Copper Control of Bacterial Nitrous Oxide Emission and Its Impact on Vitamin B12-Dependent Metabolism
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Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism Matthew J. Sullivan, Andrew J. Gates, Corinne Appia-Ayme1, Gary Rowley2, and David J. Richardson2 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom Edited by James M. Tiedje, Michigan State University, East Lansing, MI, and approved October 21, 2013 (received for review July 31, 2013) Global agricultural emissions of the greenhouse gas nitrous oxide cultures leads to up-regulation of B12 independent anabolism, a (N2O) have increased by around 20% over the last 100 y, but reg- response indicative of destruction of the B12 pool by N2O. This ulation of these emissions and their impact on bacterial cellular cytotoxicity of N2O is relieved by the addition of exogenous B12. metabolism are poorly understood. Denitrifying bacteria convert nitrate in soils to inert di-nitrogen gas (N2) via N2O and the bio- Results and Discussion chemistry of this process has been studied extensively in Paracoc- Impact of Cu-Limitation on Paracoccus denitrificans Transcription cus denitrificans. Here we demonstrate that expression of the gene Under Denitrifying Conditions. Paracoccus denitrificans was grown − encoding the nitrous oxide reductase (NosZ), which converts N2Oto under anaerobic batch culture conditions with NO3 as electron N2, is regulated in response to the extracellular copper concentra- acceptor in medium containing 13 μmol/L (Cu-H) and 0.5 μmol/L − tion. We show that elevated levels of N2O released as a consequence (Cu-L) copper. Under both culture conditions NO3 was con- of decreased cellular NosZ activity lead to the bacterium switching sumed in a growth-linked fashion, decreasing from ∼8 mmol to from vitamin B12-dependent to vitamin B12-independent biosyn- 0 mmol as the culture density increased (Fig. 1). The growth rate − thetic pathways, through the transcriptional modulation of genes and final yield was identical in both Cu-regimes and NO2 was controlled by vitamin B12 riboswitches. This inhibitory effect of observed at a maximum of 1 mmol and not detectable once N2O can be rescued by addition of exogenous vitamin B12. cultures had reached stationary phase. The key difference be- tween the two was a transient accumulation of N2O in the Cu-L denitrification | transcription | NosR | NosC culture that was not observed in the Cu-H culture. This ac- cumulation reached a maximum of around 2 mmol N·N2O (Fig. lobal atmospheric loading of the ozone-depleting green- 1). These results suggest that in the Cu-L cultures the catalytic capacity of the Cu-dependent Nos system is transiently exceeded Ghouse gas, nitrous oxide (N2O), is on the increase (1). ∼ by the rate of the reactions that generate nitrous oxide (i.e., Molecule for molecule, its radiative potential is 300-fold higher − − than carbon dioxide (2, 3), comprising ∼9% of global radiative NO3 ,NO2 , and NO reduction) and is consistent with other observations that Cu limitation can lead to nitrous oxide release forcing by greenhouse gases (4). In addition, atmospheric N2Ois by denitrifying bacteria (7, 9, 10). stable for ∼120 y. Approximately 70% of anthropogenic N2O P. denitrificans has four distinct multidomain metalloproteins loading arises from agriculture, mainly from the use of nitrogen- − − containing fertilizers by soil microbes for dissimilatory purposes. for the reduction of NO3 ,NO2 ,NO,andN2O (Fig. 2), which are encoded by narGHJI (for nitrate reductase), nirSECFDGHJN Taken together, these features make N2O an important target for mitigation strategies (5). fi N2O is an intermediate in the sequential reduction of nitrate Signi cance − − (NO3 ) to di-nitrogen (N2), via nitrite (NO2 ), nitric oxide (NO), fi and N2O, a process known as denitri cation (6). Under certain Global atmospheric loading of nitrous oxide (N2O) is on the conditions, the final step in denitrification is dispensed with and increase. This stable, long-lived greenhouse gas is a major ’ N2O is released into the atmosphere. One limiting factor in this contributor to radiative forcing by Earth s atmosphere. Here process is copper (Cu) availability, the metal cofactor required we describe the genetic regulation of N2OreductasenosZ, by the N2O reductase (NosZ) that destroys N2O (5, 7, 8). During encoding the only known N2O-removing enzyme that limits Cu-limitation the catalytic capacity of the Nos system may be the release of this denitrification intermediate during the bac- exceeded by the rate of the preceding reactions that generate terial usage of nitrogenous fertilizers. Expression of nosZ is − − N2O (i.e., NO3 ,NO2 , and NO reduction) and thus, N2Ois down-regulated in copper-limited environments, leading to net emitted by denitrifying bacteria (7, 9, 10). emission of N2O. This cytotoxic N2O emission subsequently Much attention has been given to the cytotoxic properties of modulates expression of genes controlled by vitamin B12 NO as a free-radical and oxidant, but N2O is often described as riboswitches, because N2O binds to and inactivates vitamin a relatively inert intermediate of the nitrogen cycle. However, B12.CytotoxicityofN2O can be relieved by the addition of N2O exhibits cytotoxicity, as it is known to bind to and inactivate vitamin B12. This interaction provides a role for NosZ in N2O- fi vitamin B12 (B12), an essential cellular cofactor in B12-dependent detoxi cation in nondenitrifying bacteria. enzymes involved in methionine and DNA synthesis (11, 12). B12 Author contributions: M.J.S., A.J.G., G.R., and D.J.R. designed research; M.J.S. and C.A.-A. also acts as a ligand for B12 riboswitches that modulate gene performed research; M.J.S., A.J.G., G.R., and D.J.R. analyzed data; and M.J.S., A.J.G., G.R., expression in the absence of this cofactor (13, 14). The possible and D.J.R. wrote the paper. impact of environmental N O emissions on B metabolism in 2 12 The authors declare no conflict of interest. microbiological communities has largely been ignored. As levels This article is a PNAS Direct Submission. of N2O increase in the environment, there is a compelling argu- Data deposition: The data reported in this paper have been deposited in the Gene Ex- ment for better understanding the impact of this gaseous inter- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48577). mediate on the regulation of cellular metabolism in denitrifying 1Present address: Department of Molecular Microbiology, John Innes Centre, Norwich bacteria, particularly in Cu-limited culture conditions that are NR4 7UH, United Kingdom. associated with N2O emissions (7, 9). Here we link these two 2To whom correspondence may be addressed. E-mail: [email protected] or d.richardson@ important issues in bacterial N2O research and show that the nos uea.ac.uk. genes(forN2O reduction) are strongly regulated by Cu and that the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. accumulation of only micromole amounts of N2O in Cu-deficient 1073/pnas.1314529110/-/DCSupplemental. 19926–19931 | PNAS | December 3, 2013 | vol. 110 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1314529110 Downloaded by guest on October 1, 2021 − − − Fig. 1. Growth characteristics of P. denitrificans PD1222, nosC PD2301, nosR PD2302, and nosZ PD2303 strains in anaerobic batch culture conditions in − − Cu-H media (♦) or Cu-L media (■). (Left) Optical density (OD600nm), (Center)N·NO3 (millimole N in the form of NO3 ), and (Right)N·N2O (millimole N in the form of N2O) for each respective strain. Bars represent SE between triplicates, and where not visible, these were smaller than the symbols. (for nitrite reductase), norCBQDEF (for nitric oxide re- the copper-sulfide redox centers CuA and CuZ (8). To examine ductase) and nosCRZDFYLX (for nitrous oxide reductase) if Cu limitation has an impact on the denitrification genes at the loci, respectively. NosZ is the catalytic subunit of the N2O level of transcription, global gene-expression analyses were per- reductase and binds 12 Cu ions per functional homodimer in formed using total RNA isolated from anaerobically grown cells under either Cu-H or Cu-L conditions. Cultures were compared at midexponential growth, where in Cu-L media, N2O levels had exceeded 100 μmoles per flask (Fig. 1). Under these conditions, only 41 genes were differentially expressed (Fig. 3) and the expression levels of a selection of these were verified by quanti- tative RT-PCR (qRT-PCR) (Fig. 4 and Tables S1–S3). There was MICROBIOLOGY no significant change in any of the nar, nir,ornor genes encoding − the enzymes for NO3 reduction to N2O, all of which were highly expressed under both culture conditions (Fig. S1). In contrast, nosRZDFYLX − Cu-limitation had a major effect on the genes, Fig. 2. Reactions of denitrification in which NO3 is sequentially reduced to required for the functional N2O reductase system, the expression N . Above each arrow is the metallo-enzyme complex and below is the metal 2 of which decreased by 8- to 25-fold in the Cu-limited cultures cofactor required for each reaction in P. denitrificans.N2O reduction is car- SCIENCES fi (Fig. 3 and Table S3). This result demonstrates that the nos ried out by Nos (reaction 4), the only denitri cation enzyme dependent on ENVIRONMENTAL Cu in this model denitrifier. genes are subject to regulation by Cu. Sullivan et al. PNAS | December 3, 2013 | vol. 110 | no. 49 | 19927 Downloaded by guest on October 1, 2021 − Fig. 3. Heat map representing the expression level of genes of P. denitrificans PD1222, grown anaerobically with NO3 and either 13 μmol/L (Cu-H) or 0.5 μmol/L (Cu-L) Cu in the media. (A) Genes regulated by B12 riboswiches that are modulated by N2O. (B) The Cu-responsive genes for N2O reduction and Cu- metabolism.