N 2 O production via NO detoxification under anoxic conditions may explain environmental SP values. (A) Measured in situ SP values for environmental sources (Soil, Marine, Freshwater) vs. in vitro measurements of N 2 O-producing biogenic end-members (bacterial and fungal denitrification, AOB, AOA) and N 2 O-producing abiotic reactions; black line shows median; blue lines show end-member values for AOB (8). Histogram height is normalized to each category; see SI Appendix, Fig. S11 for outlier values and more details. (B) Number of bacterial genomes hits at the phylum level for flavohemoglobin protein (Fhp) and nitrous oxide reductase (NorBC) alone or in combination from Annotree (9); minimum amino acid sequence similarity of 30% was used. See SI Appendix, Fig. S1 and Tables S2 and S3 for phylogenetic distribution. (C) Relevant N-oxide pathways of P. aeruginosa UCBPP-PA14 (Pa), the model organism used in this study. Pa possesses the full denitrification pathway as well as Fhp. (D) SP of N 2 O produced by Pa and mutant strains with fhp and/or nosZ genes deleted (ΔnosZΔfhp; ΔnosZ) in denitrifying conditions sampled at late-exponential or late-stationary growth phases; see SI Appendix, Fig. S2 for more details. (E) N 2 O SP of Pa strains with rhamnose-induced expression of norBCD (iNOR) or fhp (iFhp) alone as well as A. baumannii and S. aureus, which only have Fhp. P value was calculated via Welch's t test. Each data point in (D and E) represents an individual biological replicate.

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... median values of in situ SP measurements where microbes are present are 10.9 per mille (‰) for soils, 20.9‰ for marine systems, and 23.0‰ for freshwater habitats (Fig. 1A). These values are bounded by the median values of in vitro, pure culture studies of N 2 O-producing biogenic end-members like bacterial and fungal denitrifiers as well as ammonia-oxidizing bacteria and archaea (AOB and AOA; Fig. 1A). Bacterial and fungal denitrifiers are thought to represent two extremes of SP values for N 2 O ...

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... microbes are present are 10.9 per mille (‰) for soils, 20.9‰ for marine systems, and 23.0‰ for freshwater habitats (Fig. 1A). These values are bounded by the median values of in vitro, pure culture studies of N 2 O-producing biogenic end-members like bacterial and fungal denitrifiers as well as ammonia-oxidizing bacteria and archaea (AOB and AOA; Fig. 1A). Bacterial and fungal denitrifiers are thought to represent two extremes of SP values for N 2 O producers with median SP values of −4.3 and 32.2‰ respectively (Fig. 1A); this is assumed to reflect the activity of dissimilatory nitric oxide reductases (NOR). In AOB, the SP varies between roughly −11 and 36‰ due to multiple pathways of ...

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... of in vitro, pure culture studies of N 2 O-producing biogenic end-members like bacterial and fungal denitrifiers as well as ammonia-oxidizing bacteria and archaea (AOB and AOA; Fig. 1A). Bacterial and fungal denitrifiers are thought to represent two extremes of SP values for N 2 O producers with median SP values of −4.3 and 32.2‰ respectively (Fig. 1A); this is assumed to reflect the activity of dissimilatory nitric oxide reductases (NOR). In AOB, the SP varies between roughly −11 and 36‰ due to multiple pathways of dissimilatory N 2 O formation (8), though recent work showing abiotic N 2 O production through spontaneous hybrid formation (10) may complicate this interpretation. In ...

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... and not for energy conservation (14). Flavohemoglobin proteins (e.g., Fhp/Hmp/Yhb-henceforth referred to as "Fhp") are phylogenetically widespread and protect against NO-mediated toxicity in bacteria and yeast (15). Members of this family are roughly four times more abundant than NORs in annotated bacterial genomes [Fig. 1B and SI Appendix, Fig. S1 and Tables S1-S3; 7,109 vs. 1,854 genome hits at the phylum level for Fhp vs. NOR using 30% minimum amino acid sequence similarity (9)]. While the ability of Fhp to oxidize NO to nitrate (NO 3 − ) under oxic conditions is well known, their capacity to reduce NO to N 2 O under anoxic conditions has received less attention (15,16). Given ...

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... SP Values Reflect NOR during Denitrification. To compare the SP of Fhp to NOR in a whole-cell context (in vivo), we used the model bacterial denitrifier, Pseudomonas aeruginosa UCBPP-PA14 (Pa, Fig. 1C). Because this organism is genetically tractable, it provides a means to study the cellular processes of interest in a controlled manner (Table 1) nosZ (PA14_20200), and/or fhp (PA14_29640)-were grown anaerobically in defined medium batch cultures and sampled at late exponential and late stationary growth phases ( The SP of ΔnosZΔfhp ...

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... (WT) Pa, which can produce N 2 O through both Fhp and NOR (Fig. 1C), displayed SP values that did not vary significantly from those observed for the ΔnosZΔfhp strain across all growth phases when denitrifying (P = 0.7). In addition, the SP of WT Pa did not vary significantly by growth phase (P = 0.07). The SP of ΔnosZ was also measured because prior studies showed that NOS can increase the SP of the ...

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... addition, the SP of WT Pa did not vary significantly by growth phase (P = 0.07). The SP of ΔnosZ was also measured because prior studies showed that NOS can increase the SP of the residual N 2 O pool through preferential cleavage of the 14 N-O vs. 15 N-O bond in N 2 O (21, 22); however, SP values of ΔnosZ were similar to ΔnosZΔfhp (P = 0.7) and did not vary by growth phase (P = 0.8; Fig. 1D). Therefore, even though Fhp was likely present in all previously measured bacterial denitrifier strains for in vitro measurements (SI Appendix, Table S2), it does not affect the overall SP value when strains are grown under denitrifying conditions, suggesting that NOR dominates the isotopic signature under these conditions (Discussion). ...

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... significantly by growth phase (P = 0.07). The SP of ΔnosZ was also measured because prior studies showed that NOS can increase the SP of the residual N 2 O pool through preferential cleavage of the 14 N-O vs. 15 N-O bond in N 2 O (21, 22); however, SP values of ΔnosZ were similar to ΔnosZΔfhp (P = 0.7) and did not vary by growth phase (P = 0.8; Fig. 1D). Therefore, even though Fhp was likely present in all previously measured bacterial denitrifier strains for in vitro measurements (SI Appendix, Table S2), it does not affect the overall SP value when strains are grown under denitrifying conditions, suggesting that NOR dominates the isotopic signature under these conditions ...

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... concentrations (SI Appendix, Fig. S4) and then incubated under anoxic conditions for 24 h at 37 °C before the headspace was sampled; see Table 2 and Materials and Methods for more details. Under these conditions, iFhp displayed SP values (10.45 ± 2.17‰, n = 5) that were significantly more positive than iNOR (−2.60 ± 5.41‰, n = 5; P = 0.004; Fig. 1 E, Top). iNOR values were also consistent with both our ΔnosZΔfhp denitrifying growth SP measurements and prior in vitro NOR SP measurements (18). In addition, we observe a large variation (on the order of 10‰) in SP between biological replicates of NOR, in agreement with prior studies (−5 and −9‰; n = 2 in ref. 18). This variation neither ...

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... A. baumannii has 98.5% similarity. However, all Fhps share a common catalytic site for NO binding and reduction, a globin module with heme B (15), that is responsible for imparting the observed SP. The SP of S. aureus (5.56 ± 7.21‰, n = 3) and A. baumannii (10.38 ± 9.05‰, n = 3) were both positive and statistically indistinguishable from Pa iFhp (Fig. 1 E, Bottom). In addition, the variation in SP values for Fhp (on the order of 10‰) is similar to that of NOR, implying that there is an inherent variation in SP for these enzymes, though addressing this variation is outside the scope of this current study. ...

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... time of measurement, it is plausible that the positive spread in SP values observed in these studies (26) may reflect cryptic Fhp activity. An Fhp homolog, Yhb, exists in yeast (15) and is present in previously studied fungal denitrifiers as well (SI Appendix, Table S4), possibly contributing to the tail toward 10‰ observed from the literature (Fig. 1A), assuming the SP signature of Yhb is similar to that of Fhp. In addition, our isotopic results show that when cells are grown in denitrifying conditions, NOR dominates the SP signal (Fig. 2D). This may explain why prior studies of denitrifiers with Fhp gave SP values consistent with NOR-as a result of growing strains in conditions that ...

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... is primarily used for NO detoxification; OM and IM denote the bacterial outer vs. inner membrane. (B) After aerobic pregrowth, WT Pa was cultured anaerobically via two assay types i) to maximize growth via denitrification ("Denitrifying Growth," Left) or ii) as nongrowing cells in suspension assays ("Anoxic Suspension," Right; see SI Appendix, Fig. S12 for more details). Exogenous NO was supplied through DETA NONOate (red lines) and headspace was then sampled for SP analysis (purple lines). Culture aliquots for proteomics analysis were taken immediately prior to NO addition ("pre-") or during the same time as headspace sampling ("post-NO"). Ratio of Fhp to NOR in these conditions is ...

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... Conditions. iNOR, iFhp, and non-Pseudomonas strains were first screened for N 2 O production before scaling up the culturing process for isotopic measurement (SI Appendix, Table S2). Strains were then grown in one of two ways: i) suspension assays or ii) batch culture (SI Appendix, Fig. S12). For suspension assays, strains were first grown in shaking, aerobic pregrowths for 16 h at 37 °C (OD 600 ~3 to 4) in 150 mL SCFM-A. The aerobic pregrowths for iNOR, iFhp, A. baumannii, and S. aureus were supplemented with 100 mM KNO 3 . In WT Pa suspension assays, pregrowth was supplemented with either 100 μM DETA NONOate, 100 μM ...

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... expression of norBCD or fhp. For WT Pa suspension assays, either 500 μM DETA NONOate or 500 μM DETA NONOate and 100 mM KNO 3 were added. Following the suspension setup, vacuum flask headspace was purged with N 2 gas to establish anoxia, and flasks were incubated statically for 24 h at 37 °C before headspace sampling. See SI Appendix, Fig. S14 for more details. For batch culture assays, strains were first grown in aerobic pregrowths of 5 mL SCFM-A with 100 mM KNO 3 for 16 h at 37 °C, 250 rpm shaking (OD 600 ~ 3 to 4). Cells were then diluted to OD 600 = 0.01 in vacuum flasks with 150 mL of SCFM-A. For ΔnosZ and ΔnosZΔfhp, 100 mM of KNO 3 was added. For WT Pa, either 100 mM ...

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... of KNO 3 was added. For WT Pa, either 100 mM KNO 3 or 500 μM DETA NONOate and 100 mM KNO 3 were added. Since Pa is the only denitrifying organism tested, a concentration sweep of KNO 3 was performed ranging from 20 to 100 mM; no significant growth changes were observed, so we continued with 100 mM to ensure KNO 3 was never limited (SI Appendix, Fig. S19). Then, 100 mM KNO 3 concentrations are also consistent with prior SP studies (i.e., ref. 26), which used high KNO 3 concentrations to ensure adequate amounts of N 2 O were produced for isotopic analysis. Vacuum flask headspace was purged with N 2 gas to establish anoxia and incubated statically at 37 °C. Flasks were sampled twice: ...

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... statically at 37 °C. Flasks were sampled twice: first, approximately 12 h at end-exponential growth and second, approximately 40 h at endstationary. One WT Pa batch culture experiment, where 500 μM DETA NONOate and 100 mM KNO 3 were added to the vacuum flask, was only sampled at ~40 h after the DETA NONOate was added at ~12 h. See SI Appendix, Fig. S12 for more details. Additional moles of nitrate were accidentally added in the Aug192021 batch for a final concentration of 233 mM nitrate (SI Appendix, Table S7); however, no difference in SP was observed (Fig. ...

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... and 100 mM KNO 3 were added to the vacuum flask, was only sampled at ~40 h after the DETA NONOate was added at ~12 h. See SI Appendix, Fig. S12 for more details. Additional moles of nitrate were accidentally added in the Aug192021 batch for a final concentration of 233 mM nitrate (SI Appendix, Table S7); however, no difference in SP was observed (Fig. ...

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... to isotopic analysis. Noncondensable gases (i.e., N 2 , Ar) were removed by submerging the sample in a liquid nitrogen trap and removing the noncondensed gases; carbon dioxide was removed using an Ascarite II CO 2 Absorbent (Thermo Scientific) trap, and water was removed by condensation on an ethanol and dry ice slurry trap; see SI Appendix, Fig. S14 for more details. After headspace distillation, low-volume samples from multiple flasks were combined for subsequent isotopic analysis; see extended dataset for which samples and how many flasks were combined. Two vacuum distillation blanks (DistillationBlank-1, DistillationBlank-2) and a no-cells vacuum flask blank (FlaskBlank-1) were ...

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... measurements were corrected for background noise (17) and 13 C 16 O 2 at Mass 45; see SI Appendix for more details. Shot noise error (17) was calculated for each measurement and compared to the observed SD to ensure measurements were reaching shot noise limits (SI Appendix, Fig. S15). "Zero enrichment" tests where the reference gas is measured as a sample against itself were also regularly performed over the course of the study to ensure pressure balance across a range of sample sizes (SI Appendix, Fig. S16). ...

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... each measurement and compared to the observed SD to ensure measurements were reaching shot noise limits (SI Appendix, Fig. S15). "Zero enrichment" tests where the reference gas is measured as a sample against itself were also regularly performed over the course of the study to ensure pressure balance across a range of sample sizes (SI Appendix, Fig. S16). Two samples were also measured across both HR-IRMS instruments to ensure measurement consistency across instruments (SI Appendix, Fig. S17). Finally, all samples were corrected for "scrambling" following (5,45); see SI Appendix for more ...

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... tests where the reference gas is measured as a sample against itself were also regularly performed over the course of the study to ensure pressure balance across a range of sample sizes (SI Appendix, Fig. S16). Two samples were also measured across both HR-IRMS instruments to ensure measurement consistency across instruments (SI Appendix, Fig. S17). Finally, all samples were corrected for "scrambling" following (5,45); see SI Appendix for more ...

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... and AnnoTree Search Parameters. A phylogram of species with annotated Fhp/Hmp sequences was first made from the NCBI database (SI Appendix, Fig. S1 and Table S13). Phylogram was made to include representative strains from a range of known bacterial species. The amino acid sequence of Fhp from P. aeruginosa UCBPP-PA14 was used (PA14_29640). Default NCBI protein BLAST blastp parameters were used to identify Fhp orthologs. Protein sequences were collected, and a simple phylogeny was ...

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... Cells were collected in 5 mL aliquots from batch denitrifying or anoxic suspension assays immediately prior to DETA NONOate addition (SI Appendix, Fig. S4, red line) or after 24 to 28 h incubation with DETANONOate (SI Appendix, Fig. S12, purple line) and centrifuged at 6,800 x g, and pellets were frozen at −80 °C. Thawed pellets were processed and digested via S-Trap TM (ProtoFi, LLC, Fairport, NY) micro protocol digestion (SI Appendix). LC-MS analysis of digested peptides was performed on an EASY-nLC 1200 (Thermo Fisher Scientific, San Jose, CA) coupled to a Q ...