Crp and Arc system directly regulate the transcription of NADH dehydrogenase genes in Shewanella oneidensis nitrate and nitrite respiration

Abstract

NADH oxidation by NADH dehydrogenases (NDHs) is crucial for feeding respiratory quinone pool and maintaining the balance of NADH/NAD⁺. In the respiratory model organism Shewanella oneidensis, which possesses four NDHs, the longstanding notion had been that NDHs were not required under anoxic conditions until recent studies demonstrated their role in extracellular electron transfer. However, the role of each NDH, particularly under anoxic conditions, has not been characterized. Here, we systematically investigated the role of each NDH in aerobic and anaerobic nitrate and nitrite respiration using NDH triple mutants. We corroborated the involvement of NDHs in anaerobic nitrate/nitrite respiration, revealing different repertoires of NDHs employed by S. oneidensis in response to electron acceptor (EA) availability. The transcript levels of two nqrs were modulated by the EA conversion from nitrate to nitrite. Furthermore, we demonstrated that the global regulators Crp and the Arc system both directly controlled the transcription of four NDHs during nitrate/nitrite respiration. This study confirms the requirement of NDHs for anaerobic nitrate and nitrite respiration and sheds light on the respiratory remodeling mechanism whereby global regulators coordinate NDH genes transcription to adapt to redox-stratified environments.IMPORTANCENADH is an important electron source for the respiratory quinone pool. Multiple NADH dehydrogenases (NDHs) are widely present in prokaryotes, indicating the flexibility in NADH oxidation. As a renowned respiratory versatile model strain, Shewanella oneidensis possesses four NDHs, encompassing all three types of NDHs, with varying ion-translocating efficiencies. The redundancy of NDHs may confer advantages for S. oneidensis to survive and thrive in redox-stratified environments. However, the roles of each NDH, especially in anaerobic respiration, are less understood. Here, we evaluated the role of each NDH in aerobic and anaerobic nitrate/nitrite respiration. We found that the conversion of electron acceptor from nitrate to nitrite triggered the changes in the transcriptional levels of NDH genes, and global regulators Crp and the Arc system were involved in these processes. These findings elucidate the mechanism of the respiratory chain remodeling at the NADH oxidation step in response to different electron acceptors.

Keywords: NADH dehydrogenases; Shewanella oneidensis; global regulator; respiratory chain.

Fig 1

Nitrate and nitrite respiratory chains in S. oneidensis MR-1. Electron transfer pathways are color-coded based on respiratory conditions: brown arrows indicate the preferred electron flow during nitrate respiration; light slate gray arrows represent the electron flow during nitrite respiration. Thick arrows denote the primary electron transport mediated by CymA from the quinol pool to nitrate/nitrite reductases, while thin arrows illustrate secondary electron transfer via the cytochrome bc₁ complex. OM, outer membrane; IM, inner membrane.

Fig 2

Aerobic growths of wild type and NDH triple mutants. Growth of wild-type S. oneidensis MR-1, ΔnuoNΔndhΔnqrF1, ΔnuoNΔndhΔnqrF2, ΔnuoNΔnqrF1ΔnqrF2, and ΔndhΔnqrF1ΔnqrF2 strains in defined MS medium with lactate (20 mM) as sole electron donor in aerobic condition. The error bar represents the standard deviation of triplicate experiments. All experiments were carried out at least three times.

Fig 3

Consumption of nitrate and nitrite by wild type and NDH triple mutants under anoxic condition. Nitrate and nitrite utilization of wild-type S. oneidensis MR-1, ΔnuoNΔndhΔnqrF1, ΔnuoNΔndhΔnqrF2, ΔnuoNΔnqrF1ΔnqrF2, and ΔndhΔnqrF1ΔnqrF2 strains in defined MS medium with lactate (20 mM) as sole electron donor and 5 mM nitrate (A) or nitrite (B) as sole EA, respectively, under anoxic conditions. Cultures were sampled at indicated times for nitrate and nitrite detection. The concentrations of nitrate and nitrite reduced from nitrate were shown simultaneously when nitrate was the EA. The error bar represents standard deviation of triplicate experiments. All experiments were carried out at least three times.

Fig 4

Changes in relative transcript levels of NDH genes in response to EA conversion from nitrate to nitrite under anoxic conditions. Relative transcript levels of nqrA1 (A), nqrA2 (B), nuoA (C), and ndh (D) in wild-type strain grown in defined MS medium with nitrate (5 mM) as sole EA in anaerobic condition. The nitrate and nitrite concentration curves were embedded to indicate the EA used at indicated sampling time points. The error bar represents the standard deviation of triplicate experiments. All experiments were carried out at least three times.

Fig 5

Crp and ArcA directly regulate the transcription of nqr1 and nqr2. (A, B) Relative transcript levels of nqrA1 (A) and nqrA2 (B) in wild-type, Δcrp, and its genetically complemented strain Δcrp/crp grown in defined MS medium with 5 mM nitrate or nitrite as EA under anoxic conditions. The error bar represents standard deviation of triplicate experiments. (C, D). Relative transcript levels of nqrA1 (C) and nqrA2 (D) in wild-type, ΔarcA, and its genetically complemented strain ΔarcA/arcA grown in defined MS medium with 5 mM nitrate or nitrite as EA under anoxic conditions. The error bar represents standard deviation of triplicate experiments. (E, F) MST analysis of MBP-Crp binding to nqr1 promoter probe (E) or nqr2 promoter probe (F). (G, H) MST analysis of ArcA-P binding to nqr1 promoter probe (G) or nqr2 promoter probe (H). A two-sided Student’s t-test was used to assess statistically significant differences (^***P < 0.001; ^**P < 0.01; ^*P < 0.05). All experiments were carried out at least three times.

Fig 6

Crp and ArcA directly regulate the transcription of nuo and ndh. (A) Relative transcript levels of nuoA in wild-type, Δcrp, and its genetically complemented strain Δcrp/crp grown in defined MS medium with 5 mM nitrate or nitrite as EA under anoxic conditions. The error bar represents standard deviation of triplicate experiments. (B) MST analysis of MBP-Crp binding to nuo promoter probe. (C) MST analysis of ArcA-P binding to nuo promoter probe. (D) Relative transcript levels of ndh in wild-type, Δcrp, and its genetically complemented strain Δcrp/crp grown in defined MS medium with 5 mM nitrate or nitrite as EA under anoxic conditions. The error bar represents standard deviation of triplicate experiments. (E) MST analysis of MBP-Crp binding to ndh promoter probe. (F) MST analysis of ArcA-P binding to ndh promoter probe. A two-sided Student’s t-test was used to assess statistically significant differences (^**P < 0.01; ^*P < 0.05; ns, P > 0.05). All experiments were carried out at least three times.

Fig 7

Proposed regulatory model of Crp and ArcA on the NDH genes in response to EA conversion from nitrate to nitrite. (A) When nitrate serves as EA, cAMP-activated Crp (cAMP-Crp) binds to the promoter region of nqr1 operon as a repressor and while acting as an activator at the nqr2 operon promoter. Although phosphorylated ArcA (ArcA-P) derepresses transcription of both nqr operons, its derepression effect on nqr1 is counteracted by cAMP-Crp-mediated repression. The coordinated regulation by Crp and ArcA collectively downregulates nqr1 expression and upregulates nqr2 expression. Additional, cAMP-Crp binds to the promoter regions of nuo and ndh, repressing their transcription. (B) When nitrite serves as EA, cAMP-Crp derepresses the transcription of nqr1 while inactivating nqr2. ArcA-P represses both nqr operons; however, its repression effect on nqr1 is counteracted by cAMP-Crp-mediated derepression. This regulatory interplay results in upregulated nqr1 expression and downregulated nqr2 expression during nitrite respiration. Notably, no significant transcriptional regulation of nuo and ndh by Crp or ArcA was observed in nitrite respiration.