthesis, some genes encoding OA transporters have been identified and characterized in bacteria, such as ttdT, dct, JEN, oxlT (Kim and Unden, 2007;Iyalomhe et al, 2014;Becker, 2015;de Lima et al, 2016;Yang L et al, 2017), although their involvement in solubilizing sparingly soluble Pi is largely unknown.…”

Metabolic engineering and bio-electrochemical synthesis in Pseudomonas putida KT2440 for the production of p-hydroxybenzoate and 2-ketogluconate

“…In P. putida KT2440, the pqqABCDEFH can at least form two transcripts: the pqqABCDEH and and pqqF ( Figure 4-4 A) The PQQ synthesis is transcriptionally regulated and influenced by growth conditions (Choi et al, 2008;Gliese et al, 2009). It is interesting to notice that the inconsistent results in two publications (An and Moe, 2016;Nikel et al, 2014a), which the same strain P. putida KT2440 grown on DM9 medium with glycerol or glucose as the sole carbon sources. Apparently, it looks like both publications conducted in a similar conditions.…”