GtrS and GltR form a two-component system: the central role of 2-ketogluconate in the expression of exotoxin A and glucose catabolic enzymes inPseudomonas aeruginosa

Abstract

In the human pathogen Pseudomonas aeruginosa, the GltR regulator is required for glucose transport, whereas GtrS is a sensor kinase that plays a key role in mediating bacteria–host interaction and pathogen dissemination in the host. We show that GtrS and GltR form a two-component system that regulates the expression from the promoters Pedd/gap-1, PoprB and Pglk, which control the expression of genes involved in glucose metabolism and transport. In addition, the GtrS/GltR pair regulates the expression of toxA that encodes exotoxin A, the primary virulence factor. Microcalorimetry-based ligand screening of the recombinant GtrS ligand-binding domain revealed specific binding of 2-ketogluconate (2-KG) (KD = 5 μM) and 6-phosphogluconate (KD = 98 μM). These effectors accelerate GtrS autophosphorylation, with concomitant transphosphorylation of GltR leading to a three-fold increase in transcription. Surprisingly, in vivo a similar increase in expression from the above promoters was observed for the mutant deficient in GltR regardless of the presence of effectors. The GltR operator site was found to contain the consensus sequence 5′-tgGTTTTTc-3′. We propose that 2-KG is a key metabolite in the stringent transcriptional control of genes involved in virulence and glucose metabolism. We show that GltR is a transcriptional repressor that is released from DNA upon phosphorylation.

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

“…The genes encoding the carbohydrate catabolic pathways are organized in operons (Fig. ), which are under the control of different regulators that respond differentially to distinct pathway intermediates, suggesting a hierarchy in the control of glucose metabolism related to a tight gene expression (Rojo, ; Daddaoua et al ., ; Figs and ).…”

“…However, Daddaoua et al . () demonstrated that the GltR and GtrS form indeed a TCS. GtrS is a transmembrane sensor kinase that contains a periplasmic ligand binding domain.…”

Unification of [FeFe]-hydrogenases into three structural and functional groups

“…This motif composition differs from canonical histidine kinase proteins. Canonical histidine kinase typically has an additional two motifs including the HAMP motif [histidine kinase, adenylyl cyclase, methyl-accepting protein and phosphatase (PF00672)] which is a phosphoacceptor domain that acts as a cytoplasmic intermediate linker connecting the phosphoryl transfer (HisKA) motif [68,[70][71][72][73][74] with the sensor domains outside of cytoplasm [70,75,76]. Biochemical characterization of the G3 Hyd in A. woodii indicates that it forms a tetrameric complex that includes HydBC and a Fd-like HydD protein that contains a [2Fe-2S] cluster [21].…”

Identification of GntR as regulator of the glucose metabolism in Pseudomonas aeruginosa

“…Using microcalorimetry and EMSA the binding affinities of different effectors to the PtxS, HexR and GtrS regulators have been determined (Daddaoua et al ., ). In all cases derived constants were in the micromolar range.…”