Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120's Metabolism

Abstract

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts.IMPORTANCE While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

Keywords: NADH dehydrogenase; Pseudomonas; electron transport chain; oxidative stress; pseudomonads; redox metabolism; respiratory activity.

FIG 1

Physiological characterization of P. taiwanensis VLB120 wild type (A) and the NADH dehydrogenase deficient mutants Δndh-1 (B), Δndh-2 (C), ΔΔndh (D), Δnuo (E), and Δnuo Δndh-1 (F). The strains were cultured in MSM with 25 mM glucose. The OD_600nm (black circles), glucose levels (blue squares), and gluconate levels (green triangles) were measured over time. The shadowed area in (D) indicates the first growth phase. The data shown are the means of biological triplicates; error bars show the standard deviation. μ_recalc is the growth rate of P. taiwanensis VLB120 pSTY⁻. The wild type OD_600nm values are plotted (gray, open circles) in graphs (B) to (F) for comparison.

FIG 2

Respiratory activity of P. taiwanensis VLB120 and the ΔΔndh mutant. (A) CTR and OTR rates of the wild-type strain; the highlighted area corresponds to the surplus of consumed oxygen. The area was calculated from the sectional integrals between the OTR (dashed line) and CTR (solid line). (B) Oxygen transfer rates during cultivation of P. taiwanensis VLB120 (black dashed line) and mutant ΔΔndh (green dashed line).

FIG 3

Relative gene expression of the NADH dehydrogenase-encoding genes ndh-1, ndh-2, and nuoA in NADH dehydrogenase mutants Δndh-1 (A), Δndh-2 (B), ΔΔndh (C), Δnuo (D), and Δnuo Δndh-1 (E) at early, mid-, and late exponential growth phase normalized to the corresponding values of the wild type. mRNA abundance was determined by quantitative PCR. Values were normalized to the relative transcript levels of P. taiwanensis VLB120 in the corresponding growth phase. nuoA was used as a proxy for the expression of the nuo operon. Gene deletions in the respective mutants are marked with “X” and were not analyzed by qPCR. Experiments were performed in biological triplicates.

FIG 4

Quantification of the NADH/NAD⁺ ratio in the P. taiwanensis VLB120 (black) and the NADH dehydrogenase mutants ΔΔndh (green), Δnuo (orange), and Δnuo Δndh-1 (red) in early, mid- and late exponential growth phase.

FIG 5

Significant changes at proteome level of P. taiwanensis VLB120 NADH dehydrogenase mutants in early (A), mid- (B), and late (C) exponential growth phase relative to the wild type. Proteins are clustered into functional categories according to the KEGG classification system (39). Each bar represents the number of proteins in the depicted category, the abundance of which was either increased or decreased in response to NADH dehydrogenase deficiency. Experiments were performed in biological triplicates.

FIG 6

Proposed metabolic changes caused by type 2 NADH dehydrogenase deficiency in P. taiwanensis VLB120. An increased NADH/NAD⁺ ratio (1) might result in substrate inhibition of the Nuo complex as well as ROS production (2), which is reported for this NADH dehydrogenase (45, 46). Rerouting of the flux through the TCA cycle into the glyoxylate shunt (3) reduces redox cofactor formation (48–50) and helps to scavenge reactive oxygen species by glyoxylate (48, 52). Limited ATP provision from oxidative phosphorylation can be mitigated by upregulation of the ADI pathway, based on our proteomics data (4) (58, 72). The light representation of the Ndh dehydrogenase indicates deficiency of both isozymes. ETC, electron transport chain; ROS, reactive oxygen species; ADI, arginine deiminase pathway; Nuo, type 1 NADH dehydrogenase; Ndh, type 2 NADH dehydrogenase; Sdh, succinate dehydrogenase; bc1, cytochrome bc₁ (complex III); cbb3, cytochrome cbb₃ (complex IV); QH₂, ubiquinol; Q, ubiquinone; SUC, succinate, SUCCoA, succinyl-CoA; FUM, fumarate.