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.

Keywords: catabolism; glutarate; l-2-hydroxyglutarate; regulatory mechanism.

FIG 1

Organization and expression levels of csiR-csiD-lhgO and gcdR-gcdH-gcoT gene clusters. (A and B) Schematic representation of the csiR-csiD-lhgO (A) and gcdR-gcdH-gcoT (B) gene cluster regions of P. putida KT2440. The corresponding steps of the glutarate degradation pathways are also shown. (C) qPCR analysis of the genes in csiR-csiD-lhgO and gcdR-gcdH-gcoT gene clusters. The relative expression levels of six genes, csiR, csiD, lhgO, gcdR, gcdH, and gcoT, were measured using RNA extracted from P. putida KT2440 grown in MSM with glutarate or pyruvate as the sole carbon source. The gene expression levels are represented as expression ratios of the indicated genes in glutarate medium versus pyruvate medium, normalized to 16S rRNA. (D) The activities of CsiD and GcdH in P. putida KT2440 grown in MSM with glutarate or pyruvate as the sole carbon source. Data shown are means ± standard deviations (SD) (n = 3 independent experiments). *, P < 0.05 in two-tailed t test; **, P < 0.01 in two-tailed t test; ns, no significant difference (P ≥ 0.05 in two-tailed t test).

FIG 2

CsiR represses the transcription of csiD and lhgO. (A) Map of the csiR-csiD intergenic region. The transcriptional start site (TSS) identified in this study is shown in red letters. The predicted −10 and −35 regions are shown in bold and underlined. The start codons of csiR and csiD are shown in italics. The ribosome binding site is indicated by dotted lines. (B) The promoter activities of P_csiD in P. putida KT2440 and P. putida KT2440 (ΔcsiR) cultured in glutarate or pyruvate medium. Data shown are means ± SD (n = 3 independent experiments). (C) SDS-PAGE analysis of steps of expression and purification of CsiR. Lane M, molecular weight markers; lane 1, crude extract of E. coli BL21(DE3) harboring pETDuet-csiR; lane 2, the unbound protein of the HisTrap HP column; lane 3, CsiR (purified by the use of a HisTrap column). (D) EMSAs with csiR-csiD intergenic fragment F1 (10 nM) and purified CsiR (0, 10, 20, 30, 40, 60, 80, 100, and 120 nM). (E) DNase I footprinting analysis of CsiR binding to the csiD promoter region. F1 was labeled with FAM dye and incubated with 1 μg CsiR (blue line) or without CsiR (red line). The region protected by CsiR from DNase I cleavage is indicated with a dotted box. *, P < 0.05 in two-tailed t test; ***, P < 0.001 in two-tailed t test.

FIG 3

GcdR activates the transcription of gcdH. (A) Map of the gcdR-gcdH intergenic region. The TSS identified in this study is shown in red letters. The predicted −10 and −35 regions are shown in bold and underlined. The start codons of gcdR and gcdH are shown in italics. The ribosome binding site is indicated by dotted lines. (B) Growth of P. putida KT2440 and its derivatives on solid MSM containing 5 g liter⁻¹ glutarate as the sole carbon source. Pictures were taken at 36 h. Section 1, P. putida KT2440; section 2, P. putida KT2440 (ΔcsiD); section 3, P. putida KT2440 (ΔcsiD ΔgcdR); section 4, P. putida KT2440 (ΔgcdR). (C) SDS-PAGE analysis of steps of expression and purification of GcdR. Lane M, molecular weight markers; lane 1, crude extract of E. coli BL21(DE3) harboring pET28a-gcdR; lane 2, the unbound protein of the HisTrap HP column; lane 3, purified GcdR using a HisTrap column. (D) EMSAs with the F2 fragment containing the gcdR-gcdH intergenic region (10 nM) and purified GcdR (0, 10, 20, 30, 40, 60, 80, 100, and 120 nM). (E) DNase I footprinting analysis of GcdR binding to the gcdH promoter region. F2 was labeled with FAM dye and incubated with 5 μg GcdR (blue line) or without GcdR (red line). Each region protected by GcdR from DNase I cleavage is indicated with a dotted box.

FIG 4

CsiR and GcdR regulate their own target pathways. (A) The promoter activities of P_csiD and P_gcdH in P. putida KT2440, P. putida KT2440 (ΔgcdR) (for the determination of P_csiD data), and P. putida KT2440 (ΔcsiR) (for the determination of P_gcdH data). (B) Growth of P. putida KT2440 (ΔgcdH) and P. putida KT2440 (ΔgcdH ΔgcdR) in MSM with glutarate as the sole carbon source. The levels of growth (closed symbols) and of consumption of glutarate (open symbols) were measured. Data shown are means ± SD (n = 3 independent experiments). (C) EMSAs with F2 (10 nM) and purified CsiR (0, 20, 40, 60, 80, 60, 100, 150, and 200 nM). (D) EMSAs with F1 (10 nM) and purified GcdR (0, 20, 40, 60, 80, 60, 100, 150, and 200 nM). A 148-bp internal fragment of csiD (10 nM) was used as a negative control (C rows). ns, no significant difference (P ≥ 0.05 in two-tailed t test).

FIG 5

Characterization of the effectors of CsiR. (A) Schematic representation of l-lysine catabolism in P. putida KT2440 and the influences of davT and alr deletions. Pathways whose activity could not continue after the deletions of davT and alr are indicated by solid dashed arrows. davB, l-lysine monooxygenase; davA, 5-aminovaleramide amidohydrolase; davT, 5-aminovalerate aminotransferase; davD, glutaric semialdehyde dehydrogenase; alr, alanine racemase; amaC, d-lysine aminotransferase; dpkA, Δ¹-piperideine-2-carboxylate reductase; amaB, l-pipecolate oxidase; amaA, l-piperidine-6-carboxylate dehydrogenase. (B and C) The β-galactosidase assays were performed with P. putida KT2440-pME6522-P_csiD (B) and P. putida KT2440 (ΔdavT Δalr)-pME6522-P_csiD (C) grown in MSMs with 2.5 g liter⁻¹ pyruvate and different compounds as the carbon sources. Data shown are means ± SD (n = 3 independent experiments). (D) Glutarate and l-2-HG prevent CsiR binding to F1. EMSAs were performed with F1 (10 nM) and a 5-fold molar excess of CsiR in the absence of any other tested compounds (0) and in the presence of 40 mM l-lysine, 5-aminovalerate, glutarate, and l-2-HG. The leftmost lane shows the migration of free DNA (no CsiR).

FIG 6

Characterization of the effector of GcdR. The β-galactosidase assays were performed with P. putida KT2440-pME6522-P_gcdH (A) and P. putida KT2440 (ΔdavT Δalr)-pME6522-P_gcdH (B) grown in MSMs with 2.5 g liter⁻¹ pyruvate and different compounds as the carbon sources. Data shown are means ± SD (n = 3 independent experiments).

FIG 7

CsiR regulates the catabolism of l-2-HG. (A) Growth of P. putida KT2440 and its lhgO mutant in MSM with l-2-HG as the sole carbon source. Growth (closed symbols) and the consumption of l-2-HG (open symbols) of wild-type P. putida KT2440 (black lines with squares) and its lhgO mutant (red lines with circles) were measured in MSM supplemented with 5 g liter⁻¹ l-2-HG as the sole carbon source. (B) Growth (closed symbols) and consumption of l-2-HG (open symbols) of P. putida KT2440 (ΔcsiR) in MSM with l-2-HG as the sole carbon source. (C) The activity of LhgO in P. putida KT2440 cultured in MSMs with different compounds as the sole carbon sources. (D) The activity of LhgO in P. putida KT2440 (ΔcsiR) cultured in MSMs with different compounds as the sole carbon sources. Data shown are means ± SD (n = 3 independent experiments).

FIG 8

The proposed model for the regulation of glutarate catabolism by CsiR and GcdR in P. putida KT2440. The CsiR regulator represses the expression of csiD-lhgO genes in the glutarate hydroxylation pathway. Glutarate and l-2-HG from metabolism of their respective precursors or extracellular transport are effectors of CsiR and prevent CsiR binding to the csiD promoter region (red arrows). The GcdR regulator is activated by glutarate, thereby initiating expression of gcdH-gcoT genes in the glutaryl-CoA dehydrogenation pathway (green arrows).