Copper Control of Bacterial Nitrous Oxide Emission and Its Impact on Vitamin B12-Dependent Metabolism

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 between 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 accumulation 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. Colors indicate average log2 normalized expression values between three biological replicates. Black arrows indicate gene orientation. Bold text denotes those genes ratified using qRT-PCR. ID represents unique locus tag of each gene in the P. denitrificans genome (Accession: NC_008686–008688).

NosC and NosR Are both Required for Cu-Mediated Regulation NosR contains the necessary residues for binding Cu, we rea- of nosRZDFYLX. Interestingly and in contrast to the adjacent soned that these proteins might be involved the Cu-dependency nosRZDFYLX nos nosC , the proximal gene in the cluster, was of N2O reduction. up-regulated in response to Cu-limitation. The gene’s product, To test this hypothesis, we deleted nosC and nosR in- NosC, is a hypothetical protein with unknown function and close dependently and examined the phenotypes of these strains dur- (>50% identical) homologs appear to be only distributed among ing anaerobic growth with nitrate as the electron acceptor. In the Paracoccus − other species. Notably, all known homologs of NosC nosR mutant (PD2302), we noted that N2O reduction was vastly contain a CXXCXXC motif that may bind a redox active co- reduced, but not completely abolished in both Cu-H and Cu-L fi − factor, the signi cance of which is unknown. NosR is a trans- conditions, because 50–70% (∼4.5–7 mmol) of the NO3 con- fl − membrane iron-sulfur cluster containing avoprotein required sumed was detected as N2O (Fig. 1). In the nosC mutant for reduction of N2O that also contains two putative metal (PD2301), we noted that N2O reduction was altered in Cu-H – binding CXXXCP motifs (15 17), noted for their ability to bind media, because up to 2 mmols of N2O were detected (Fig. 1). As Cu in some proteins, as discussed below. NosDFYLX are all a control, we generated a nosZ-deficient strain (PD2303) and − thought to be important for NosZ assembly and activation, but saw that ∼90–100% of NO3 was detectable as N2O following − a clear function for these polypeptides is yet to be demonstrated anaerobic growth using NO3 . To further investigate the role of (16). Similarly, previous studies have not formally identified the nosC and nosR genes, we measured the transcription of nosZ a function for the NosR protein (15–17) and its involvement in under both Cu regimes in both mutant strains. Strikingly, we − the Cu-dependency of the Nos system has never been reported. found that nosZ transcript levels in both the nosC strain − Nevertheless, nosR occurs adjacent to nosZ in the vast majority (PD2301) and the nosR strain (PD2302) were similar in Cu-H of denitrifier genomes, underpinning its involvement in N2O and Cu-L conditions (Fig. 4), providing strong evidence that reduction. Given that nosC was differentially expressed in Cu- repression of nosZ during Cu-limitation had become deregu- limited conditions compared with the rest of the nos genes, and lated. This result identifies a role for both NosC and NosR in the

Fig. 4. Expression of selected genes from P. denitrificans, as monitored by qRT-PCR. (A) The effects of either Cu-limitation (Cu-L/Cu-H) or N2O (Cu-H+N2O/Cu-H) − exposure on the expression of a selection of genes identified from transcriptomic studies. (B) Expression of nosZ in wild-type P. denitrificans PD1222, nosC − PD2301, or the nosR PD2302-deficient strains during anaerobic growth and Cu-limitation. (C) The expression of the three-gene cluster involved in Cu-metabolism, during Cu-limitation in aerobic and anaerobic conditions. Bars show SE of three biological replicates.

19928 | www.pnas.org/cgi/doi/10.1073/pnas.1314529110 Sullivan et al. Downloaded by guest on October 1, 2021 Cu-regulation of nosZ, although the mechanisms by which these even in Cu-H media the catalytic activity of NosZ is compro- proteins mediate this Cu-response remains to be established and mised by the loss of pcuC. Given the observed phenotypes of the warrants further investigation. respective mutants (PD2304 – 2306), we predict that the proteins encoded by Pden_4445, pcuC, and senC2 are involved in either Cu-Responsive senC2 and pcuC Are Involved in Cu-Metabolism in insertion or maintenance of the Cu-centers of NosZ. Further- P. denitrificans. The remaining genes highlighted by our tran- more, these proteins may have a role in aerobic Cu metabolism, scriptomic studies were all up-regulated during Cu limitation given that they are also up-regulated in aerobic Cu-limited (Fig. 3). Of these genes, we found a three-gene cluster that plays a conditions (Fig. 4). However, the functions and activities of the pivotal role in anaerobic Cu-metabolism, encoded by Pden_4445, putative Cu-binding proteins PcuC and SenC2 and the role of 4444 (here we term pcuC), and 4443 (here we term senC2). the product of Pden_4445 are yet to be fully established. Pden_4445 encodes a hypothetical protein with unknown function. The product of pcuC is predicted to be secreted to the N2O Modulates Expression of Vitamin B12-Independent Metabolic periplasm by the Sec system and is homologous to the bacterial Pathways. Upon interrogation of the remaining up-regulated Cu(I) protein PCuAC (periplasmic CuA chaperone), including genes, we observed that many have an association with B12- the conserved Cu(I)-binding H(M)X10MX21HXM motif. In dependent metabolism and may be under the control of B12 other bacterial species, PCuAC directs the insertion of Cu(I) into riboswitches, known for their ability to modulate transcription the CuA site of cytochrome ba3 oxidase (18). The product of senC2 of downstream genes dependent on the presence of the B12 ligand is a member of the bacterial homologs of Sco1, involved in the (13, 14, 22–24). Nine putative gene clusters downstream of B12 assembly of the CuA center of cytochrome oxidases in mitochon- riboswitches had elevated expression in Cu-limited conditions dria. The sensor of Cox (SenC)/Sco1 family, including SenC2, (Fig. 3, Fig. S2,andTable S3). These clusters included genes for have a CXXXCP Cu-binding motif that provides for cysteinyl the B12-independent versions of methionine synthase (metEF) coordination of Cu, completed by an axial histidinyl ligand (19–21). and both the class Ib and class III ribonucleotide reductases, Deletions in each of these genes significantly decreased N2O nrdHIEF and nrdDG respectively. Other genes encoded products reduction in Cu-L cultures, reduced the final biomass yield, and in associated with B12-scavenging apparatus, including a cobalt trans- pcuC− the case of the strain PD2305, severely delayed the tran- porter (cbtAB), several iron/B12 ABC-type transporter systems (e.g., − sition into anaerobic metabolism using NO3 (Fig. 5). Interest- btuBCDF), and several hypothetical proteins of unknown function. ingly, mutations in Pden_4445 and senC2 had no effect in Cu-H Because Cu-limited cultures accumulate significant amounts conditions, suggesting that these mutants can be complemented of N2O, a molecule that directly interacts with B12 or B12- − by micromolar Cu levels. However, the pcuC strain PD2305 dependent enzymes (11, 12, 25, 26), we hypothesized that the accumulatedupto1mmolN·N2O in Cu-H media, indicating that up-regulation of these riboswitch-controlled genes was being MICROBIOLOGY

− − − Fig. 5. Growth characteristics of Pden_4445 PD2304, pcuC PD2305, and senC2 PD2306 strains in anaerobic batch culture conditions in Cu-H media (♦)or SCIENCES ■ · − − · Cu-L media ( ). (Left) Optical density (OD600nm), (Center)NNO3 (millimole N in the form of NO3 ), and (Right)NN2O (millimole N in the form of N2O) for ENVIRONMENTAL each respective strain. Bars represent SE between triplicates, and where not visible, these were smaller than the symbols.

Sullivan et al. PNAS | December 3, 2013 | vol. 110 | no. 49 | 19929 Downloaded by guest on October 1, 2021 modulated not by Cu, but by the consequential N2O emitted Vitamin B12-Independent Methionine Synthase MetE Counteracts the during Cu-limitation. To test this theory, we cultured P. deni- Toxicity of N2O. Given that the B12-independent methionine synthase trificans under anaerobic conditions in Cu-H media, in the pres- MetE was up-regulated in the presence of N2O, we postulated that ence and absence of excess N2O, and monitored the response of this enzyme compensates for the loss of functional MetH, which is the nos and other genes by qRT-PCR. Indeed, all of the gene inactivated by N2O. In other bacteria MetE is regulated by a B12 riboswitch (27). To examine the role of MetE, we generated clusters downstream of B12 riboswitches were up-regulated upon − a metE strain (PD2307) and assessed the impact of anaerobic exposure to N2O. Conversely, expression of the nos genes and the senC2 pcuC growth in N2O-genic, Cu-L media on the survival of this mutant. / gene clusters did not change (Fig. 4 and Table S3). metE− fi This finding suggested that up-regulation of B -linked gene The -de cient strain was hampered by growth in Cu-L me- 12 dia, but not in Cu-H media (Fig. 6 and Fig. S3), suggesting that in clusters is linked to the presence of N O, consistent with this 2 conditions in which N O accumulated, growth was signifi- gaseous intermediate accumulating during exponential growth in 2 cantly perturbed. To further demonstrate this phenomenon, we − − the Cu-limited media. Such an interaction between B12 and N2O analyzed the growth of a nosZ metE double-mutant (PD2308), would diminish the active B12 pool and lead to the synthesis of which would accumulate N2O during anaerobic metabolism, irre- B12-independent enzymes to counteract this loss of cofactor. spective of Cu levels. Growth of PD2308 was impaired compared with the wild-type, regardless of Cu-content, with an extended lag Cytotoxicity of N2O Exposure Is Alleviated by the Addition of Vitamin fi fi fi phase and lower nal biomass. This nding further demonstrates B12. To con rm the phenomenon that N2O exhibited a cytotoxic the influence of N2OonaspectsofvitaminB12-linked metabolism effect mediated by the destruction of B12, the atmospheres of (Fig. 6 and Fig. S4). sealed flasks containing Cu-H media was replaced with N2O, In support of our findings, B12-deficiency and hyperhomo- such that each vessel had media and atmosphere completely cysteinemia have been previously reported in humans as a conse- saturated with N2O. These vessels were then inoculated with quence of N O-based anesthesia (28). Under these circumstances P. denitrificans 2 and incubated for 30 h at 30 °C. During this initial both patients and clinicians are exposed to N2O. B12 is essential incubation cultures grew poorly in an N2O atmosphere. After for a variety of mammalian enzymes, which in humans is obtained 30 h additions of B12, L-methionine (L-met), or unsupplemented primarily through diet and from the gastrointestinal microflora. media was made to the vessels, and growth was monitored for Hyperhomocysteinemia and methylmalonic acidemia arise from a further 30 h. Notably, following addition of either B12 or L-met, the malfunction of B12-dependent enzymes required for methi- the growth of P. denitrificans rapidly entered exponential phase onine synthesis and fatty acid catabolism (28, 29), and can be and reached high cell densities, but the culture without either of treated with dietary supplements of B12. these compounds was unable to grow (Fig. 6). This restoration of Concluding Remarks growth confirms that the cytotoxicity of N2O is inextricably linked Bacterial anaerobic denitrification is well characterized at the to the status of L-met/B12 cellular pools, likely mediated through − − structural and biochemical level. Expression of the NO ,NO , the inactivation of the B12-requiring methionine synthase MetH 3 2 by this potent greenhouse gas, as previously reported (11, 12). NO, and N2O reductases are subject to regulation by gas-sensi- tive regulatory proteins (30), which sense O2, NO, or the anion − NO3 (31–33), and regulate a variety of genes involved in the transition to anaerobic respiration (34). For nearly three decades it has been known that Cu concentrations can drastically affect the activity of N2O reductase and the subsequent yield of N2O from denitrifying cells (7, 9, 10). Emerging from this study are two additional features in the biology of this potent green-house gas. Here, we describe evidence for transcriptional modulation of nos expression by Cu-status, mediated through the NosC and NosR proteins. In addition, we reveal a remarkable interplay between N2O emission and the expression of B12 riboswitch- regulated gene clusters that has far-reaching implications. In P. denitrificans,N2O modulates the expression of genes for anaerobic anabolism that are controlled by B12 riboswitches. This result in turn impacts on growth under denitrifying conditions. In other bacteria, such as the human pathogens Enterococcus and Listeria,B12 riboswitches control a variety of responses including pathogenesis and virulence (22, 23). Our data suggest that N2O accumulation is toxic to cellular metabolism at extracellular concentrations as low as ∼0.1 mmol/L. Thus, NosZ may play a previously unrecognized role in N2O resistance in microbial communities and this provides an insight into the function of NosZ in nondenitrifier bacterial genomes (35). The implications are that organisms that live in microbial communities and do not have B12-independent mechanisms, or NosZ, could be compromised by the release of N2O following incomplete denitrification by partial denitrifiers or Fig. 6. Cytotoxicity of N2O can be alleviated by the addition of vitamin B12 fi − during Cu-limitation. This result may give rise to a selection pres- or L-met. (A) Growth of P. denitri cans in anaerobic conditions with NO3 in sure in which nosZ is conserved for reasons other than for the Cu-H conditions with N O-saturated atmospheres. After 30 h (black arrow) 2 generation of proton motive force during denitrification. additions of either vitamin B12 (▲), L-met (■), or unsupplemented media (♦) was made to the cultures, and growth monitored for a further 30 h. B and C − compare the growth of P. denitrificans wild-type (PD1222; ♦), metE (PD2307; Materials and Methods − − ▲), or metE nosZ (PD2308; ■) cultures, grown either in Cu-H conditions (B) For detailed methods, refer to the SI Materials and Methods. Batch cultures

or the N2O-genic Cu-L conditions (C). Bars represent SE between triplicates, of P. denitriﬁcans were grown anaerobically in minimal media supplemented − and where not visible, these were smaller than the symbols. with 30 mmol/L succinate, 10 mmol/L NH4Cl, and 20 mmol/L NO3 , containing

19930 | www.pnas.org/cgi/doi/10.1073/pnas.1314529110 Sullivan et al. Downloaded by guest on October 1, 2021 either 13 μmol/L (Cu-H) or <0.5 μmol/L Cu (Cu-L) in the form of CuSO4,determined by qRT-PCR as described previously (36), conforming to the Minimum In- by inductively coupled plasma optical emission spectrometry. N-oxyanions were formation of Quantitative Real-Time PCR Experiment guidelines. quantified as previously described (7) with minor changes. Mutant strains were generated using pK18mobsacB and selected using a modified Luria-Bertani recipe ACKNOWLEDGMENTS. We thank the US Department of Energy for providing fi containing sucrose. For N2O exposure, vessels containing Cu-H media were satu- the genome sequence of Paracoccus denitri cans PD1222; Verity Lyall and P. Malaka P. De Silva for technical support; and Stuart Ferguson, Rob van rated by sparging with N2 and subsequently with N2O for 15 min. Additions of Spanning, Georgios Giannopoulos, and Sebastian Runkel for discussions. This L-met, AdoCbl, or blank media were added anaerobically after 30 h of N O 2 work was funded by the Biotechnology and Biological Sciences Research exposure. Total RNA was harvested from midexponential phase batch cultures. Council (United Kingdom) (BB/D012384/1, BB/D010942/1, and BB/H012796/1); Minimum Information About a Microarray Experiment-compliant microarrays a Royal Society and Wolfson Foundation Merit award (to D.J.R.); and capital were carried out using RNA from three independent replicates using custom funds for the Wolfson Bioenergy and Fermentation Laboratory at the University designed tiled 44 K microarray slides (Agilent), and transcripts were quantified of East Anglia (D.J.R.).

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