Copper control of bacterial nitrous oxide emission and its impact on vitamin B ₁₂ -dependent metabolism

Abstract

Global agricultural emissions of the greenhouse gas nitrous oxide (N 2 O) have increased by around 20% over the last 100 y, but regulation of these emissions and their impact on bacterial cellular metabolism are poorly understood. Denitrifying bacteria convert nitrate in soils to inert di-nitrogen gas (N 2 ) via N 2 O and the biochemistry of this process has been studied extensively in Paracoccus denitrificans. Here we demonstrate that expression of the gene encoding the nitrous oxide reductase (NosZ), which converts N 2 O to N 2 , is regulated in response to the extracellular copper concentration. We show that elevated levels of N 2 O released as a consequence of decreased cellular NosZ activity lead to the bacterium switching from vitamin B 12 -dependent to vitamin B 12 -independent biosynthetic pathways, through the transcriptional modulation of genes controlled by vitamin B 12 riboswitches. This inhibitory effect of N 2 O can be rescued by addition of exogenous vitamin B 12 .denitrification | transcription | NosR | NosC G lobal atmospheric loading of the ozone-depleting greenhouse gas, nitrous oxide (N 2 O), is on the increase (1). Molecule for molecule, its radiative potential is ∼300-fold higher than carbon dioxide (2, 3), comprising ∼9% of global radiative forcing by greenhouse gases (4). In addition, atmospheric N 2 O is stable for ∼120 y. Approximately 70% of anthropogenic N 2 O loading arises from agriculture, mainly from the use of nitrogencontaining fertilizers by soil microbes for dissimilatory purposes. Taken together, these features make N 2 O an important target for mitigation strategies (5).N 2 O is an intermediate in the sequential reduction of nitrate (NO 3 − ) to di-nitrogen (N 2 ), via nitrite (NO 2 − ), nitric oxide (NO), and N 2 O, a process known as denitrification (6). Under certain conditions, the final step in denitrification is dispensed with and N 2 O is released into the atmosphere. One limiting factor in this process is copper (Cu) availability, the metal cofactor required by the N 2 O reductase (NosZ) that destroys N 2 O (5, 7, 8). During Cu-limitation the catalytic capacity of the Nos system may be exceeded by the rate of the preceding reactions that generate N 2 O (i.e., NO 3 − , NO 2 − , and NO reduction) and thus, N 2 O is emitted by denitrifying bacteria (7, 9, 10).Much attention has been given to the cytotoxic properties of NO as a free-radical and oxidant, but N 2 O is often described as a relatively inert intermediate of the nitrogen cycle. However, N 2 O exhibits cytotoxicity, as it is known to bind to and inactivate vitamin B 12 (B 12 ), an essential cellular cofactor in B 12 -dependent enzymes involved in methionine and DNA synthesis (11,12). B 12 also acts as a ligand for B 12 riboswitches that modulate gene expression in the absence of this cofactor (13,14). The possible impact of environmental N 2 O emissions on B 12 metabolism in microbiological communities has largely been ignored. As levels of N 2 O increase in the environment, there is a compelling argument for ...

AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans

“…Primers were designed to amplify 100 -150-bp regions of each gene with a T m of ϳ60°C and used at a final concentration of 0.3 M. Quantitative real time PCR was done using Power SYBR Green PCR Master Mix (Applied Biosystems) CFX96 RT-PCR real time system combined with a C1000 thermal cycler (Bio-Rad). Relative transcript abundance was normalized to dnaN, encoding the ␤-subunit of DNA-polymerase III (Pden_0970), a housekeeping gene previously used for P. denitrificans RT-PCR experiments (25). Standard PCR was performed using the forward RT-PCR primer for aztC and the reverse primer for aztD to amplify a 1157-bp product spanning the intergenic region.…”

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

“…Primers were designed to amplify 100 -150 bp regions of each gene with a T m ϳ60°C and used at a final concentration of 0.3 M. Quantitative real-time PCR was done using Power SYBR Green PCR Master Mix (Applied Biosystems) and a CFX96 RT-PCR real-time system combined with a C1000 Thermal cycler (Bio-Rad). Relative transcript abundance was normalized to dnaN, encoding the ␤-subunit of DNA-polymerase III (Pden_0970), a housekeeping gene previously used for P. denitrificans RT-PCR experiments (32).…”

Nitrous oxide and methane emissions from a vetch cropping season are changed by long-term tillage practices in a Mediterranean agroecosystem

“…To gain a better understanding of the underlying processes associated with tillage management practices on N 2 O emissions, the abundance of denitrification bacterial genes (nirK and nirS), denitrification activity and some microbiological characteristics for each tillage were also analyzed in spring season. In addition, soil Cu content was measured because of the important role that it seems to play in the activity of nitrite reductase (Enwall et al 2010) and N 2 O reductase enzymes (Sullivan et al 2013).…”