Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
1
Title: Widespread detoxifying NO reductases impart a distinct isotopic
fingerprint on N2O under anoxia
Authors: Renée Z. Wang1†, Zachery R. Lonergan2†, Steven A. Wilbert2,3, John M. Eiler*1,
Dianne K. Newman1,2*
Affiliations:
5
1Division of Geological and Planetary Sciences, Caltech; Pasadena, 91101, USA.
2Division of Biology and Biological Engineering, Caltech; Pasadena, 91101, USA.
3Current Address: Department of Environmental Health and Engineering, Johns Hopkins;
Baltimore, 21218, USA
*Corresponding authors. Email: eiler@caltech.edu, dkn@caltech.edu
10
†These authors contributed equally to this work.
Abstract: Nitrous oxide (N2O), a potent greenhouse gas, can be generated by compositionally
complex microbial populations in diverse contexts. Accurately tracking the dominant biological
sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well 15 as inform the diagnosis of human infections. Isotopic “Site Preference” (SP) values have been
used towards this end, as bacterial and fungal nitric oxide reductases produce N2O with different
isotopic fingerprints. Here we show that flavohemoglobin, a hitherto biogeochemically neglected
yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N2O
under anoxic conditions that correlates with typical environmental N2O SP measurements. We 20 suggest a new framework to guide the attribution of N2O biological sources in nature and
disease.
One-Sentence Summary: Detoxifying nitric oxide reductases impart a distinct isotopic
biosignature on nitrous oxide.
25
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
2
Main Text:
Nitrous oxide (N2O) is a ubiquitous metabolite present in myriad environments ranging from
soils, marine and freshwater systems, and the atmosphere to the human body. Because N2O can
be produced and consumed by multiple microbial nitrogen-cycling processes (1), tracking its
sources and fates is challenging. One motivation to do so springs from the fact that N2O is a 5 potent greenhouse gas, whose current atmospheric concentration is more than 20% compared to
preindustrial levels (2); a better understanding of N2O sources could help facilitate mitigation
efforts. Analogously, because N2O has been measured in chronic pulmonary infections (3),
clarity on which pathogens are metabolically active in disease contexts could inform treatment
strategies (4). 10
An intramolecular isotopic fingerprint called “Site Preference” (SP), which measures the relative
enrichment of natural abundance 15N in the central (
) versus terminal (
) nitrogen position in
N2O (Fig. 1A; (5)) may be applied for such purposes. Unlike traditional natural abundance
isotopic measurements of the total 15N in the bulk molecule (6), SP does not rely on the isotopic
composition of the source substrate but instead reflects the reaction mechanism (7), making it a 15 potentially powerful tool to disentangle N2O sources in different contexts.
The median values of in situ SP measurements where microbes are present are 10.9, 20.9 and
23.0 per mille (‰) for soils, marine and freshwater systems, respectively (Fig. 1A). These values
are bounded by the median values of in vitro, pure culture studies of N2O-producing biogenic
end-members like bacterial and fungal denitrifiers as well as ammonia-oxidizing bacteria (AOB;
20 Fig. 1A). Bacterial and fungal denitrifiers are thought to represent two extremes of SP values for
N2O producers with median SP values of -4.3 and 32.2‰ respectively (Fig. 1A), which are
assumed to reflect the activity of dissimilatory Nitric Oxide Reductases (NOR); in AOBs, the SP
varies between roughly -11 and 36‰ due to multiple dissimilatory N2O formation pathways (8).
Because the vast majority of in situ environmental observations lie between end-member values
25 for bacterial and fungal NORs and AOBs, the SP values of biogenic N2O produced in the
environment have been rationalized by mixing of these end-members, which assume the activity
of catabolic pathways that are tied to microbial growth.
However, an entire other class of enzymes exists that produce N2O as a consequence of nitric
oxide (NO) detoxification and not for energy-conservation (9). Flavohemoglobin proteins (e.g. 30 Fhp/Hmp/Yhb–henceforth referred to as “Fhp”) are phylogenetically widespread and protect
against nitrosative stress in bacteria and yeast (10). Members of this family are roughly four times
more abundant than NORs in annotated bacterial genomes (Fig. 1B, Fig. S1, Tables S1-3; 7109
vs. 1854 genome hits at the phylum level for Fhp vs. NorBC using 30% minimum amino acid
sequence similarity (11)). While their ability to oxidize NO to nitrate (NO3-) under oxic 35 conditions is well known, their ability to reduce NO to N2O under anoxic conditions has received
less attention (10, 12). Given that bacterial denitrifiers commonly possess both Fhp and NOR (Fig.
1B and Table S1), we hypothesized that Fhp might play a role in N2O emissions and set out to
determine whether it imparts a SP onto N2O distinct from that of bacterial or fungal NORs.
Overall SP values reflect NOR during denitrification
40
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). To determine SP values under denitrifying conditions, wild type (WT) Pa,
Δ
nosZ and
Δ
nosZ
Δ
fhp – strains with deletions of the nitrous oxide reductase (NOS) gene, nosZ 45
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
3
(PA14_20200) and/or fhp (PA14_29640) – were grown anaerobically in defined medium batch
cultures and sampled at late exponential and late stationary growth phase (Table 2, Fig. S2 and
Methods). N2O was cryogenically distilled and analyzed for nitrogen and oxygen isotopes on the
Thermo Scientific Ultra High-Resolution Isotope Ratio Mass Spectrometer (HR-IRMS; (13);
Methods). All isotope data is reported in the delta (
δ
) notation in units of per mille (‰) where 5
δ
15N = [(15N/14N)sample / (15N/14N)reference - 1]*1000 and SP =
δ
15N
-
δ
15N
. Values are reported
relative to the international reference of AIR for nitrogen; see Methods for more detail.
The SP of
Δ
nosZ
Δ
fhp should only reflect NOR, since all other known pathways for N2O
production and consumption were deleted. The in vivo SP of this strain did not vary significantly
by growth phase (Welch’s t-test, P=0.2), and its average value across all growth phases (-2.53 ±
10 2.90, mean ± s.d. throughout, n = 10) was consistent with prior in vitro measurements of NOR
purified from Paracoccus denitrificans ATCC 35512 (-5.9 ± 2.1‰, (14)). The SP of the
Δ
nosZ
Δ
norBC strain, which only has fhp, was not measured because it did not grow appreciably
under these conditions (Fig. S2) presumably due to growth suppression when NO build-up is too
high (15, 16). 15
WT Pa, which can produce N2O through both Fhp and NOR (Fig. 1C), displayed SP values that
did not vary significantly from those observed for the
Δ
nosZ
Δ
fhp 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
Δ
nosZ was also measured because prior studies showed that NOS can
increase the SP of the residual N2O pool through preferential cleavage of the 14N-O vs. 15N-O 20 bond in N2O (17, 18); however, SP values of
Δ
nosZ were similar to
Δ
nosZ
Δ
fhp (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 (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. However, the potential for Fhp to
25 impact the SP of N2O under other conditions remained open.
Fhp has a intermediate, positive SP value compared to bacterial and fungal NORs
To distinguish the SP of Fhp and NOR, we engineered two Pa strains possessing only Fhp or
NOR that could be induced in the presence of rhamnose; inducible Fhp (“iFhp”) and NOR
(“iNOR”) functionality was validated by complementation experiments (Table 1, Fig. S3). Since
30 these strains lack denitrification enzymes and are incapable of anaerobic growth, suspension
assays were developed to culture bacteria aerobically while inducing gene expression prior to
placement in non-growing, anoxic conditions. Strains were provided exogenous NO via the small
molecule donor DETA NONOate (C4H13N5O2) at sub-toxic concentrations (Fig. S4) and then
incubated under anoxic conditions for 24 hours at 37°C before the headspace was sampled; see
35 Table 2 and Methods for more detail.
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. 1E, top). iNOR values were also
consistent with both our
Δ
nosZ
Δ
fhp measurements and prior in vitro NOR measurements (14). In
addition, we observe a large variation (on the order of 10‰) in SP between biological replicates
40 of NOR, in agreement with prior studies (-5 and -9‰; n = 2 in (14)). This variation neither
correlates with the degree of nitrate consumption for
Δ
nosZ
Δ
fhp, nor N2O production for
Δ
nosZ
Δ
fhp and iNOR (Fig. S5), indicating that this variation in SP may be inherent to NOR.
Next, to validate Fhp SP values outside Pa, two WT, non-denitrifying strains with only Fhp,
Staphylococcus aureus USA300 LAC and Acinetobacter baumannii ATCC 17978 were also
45
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
4
measured. Fhp from S. aureus has 31.6% amino acid sequence similarity to Fhp from P.
aeruginosa, while Fhp from 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 (10), 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
5 iFhp (Fig. 1E, bottom).
Exogenous NO shifts SP values towards Fhp
Given the potential for Fhp to impart a positive SP distinct from NOR, we next sought to identify
physiological conditions where it might dominate the N2O isotopic fingerprint in the WT.
Historically, N2O isotopic measurements from pure cultures have been made for actively 10 growing cells, which would be expected to amplify isotopic signatures imparted by catabolic
enzymes like NOR. Yet evidence is mounting that slow, survival physiology dominates
microbial existence in diverse habitats (19, 20), motivating N2O SP measurement during non-
growth conditions.
To test if Pa can produce positive SP values indicative of Fhp activity, we grew WT Pa in
15 denitrifying batch cultures and non-growing, anoxic suspensions with varying combinations of
nitrate (NO3-) and DETA NONOate to provide NO endogenously via denitrification and/or
exogenously via small molecule-mediated NO release (Fig. 2A), which we hypothesized would
promote NOR or Fhp activity, respectively. We validated the induction of NOR and Fhp using
quantitative unlabeled proteomics (Methods) and calculated the ratios of Fhp to NOR to quantify
20 relative changes of each NO reductase. In denitrifying, batch culture conditions (Fig. 2B), the
ratio of Fhp to NOR was less than one (~0.25) and did not significantly change upon addition of
NO (P = 0.09; Fig. 2B). By contrast, NorB, which contains the catalytic subunit of NOR, was
undetectable before NO addition in the suspension assays (Fig. S6), which were performed by
shifting oxic pre-grown cultures to non growing, anoxic conditions. Although NorB increased to
25 detectable levels upon the addition of DETA NONOate (Fig. S6), Fhp was far more abundant,
leading to a high ratio of Fhp to NOR (~3, Fig. 2C).
Paired SP and
δ
15Nbulk data allowed us to track which pool of NO was used by Fhp or NOR for
N2O production (Fig. S7, Fig. 2C,D). When N-oxides are reduced to N2O,
δ
15Nbulk retains the
isotopic signature of the original N(21)(21). The NO3- and DETA NONOate used in our 30 experiments had distinct
δ
15N values (0.40 ± 1.28 vs. -22.95 ± 0.15‰ respectively); non-WT Pa
strains grown as batch cultures with only nitrate or incubated as suspension assays with DETA
NONOate retained these distinct signatures in their
δ
15Nbulk values (-27.4 ± 1.4 vs. -91.0 ± 6.5‰
respectively, Fig. S8). All strains exhibit a large (roughly -70‰) of bulk
δ
15N when producing
N2O from exogenous NO sourced from DETA NONOate, whether NOR or Fhp is utilized; this 35 compares with typical bulk
δ
15N fractionations of -10 to -30 ‰ between source nitrate and
product N2O in prior studies of biological nitrate reduction (22). Thus, the large amplitude
contrast in
δ
15N between N2O produced from nitrate vs. exogeneous NO in our experiments
reflects the combined influences of the difference in
δ
15N between our nitrate and NO substrates,
and the difference in overall isotopic fractionations between the nitrate-to-N2O and the NO-to-40 N2O reactions. When WT Pa was incubated anoxically with either NO3- or DETA NONOate,
δ
15Nbulk values correspondingly showed only one NO source (Fig. 2C); when given both
substrates simultaneously, N2O could be made from varying ratios of both exogenous and
endogenous NO.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
5
SP data (Fig. 2D) was consistent with denitrifying batch cultures favoring NOR production, and
non-growing, anoxic suspension assays favoring Fhp. When WT Pa was grown under
denitrifying conditions, SP values were more negative and within the range of iNOR. However,
in suspension assays, SP values spanned the range from iNOR to iFhp, consistent with increased
Fhp abundance in these conditions. The most positive SP values, within the range of iFhp, were
5 seen when WT Pa was given a high dose of both endogenous and exogenous NO in oxic pre-
growth (NO3- and DETA NONOate, blue stars, Fig. S7) followed by anoxic incubation with
exogenous NO (DETA NONOate only, blue circles, Fig. 2D).
Consequences for interpreting existing SP data
Because Fhp homologs are present in many denitrifying bacteria and AOB (Fig. S1, S9, Tables
10 S1-S3), it is possible that Fhp may have contributed to the SP values measured in previous pure
culture studies. Notably, all prior reports of SP from bacterial denitrifiers used strains that also
have Fhp (Table S1); given the sensitivity of enzyme abundance to the physiological state during
the time of measurement, it is plausible that the positive spread in SP values observed in these
studies (23) may reflect cryptic Fhp activity. An Fhp homologue, Yhb, exists in yeast (10) and is 15 present in previously studied fungal denitrifiers as well (Table S3), possibly contributing to the
tail towards 10‰ observed from the literature (Fig. 1A) if the SP signature of Yhb is similar to
that of Fhp.
Fhp is phylogenetically widespread and more abundant than NOR; therefore, measuring Fhp
values from a representative group of diverse bacteria may illuminate the natural variation in SP
20 values. In addition, measuring other NO-detoxifying proteins may shed further light on the SP
values of this neglected class of non-catabolic enzymes. Flavo-diiron proteins, which only
operate in anoxic conditions and only reduce NO to N2O for detoxification (9) present an
attractive next target for SP measurements. Finally, further detailed studies of Fhp’s reaction
mechanism paired with SP data is needed to reveal what determines the SP of N2O formation 25 through NO reduction, for both abiotic and biotic reactions (7, 24–26).
Beyond helping to explain the N2O SP variation seen in prior pure-culture studies, our finding
that Fhp produces an intermediate SP value that overlaps with many natural SP measurements,
particularly those found in soil (median Fhp SP=9.5‰, median soil SP=10.9‰; Fig. S10). This
begs the question: How can we distinguish Fhp-generated N2O from that produced by a mixture 30 of other enzyme sources in complex environments such as soils or infected tissues? This is a
difficult task. Though we can infer whether certain enzymes may be present and active based on
knowledge of what regulates their expression, in order to predict whether they are active in any
given sample, we need to know the conditions experienced by cells in situ. For example, our
work indicates that Fhp dominates the SP fingerprint when cells grown under oxic conditions
35 subsequently encounter a concentrated pulse of NO under anoxia. Intriguingly, pulses of NO and
N2O have been detected after wetting of dryland soils (27, 28) nd opportunistic pathogens are
thought to experience NO bursts from different cell types in the human immune system (29). Yet
to speculate on whether such pulses may trigger Fhp activity, we would need to be able to track
in situ NO and oxygen concentrations at the microscale. Ultimately, knowledge of the relative
40 abundance of NO reductases present at the protein level in any given sample where N2O SP is
measured, paired with knowledge of microscale environmental states, will aid in source
attribution.
Though this manuscript focuses on N2O, the general approach presented here exemplifies how
particular microbial metabolic pathways may be forensically distinguished by means of
45
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
6
intramolecular isotopic biosignatures within their products. Going forward, we envision such an
approach, when applied to other metabolites of interest, may help us better understand how
microbial activities shape diverse habitats, from soils to animal hosts.
References and Notes
5
1. M. M. M. Kuypers, H. K. Marchant, B. Kartal, The microbial nitrogen-cycling network.
Nat. Rev. Microbiol. 16, 263–276 (2018).
2. H. Tian, R. Xu, J. G. Canadell, R. L. Thompson, W. Winiwarter, P. Suntharalingam, E.
A. Davidson, P. Ciais, R. B. Jackson, G. Janssens-Maenhout, M. J. Prather, P. Regnier, N.
Pan, S. Pan, G. P. Peters, H. Shi, F. N. Tubiello, S. Zaehle, F. Zhou, A. Arneth, G.
10 Battaglia, S. Berthet, L. Bopp, A. F. Bouwman, E. T. Buitenhuis, J. Chang, M. P.
Chipperfield, S. R. S. Dangal, E. Dlugokencky, J. W. Elkins, B. D. Eyre, B. Fu, B. Hall,
A. Ito, F. Joos, P. B. Krummel, A. Landolfi, G. G. Laruelle, R. Lauerwald, W. Li, S.
Lienert, T. Maavara, M. MacLeod, D. B. Millet, S. Olin, P. K. Patra, R. G. Prinn, P. A.
Raymond, D. J. Ruiz, G. R. van der Werf, N. Vuichard, J. Wang, R. F. Weiss, K. C.
15 Wells, C. Wilson, J. Yang, Y. Yao, A comprehensive quantification of global nitrous
oxide sources and sinks. Nature. 586, 248–256 (2020).
3. M. Kolpen, M. Kühl, T. Bjarnsholt, C. Moser, C. R. Hansen, L. Liengaard, A. Kharazmi,
T. Pressler, N. Høiby, P. Ø. Jensen, Nitrous oxide production in sputum from cystic
fibrosis patients with chronic Pseudomonas aeruginosa lung infection. PLoS ONE. 9,
20 e84353 (2014).
4. G. M. Cook, C. Greening, K. Hards, M. Berney, Energetics of pathogenic bacteria and
opportunities for drug development. Adv. Microb. Physiol. 65, 1–62 (2014).
5. S. Toyoda, N. Yoshida, Determination of Nitrogen Isotopomers of Nitrous Oxide on a
Modified Isotope Ratio Mass Spectrometer. Anal. Chem. 71, 4711–4718 (1999).
25
6. T. R. A. Denk, J. Mohn, C. Decock, D. Lewicka-Szczebak, E. Harris, K. Butterbach-
Bahl, R. Kiese, B. Wolf, The nitrogen cycle: A review of isotope effects and isotope
modeling approaches. Soil Biol. Biochem. 105, 121–137 (2017).
7. Z. Wang, E. A. Schauble, J. M. Eiler, Equilibrium thermodynamics of multiply
substituted isotopologues of molecular gases. Geochim. Cosmochim. Acta. 68, 4779–4797
30 (2004).
8. C. H. Frame, K. L. Casciotti, Biogeochemical controls and isotopic signatures of nitrous
oxide production by a marine ammonia-oxidizing bacterium. Biogeosciences. 7, 2695–
2709 (2010).
9. C. Ferousi, S. H. Majer, I. M. DiMucci, K. M. Lancaster, Biological and Bioinspired
35 Inorganic N-N Bond-Forming Reactions. Chem. Rev. 120, 5252–5307 (2020).
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
7
10. R. K. Poole, M. N. Hughes, New functions for the ancient globin family: bacterial
responses to nitric oxide and nitrosative stress. Mol. Microbiol. 36, 775–783 (2000).
11. K. Mendler, H. Chen, D. H. Parks, B. Lobb, L. A. Hug, A. C. Doxey, AnnoTree:
visualization and exploration of a functionally annotated microbial tree of life. Nucleic
Acids Res. 47, 4442–4448 (2019).
5
12. A. Bonamore, A. Boffi, Flavohemoglobin: structure and reactivity. IUBMB Life. 60, 19–
28 (2008).
13. J. M. Eiler, M. Clog, P. Magyar, A. Piasecki, A. Sessions, D. Stolper, M. Deerberg, H.-J.
Schlueter, J. Schwieters, A high-resolution gas-source isotope ratio mass spectrometer.
Int. J. Mass Spectrom. 335, 45–56 (2013).
10
14. T. Yamazaki, T. Hozuki, K. Arai, S. Toyoda, K. Koba, T. Fujiwara, N. Yoshida,
Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted
from nitrifying and denitrifying bacteria. Biogeosciences. 11, 2679–2689 (2014).
15. S. A. Wilbert, D. K. Newman, The contrasting roles of nitric oxide drive microbial
community organization as a function of oxygen presence. Curr. Biol. 32, 5221-5234.e4
15 (2022).
16. S. S. Yoon, R. F. Hennigan, G. M. Hilliard, U. A. Ochsner, K. Parvatiyar, M. C. Kamani,
H. L. Allen, T. R. DeKievit, P. R. Gardner, U. Schwab, J. J. Rowe, B. H. Iglewski, T. R.
McDermott, R. P. Mason, D. J. Wozniak, R. E. W. Hancock, M. R. Parsek, T. L. Noah,
R. C. Boucher, D. J. Hassett, Pseudomonas aeruginosa anaerobic respiration in biofilms:
20 relationships to cystic fibrosis pathogenesis. Dev. Cell. 3, 593–603 (2002).
17. K. L. Casciotti, M. Forbes, J. Vedamati, B. D. Peters, T. S. Martin, C. W. Mordy, Nitrous
oxide cycling in the Eastern Tropical South Pacific as inferred from isotopic and
isotopomeric data. Deep Sea Res. Part II Top. Stud. Oceanogr. 156, 155–167 (2018).
18. N. E. Ostrom, A. Pitt, R. Sutka, P. H. Ostrom, A. S. Grandy, K. M. Huizinga, G. P.
25 Robertson, Isotopologue effects during N2 O reduction in soils and in pure cultures of
denitrifiers. J. Geophys. Res. 112 (2007), doi:10.1029/2006JG000287.
19. A. Bodor, N. Bounedjoum, G. E. Vincze, Á. Erdeiné Kis, K. Laczi, G. Bende, Á.
Szilágyi, T. Kovács, K. Perei, G. Rákhely, Challenges of unculturable bacteria:
environmental perspectives. Rev Environ Sci Biotechnol. 19, 1–22 (2020).
30
20. M. Bergkessel, D. W. Basta, D. K. Newman, The physiology of growth arrest: uniting
molecular and environmental microbiology. Nat. Rev. Microbiol. 14, 549–562 (2016).
21. D. M. Sigman, K. L. Casciotti, M. Andreani, C. Barford, M. Galanter, J. K. Böhlke, A
bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater.
Anal. Chem. 73, 4145–4153 (2001).
35
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
8
22. S. Toyoda, N. Yoshida, K. Koba, Isotopocule analysis of biologically produced nitrous
oxide in various environments. Mass Spectrom. Rev. 36, 135–160 (2017).
23. S. Toyoda, H. Mutobe, H. Yamagishi, N. Yoshida, Y. Tanji, Fractionation of N2O
isotopomers during production by denitrifier. Soil Biol. Biochem. 37, 1535–1545 (2005).
24. C. L. Stanton, C. T. Reinhard, J. F. Kasting, N. E. Ostrom, J. A. Haslun, T. W. Lyons, J.
5 B. Glass, Nitrous oxide from chemodenitrification: A possible missing link in the
Proterozoic greenhouse and the evolution of aerobic respiration. Geobiology. 16, 597–609
(2018).
25. L. Y. Yeung, Combinatorial effects on clumped isotopes and their significance in
biogeochemistry. Geochim. Cosmochim. Acta. 172, 22–38 (2016).
10
26. H.-L. Schmidt, R. A. Werner, N. Yoshida, R. Well, Is the isotopic composition of nitrous
oxide an indicator for its origin from nitrification or denitrification? A theoretical
approach from referred data and microbiological and enzyme kinetic aspects. Rapid
Commun. Mass Spectrom. 18, 2036–2040 (2004).
27. A. H. Krichels, P. M. Homyak, E. L. Aronson, J. O. Sickman, J. Botthoff, H. Shulman, S.
15 Piper, H. M. Andrews, G. D. Jenerette, Rapid nitrate reduction produces pulsed NO and
N2O emissions following wetting of dryland soils. Biogeochemistry. 158, 233–250
(2022).
28. P. M. Homyak, J. C. Blankinship, K. Marchus, D. M. Lucero, J. O. Sickman, J. P.
Schimel, Aridity and plant uptake interact to make dryland soils hotspots for nitric oxide
20 (NO) emissions. Proc Natl Acad Sci USA. 113, E2608-16 (2016).
29. M. Kolpen, T. Bjarnsholt, C. Moser, C. R. Hansen, L. F. Rickelt, M. Kühl, C. Hempel, T.
Pressler, N. Høiby, P. Ø. Jensen, Nitric oxide production by polymorphonuclear
leucocytes in infected cystic fibrosis sputum consumes oxygen. Clin. Exp. Immunol. 177,
310–319 (2014).
25
Acknowledgments: We thank Colette L. Kelly for valuable guidance and help with the
scrambling correction; Nami Kitchen for assistance with IRMS measurements; and Nathan Hart
at the Caltech Glass Shop for building the vacuum flasks. We thank the Dr. Tsui-Fen Chou and
Baiyi Quan at the Caltech Proteome Exploration Laboratory for assistance with proteomics-
based experiments.
30
Funding: National Science Foundation Graduate Research Fellowship Program (RZW), Jane
Coffin Childs Memorial Fund for Medical Research Fellowship (ZRL), National Institutes of
Health grant R01 HL152190-03 (DKN, JML)
Author contributions:
Conceptualization: DKN, JME, RZW, ZRL
35
Methodology: RZW, ZRL, SAW
Investigation: RZW, ZRL, SAW, DKN
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
9
Visualization: RZW, ZRL, DKN, JME
Funding acquisition: DKN, JME, RZW, ZRL
Project administration: DKN
Supervision: DKN, JME
Writing – original draft: RZW, ZRL
5
Writing – review & editing: RZW, ZRL, DKN, JME
Competing interests: Authors declare that they have no competing interests.
Data and materials availability: All data are available in the main text or the supplementary
materials.
Supplementary Materials
10
Materials and Methods
Supplementary Text
Figs. S1 to S18
Tables S1 to S13
15
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
10
Fig. 1. N
2
O production via NO detoxification under anoxic conditions may explain
environmental SP values. (A) Measured in situ SP values for environmental (Soil, Marine,
Freshwater) vs. in vitro measurements of biogenic end-members (Bacterial and Fungal
5
Denitrification, Ammonia Oxidizing Bacteria (AOB)); black line shows median; blue lines show
end-member values for AOB (8). Histogram height is normalized to each category; see Fig. S11
for outlier values and more detail. (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 (11); minimum amino acid sequence similarity of 30% was used. See Fig. S2,
10
Tables S1-S4 for phylogenetic distribution. (C) Relevant N-oxide pathways of Pseudomonas
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 (
Δ
nosZ
Δ
fhp;
Δ
nosZ) in denitrifying conditions; see Fig. S2 for
more detail. (E) of Pa strains with rhamnose-induced expression of norBC (iNor) or fhp (iFhp)
15
alone as well as Acinetobacter baumannii and Staphylococcus aureus, which only have Fhp. P
value was calculated via Welch’s t-test. Each data point represents an individual biological
replicate in (D) and (E).
w
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
11
Fig. 2. High concentrations of NO shift SP values towards Fhp. (A) In Pa
, NorBC contributes
to overall cell energetics as part of the denitrification pathway; Fhp does not and is primarily
used for NO detoxification. (B) WT Pa was cultured anaerobically via two assay types after
5
aerobic pre-growth with nitrate to either maximize growth via denitrification (left) or be re-
suspended as non-growing cells (right). 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 are shown as
10
bar charts below; see Fig. S6 for full results. P values were calculated via Welch’s t-test. (C)
δ
15
N
bulk
values for WT Pa incubated anoxically with DETA (blue), nitrate (yellow) or both
(green); end-member values are from non-WT Pa strains incubated w
ith only nitrate or DETA as
an NO source (Fig. S8). (D) SP measurements for WT Pa grown as denitrifying growths or
anoxic suspensions, as illustrated in (B). Colors indicate anoxic incubation substrate and are the
15
same as panel (C). iNOR and iFhp SP values are from Fig. 1E. For (C, D), box plots indicate
median, upper and lower quartiles, and extreme values.
tes
as
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
12
Name Strain Description Fhp? Nor? Source
WT Pa Wild-type Pseudomonas aeruginosa UCBPP-PA14 Yes Yes
Lab
Collectio
n
Δ
nosZ Deletion of nitrous oxide reductase gene (nosZ, PA14_20200) from
WT Pa Yes Yes This
study
Δ
nosZ
Δ
fhp Deletion of nosZ and flavohemoglobin protein (fhp, PA14_29640)
from WT Pa Yes No This
study
iFhp Rhamnose-induced expression of fhp integrated into the
chromosome of WT Pa with deletion of native norBC, fhp, and nosZ.
Yes No This
study
iNOR
Rhamnose
-
induced expression of the nitric oxide reductase operon,
norBCD (PA14_16810, PA14_16830, PA14_06840), integrated into
the att neutral chromosomal site of Pa with deletion of native nitrate
reductase (narGHJI; PA14_13780-13830), nitrite reductase (nirS;
PA14_06750), norBC, nosZ, and fhp.
No Yes
This
study
S. aureus Wild-type Staphylococcus aureus USA300 LAC Yes No Gift
A. baumannii Wild-type Acinetobacter baumannii ATCC 17978 Yes No Gift
Table 1. Strains studied. The SP of N2O produced by five strains of Pseudomonas aeruginosa
(WT Pa,
Δ
nosZ,
Δ
nosZ
Δ
fhp, iFhp, iNOR) and two wild-type strains of Staphylococcus aureus
and Acinetobacter baumannii were measured. See Materials and Methods for further detail. S.
aureus and A. baumannii were both kindly provided by Eric Skaar, Vanderbilt University
5 Medical Center.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
13
Strain Assay Type Aerobic pre-growth Anaerobic incubation SP (‰) n
iNOR Suspension 100 mM nitrate 100 mM nitrate, 500 uM
DETA NONOate, 305 uM
rhamnose
-2.60 ± 5.41
5
iFhp Suspension 100 mM nitrate 100 mM nitrate, 500 uM
DETA NONOate, 305 uM
rhamnose
10.45 ± 2.17
5
A. baumannii
Suspension 100 mM nitrate 100 mM nitrate, 500 uM
DETA NONOate
10.38 ± 9.05
3
S. aureus Suspension 100 mM nitrate 100 mM nitrate, 500 uM
DETA NONOate
5.56 ± 7.21
3
nosZ
Batch; End-
exponential
100 mM nitrate 100 mM nitrate -1.56 ± 5.04
4
Batch; End-
stationary
100 mM nitrate 100 mM nitrate -2.21 ± 4.10
5
nosZ
fhp
Batch; End-
exponential
100 mM nitrate 100 mM nitrate -1.39 ± 2.78
5
Batch; End-
stationary
100 mM nitrate 100 mM nitrate -3.68 ± 2.81
5
WT Pa
Batch; End-
exponential
100 mM nitrate 100 mM nitrate -0.70 ± 4.19
5
Batch; End-
stationary
100 mM nitrate 100 mM nitrate -5.43 ± 2.04
5
Suspension
100
M DETA NONOate
500
M DETA NONOate
-2.59 ± 7.53
2
Suspension 100
M DETA NONOate
+ 100 mM nitrate
500
M DETA NONOate 9.14 ± 3.70
2
Suspension 100 mM nitrate 500
M DETA NONOate +
100 mM nitrate
2.61 ± 9.31
5
Batch; End-
stationary
100 mM nitrate 500
M DETA NONOate +
100 mM nitrate
-3.34 ± 0.83
2
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
14
Table 2. Culturing conditions and SP results. All strains were grown in aerobic pre-growths
before being resuspended in fresh media and anoxically incubated for headspace sampling as
batch culture or suspension assays (Fig. S12); nitrate and/or DETA NONOate (C4H13N5O2) was
supplemented to provide endogenous vs. exogenous NO respectively. See Methods for more
5 detail. SP values (mean ± s.d.) of n biological replicates; see Supplemental for full data table.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 14, 2023. ; https://doi.org/10.1101/2023.10.13.562248doi: bioRxiv preprint
