Cyanate as energy source for nitrifiers

Ammoniaand nitrite-oxidizers are collectively responsible for the aerobic oxidation of ammonia via nitrite to nitrate and play essential roles for the global biogeochemical nitrogen cycle. The physiology of these nitrifying microbes has been intensively studied since the first experiments of Sergei Winogradsky more than a century ago. Urea and ammonia are the only recognized energy sources that promote the aerobic growth of ammonia-oxidizing bacteria and archaea. Here we report the aerobic growth of a pure culture of the ammonia-oxidizing thaumarchaeote Nitrososphaera gargensis1 on cyanate as the sole source of energy and reductant, the first organism known to do so. Cyanate, which is a potentially important source of reduced nitrogen in aquatic and terrestrial ecosystems2, is converted to ammonium and CO2 by this archaeon using a cyanase that is induced upon addition of this compound. Within the cyanase gene family, this cyanase is a member of a distinct clade that also contains cyanases of nitrite-oxidizing bacteria of the genus Nitrospira. We demonstrate by co-culture experiments that these nitrite-oxidizers supply Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http:// www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials should be addressed to Michael Wagner, Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria, [email protected], Phone: +43 1 4277 76600, Fax: +43 1 4277 876601. †current address: ZIEL Research Center for Nutrition and Food Sciences, TU München, Gregor-Mendel-Str. 2, 85354 FreisingWeihenstephan, Germany ‡current address: Agrophysical Research Institute, 195220, Saint-Petersburg, Grazhdanskiy pr., 14, Russia Author Contributions. M.Pa., P.H. and A.G. performed experiments with N. gargensis; C.H. and I.L. carried out analysis of metagenomic datasets and proteomics data; N.J. and M.vB. performed proteomics measurements and data analysis; M.Po. and H.K. performed all experiments with N. moscoviensis; H.K. did genomic analysis of ammoniaand nitrite-oxidizing organisms; S.M.K. contributed to metagenomic data analysis; D.B. performed cyanate decomposition modeling; M.W., H.D., and M.Pa. designed the study and analysed data. M.W. wrote the paper. All authors discussed the results and commented the manuscript. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. The authors declare no competing financial interests. Europe PMC Funders Group Author Manuscript Nature. Author manuscript; available in PMC 2016 February 06. Published in final edited form as: Nature. 2015 August 6; 524(7563): 105–108. doi:10.1038/nature14856. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts ammonia-oxidizers lacking cyanase with ammonium from cyanate, which is fully nitrified by this consortium through reciprocal feeding. Screening of a comprehensive set of more than 3,000 publically available metagenomes from environmental samples revealed that cyanase-encoding genes clustering with the cyanases of these nitrifiers are widespread in the environment. Our results demonstrate an unexpected metabolic versatility of nitrifying microbes and suggest a previously unrecognized importance of cyanate for N-cycling in the environment. Cyanate is a small molecule containing carbon, nitrogen, and oxygen atoms. It is formed spontaneously within cells from urea and carbamoylphosphate3,4, but also occurs in the environment where it may be produced from the (physico)chemical decomposition of urea or cyanide5,6. Until recently, environmental cyanate concentrations were virtually unavailable as analytical methods were inadequate for sub-micromolar detection. Furthermore, cyanate is not chemically stable and decomposes relatively slowly to ammonium and carbon dioxide. This decomposition rate is linearly related to the concentration of cyanate and thus cyanate is rather stable at low concentrations (Extended Data Figure 1). A more sensitive chromatographic method for the detection of cyanate in aquatic samples was very recently developed and revealed nanomolar-range cyanate concentrations in seawater6. These cyanate levels are in the same order of magnitude as ammonium concentrations typically found in oligotrophic marine environments7. Consistently, cyanate has been postulated to serve as a nitrogen source for the growth of certain marine cyanobacteria under nitrogen limitation2,8. For the assimilation of cyanate, these phototrophic bacteria convert it to ammonium (and CO2) by a dedicated enzyme called cyanate lyase or cyanase. Cyanases are also found in a variety of other bacteria and archaea where they have been reported to play a role in nitrogen assimilation or detoxification as cyanate chemically modifies proteins via carbamylation9,10. However, no microbe has been described that can grow on cyanate as source of energy and reductant. Nitrifying microbes are generally considered to be highly specialized chemolithoautotrophs that oxidize either ammonia or nitrite for generating energy and reductant for growth and use CO2 as carbon source. Over the last decades, this perception has been challenged by several studies11-13. For example it was reported that uncultured thaumarchaeota closely related to the described ammonia-oxidizer N. gargensis thrive in wastewater treatment plants by using other (unknown) sources of energy and reductant than ammonium or urea14 and that nitrite-oxidizers of the genus Nitrospira can derive energy for growth by aerobic hydrogen oxidation15. Furthermore, the growth of some thaumarchaeotal ammonia-oxidizers is stimulated by the addition of organic compounds16 and others may be obligate mixotrophs17. However, aerobic growth of ammonia-oxidizing microbes has still only been demonstrated in the presence of urea or ammonium. Recently we sequenced the genome of the thaumarchaeotal ammonia-oxidizer N. gargensis that was enriched from a thermal spring sample1. Unexpectedly, a gene encoding a putative cyanase was detected in this genome close to the gene of a putative cyanate/nitrite/formate transporter18. In contrast, all other sequenced genomes of archaeal or bacterial ammoniaoxidizers including its closest relative N. viennensis19 do not contain a cyanase-encoding gene. As N. gargensis shares most central metabolic pathways with other thaumarchaeotes it Palatinszky et al. Page 2 Nature. Author manuscript; available in PMC 2016 February 06. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts is very unlikely that its cyanase is required for detoxification of internally produced cyanate. We therefore hypothesized that N. gargensis might use cyanate as a source of energy and reductant for growth. Prior to testing our hypothesis a pure culture of N. gargensis was obtained by repeated serial dilutions over a period of 16 months (Supplementary Information 1). The pure culture of N. gargensis grew well in the presence of 2 mM ammonium and growth was not inhibited by addition of 0.5 mM cyanate. After a few days of growth in the presence of both compounds, biomass of N. gargensis was transferred to a medium containing cyanate as the only source of energy, reductant and nitrogen. In this medium, N. gargensis stochiometrically converted cyanate via ammonium to nitrite (Figure 1) and cyanate degradation was the rate-limiting step of the overall process (Extended Data Figure 1). A much slower cyanate conversion to ammonium reflecting chemical decay was observed in control experiments with equal amounts of dead biomass of N. gargensis (Figure 1). Importantly, growth of N. gargensis in the medium containing cyanate as the sole source of energy and reductant was demonstrated by total protein measurements (Figure 1) and by a qPCR assay targeting its 16S rRNA gene (Extended Data Figure 2). During growth on 0.5 mM cyanate, N. gargensis showed according to total protein measurements a mean generation time of 136.3 h (+/− 11.4 SD), which is slightly higher than the mean generation time observed during growth on 0.5 mM ammonium, which was determined to be 113.4 h (+/− 6.1 SD). This difference might reflect toxicity of cyanate despite the presence of a cyanase or the additional energy demand for the synthesis of cyanase during growth on this compound. Proteomic analyses revealed that upon exposure of N. gargensis (that had not been exposed to cyanate) to 0.5 mM cyanate for 48h, the cyanase of N. gargensis was the most strongly induced protein (Extended Data Figure 3; 32× fold change; mean from triplicates), confirming its key role for growth on this source of energy and reductant. Interestingly however, the putative cyanate/nitrite/formate transporter encoded in the same genomic region was not detected, despite the fact that a protocol optimized for extraction of membrane proteins was applied. This is likely caused by the fact that cyanate at mM concentration diffuses through biological membranes20. Interestingly, cyanate conversion by N. gargensis was also observed without a previous growth period in the presence of ammonium and cyanate. In addition, cyanate conversion to nitrite by N. gargensis could also be detected at a 10x lower concentration of the compound (0.05 mM) (Extended Data Figure 4). While N. gargensis is the only ammonia-oxidizing microbe with a sequenced genome in which a cyanase is present that was likely acquired from a Nitrospira strain via lateral gene transfer18, all nitrite-oxidizers for which a genome sequence is available contain a gene annotated as cyanase (Extended Data Table 1). To test whether these genes are functional, experiments were performed with a pure culture of the nitrite-oxidizer Nitrospira moscoviensis that possesses a cyanase closely related to the respective enzyme of N. gargensis. After 96 h of incubation in the presence of around 1 mM cyanate N. moscoviensis degraded significantly more cyanate leading to ammonium released from the cells than a negative control that included an identical amount of dead biomass of this strain (Extended Data Figure 5). Consequently, N. moscoviensis is capable of cyanate degradation. In a separate experiment addition of 1 mM cyanate only slightly decreased nitrite oxidation rates in N. moscoviensis, while higher concentrations showed a stronger effect (Extended Data Palatinszky et al. Page 3 Nature. Author manuscript; available in PMC 2016 February 06. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts Figure 6). The presence of a cyanase in the genomes of all nitrite-oxidizers might reflect that these nitrifiers make more cyanate as a side product of their metabolism than ammoniaoxidizing microbes. Cyanate is produced from both carbamoylphosphate metabolism and urea formation, and while the enzymatic repertoire involved in these processes is highly similar between ammoniaand nitrite-oxidizers, many members of the latter group (but also some thaumarchaeotes) do not contain enzymes for degradation of internally produced urea (Extended Data Table 1). In addition, nitrite-oxidizers might continuously import cyanate from the environment as some of their transporters for the uptake of nitrite from the environment also transport cyanate21. In both scenarios the presence of a cyanase is beneficial for nitrite-oxidizers as it allows them to detoxify cyanate and as the formed ammonium is not only available for assimilation, but after secretion (Extended Data Figure 5) might also serve as source of energy and reductant for ammonia-oxidizers which typically grow in close vicinity to nitrite-oxidizers22,23. The activity of the ammonia-oxidizers will lead to nitrite formation that can then be consumed by the nitrite-oxidizers (Figure 2a). This reciprocal feeding would enable nitrite-oxidizers as well as ammonia-oxidizers without a cyanase to convert cyanate for energy and reductant generation. We experimentally tested this hypothesis by establishing a co-culture of the ammonia-oxidizing bacterium Nitrosomonas nitrosa Nm9024 that has no cyanase activity but is not inhibited in its activity by 1 mM cyanate (Extended Data Figure 7) with the cyanase-encoding nitrite-oxidizer N. moscoviensis. Consistent with the reciprocal feeding hypothesis, this co-culture stoichiometrically converted cyanate to nitrate (Figure 2c; Extended Data Figure 8) and fluorescence in situ hybridization with specific 16S rRNA-targeted probes revealed that dense clusters containing both nitrifiers had formed (Figure 2b). Cyanate conversion rates to nitrate by this consortium could be accelerated by adding ammonium at the start of the experiment in order to allow consortium members to gain energy and reductant before interspecies cyanate degradation was fully established (Figure 2d). In contrast no nitrate formation was observed in abiotic control experiments using the same medium (Extended Data Figure 9). The cyanases found in N. gargensis and members of the genus Nitrospira form a deepbranching clade to the exclusion of other cultured organisms18. We searched a collection of 3,000 metagenomic datasets available from IMG25 and identified 225 additional metagenomic cyanase gene (fragments) that are related to the cyanases of these known nitrifiers (Figure 3). These findings show that the novel cyanase family is widespread in the environment. Most of these cyanases were located on very small contigs preventing an independent phylogenetic classification of the organisms carrying these genes. The metagenomic cyanase fragments most closely related to N. gargensis (47-55 % amino acid similarity) were retrieved from three different peat and permafrost soils in Alaska, while the sequences most closely affiliated with Nitrospira cyanases (67-80 % amino acid similarity) were mostly found in temperate forest and agricultural soil from lower latitudes as well as in lakes, freshwater sediment and groundwater, matching the known distribution of Nitrospira in a broad range of different ecosystems26 (Figure 3). Our findings show that an archaeal ammonia-oxidizer can grow on cyanate as the sole source of energy, reductant and nitrogen. Furthermore, nitrite-oxidizers of the genus Palatinszky et al. Page 4 Nature. Author manuscript; available in PMC 2016 February 06. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts Nitrospira (and likely all nitrite-oxidizers) convert cyanate to ammonium and are capable of fully nitrifying it by a new type of reciprocal feeding with cyanase-negative ammoniaoxidizers. This metabolic capability potentially provides them with a selective advantage in environments where cyanate is present, in particular if ammonium concentrations are low, and thus might be an important facet of the ecology of nitrifiers. Cyanate forms spontaneously by isomerization of urea in aqueous solution. The high concentration of urea in many ecosystems (ranging from polar seawater and sea ice27 to the huge areas of ureafertilized soils in global agriculture) combined with the wide distribution of nitrifier-related cyanase genes underscores the potential environmental ubiquity of this unique physiology.