bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 available under aCC-BY-NC 4.0 International license.

1 Metabolic versatility of the -oxidizing bacterium

2 marina and its proteomic response to oxygen-limited conditions

3 Barbara Bayer1*, Mak A. Saito2, Matthew R. McIlvin2, Sebastian Lücker3, Dawn M. Moran2,

4 Thomas S. Lankiewicz1, Christopher L. Dupont4, and Alyson E. Santoro1*

5

6 1 Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara,

7 CA, USA

8 2 Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution,

9 Woods Hole, MA, USA

10 3 Department of Microbiology, IWWR, Radboud University, Nijmegen, The Netherlands

11 4 J. Craig Venter Institute, La Jolla, CA, USA

12

13 *Correspondence:

14 Barbara Bayer, Department of Ecology, Evolution and Marine Biology, University of California,

15 Santa Barbara, CA, USA. E-mail: [email protected]

16 Alyson E. Santoro, Department of Ecology, Evolution and Marine Biology, University of

17 California, Santa Barbara, CA, USA. E-mail: [email protected]

18

19 Running title: and proteome of Nitrospira marina

20

21 Competing Interests: The authors declare that they have no conflict of interest.

22

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23 Abstract

24 The genus Nitrospira is the most widespread group of chemolithoautotrophic nitrite-oxidizing

25 that thrive in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T

26 represents the type genus and was isolated from the oceanic water column over 30 years ago,

27 however, its genome has not yet been analyzed. Here, we analyzed the complete genome

28 sequence of N. marina and performed select physiological experiments to test genome-derived

29 hypotheses. Our data confirm that N. marina benefits from additions of undefined organic

30 carbon substrates, has adaptations to combat oxidative, osmotic and UV-light induced stress

31 and low dissolved pCO2, and is able to grow chemoorganotrophically on formate. We further

32 investigated the metabolic response of N. marina to low (∼5.6 µM) O2 concentrations commonly

33 encountered in marine environments with high nitrite concentrations. In response to O2-limited

34 conditions, the abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin

35 (POR) complex and a high affinity cbb3-type terminal oxidase increased,

36 suggesting a role in sustaining nitrite oxidation-driven autotrophy under O2 limitation.

37 Additionally, a Cu/Zn-binding superoxide dismutase increased in abundance potentially

38 protecting this putatively more O2-sensitive POR complex from oxidative damage. An increase

39 in abundance of proteins involved in alternative energy metabolisms, including type 3b [NiFe]

40 hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive

41 conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of

42 the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides

43 important insights into the metabolic diversity and niche differentiation of NOB in marine

44 environments.

45

46

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47 Introduction

- - 48 Aerobic nitrite (NO2 ) oxidation is the main biochemical nitrate (NO3 )-forming reaction, carried

49 out during the second step of [1]. In marine ecosystems, nitrate is the dominant form

50 of biologically available nitrogen, which is rapidly assimilated by phytoplankton in surface waters

51 and accumulates in the deep sea [2].

52 Nitrite-oxidizing bacteria (NOB) are chemolithoautotrophic microorganisms comprising

53 seven known genera (, Nitrococcus, Nitrospina, Nitrospira, Nitrotoga, Nitrolancea,

54 and Candidatus Nitromaritima) in four phyla [3]. The genus Nitrospira consists of at least six

55 phylogenetic sublineages and is the most diverse NOB genus [3]. Nitrospira are ubiquitously

56 present in natural and engineered ecosystems, including oceans [4, 5], freshwater [6],

57 soils [7, 8], saline-alkaline lakes [9], hot springs [10], wastewater treatment plants [11–13], and

58 aquaculture biofilters [14, 15]. In human-made ecosystems, Nitrospira is generally considered to

- - 59 be adapted to low NO2 concentrations [16]. In the open ocean, however, where NO2

60 concentrations are exceedingly low, NOB affiliated with the appear to be the

- 61 dominant nitrite oxidizers [4], whereas Nitrospira bacteria are found in relatively high NO2

62 environments such as sediments and deep sea hydrothermal vent plumes [17, 18]. Nitrospira

63 also dominate over Nitrospinae-affiliated bacteria in some deep-sea trench environments [19,

- 64 20]. High NO2 concentrations are found coincident with low O2 concentrations in oxygen

65 minimum zone (OMZ) waters [21, 22] in a feature known as the secondary nitrite maximum.

- 66 Despite the O2-dependency of all known NOB, NO2 oxidation can still be detected at nanomolar

67 O2 concentrations [23].

68 Some metabolic features appear to be common among Nitrospira, based on genomic

69 analyses to date. Metagenomic analysis of Ca. Nitrospira defluvii, a representative of Nitrospira

70 sublineage I, indicated a periplasmic location of nitrite oxidoreductase (NXR), the key of

- 71 the NO2 oxidation, and the presence of the O2-sensitive reductive tricarboxylic acid (rTCA)

72 cycle for [24]. These results suggest that Nitrospira evolved from microaerophilic

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73 or anaerobic ancestors [24]. Ca. N. defluvii lacks genes for classical oxidative stress defense

74 present in most aerobic organisms including catalase and superoxide dismutase,

75 indicative for adaptations to low O2 environmental niches [24]. However, other Nitrospira

76 species encode both types of enzymes [25, 26], suggesting different O2 tolerances within

- 77 members of the Nitrospira genus. In addition to NO2 oxidation, Nitrospira bacteria have been

78 shown to exhibit a high metabolic versatility, growing aerobically on hydrogen [27] or

79 anaerobically on organic acids while respiring nitrate [25]. Recently, the capability for the

80 complete oxidation of to nitrate (comammox) was identified in representatives of

81 sublineage II Nitrospira [14, 28], however, comammox Nitrospira appear to be absent in marine

82 systems [28].

83 N. marina Nb-295T, the type species of the genus Nitrospira, was isolated from a water

84 sample collected at a depth of 206 m from the Gulf of Maine in the Atlantic Ocean over 30 years

85 ago [5]. It is the only Nitrospira species isolated from the oceanic water column, however, its

86 genome has not yet been analyzed. Here, we analyzed the complete genome sequence of N.

87 marina Nb-295T and compared the proteome signatures of cultures grown under atmospheric

88 O2 tension and under low O2 conditions.

89

90 Materials and Methods

91 Cultivation of N. marina Nb-295

92 Nitrospira marina Nb-295T was obtained from the culture collection of John B. Waterbury at the

93 Woods Hole Oceanographic Institution (WHOI). To test the effect of different organic substrates

94 on strain Nb-295, cultures were grown in 50 ml polycarbonate bottes in autotrophic mineral salts

95 medium at pH 7.8 containing 2 mM NaNO2 (Table S1), and bottles were incubated at 25°C in

96 the dark without agitation. The following organic carbon substrates were individually added to

97 the culture medium of duplicate cultures: 150 mg L-1 yeast extract, 150 mg L-1 tryptone, 1 mM

-1 - 98 pyruvate, 1 mM formate, or 1 g L glycerol. NO2 consumption was measured as previously

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99 described [29] and growth was monitored by flow cytometry (see Supplementary Methods). To

- 100 test for chemoorganotrophic growth, NO2 was omitted from the culture medium.

101 For incubations at different O2 concentrations, triplicate cultures of N. marina Nb-295

102 were grown at 22°C in 400 mL of mineral salts medium containing 70% seawater and 2 mM

103 NaNO2 as described by Watson et al. [5] in 500 mL polycarbonate bottles with a custom-made

104 sparging rig. Bottles were constantly bubbled with one of two sterile custom gas mixes

- 105 containing either 0.5% or 20% oxygen, 300 ppm CO2, and a balance of high-purity N2. NO2

106 concentrations were measured as a proxy for growth as described above and 10 mL aliquot of

107 each culture was fixed at the last time point (2% formaldehyde, 1 h, 4°C) for cell enumeration

108 on an epifluorescence microscope as previously described [30].

109

110 DNA extraction, genome sequencing and annotation

111 High molecular weight genomic DNA was extracted from stationary phase cultures using a

112 CTAB extraction protocol [31] and sequenced on the PacBio platform at the US Department of

113 Energy Joint Genome Institute (JGI). 754,554 reads were produced, with 209,987 passing

114 quality control. The assembly was conducted using HGAP (v 2.2.0.p1) with improvement with

115 Quiver [32] resulting in a single contig.

116 Gene annotation was conducted using JGI's Integrated Microbial and

117 Microbiomes (IMG/M) pipeline [33] and the MicroScope platform [34]. Manual curation included

118 sequence similarity searches using BLASTP [35] against the Transporter Classification

119 database [36] and protein searches using InterProScan (release 72.0) [37]. Signal

120 peptides were identified with SignalP 5.0 [38] to determine if proteins were potentially addressed

121 to the membrane and/or released to the periplasmic space. A list of manually curated

122 annotations can be found in Table S5.

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123 Phylogenomic analysis was performed using 120 concatenated phylogenetic marker

124 genes of representatives of the phylum /Nitrospirota as implemented in the Genome

125 Database Toolkit (GTDB-tk) version 1.1.1. [39] (see Supplementary Methods).

126

127 Protein extraction and proteome analyses

- 128 Cells were harvested for proteomic analysis during exponential growth when [NO2 ] dropped to

129 ~ 500 µM. Each culture was mixed with an equal volume of a house-made fixative [40] similar to

130 the commercially available solution RNALater (Thermo Fisher), and filtered by vacuum filtration

131 onto 25 mm, 0.2 µm pore size Supor filters (Pall). 200 mL of fixed culture (equivalent to 100 mL

132 of growth medium) were filtered for protein extraction and proteomic analysis. Filters were

133 frozen at -80°C until extraction. A detailed description of the procedures used for protein

134 extraction and purification can be found in the Supplementary Methods. Procedures for mass

135 spectrometry analyses can be found in Saito et al. [41]. In addition, targeted quantitative

136 proteomics using custom-made isotopically-labelled (15N) peptide standards was performed as

137 previously described [41]. Tryptic peptides from different proteins, including nitrite

138 oxidoreductase (NXR), were targeted for absolute quantitation (see Table S2). Cellular NXR

139 concentrations were calculated based on NxrA peptide concentrations using a cellular carbon

140 content of 152 fg cell-1 (Santoro et al, unpublished) and an estimated cellular protein content of

141 50% (Table 1). NXR complex density on the cellular membrane was calculated using an

142 estimated NXR complex size of 63 nm2 [42] and an estimated surface area of 2.14 µm2

143 (assuming a cylindrical shape with a length of 1.5 µm and a radius of 0.2 µm).

144 Differential levels of expression between the two treatments (i.e., atmospheric O2 and

145 low O2 conditions) were tested with the DESeq2 Bioconductor package (version 1.20.0) [43] in

146 the R software environment (version 3.5.0) [44] using spectral counts as input data as

147 previously described [45]. Proteins with a mean spectral count below 6 across all treatments

148 were excluded from the analysis. In DESeq2, only proteins that increased in abundance under

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149 low O2 conditions were considered (‘altHyphothesis=greater’), P values were adjusted using the

150 Benjamini-Hochberg method (‘pAdjustmethod=BH’) and independent filtering was omitted

151 (‘independentFiltering=FALSE’). Changes in protein abundance were considered statistically

152 significant when adjusted P values were lower than or equal to 0.05 (see Table S3). While

153 DESeq2 has a high precision and accuracy [46], it is more conservative than other methods on

154 low-count transcripts/proteins [47]. Protein abundances were visualized with the pheatmap

155 package (version 1.0.12) [48] in the R software environment [44]. The normalized spectral

156 abundance factor (NSAF) was calculated as proxy for relative protein abundances [49], and

157 values were square-root transformed to improve visualization of low abundant proteins.

158

159 Results and Discussion

160 Genome analysis

161 The genome of N. marina Nb-295 is a single element of 4,683,627 bp and contains 4272 coding

162 DNA sequences (CDS) including one rRNA operon. A 5578 bp region of 99.7% identity on each

163 end of the scaffold suggests circularization into a single chromosome. No plasmids or extra-

164 chromosomal elements were identified. The G+C content is 50.04%, which is lower than other

165 Nitrospira species and the marine nitrite-oxidizers Nitrospina gracilis and Nitrococcus mobilis

166 (Table S4). Phylogenomic analysis of available closed genomes, metagenome-assembled

167 genomes (MAGs) and single-amplified genomes (SAGs) affiliated with the phylum

168 Nitrospirae/Nitrospirota (see Supplementary Methods) placed N. marina Nb-295 within a cluster

169 of genomes derived from marine and saline environments (Fig. 1). The most closely related

170 Nitrospira MAG (UBA8639) was obtained from a laboratory-scale nitrification reactor, however,

171 the reactor influent consisted to 33% of untreated seawater [50], suggesting a marine origin of

172 this MAG. The 16S rRNA gene sequence of N. marina Nb-295 clustered together with

173 environmental sequences derived from marine sediments and marine aquaculture biofilters (Fig.

174 S1), and shared 99.1% and 97.9% sequence identity with the cultured lineage IV

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175 representatives Nitrospira sp. Ecomares 2.1 [15] and Ca. Nitrospira alkalitolerans [9],

176 respectively.

177

178 Nitrogen metabolism and respiratory chain

- 179 The genome of N. marina encodes orthologs of all known proteins required for NO2 oxidation,

180 including the putatively membrane-associated periplasmic nitrite oxidoreductase (NXR). Three

181 candidates each of NxrA and NxrB, three putative NxrC candidates and two NxrC-like proteins

182 were identified in the N. marina genome (Table S4 and S5). The genes nxrA and nxrB,

183 encoding the alpha and beta subunits of NXR, are co-localized in three clusters whereas all

184 nxrC candidate genes are localized separately from nxrAB, as previously described for N.

185 moscoviensis [51]. The NxrA subunits share 87.3-88.9% amino acid identity, whereas the NxrB

186 subunits share 98.8-99.5% amino acid identity with each other. The putative NxrC subunits are

187 less conserved, sharing between 33.7.7-86.9% amino acid identity.

188 Like all other analyzed Nitrospira genomes, N. marina encodes a putative copper-

189 dependent NO-forming (NirK), yet its function in Nitrospira and other NOB

190 remains unknown [52]. N. marina also encodes the ferredoxin-dependent nitrite reductase

191 (NirA) for assimilatory nitrite reduction, which appears to be conserved in Ca. Nitrospira lenta

192 and Ca. N. defluvii but absent in other Nitrospira species [52]. Additionally, N. marina encodes

193 three high affinity ammonium transporters (Amt) enabling direct uptake of reduced N for

194 assimilation, and cyanate to hydrolyze cyanate to ammonium and CO2 (Table S5). In

195 contrast to some Nitrospira species [13, 25, 28], N. marina does not encode a operon or

196 a transporter, which would catabolize urea to ammonia for N assimilation (Table S4).

197 Previous genome studies of Nitrospira revealed multiple copies of several complexes of

198 the respiratory chain [24–26, 51]. N. marina encodes two paralogous copies of complex I, one of

199 which contains a duplication of NuoM and lacks genes for NuoE, NuoF and NuoH (Table S5),

200 which is a characteristic feature of Nitrospira genomes [51, 53]. Further, the N. marina genome

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201 contains two copies of complex III, two cytochrome bd oxidases and seven putative cytochrome

202 bd-like oxidases (Table S5), which show limited partial similarity to canonical cytochrome bd

203 oxidase as described for N. moscoviensis [51]. One of these cytochrome bd-like oxidases (bd-

204 like_6) contains putative b and copper binding sites potentially functioning as a novel

205 terminal oxidase as previously proposed [24]. Additionally, N. marina encodes for a cbb3-type

206 terminal oxidase, which usually exhibit high affinities for O2 [54]. This feature is shared with the

207 closely related Ca. N. alkalitolerans [9] and with the more distantly related marine NOB

208 Nitrospina gracilis [55], but absent in all other thus far sequenced Nitrospira species. In addition

+ 209 to the canonical H -translocating F1FO-ATPase (complex V), N. marina also encodes a putative

210 alternative Na+-translocating N-ATPase (Table S5), which potentially contributes to the

211 maintenance of the membrane potential and the generation of a sodium motive force (SMF) as

212 suggested for Ca. N. alkalitolerans [9]. Furthermore, a H+-translocating pyrophosphatase (H+-

213 PPase) with homology to Leptospira/protozoan/plant-type enzymes [56] was identified. H+-

214 PPases couple the translocation of H+ to the hydrolysis of the biosynthetic by-product

215 pyrophosphate (PPi), which is suggested to be an adaptation to life under energy limitation [57].

216 The N. marina genome also contains an alternative complex III (ACIII) module, which shares

217 similarity with that from sulfur-reducing [58]. Like canonical complex III, ACIII also

218 functions as a quinol oxidase transferring electrons to cytochrome c and contributes to energy

219 conservation (Refojo et al., 2012). With the exception of the comammox bacterium Ca.

220 Nitrospira nitrificans, no homologues of ACIII modules were identified in any other NOB

221 genome.

222 N. marina encodes a putative hyb-like operon containing four subunits of a cytoplasmic

223 type 3b [NiFe] hydrogenase and six accessory proteins involved in hydrogenase assembly and

224 maturation (Table S5). This type of hydrogenase appears to be conserved in the marine NOBs

225 Nitrospina gracilis 3/211 and Nitrococcus mobilis Nb-231 [55, 59], Ca. N. alkalitolerans [9] and

226 in comammox Nitrospira [14, 28] (Table S4). In addition to catalyzing the reversible, NAD+-

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227 dependent oxidation of hydrogen, these so-called sulfhydrogenases are able to reduce

o 228 elemental sulfur (S ) or polysulfides to hydrogen sulfide (H2S) [60]. Furthermore, the N. marina

229 genome encodes a putative periplasmic sulfite:cytochrome c oxidoreductase which might

2- 2- 230 couple sulfite (SO3 ) oxidation to sulfate (SO4 ) with the reduction of two cytochromes as

231 previously suggested for Nitrospina gracilis [55] whereas sulfide/quinone oxidoreductase, which

232 is speculated in to mediate sulfide oxidation in Nitrococcus [59], is lacking. However, whether or

2- 233 not these enzymes are involved in energy conservation using H2S and SO3 as alternative

234 substrates remains to be experimentally validated in NOB.

235

236 Central carbon metabolism

237 In agreement with previously characterized Nitrospira genomes [13, 24, 51, 52], N. marina

238 encodes the complete gene repertoire for the reductive tricarboxylic acid (rTCA) cycle for

239 (CO2) fixation, including the key enzymes ATP-citrate lyase and 2-

240 oxoglutarate/pyruvate:ferredoxin oxidoreductase (OGOR/POR) (Table S5). In the ocean,

- 241 inorganic carbon is predominately available in the form of bicarbonate (HCO3 ) and to a much

242 lesser extent as CO2. Five inorganic anion transporters (SulP family) with homology to BicA

- 243 HCO3 uptake systems of the cyanobacterium Synechococcus [61] were identified in the N.

244 marina genome (Table S5). Two of these putative BicA-like transporters are co-localized with

+ + - + 245 genes encoding Na /H antiporters (NhaB family), which could drive the uptake of HCO3 via Na

246 extrusion under alkaline conditions as suggested for the cyanobacterium Aphanothece

247 halophytica [62] and Ca. N. alkalitolerans [9]. N. marina also encodes one putative SulP-related

248 bicarbonate transporter fused to a carbonic anhydrase and four genes encoding putative alpha,

- 249 beta and gamma carbonic anhydrases (Table S5), which can convert the imported HCO3 to

250 CO2 for inorganic carbon fixation via the rTCA cycle.

251 In addition to the rTCA cycle, N. marina encodes all required genes for the oxidative

252 TCA cycle for pyruvate oxidation via acetyl-CoA, complete gluconeogenesis and glycolysis

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253 pathways, and the oxidative and non-oxidative branches of the pentose phosphate pathway

254 (Table S5), which are common features of all sequenced Nitrospira genomes [9, 24–26, 52].

255 Furthermore, biosynthetic pathways for all amino acids except methionine were identified in the

256 N. marina genome. Although N. marina encodes a -dependent methionine synthase

257 (MetH) (Table S5), it appears to lack additional enzymes of known methionine biosynthesis

258 pathways, a trait shared by all other sequenced Nitrospira species and Nitrospina gracilis [9,

259 24–26, 52, 55]. However, as N. marina can grow in artificial seawater medium without added

260 methionine, we hypothesize that an alternative unknown pathway for the early steps of

261 methionine biosynthesis functions in Nitrospira and Nitrospina. The N. marina genome contains

262 genes for the biosynthesis and degradation of the storage compounds glycogen and

263 polyphosphate (Table S5). In contrast to other Nitrospira and the marine NOBs N. gracilis and

264 N. mobilis that encode a glgA-type glycogen synthase, N. marina encodes alpha-maltose-1-

265 phosphate synthase (glgM) and alpha-1,4-glucan:maltose-1-phosphate maltosyltransferase

266 (glgE) for the synthesis of glycogen via alpha-maltose-1-phosphate.

267

268 Use of organic substrates

269 N. marina has been reported to be an obligate chemolithotroph that grows best in medium

270 supplemented with low concentrations of organic compounds including pyruvate, glycerol, yeast

271 extract and peptone [5]. Thus, we investigated the genomic basis for this observation and

272 conducted additional physiological experiments.

273 In addition to its complete glycolysis pathway and oxidative TCA cycle, a putative

274 carbohydrate degradation operon was identified in the genome of N. marina, consisting of a

275 sugar ABC transporter module, beta-glucosidase, a putatively secreted glycoside

276 (GH15) and a carbohydrate-binding protein (Table S5). N. marina also encodes two putative

277 carbohydrate-selective porins (OprB), a sugar:sodium symporter (SSS family), a putative

278 galactonate/glucarate transporter (MFS superfamily) and a putative carboxylate transporter

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279 (DASS family). Additionally, the genomic repertoire for the catabolic degradation and

280 assimilation of peptides and amino acids, including transporter proteins for di- and oligopeptides

281 (ABC and POT/PTR families), multiple amino acid:cation symporter (SSS, DAACS and AGCS

282 families), amino acid/polyamine transporters (APC superfamily) and multiple putatively secreted

283 peptidases are present in the N. marina genome (Table S5).

- 284 In agreement with Watson et al. [5], NO2 oxidation activity was enhanced when

285 undefined organic compound mixtures such as tryptone and yeast extract were added to the

286 culture medium (Fig. 2). Interestingly, growth was greatly stimulated by tryptone (Fig. 2), while

- 287 amendment with defined organic carbon compounds had no effect on NO2 oxidation or growth

288 (Table S6). Parallel incubations with ammonium as an N source did not increase activity or

289 growth (Table S6), suggesting that the stimulating effect of tryptone, and to a lesser extent of

290 yeast extract, most likely can be attributed to direct amino acid assimilation and does not reflect

- 291 only the abolished energy demand for assimilatory NO2 reduction. No growth on yeast and

- 292 tryptone was observed in the absence of NO2 (Fig. 3), corroborating their use as source of

293 amino acids rather than for energy conservation.

294 In addition to undefined organic substrates, defined organic compounds such as glycerol

295 and pyruvate have been reported to enhance the growth of N. marina and Ca. N. defluvii,

296 respectively [5, 12]. Furthermore, formate has been shown to serve as electron donor and

297 carbon source for some lineage I and II Nitrospira [11, 25]. N. marina encodes a putative

298 formate dehydrogenase (FdhA) (Table S5), which is divergent from those found in N.

299 moscoviensis and Ca. N. defluvii (∼24 and 27% amino acid identity, respectively), but shares a

300 relatively high sequence similarity (∼48% amino acid identity) to the functionally characterized

301 formate dehydrogenase Fdh4 from Methylobacterium extorquens [63]. N. marina was able to

- 302 grow chemoorganotrophically on 1 mM formate in the absence of NO2 (Fig. 3), suggesting that

- 303 it is not an obligate chemolithotrophic organism. However, the use of formate instead of NO2 as

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304 electron donor resulted in slower growth rates as previously shown for N. moscoviensis [25]. In

- 305 contrast to earlier reports [5], additions of glycerol did not stimulate NO2 oxidation activity or

306 growth (Table S6). Furthermore, pyruvate could neither be used as alternative energy source

- 307 (Fig. 3), nor did it stimulate metabolic activity in the presence of NO2 (Table S6). We thus

308 hypothesize that the stimulating effect of pyruvate on Ca. N. defluvii could potentially be

309 attributed to H2O2 detoxification as shown for AOA that lack catalases [45, 64].

310

311 Oxidative stress defense and osmoregulation

312 The formation of reactive oxygen species (ROS) is prevalent in oxic environments and oxidative

313 stress defense is an important component of the stress response in marine organisms [65]. N.

314 marina encodes multiple enzymes to reduce oxidative stress, including a cytoplasmic Mn/Fe-

315 binding superoxide dismutase, a periplasmic Cu/Zn-binding SOD, two heme-containing

316 catalases (of which KatE contains a signal peptide suggesting secretion into the or

317 extracellular space) and various peroxiredoxins (Table S5). In contrast, Nitrospina gracilis and

318 marine ammonia-oxidizing archaea lack catalase [45, 55], suggesting that N. marina is less

319 susceptible to oxidative stress compared to other marine nitrifiers. In addition to its plethora of

320 oxidative stress defense-related proteins, N. marina also encodes two putative photolyases,

321 enzymes known to be involved in the repair of UV induced DNA damage [66], suggesting that it

322 is well adapted to conditions characteristic for euphotic environments.

323 Marine microorganisms need to counteract the external osmotic stress from high salt

324 concentrations by accumulating a variety of small molecules in the cytoplasm. These organic

325 solutes (=osmolytes) can either be synthesized de-novo or transported into the cell from the

326 surrounding environment [67]. In addition to select amino acids that can serve as compatible

327 solutes (e.g. proline and glutamate) [68], biosynthesis pathways for the osmolytes glycine

328 betaine and trehalose were identified in the N. marina genome (Table S5). N. marina is able to

329 synthesize trehalose via two different pathways (either directly from maltose using trehalose

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330 synthase or from glycogen via maltodextrin and maltooligosyl-trehalose) and encodes two

331 putative glycine betaine/osmolyte transport systems (ABC and BCCT family) to import

332 exogenous glycine betaine (Table S5). Furthermore, despite their diverse roles and intracellular

333 functions, polyamines can also contribute to osmotic stress protection [69, 70]. N. marina can

334 synthesize the polyamines putrescine and spermidine, and encodes a putative polyamine ABC

335 transport module (Table S5). Protection against hypo-osmotic shock in N. marina might also be

336 conferred by one large- and seven small-conductance mechanosensitive ion channels, Na+:H+

337 antiporters and voltage-dependent K+ and Na+ channels (Table S5). The production and

338 concomitant release of osmolytes (i.e. via diffusion, excretion, predation or cell lysis) could

339 potentially fuel heterotrophic metabolism in the ocean [71] representing a link between

340 chemolithoautotrophic production and heterotrophic consumption of DOM as recently suggested

341 for ammonia-oxidizing archaea [72].

342

343 Vitamin biosynthesis and metal acquisition

344 B vitamins are important biochemical co-factors required for cellular metabolism and their

345 concentrations are depleted to near zero in large areas of the global ocean [73]. N. marina

346 encodes the complete biosynthetic pathways for the B vitamins thiamin (B1), riboflavin (B2),

347 pantothenate (B5), pyridoxine (B6), biotin (B7) and tetrahydrofolate (B9). An incomplete

348 cobalamin (vitamin B12) biosynthesis pathway was also identified, lacking genes for multiple

349 precorrin conversion reactions that ultimately lead to the biosynthesis of the molecule’s

350 ring [74]. However, N. marina encodes for a putative bifunctional precorrin-2

351 dehydrogenase/sirohydrochlorine ferrochelatase converting precorrin-2 to , a

352 at the active sites of many enzymes, including assimilatory nitrite reductases [75]. Since N.

353 marina only encodes the cobalamin-dependent versions of methionine synthase, ribonucleotide

354 reductase and methylmalonyl-CoA mutase, it must rely on the supply of cobalamin or its

355 precursors by other members of the microbial community. This auxotrophy may explain why N.

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356 marina has previously been difficult to cultivate in a mineral medium [5]. Interestingly, the N.

357 marina genome contains genes for multiple reactions that convert the precursor

358 cobyrinate/hydrogenobyrinate to cobalamin, and encodes all genes required for cobalamin

359 salvage from cobinamide (Table S5). Hence, in contrast to many other bacteria that lack the

360 complete pathway [76], N. marina appears to obtain its B12 solely from

361 salvage of multiple intermediates from the environment. Cobalamin uptake is likely mediated by

362 a TonB-dependent receptor and an ABC-type cobalamin/Fe3+ siderophore uptake system co-

363 localized with cobalamin biosynthesis and salvage genes (Table S5). This absolute B12

364 requirement for three enzymes coupled to an incomplete biosynthetic pathway implies that B12

365 nutrition is an important aspect for the physiology of nitrite oxidizers. When N. marina was

366 grown in natural seawater, cellular concentrations of ribonucleotide reductase were

367 approximately 39-fold higher compared to the cyanobacterium Prochlorococcus [79] (Table 1)

368 despite only having a ~two-fold greater cell volume, consistent with the importance of B12

369 nutrition to N. marina. Moreover, this salvage acquisition mode is interesting in the context of

370 dissolved cobalt speciation that is complexed by strong organic ligands hypothesized to also be

371 B12 precursors or degradation products based on their thermodynamic properties [77]. In the

372 Northwest Atlantic near where strain Nb-295 was isolated organic cobalt complexes are

373 abundant, comprising about half the dissolved cobalt inventory [78].

374 In addition to B vitamins, low concentrations of iron limit ocean productivity in many parts

375 of the global ocean [80, 81] and could limit NOB due to the high iron requirements of their

376 respiratory chain [41, 82]. N. marina encodes multiple metal transport systems, including a

377 molybdenum ABC transporter module, two putative zinc transporters (ZIP family), a putative

378 Fe2+/Mn2+ transporter (VIT1/CCC1 family), two putative copper-transporting P-type ATPases,

379 and a putative high-affinity nickel transport protein (Table S5), however, it lacks an ABC-type

380 uptake system for Fe3+ present in other characterized Nitrospira species [24]. Furthermore, no

381 known proteins associated with siderophore production were identified in the N. marina

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382 genome. However, the presence of multiple TonB-like receptors suggests the potential for the

383 uptake of iron-bound-siderophores produced by other microbes. Additionally, the N. marina

384 genome contains three putative bacterioferritin-related proteins for iron storage. While only one

385 complete ABC-type cobalamin/Fe3+ siderophore uptake system that likely transports cobalamin

386 (discussed above) was identified in the N. marina genome, an additional periplasmic substrate

387 binding protein of a putative ABC-type cobalamin/Fe3+-siderophore transport system is located

388 in proximity to iron uptake-related genes including a ferric uptake regulator (Fur) and a ferritin-

389 like protein (Table S5). Additionally, some major facilitator superfamily-type permeases have

390 been shown to promote bacterial iron-siderophore import from the periplasm into the cytoplasm

391 [83]. Alternatively, iron might be released from the siderophore by a reduction process in the

392 periplasm and subsequently imported into the cytoplasm by one of the divalent cation

393 transporters or the putative VIT1/CCC1 family Fe2+/Mn2+ transporter, which would enable re-

394 utilization of intact siderophores [83].

395

396 Metabolic response to low oxygen concentrations

397 Given the presence of multiple signatures of microaerophilic adaptation and metabolic diversity

398 of nitrite oxidizers [24, 25, 55, 59] and their occurrence in low oxygen environments [23, 84, 85],

399 we sought to further investigate potential adaptations of Nitrospira marina Nb-295 to low O2

400 conditions. N. marina was grown at O2 concentrations characteristic for most parts of the oxic

401 ocean (∼200 µM) and at O2-limiting conditions (∼5.6 µM O2) found in environments with high

- 402 NO2 concentrations such as oxygen minimum zones or sediments [86, 87].

- 403 When grown under atmospheric O2 concentration, N. marina oxidized 1.5 mM NO2

- 404 within 12 days whereas NO2 oxidation activity was decreased under O2-limiting conditions and

405 depletion of 1.5 mM substrate took 27 days (Fig. 4). This result is in agreement with the initial

406 description by Watson et al. [5], who reported partial inhibition at low O2 partial pressure. Cell

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- 407 abundances at the final timepoint (after 1.5 mM NO2 was oxidized) were comparable for both

- 408 treatments (Fig. S2), indicating that the reduced NO2 oxidation activity during O2-limiting

409 conditions ultimately resulted in similar cell yields.

410

411 General proteomic response and upregulation of gene clusters

412 Cultures grown under atmospheric O2 tension and under low O2 concentration were harvested

413 for proteomic analysis during exponential growth (see Material and Methods). A total of 2,086

414 and 2,100 proteins were identified by liquid chromatography-tandem mass spectrometry (LC-

415 MS/MS) in the atmospheric and low O2 treatments, respectively, accounting for 48.8 and 49.1 %

416 (49.9 % combined from a total of 175,653 peptides at a false discovery rate of 4.6%) of the

417 predicted protein coding sequences (CDS) in the N. marina genome. As previously reported for

418 N. marina and Nitrococcus mobilis, proteins exhibiting the highest abundances were associated

- 419 with NO2 oxidation [41] (Table S5). NXR made up on average 4% of all peptide spectral counts

420 and cellular NXR concentrations were ~13,500 copies cell-1 (Table 1), approximately covering

421 one-fourth of the membrane surface (see Material and Methods). All three NxrA copies were

422 detected in the proteome as determined by detection of unique peptides in each, and NxrA_3

423 appeared to be more abundant compared to NxrA_1 and NxrA_2 (Table 1). Under low O2

424 conditions, NxrA_1 increased in abundance (Table 1, Fig. 5) indicating different metabolic or

425 regulatory roles of the highly similar subunits.

426 Proteins involved in CO2 fixation, DNA replication, electron transport and central carbon

427 metabolism were also highly abundant under both conditions, indicating that N. marina retained

428 its central metabolic capacities during O2 deficiency (Table S5). Although the relative

429 abundances of the majority of proteins remained constant during both treatments, 93 proteins

430 significantly increased in abundance (adjusted P value ≤0.05) during growth at low O2

431 concentrations (Fig. 5, Table S3). These results are supported by the targeted quantitative

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432 proteomics analysis (Fig. S3, Table S2), suggesting that differences in spectral counts are a

433 good proxy for changes in absolute protein abundances in our dataset.

434 Multiple universal stress proteins (UspA superfamily) were among the proteins that

435 showed the highest increase in abundance under low O2 conditions compared to the control

436 treatment (Fig. 5, Table S3). UspA proteins have versatile regulatory and protective functions to

437 enable survival under diverse external stresses [88] and are induced during growth inhibition

438 [89]. They are also induced in response to oxygen starvation in smegmatis [90]

439 and contribute to the survival of Pseudomonas aeruginosa under anaerobic conditions [91]. In

440 N. marina, all four upregulated UspA proteins are located upstream or downstream of operons

441 containing genes that also increased in abundance under low O2 conditions (Fig. 5), suggesting

442 a regulatory role of UspA-related proteins upon O2 limitation. These upregulated gene clusters

443 include proteins involved in electron transport, carbon metabolism and alternative energy

444 metabolism (Fig. 5).

445 In addition to putative UspA-regulated gene clusters, a gene cluster containing type VI

446 secretion system (T6SS)-related proteins exhibited higher abundances under low O2 conditions

447 (Fig. 5). The T6SS is typically involved in the secretion of effectors required for pathogenesis,

448 bacterial competition, formation, and cell communication (e.g., quorum sensing) [92, 93].

449 While quorum sensing has recently been shown for diverse NOB including Nitrospira

450 moscoviensis [94], no LuxI autoinducer synthases and/or LuxR signal receptor homologues

451 were identified in the N. marina genome. Hence, the role of T6SS in N. marina remains to be

452 determined.

453

454 Induction of a putative O2-sensitive 2-oxoacid:ferredoxin oxidoreductase complex

455 The key enzymes of the rTCA cycle, pyruvate:ferredoxin oxidoreductase (POR) and 2-

456 oxoglutarate:ferredoxin oxidoreductase (OGOR), are typically highly O2 sensitive because they

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457 contain easily oxidized iron-sulfur clusters [95]. In thermophilus, five-subunit

458 O2-tolerant forms of POR and OGOR mainly support aerobic growth, while a O2-sensitive two-

459 subunit form is used under anaerobic conditions [96]. N. marina encodes three 2-

460 oxoacid:ferredoxin oxidoreductase gene clusters that could exhibit POR or OGOR activity

461 (Table S5). Two of these gene clusters consist of five CDS which exhibit a high sequence

462 similarity to the O2-tolerant five-subunit POR/OGOR of H. thermophilus [97, 98], as previously

463 described for Ca. N. defluvii [24]. Both complexes were highly abundant in N. marina proteomes

464 from atmospheric and low O2 treatments (Table S5, Table 1) confirming their important role in

465 central carbon metabolism. The third cluster contains alpha, beta and gamma subunits of a

466 putative POR with homology to the functionally characterized four-subunit PORs of anaerobic

467 thermophiles Pyrococcus and [99] and is absent in all other Nitrospira with the

468 exception of Ca. N. alkalitolerans [9]. A protein with a 4Fe-4S binding domain was identified in

469 the same operon, potentially representing the missing delta subunit of the POR complex (Table

470 S5). This putative four-subunit POR was among the proteins that showed the highest increase

471 in abundance under low O2 conditions in N. marina (Fig. 5). The O2-tolerant POR/OGOR

472 isoforms were reported to have a >5 times lower specific activity [96] and might therefore

473 constitute a substantial part of the cellular soluble protein content in H. thermophilus [100].

474 Hence, it is tempting to speculate that N. marina increases the expression of a more efficient

475 (i.e. higher specific activity), O2-sensitive four-subunit POR under O2 limitation. While oxidative

476 stress typically decreases under low O2 conditions, it might still be high enough to damage O2-

477 sensitive enzymes. In N. marina, the abundance a periplasmic Cu/Zn-binding superoxide

478 dismutase (SOD) and a cytoplasmic catalase (KatG) increased under O2-limited conditions (Fig.

479 5), while the abundances of other oxidative stress defense-related proteins remained constant

480 (Table S5). SOD has been shown to be efficient in protecting POR activity from oxidative

481 damage in Entamoeba histolytica [101]. In N. marina, SOD was among the proteins with the

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482 highest increase in abundance under low O2 conditions (foldchange: 27.9), suggesting a role in

483 POR protection.

484

485 Expression of a high O2-affinity cbb3-type terminal oxidase

486 The majority of proteins related to electron transport showed similar abundance levels during

487 growth at atmospheric and limiting O2 concentrations (Table S5). Despite its overall low

488 abundance, a putative high-affinity cytochrome cbb3-type terminal oxidase was 3.6-times more

489 abundant under low O2 concentrations compared to atmospheric O2 tension (Fig. 5). The cbb3-

490 type terminal oxidase of Bradyrhizobium japonicum was reported to have a Km value of 7 nmol

-1 - 491 L O2 [102] and NO2 oxidation rates have been detected at O2 concentrations in the low

−1 492 nanomolar range (5-33 nmol L O2) [23]. This suggests that the cbb3-type terminal oxidase

493 might enable N. marina to retain aerobic respiration at low O2 concentrations, albeit at lower

- 494 NO2 oxidation rates (Fig. 4). Low-affinity terminal oxidases are typically more efficient in energy

495 conservation [103], indicating that N. marina benefits from the presence of a terminal oxidase

496 with lower O2 affinity in well-oxygenated environments. While N. gracilis encodes a highly similar

497 high affinity cbb3-type terminal oxidase as N. marina [55], this enzyme is lacking in most

498 Nitrospinae, including those identified in oxygen minimum zones (OMZ) and sediments [85, 104,

499 105]. Interestingly, these genomes also lack the putative terminal oxidase proposed for

- 500 Nitrospira [24]. Still, in an OMZ where Nitrospinae bacteria were the only detected NOB, NO2

−1 501 oxidation rates already approached saturation at ∼1 μmol L O2 [23], indicating that Nitrospinae

502 bacteria might be better adapted to low O2 concentrations compared to N. marina, but the

503 enzyme conferring high O2 affinity in Nitrospinae remains to be identified.

504

505 Upregulation of genes involved in alternative energy metabolism

506 The abundance of proteins putatively involved in alternative energy metabolisms increased

507 under low O2 concentrations. These included type 3b [NiFe] hydrogenase and formate

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508 dehydrogenase (Fig. 5). Alternative electron donors such as H2 and formate are common

509 reaction products at oxic/anoxic interfaces [106]. N. moscoviensis can couple H2 and formate

- 510 oxidation to NO3 reduction to remain active under anoxia [25, 27] (whereas no net growth was

511 observed for the latter [25]), however, it is unlikely that H2 or formate were present at the culture

512 conditions in this study. While abundances of hydrogenase and formate dehydrogenase

513 increased under low O2 conditions, they were overall still comparably low (Fig. 5), suggesting

514 that their expression might be regulated when conditions become more unfavorable for aerobic

– + 515 NO2 oxidation. Curiously, multiple subunits of a Na -translocating ATPase (alternative complex

+ 516 V) exhibited higher abundances at low O2 concentration (Fig. 5). Na -translocating ATPases are

517 suggested to be ancient enzymes that were later replaced by energetically more favorable H+-

518 translocating ATPases [107]. While only few obligate anaerobes with very tight energy budgets

519 (which cannot cover the losses caused by proton leaks) primarily use Na+ energetics, many

520 organisms retained Na+ pumps and utilize them under energetically less favorable conditions

521 such as anaerobiosis [107]. Hence, expression of a Na+-translocating ATPase suggests an

522 adaptation of N. marina to overcome periods of starvation when energetically favorable electron

523 donors or acceptors are short in supply.

524

525 Conclusions

526 Although the vast majority of the ocean is well oxygenated, oxygen-depleted zones exist within

527 the oceanic water column and in marine sediments [86, 87] with consequences for microbial

528 adaptation and evolution. Our results show that, in contrast to some marine NOB populations,

- -1 529 NO2 oxidation activity of Nitrospira marina was reduced when grown at ∼5.6 µM L O2,

530 suggesting different O2 adaptations among different NOB. In accordance with its faster growth

531 rates at atmospheric O2 tension, N. marina encodes a plethora of proteins involved in oxidative

532 stress defense. We confirm that N. marina benefits from the addition of undefined organic

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533 substrates, which were shown to be inhibitory for Nitrospina gracilis [108], potentially further

534 contributing to ecological niche partitioning within marine NOB. Our results indicate that N.

535 marina is highly metabolically versatile, which might enable it to survive under unfavorable

536 conditions with fluctuating levels of electron donors and acceptors. Hence, while Nitrospinae

537 bacteria are the dominant nitrite oxidizers in oligotrophic oceanic regions and OMZs, Nitrospira

538 might be better adapted to well-oxygenated high productivity regions including coastal systems,

539 deep-sea trenches and hydrothermal vents. Finally, our results also indicate several previously

540 unreported ways that NOB may interact with other members of the marine microbial

541 community—through the supply of organic carbon-containing osmolytes and their requirement

542 for exogenous cobalamin.

543

544 Acknowledgements

545 We thank John B. Waterbury and Frederica Valois for providing the culture of Nitrospira marina

546 Nb-295T. The N. marina genome was sequenced as part of US Department of Energy Joint

547 Genome Institute Community Sequencing Project 1337 to CLD, AES, and MAS in collaboration

548 with the user community. We thank Claus Pelikan for bioinformatic assistance. This research

549 was supported by a Simons Foundation Early Career Investigator in Marine Microbiology and

550 Evolution Award (345889) and US National Science Foundation (NSF) award OCE-1924512 to

551 AES. Proteomics analysis was supported by NSF awards OCE-1924554 and OCE-1850719,

552 and NIH award 1R01GM135709-01A1 to MAS. BB was supported by the Austrian Science Fund

553 (FWF) Project Number: J4426-B (“The influence of nitrifiers on the oceanic carbon cycle”), SL

554 by the Netherlands Organization for Scientific Research (NWO) grant 016.Vidi.189.050, and

555 CLD by NSF award OCE-125999.

556

557 Data availability

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (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 available under aCC-BY-NC 4.0 International license.

558 The genome of Nitrospira marina Nb-295T is available in the JGI IMG/M repository under

559 genome ID number 2596583682. Targeted peptide concentrations are available in

560 Supplementary Table 2, global proteomic spectral counts and differential expression analysis

561 are available in Supplementary Table 3. Raw mass spectra are available in PRIDE as project

562 number XXXX.

563

564

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565 Figure and table legends

566

567 Fig 1. Maximum likelihood phylogenetic tree of representatives of the phylum

568 Nitrospirae/Nitrospirota. The multiple sequence alignment consisting of 120 concatenated

569 phylogenetic marker genes contained 95 genomes and metagenome-assemble genomes

570 (MAGs) from the Genome Taxonomy Database (GTDB) (Release 04-RS89, 19 June 2019), the

571 genome of N. marina, the MAG of Ca. N. alkalitolerans and two open ocean SAGs AC-738-G23

572 and AC-732-L14. All MAGs and SAGs were estimated to be ≥50% complete with ≤5%

573 contamination. Nodes with UFBoot support of at least 95% are indicated as black filled circles.

574 The scale bar represents 0.1 substitutions per site.

575

576 Fig 2. The effect of undefined organic carbon substrates on nitrite oxidation activity and growth

577 of N. marina Nb-295. Error bars represent the range of measurements from duplicate cultures.

578

579 Fig 3. Growth of N. marina Nb-295 on formate (1mM), pyruvate (1mM) and yeast+tryptone (150

580 mg L-1 each) in the absence of nitrite. Error bars represent the range of measurements from

581 duplicate cultures.

582

583 Fig 4. Nitrite consumption of N. marina Nb-295 grown under atmospheric (filled circles) and low

584 O2 conditions (open circles). Cells for proteome analysis were harvested after 12 and 27 days

585 (indicated by red arrows), respectively. Error bars represent standard deviations from

586 measurements of triplicate cultures.

587

588 Fig 5. Heat map of N. marina Nb-295 proteins that were more abundant under low O2

589 concentrations compared to atmospheric O2 concentration. Relative protein abundance values

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590 were square-root transformed and hypothetical proteins were excluded to improve readability.

591 The complete set of untransformed values can be found in Table S3. Fold-changes and

592 significance values (adj. P value ≤ 0.001, ***; ≤ 0.01, **; ≤ 0.05, *) are shown in white boxes next

593 to the corresponding protein. Select low abundant proteins of interest with high Fold-changes

594 were included despite being not statistically significant (see Material and Methods). Fold-

595 changes of proteins that were not detected under atmospheric O2 conditions are omitted to

596 avoid dividing by zero (not available, na). Functional categories of depicted proteins are

597 indicated by different colors. Gene clusters are indicated by black brackets.

598

599 Table 1. Quantitative proteomics of selected proteins using isotopically-labelled (15N) peptide

600 standards. Nitrite oxidoreductase peptide targets were designed to match a specific NxrA copy

601 (1, 2 or 3), or to match all three as indicated in the target protein column. Values represent the

602 mean of triplicate measurements and standard deviations are shown in brackets.

603

604

605

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Other Nitrospirae / Nitrospirota

Nitrospiraceae bacterium NP85 (GCA_002726235.1) Nitrospiraceae bacterium UBA2166 (GCA_002328825.1) Nitrospira sp. bin75 (GCA_002238765.1) Nitrospirae bacterium SPGG5 (GCA_001643555.1) Nitrospirae bacterium SCGC AC-728-G23 (2634166671) Nitrospirae bacterium SCGC AC-732-L14 (2634166674) Nitrospira marina Nb-295 Nitrospirae bacterium UBA8639 (GCA_003523945.1) Ca. Nitrospira alkalitolerans KS Nitrospirae bacterium SZUA-312 (GCA_003235785.1) Nitrospirae bacterium SZUA-4 (GCA_003233615.1) Nitrospirae bacterium SZUA-215 (GCA_003228495.1) Nitrospirae bacterium 13_2_20CM_2_62_8 (GCA_001914955.1) Nitrospirae bacterium RIFCSPLOWO2 (GCA_001805245.1) Nitrospira sp. OLB3 (GCA_001567445.1) Nitrospira sp003456605 (GCA_003456605.1) Ca. Nitrospira sp. strain ND1 (GCF_900170025.1) Ecosystem type Ca. Nitrospira defluvii (GCF_000196815.1) Nitrospira japonica NJ11 (GCF_900169565.1) marine/saline Nitrospira sp. UBA5699 (GCA_002420105.1) freshwater/terrestrial Nitrospirae bacterium Baikal-G1 (GCA_002737345.1) human-made Nitrospira sp. palsa_1310 (GCA_003135435.1) Nitrospira sp. CG24C (GCA_002869885.1) Nitrospira sp. CG24E (GCA_002869895.1) Specific environment Nitrospira sp. CG24A (GCA_002869925.1) Nitrospira sp. UBA6493 (GCA_002435325.1) Marine water column Nitrospira lenta BS10 (GCA_900403705.1) Marine hydrothermal vent Nitrospira sp. ST-bin5 (GCA_002083555.1) Marine sediment Nitrospira sp. CG24D (GCA_002869855.1) Nitrospira moscoviensis NSP M-1 (GCF_001273775.1) Marine sponge Nitrospira sp. UBA2082 (GCA_002331335.1) Saline alkaline lake Nitrospira inopinata ENR4 (GCF_001458695.1) Nitrospira sp. UBA6909 (GCA_002451055.1) Freshwater Nitrospira sp. ST-bin4 (GCA_002083565.1) Soil Nitrospira sp. SBR1015 (GCF_900078535.2) Activated sludge Nitrospira sp. SG-bin1 (GCA_002083365.1) Ca. Nitrospira nitrificans COMA2 (GCF_001458775.1) Aquaculture/drinking water biofilter Nitrospira sp. CG24B (GCA_002869845.1) Urban waterways Nitrospira sp. Ga0074138 (GCA_001464735.1) Nitrospira sp. UBA2083 (GCA_002331625.1) Heating system pipe Nitrospira sp. SBR1015 (GCF_900078515.1) Subsurface aquifer Nitrospira sp. UBA5698 (GCA_002420115.1) Ca. Nitrospira nitrosa COMA1 (GCF_001458735.1) Nitrospira sp. UW-LDO-01 (GCA_002254365.1) Tree scale: 0.1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (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 available under aCC-BY-NC 4.0 International license.

2000 - NO2 - NO2 + tryptone - 1500 NO2 + yeast extract

1000

Nitrite (µM) 500

0 3×107

) 7 -1 2×10

107 Cells (ml

0 0 3 6 9 12 15 Time (d) bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (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 available under aCC-BY-NC 4.0 International license.

107 Formate Pyruvate 7.5×106 Yeast+Tryptone ) -1

5×106 Cells (ml 2.5×106

0 0 10 20 30 Time (d) bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (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 available under aCC-BY-NC 4.0 International license.

2000 atm. O2

low O2 1500

1000 Nitrite (µM) Nitrite

500

0 4 8 12 16 20 24 28 Time (d) 1.5 *** 2597406480 Nitrite oxidoreductase, alpha subunit (NxrA_1) 6.8 ** 2597406746 Glucose / Sorbosone dehydrogenase 2.6 * 2597407001 Polyphosphate glucokinase

3.7 2597407265 Cbb3 −type cytochrome oxidase, fused subunits I, II, and III 2.9 ** 2597407299 Formate / Nitrite transporter, FNT family 2 *** 2597407300 Ferredoxin−nitrite reductase NirA 3.3 * 2597407379 putative Quercetin 2,3−dioxygenase 3.2 * 2597407380 RND efflux system, membrane fusion protein 27.9 *** 2597407590 Cu/Zn−binding superoxide dismutase na ** 2597407591 putative Cytochrome C 5 * 2597407609 Succinate dehydrogenase / Fumarate reductase cytochrome b subunit na 2597407610 Succinate dehydrogenase / Fumarate reductase iron−sulfur subunit na 2597407811 putative Formate dehydrogenase 18.2 ** 2597408031 putative Oxidoreductase, molybdopterin−binding 2.8 *** 2597408085 Type VI secretion system protein ImpJ 3.3 *** 2597408087 Type VI secretion system protein ImpL 3.2 * 2597408089 Type VI secretion system protein ImpE 3.2 *** 2597408090 Type VI secretion system protein ImpA bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.1855041.7 *** 2597408092; this version posted Type July VI secretion4, 2020. The system copyright protein holder for ImpC this preprint (which was not certified by peer review) is the author/funder, who2597408106 has granted bioRxiv Type a licenseVI secretion to display system the preprint protein in perpetuity. ImpG It is made 10.5available under aCC-BY-NC 4.0 International license. 4.1 2597408107 Type VI secretion system protein ImpH 2 *** 2597408108 Type VI secretion system protein VasG 8.2 *** 2597408110 Type VI secretion system secreted protein VgrG na * 2597408115 putative 3−oxoacyl−ACP−synthase 1.5 * 2597408190 putative Porin 2.7 * 2597408197 Glutamate−ammonia− adenylyltransferase GlnE 2.4 *** 2597408359 Translation elongation factor 2 (EF−2/EF−G) 2 * 2597408607 Ribosome recycling factor 2.1 ** 2597408676 putative Malto−oligosyltrehalose synthase na * 2597408699 putative Peptidoglycan−associated lipoprotein 1.5 * 2597408986 Catalase (hydroperoxidase II) KatG 16 *** 2597409086 Multicopper oxidase 9.2 * 2597409113 FAD/FMN−containing dehydrogenase 5.3 * 2597409177 PAS domain S−box−containing protein 4 * 2597409271 CBS domain−containing protein 6.4 ** 2597409318 putative Alpha−amylase 2.7 * 2597409327 putative Alpha−amylase 2.2 *** 2597409329 Xylulose−5−phosphate / Fructose−6−phosphate phosphoketolase 1.8 * 2597409331 Universal stress protein, UspA family 2.1 * 2597409377 Cold shock protein, CspA family 3.5 * 2597409384 6−Phosphofructokinase PfkA 15.3 *** 2597409388 Universal stress protein, UspA family

18 ** 2597409389 alternative F1F0 ATPase, subunit beta na *** 2597409395 alternative F1F0 ATPase, subunit B 7.5 *** 2597409396 alternative F1F0 ATPase, subunit alpha na *** 2597409398 Polyphosphate:nucleotide phosphotransferase, PPK2 family 7.1 ** 2597409401 PAS domain S−box−containing protein 4.1 *** 2597409403 Response regulator receiver protein na *** 2597409404 Universal stress protein, UspA family na * 2597409406 Cupredoxin family copper−binding protein na * 2597409409 [NiFe] Hydrogenase, beta subunit 2.8 * 2597409415 Glutamine−amidotransferase domain−containing protein na 2597409416 [NiFe] Hydrogenase, gamma subunit 6.3 *** 2597409418 [NiFe] Hydrogenase, alpha subunit 5.7 *** 2597409427 Universal stress protein, UspA family 10.2 ** 2597409428 alternative Pyruvate:ferredoxin oxidoreductase, delta subunit (alt_PorD) na ** 2597409429 alternative Pyruvate:ferredoxin oxidoreductase, beta subunit (alt_PorB) 17.1 * 2597409430 alternative Pyruvate:ferredoxin oxidoreductase, alpha subunit (alt_PorA) na ** 2597409431 alternative Pyruvate:ferredoxin oxidoreductase, gamma subunit (alt_PorC) 2.6 * 2597409460 Signal transduction histidine kinase 5.3 ** 2597409465 Transcriptional regulator, Fis family 8.4 ** 2597409716 OsmC family protein 3.7 * 2597409744 Cytochrome C 2 *** 2597409910 putative (Protein PII) uridylyltransferase GlnD 2.9 ** 2597409911 Glutamine−dependent NAD(+) synthase NadE 1.9 * 2597410076 UDP−N−acetylmuramoylalanyl−D−glutamate−−2,6−diaminopimelate ligase 2 *** 2597410120 Hopanoid biosynthesis associated radical SAM protein HpnH na *** 2597410638 PlsC domain−containing protein 2.3 ** 2597410685 putative Peptidase M20 22.3 * 2597410732 Cytochrome P450 A B C D E F

atm. O2 low O2 Functional category: Regulatory function Protein synthesis Normalized protein abundance Electron transport chain Molecular transport Carbon metabolism Alternative metabolism 0 0.2 0.4 0.6 0.8 1 Nitrogen metabolism Cell wall synthesis and associated proteins Oxidative stress defense Unknown function bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (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 available under aCC-BY-NC 4.0 International license.

Table 1. Quantitative proteomics of selected proteins using isotopically-labelled (15N) peptide standards. Nitrite oxidoreductase peptide targets were designed to match a specific NxrA copy (1, 2 or 3), or to match all three as indicated in the target protein column. Values represent the mean of triplicate measurements and standard deviations are shown in brackets.

Target protein* 15N-Peptide sequence Peptide concentration (copies cell -1)

atm. O2 low O2

Nitrite oxidoreductase, alpha subunit 1 LVVITPEYNPTAYR 845 (52) 1,924 (275) DYAFPDFANSYSGK 241 (40) 451 (91)

HPFWEETNESKPQWTR 673 (39) 1,571 (388)

Nitrite oxidoreductase, alpha subunit 2 VVVITPEYNPTAQR 1,154 (27) 851 (187) Nitrite oxidoreductase, alpha subunit 3 DYQFPDFTSTYSGK 1,761 (159) 1,261 (242) SGIDPALTGTHR 4,519 (516) 3,400 (526)

IAVITPEYNPTAYR 4,860 (496) 3,741 (710)

Nitrite oxidoreductase, alpha subunit (all) GWKPSDPYYK 11,467 (1,957) 9,564 (1,083) AIALDTGYQSNFR 13,996 (962) 13,116 (2,934)

Pyruvate:ferredoxin oxidoreductase, TPSFFTGSEVIK 2,026 (192) 1,550 (248) alpha subunit (PorA) EAIAILEEEGIR 1,362 (80) 1,054 (170)

EVSATVPNNER 2,453 (169) 1,736 (215)

Ribonucleotide reductase TGESPYQTIPFSHR 300 (46) 198 (30) EAAVPEPYIHR 705 (132) 472 (77)

IINQSLPPALR 750 (88) 546 (68)

*A complete list of quantified proteins can be found in Supplementary table 2.