Role of norEF in Denitrification, Elucidated by Physiological Experiments with Rhodobacter sphaeroides

Abstract

Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (V max and K s ), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ϳ50% reduction in V max but no change in apparent K s . These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources.

Highly diverse nirK genes comprise two major clades that harbour ammonium-producing denitrifiers

“…The same bifurcating tree in cladogram format with an estimation of reliability based on bootstrap analysis can be found in supplementary information (Additional file 1 ). Physiologically characterized denitrifying strains are indicated by ▼: Bacillus azotoformans LMG 9581, Bacillus bataviensis LMG 21833 [ 39 ], Haloferax denitrificans ATCC 35960 [ 40 ], Hyphomicrobium denitrificans ATCC 51888 [ 77 ], Shewanella loihica PV-4 [ 44 ], Shewanella denitrificans OS217 [ 78 ], Rhodobacter sphaeroides ATCC 17025 [ 79 ], Alcaligenes faecalis S-6 [ 80 ]. The grey ovals represent the two previously proposed clades of NirK sequences

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Modularity of nitrogen‐oxide reducing soil bacteria: linking phenotype to genotype

“…The nitrite accumulation in these isolates was likely due to a temporary imbalance in nitrate and nitrite reducing activities. It has been observed in other bacteria that nitrate reductase activity is the rate limiting step in the pathway, therefore only once Nir becomes active will nitrite be kept at below detectable levels (Bergaust et al , ). In both isolates the NO pulse was likely due to Nir activity preceding Nor (Bergaust et al , ).…”

Nitrous Oxide Metabolism in Nitrate-Reducing Bacteria

“…Inactivation of norEF genes has been shown to slow NO reduction in both Pa. denitrificans and R. sphaeroides 2.4.3 (de Boer et al, 1996;Hartsock & Shapleigh, 2010). Recent physiological experiments have shown that norEF are not essential for Nor activity; however, their absence does affect activity under conditions where endogenous Nir activity generates prolonged exposure to NO (Bergaust, Hartsock, Liu, Bakken, & Shapleigh, 2014).…”