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. 2025 Jun 15;13(6):1395.
doi: 10.3390/microorganisms13061395.

Molecular Characterization of a Transcriptional Regulator GntR for Gluconate Metabolism in Industrial 2-Ketogluconate Producer Pseudomonas plecoglossicida JUIM01

Mengxin Qu  1 , Lulu Li  1 , Xinyi Zan  1 , Fengjie Cui  1   2 , Lei Sun  1 , Wenjing Sun  1   2
Affiliations

Molecular Characterization of a Transcriptional Regulator GntR for Gluconate Metabolism in Industrial 2-Ketogluconate Producer Pseudomonas plecoglossicida JUIM01

Mengxin Qu et al. Microorganisms. .

Abstract

The GntR is a transcriptional regulator generally known as a gluconate-operon repressor to specifically regulate the transportation and phosphorylation of gluconate. In the present study we report the cloning of the GntR-encoding gene of the industrial 2-ketogluconate (2KGA)-producer Pseudomonas plecoglossicida JUIM01, which is involved in the regulation of gluconate metabolism, along with the identification of some of its target genes and its operator sequence. GntR is a 36.36-kDa cytoplasmic and hydrophobic DNA-binding transcriptional regulator belonging to the LacI family. The knockout of gntR resulted in the significant upregulation of the transcription of the gluconate kinase gene gntK and, to a lesser extent, the permease gene gntP, as well as downregulation of genes involved in glucose uptake (oprB-1, gltB, gltF, gltG, and gltK) and those involved in 2-ketogluconate (2KGA) transport (kguT) and catabolism (kguE, kguK, and kguD). These results indicated that GntR positively regulated glucose and 2KGA transport and catabolism, while negatively affecting GntP-mediated gluconate uptake and gluconate phosphorylation by GntK. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting analyses confirmed that GntR interacted with operator sequences in the divergent promoter regions of gntK and gntP, as well as in the gntR promoter region. A putative operator sequence (consensus 5'-AG-N2-AGCGCT-N-TCT-3') was identified. These data suggest that GntR positively regulates genes involved in glucose uptake/transport and 2KGA transport/catabolism, while repressing its own expression as well as that of genes involved in gluconate transport/catabolism. These findings not only elucidate the regulation of GntR and its target genes in P. plecoglossicida, but also provide valuable insights for optimizing industrial 2KGA production.

Keywords: 2-ketogluconate (2KGA); LacI family transcriptional regulator GntR; Pseudomonas plecoglossicida; gluconate metabolism; specific binding sites.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Glucose metabolism in Pseudomonas deduced from gene annotations and functional analysis.
Figure 2
Figure 2
Comparison of the expression levels of related genes involved in glucose/gluconate/2KGA metabolism between P. plecoglossicida JUIM01 and JUIM01ΔgntR. The yellow and orange arrows represent the genes encoding transcriptional regulators.
Figure 3
Figure 3
Electrophoretic mobility shift assays of PgntR, PgntK, and PgntP promoter fragments with PpGntR.
Figure 4
Figure 4
DNase I footprinting assays of (A) PgntR, (B) PgntK, and (C) PgntP promoter fragments with PpGntR, (D) analysis of conserved GntR-binding motif, and (E) prediction of the regulatory mechanism of GntR in P. plecoglossicida.
Figure 5
Figure 5
The tertiary-structure and protein-DNA binding prediction of GntR. (A) Monomer; (B) Dimer; (C) The binding of GntR dimer to the conserved binding motif (5′-AG-N2-AGCGCT-N-TCT-3′); (D) The dimerization interfaces of GntR.
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