Regulation of Glutarate Catabolism by GntR Family Regulator CsiR and LysR Family Regulator GcdR in Pseudomonas putida KT2440

Abstract

Glutarate, a metabolic intermediate in the catabolism of several amino acids and aromatic compounds, can be catabolized through both the glutarate hydroxylation pathway and the glutaryl-coenzyme A (glutaryl-CoA) dehydrogenation pathway in Pseudomonas putida KT2440. The elucidation of the regulatory mechanism could greatly aid in the design of biotechnological alternatives for glutarate production. In this study, it was found that a GntR family protein, CsiR, and a LysR family protein, GcdR, regulate the catabolism of glutarate by repressing the transcription of csiD and lhgO, two key genes in the glutarate hydroxylation pathway, and by activating the transcription of gcdH and gcoT, two key genes in the glutaryl-CoA dehydrogenation pathway, respectively. Our data suggest that CsiR and GcdR are independent and that there is no cross-regulation between the two pathways. l-2-Hydroxyglutarate (l-2-HG), a metabolic intermediate in the glutarate catabolism with various physiological functions, has never been elucidated in terms of its metabolic regulation. Here, we reveal that two molecules, glutarate and l-2-HG, act as effectors of CsiR and that P. putida KT2440 uses CsiR to sense glutarate and l-2-HG and to utilize them effectively. This report broadens our understanding of the bacterial regulatory mechanisms of glutarate and l-2-HG catabolism and may help to identify regulators of l-2-HG catabolism in other species. IMPORTANCE Glutarate is an attractive dicarboxylate with various applications. Clarification of the regulatory mechanism of glutarate catabolism could help to block the glutarate catabolic pathways, thereby improving glutarate production through biotechnological routes. Glutarate is a toxic metabolite in humans, and its accumulation leads to a hereditary metabolic disorder, glutaric aciduria type I. The elucidation of the functions of CsiR and GcdR as regulators that respond to glutarate could help in the design of glutarate biosensors for the rapid detection of glutarate in patients with glutaric aciduria type I. In addition, CsiR was identified as a regulator that also regulates l-2-HG metabolism. The identification of CsiR as a regulator that responds to l-2-HG could help in the discovery and investigation of other regulatory proteins involved in l-2-HG catabolism.

Coordinated regulation of anthranilate metabolism and bacterial virulence by the GntR family regulator MpaR in Pseudomonas aeruginosa

“…The GntR family regulators are involved in a variety of carbon metabolic pathways in bacteria (Hoskisson and Rigali, 2009). For example, CsiR regulates the glutarate hydroxylation and glutaryl‐CoA dehydrogenation metabolism in Pseudomonas putida KT2440 (Zhang et al ., 2019). Pseudomonas aeruginosa GntR (PA2320) has been identified as a regulator of the glucose metabolism (Daddaoua et al ., 2017).…”

An l-2-hydroxyglutarate biosensor based on specific transcriptional regulator LhgR

“…1a ). In Pseudomonas putida KT2440, the glutarate regulon is regulated by allosteric transcription factor CsiR, which is encoded upstream of csiD 27 . The glutarate sensing allosteric transcription factor CsiR and its cognate promoter were cloned into broad host range vectors to create a glutarate biosensor 28 .…”

Dehydrogenation Mechanism of Three Stereoisomers of Butane-2,3-Diol in Pseudomonas putida KT2440

“…The native molecular weight of R , R -BDH was estimated by using a gel filtration column (Superdex 200 10/300 GL; GE Healthcare, United States) as described previously ( Zhang et al, 2019 ) with the eluent buffer (pH 7.2, 50 mM sodium phosphate and 150 mM sodium chloride) at a flow rate of 0.5 mL min −1 . Thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa) were used as standard proteins.…”