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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 nitrite-oxidizing bacterium Nitrospira
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: Genome and proteome of Nitrospira marina
20
21 Competing Interests: The authors declare that they have no conflict of interest.
22
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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.
23 Abstract
24 The genus Nitrospira is the most widespread group of chemolithoautotrophic nitrite-oxidizing
25 bacteria 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 oxidoreductase (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
2 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.
47 Introduction
- - 48 Aerobic nitrite (NO2 ) oxidation is the main biochemical nitrate (NO3 )-forming reaction, carried
49 out during the second step of nitrification [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 (Nitrobacter, 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 habitats [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 phylum Nitrospinae 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 enzyme of
- 71 the NO2 oxidation, and the presence of the O2-sensitive reductive tricarboxylic acid (rTCA)
72 cycle for carbon fixation [24]. These results suggest that Nitrospira evolved from microaerophilic
3 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.
73 or anaerobic ancestors [24]. Ca. N. defluvii lacks genes for classical oxidative stress defense
74 enzymes 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 ammonia 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
4 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.
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 Genomes 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 domain 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.
5 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.
123 Phylogenomic analysis was performed using 120 concatenated phylogenetic marker
124 genes of representatives of the phylum Nitrospirae/Nitrospirota as implemented in the Genome
125 Taxonomy 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
6 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.
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
7 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.
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 nitrite reductase (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 lyase 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 urease operon or
196 a urea 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
8 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.
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 heme 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 Acidobacteria [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+-
9 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.
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 carbon dioxide (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
10 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.
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 vitamin B12-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 hydrolase
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
11 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.
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
12 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.
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 periplasm 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
13 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.
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 corrin
350 ring [74]. However, N. marina encodes for a putative bifunctional precorrin-2
351 dehydrogenase/sirohydrochlorine ferrochelatase converting precorrin-2 to siroheme, a cofactor
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.
14 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.
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 cobalamin biosynthesis 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
15 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.
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
16 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.
- 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
17 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.
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 Mycobacterium 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, biofilm 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
18 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.
457 contain easily oxidized iron-sulfur clusters [95]. In Hydrogenobacter 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 Thermotoga [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
19 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.
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
20 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.
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
21 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.
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
23 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.
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
24 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.
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
25 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.
606 References
607
608 1. Kuypers MMM, Marchant HK, Kartal B. The microbial nitrogen-cycling network. Nat Rev
609 Microbiol 2018; 16: 263–276.
610 2. Gruber N. The marine nitrogen cycle: Overview and challenges. In: G. Capone, D.A.
611 Bronk, M.R. Mulholland EJC (ed). Nitrogen in the Marine Environment, 2nd ed. 2008.
612 Academic Press, pp 1–50.
613 3. Daims H, Lücker S, Wagner M. A New Perspective on Microbes Formerly Known as
614 Nitrite-Oxidizing Bacteria. Trends Microbiol 2016; 24: 699–712.
615 4. Pachiadaki MG, Sintes E, Bergauer K, Brown JM, Record NR, Swan BK, et al. Major role
616 of nitrite-oxidizing bacteria in dark ocean carbon fixation. Science 2017; 358: 1046–1051.
617 5. Watson SW, Bock E, Valois FW, Waterbury JB, Schlosser U. Nitrospira marina gen. nov.
618 sp. nov.: a chemolithotrophic nitrite-oxidizing bacterium. Arch Microbiol 1986; 144: 1–7.
619 6. Altmann D, Stief P, Amann R, De Beer D, Schramm A. In situ distribution and activity of
620 nitrifying bacteria in freshwater sediment. Environ Microbiol 2003; 5: 798–803.
621 7. Freitag TE, Chang L, Clegg CD, Prosser JI. Influence of inorganic nitrogen management
622 regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils. Appl
623 Environ Microbiol 2005; 71: 8323–8334.
624 8. Pester M, Maixner F, Berry D, Rattei T, Koch H, Lücker S, et al. NxrB encoding the beta
625 subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing
626 Nitrospira. Environ Microbiol 2014; 16: 3055–3071.
627 9. Daebeler A, Kitzinger K, Koch H, Herbold CW, Steinfeder M, Schwarz J, et al. Exploring
628 the upper pH limits of nitrite oxidation: diversity, ecophysiology, and adaptive traits of
629 haloalkalitolerant Nitrospira. bioRxiv 2020; doi:
630 https://doi.org/10.1101/2020.03.05.977850.
26 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.
631 10. Lebedeva E V., Alawi M, Fiencke C, Namsaraev B, Bock E, Spieck E. Moderately
632 thermophilic nitrifying bacteria from a hot spring of the Baikal rift zone. FEMS Microbiol
633 Ecol 2005; 54: 297–306.
634 11. Gruber-Dorninger C, Pester M, Kitzinger K, Savio DF, Loy A, Rattei T, et al. Functionally
635 relevant diversity of closely related Nitrospira in activated sludge. ISME J 2015; 9: 643–
636 655.
637 12. Spieck E, Hartwig C, McCormack I, Maixner F, Wagner M, Lipski A, et al. Selective
638 enrichment and molecular characterization of a previously uncultured Nitrospira-like
639 bacterium from activated sludge. Environ Microbiol 2006; 8: 405–415.
640 13. Ushiki N, Fujitani H, Aoi Y, Tsuneda S. Isolation of Nitrospira belonging to sublineage II
641 from a wastewater treatment plant. Microbes Environ 2013; 28: 346–353.
642 14. Van Kessel MAHJ, Speth DR, Albertsen M, Nielsen PH, Op Den Camp HJM, Kartal B, et
643 al. Complete nitrification by a single microorganism. Nature 2015; 528: 555–559.
644 15. Keuter S, Kruse M, Lipski A, Spieck E. Relevance of Nitrospira for nitrite oxidation in a
645 marine recirculation aquaculture system and physiological features of a Nitrospira
646 marina-like isolate. Environ Microbiol 2011; 13: 2536–2547.
647 16. Nowka B, Off S, Daims H, Spieck E. Improved isolation strategies allowed the phenotypic
648 differentiation of two Nitrospira strains from widespread phylogenetic lineages. FEMS
649 Microbiol Ecol 2015; 91: fiu031.
650 17. Hongxiang X, Min W, Xiaogu W, Junyi Y, Chunsheng W. Bacterial diversity in deep-sea
651 sediment from northeastern Pacific Ocean. Acta Ecol Sin 2008; 28: 479–485.
652 18. Baker BJ, Sheik CS, Taylor CA, Jain S, Bhasi A, Cavalcoli JD, et al. Community
653 transcriptomic assembly reveals microbes that contribute to deep-sea carbon and
654 nitrogen cycling. ISME J 2013; 7: 1962–1973.
655 19. Nunoura T, Takaki Y, Hirai M, Shimamura S, Makabe A, Koide O, et al. Hadal biosphere:
656 Insight into the microbial ecosystem in the deepest ocean on Earth. Proc Natl Acad Sci U
27 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.
657 S A 2015; 112: E1230–E1236.
658 20. Hiraoka S, Hirai M, Matsui Y, Makabe A, Minegishi H, Tsuda M, et al. Microbial
659 community and geochemical analyses of trans-trench sediments for understanding the
660 roles of hadal environments. ISME J 2020; 14: 740–756.
661 21. Lam P, Kuypers MMM. Microbial nitrogen cycling processes in oxygen minimum zones.
662 Ann Rev Mar Sci 2011; 3: 317–345.
663 22. Codispoti LA, Friederich GE, Packard TT, Glover HE, Kelly PJ, Spinrad RW, et al. High
664 nitrite levels off northern Peru: A signal of instability in the marine denitrification rate.
665 Science 1986; 233: 1200–1202.
666 23. Bristow LA, Dalsgaard T, Tiano L, Mills DB, Bertagnolli AD, Wright JJ, et al. Ammonium
667 and nitrite oxidation at nanomolar oxygen concentrations in oxygen minimum zone
668 waters. Proc Natl Acad Sci U S A 2016; 113: 10601–10606.
669 24. Lücker S, Wagner M, Maixner F, Pelletier E, Koch H, Vacherie B, et al. A Nitrospira
670 metagenome illuminates the physiology and evolution of globally important nitrite-
671 oxidizing bacteria. Proc Natl Acad Sci U S A 2010; 107: 13479–13484.
672 25. Koch H, Lücker S, Albertsen M, Kitzinger K, Herbold C, Spieck E, et al. Expanded
673 metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira. Proc
674 Natl Acad Sci U S A 2015; 112: 11371–11376.
675 26. Ushiki N, Fujitani H, Shimada Y, Morohoshi T, Sekiguchi Y, Tsuneda S. Genomic
676 analysis of two phylogenetically distinct Nitrospira species reveals their genomic plasticity
677 and functional diversity. Front Microbiol 2018; 8: 2637.
678 27. Koch H, Galushko A, Albertsen M, Schintlmeister A, Gruber-Dorninger C, Lücker S, et al.
679 Growth of nitrite-oxidizing bacteria by aerobic hydrogen oxidation. Science 2014; 345:
680 1052–1054.
681 28. Daims H, Lebedeva E V., Pjevac P, Han P, Herbold C, Albertsen M, et al. Complete
682 nitrification by Nitrospira bacteria. Nature 2015; 528: 504–509.
28 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.
683 29. Strickland JDH, Parsons TR. A practical handbook of seawater analysis, 2nd ed. 1972.
684 Fisheries Research Board of Canada.
685 30. Porter KG, Feig YS. The use of DAPI for identifying aquatic microflora. Limnol Oceanogr
686 1980; 25: 943–948.
687 31. Wilson K. Preparation of Genomic DNA from Bacteria. Curr Protoc Mol Biol 2001; 56:
688 2.4.1-2.4.5.
689 32. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. Nonhybrid,
690 finished microbial genome assemblies from long-read SMRT sequencing data. Nat
691 Methods 2013; 10: 563–569.
692 33. Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Grechkin Y, et al. IMG: the
693 Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids
694 Res 2012; 40: D115-22.
695 34. Vallenet D, Calteau A, Dubois M, Amours P, Bazin A, Beuvin M, et al. MicroScope: an
696 integrated platform for the annotation and exploration of microbial gene functions through
697 genomic, pangenomic and metabolic comparative analysis. Nucleic Acids Res 2020; 48:
698 D579–D589.
699 35. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool.
700 J Mol Biol 1990; 215: 403–410.
701 36. Saier MH. TCDB: the Transporter Classification Database for membrane transport protein
702 analyses and information. Nucleic Acids Res 2006; 34: D181–D186.
703 37. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, et al. InterProScan:
704 Protein domains identifier. Nucleic Acids Res 2005; 33: W116-120.
705 38. Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S,
706 et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat
707 Biotechnol 2019; doi:10.1038/s41587-019-0036-z.
708 39. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify
29 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.
709 genomes with the Genome Taxonomy Database. Bioinformatics 2019; 36: 1925–1927.
710 40. Malmstrom R. RNAlater Recipe. protocols.io 2015;
711 dx.doi.org/10.17504/protocols.io.c56y9d.
712 41. Saito MA, Mcilvin MR, Moran DM, Santoro AE, Dupont CL, Rafter PA, et al. Abundant
713 nitrite-oxidizing metalloenzymes in the mesopelagic zone of the tropical Pacific Ocean.
714 Nat Geosci 2020; 13: 355–362.
715 42. Spieck E, Ehrich S, Aamand J, Bock E. Isolation and immunocytochemical location of the
716 nitrite-oxidizing system in Nitrospira moscoviensis. Arch Microbiol 1998; 169: 225–230.
717 43. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for
718 RNA-seq data with DESeq2. Genome Biol 2014; 15: 550.
719 44. R Core Team. R: A language and environment for statistical computing. R Foundation for
720 Statistical Computing, Vienna, Austria. 2013; http://www.R-project.org.
721 45. Bayer B, Pelikan C, Bittner MJ, Reinthaler T, Könneke M, Herndl GJ, et al. Proteomic
722 Response of Three Marine Ammonia-Oxidizing Archaea to Hydrogen Peroxide and Their
723 Metabolic Interactions with a Heterotrophic Alphaproteobacterium. mSystems 2019; 4:
724 e00181-19.
725 46. Langley SR, Mayr M. Comparative analysis of statistical methods used for detecting
726 differential expression in label-free mass spectrometry proteomics. J Proteomics 2015;
727 129: 83–92.
728 47. Raithel S, Johnson L, Galliart M, Brown S, Shelton J, Herndon N, et al. Inferential
729 considerations for low-count RNA-seq transcripts: A case study on the dominant prairie
730 grass Andropogon gerardii. BMC Genomics 2016; 17: 140.
731 48. Kolde R. pheatmap: Pretty Heatmaps. R package version 1.0.8. 2015; http://CRAN.R-
732 project.org/package_pheatmap.
733 49. Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP. Statistical
734 Analysis of Membrane Proteome Expression Changes in Saccharomyces cerevisiae. J
30 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.
735 Proteome Res 2006; 5: 2339–2347.
736 50. Ye L, Shao MF, Zhang T, Tong AHY, Lok S. Analysis of the bacterial community in a
737 laboratory-scale nitrification reactor and a wastewater treatment plant by 454-
738 pyrosequencing. Water Res 2011; 45: 4390–4398.
739 51. Mundinger AB, Lawson CE, Jetten MSM, Koch H, Lücker S. Cultivation and
740 transcriptional analysis of a canonical nitrospira under stable growth conditions. Front
741 Microbiol 2019; 10: 1325.
742 52. Sakoula D, Nowka B, Spieck E, Daims H, Lücker S. The draft genome sequence of
743 “Nitrospira lenta” strain BS10, a nitrite oxidizing bacterium isolated from activated sludge.
744 Stand Genomic Sci 2018; 13: 32.
745 53. Chadwick GL, Hemp J, Fischer WW, Orphan VJ. Convergent evolution of unusual
746 complex I homologs with increased proton pumping capacity: energetic and ecological
747 implications. ISME J 2018; 12: 2668–2680.
748 54. Cosseau C, Batut J. Genomics of the ccoNOQP-encoded cbb3 oxidase complex in
749 bacteria. Arch Microbiol 2004; 181: 89–96.
750 55. Lücker S, Nowka B, Rattei T, Spieck E, Daims H. The genome of Nitrospina gracilis
751 illuminates the metabolism and evolution of the major marine nitrite oxidizer. Front
752 Microbiol 2013; 4: 27.
753 56. Luoto HH, Belogurov GA, Baykov AA, Lahti R, Malinen AM. Na+-translocating membrane
754 pyrophosphatases are widespread in the microbial world and evolutionarily precede H+-
755 translocating pyrophosphatases. J Biol Chem 2011; 286: 21633–21642.
756 57. Luoto HH, Baykov AA, Lahti R, Malinen AM. Membrane-integral pyrophosphatase
757 subfamily capable of translocating both Na+ and H+. Proc Natl Acad Sci U S A 2013;
758 110: 1255–1260.
759 58. Hausmann B, Pelikan C, Herbold CW, Köstlbacher S, Albertsen M, Eichorst SA, et al.
760 Peatland Acidobacteria with a dissimilatory sulfur metabolism. ISME J 2018; 12: 1729–
31 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.
761 1742.
762 59. Füssel J, Lücker S, Yilmaz P, Nowka B, van Kessel MAHJ, Bourceau P, et al.
763 Adaptability as the key to success for the ubiquitous marine nitrite oxidizer Nitrococcus.
764 Sci Adv 2017; 3: 2–10.
765 60. Ma K, Weiss R, Adams MWW. Characterization of hydrogenase II from the
766 hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur
767 reduction. J Bacteriol 2000; 182: 1864–1871.
768 61. Price GD, Woodger FJ, Badger MR, Howitt SM, Tucker L. Identification of a SulP-type
769 bicarbonate transporter in marine cyanobacteria. Proc Natl Acad Sci U S A 2004; 101:
770 18228–18233.
771 62. Fukaya F, Promden W, Hibino T, Tanaka Y, Nakamura T, Takabe T. An Mrp-like cluster
772 in the halotolerant cyanobacterium Aphanothece halophytica functions as a Na+/H+
773 antiporter. Appl Environ Microbiol 2009; 75: 6626–6629.
774 63. Chistoserdova L, Crowther GJ, Vorholt JA, Skovran E, Portais JC, Lidstrom ME.
775 Identification of a fourth formate dehydrogenase in Methylobacterium extorquens AM1
776 and confirmation of the essential role of formate oxidation in methylotrophy. J Bacteriol
777 2007; 189: 9076–9081.
778 64. Kim J-G, Park S-J, Damsté JSS, Schouten S, Rijpstra WIC, Jung M-Y, et al. Hydrogen
779 peroxide detoxification is a key mechanism for growth of ammonia-oxidizing archaea.
780 Proc Natl Acad Sci USA 2016; 113: 7888–7893.
781 65. Lesser MP. Oxidative stress in marine environments: Biochemistry and physiological
782 ecology. Annu Rev Physiol 2006; 68: 253–278.
783 66. Heelis PF, Kim ST, Okamura T, Sancar A. The photo-repair of pyrimidine dimers by DNA
784 photolyase and model systems. J Photochem Photobiol B 1993; 17: 219–228.
785 67. Roberts M. Organic compatible solutes of halotolerant and halophilic microorganisms.
786 Saline Systems 2005; 1: 5.
32 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.
787 68. Csonka L. Prokaryotic Osmoregulation: Genetics And Physiology. Annu Rev Microbiol
788 1991; 45: 569–606.
789 69. Miller-Fleming L, Olin-Sandoval V, Campbell K, Ralser M. Remaining Mysteries of
790 Molecular Biology: The Role of Polyamines in the Cell. J Mol Biol 2015; 427: 3389–3406.
791 70. Groppa MD, Benavides MP. Polyamines and abiotic stress: Recent advances. Amino
792 Acids 2008; 34: 35–45.
793 71. Boysen AK, Carlson LT, Durham BP, Groussman RD, Aylward FO, Heal KR, et al. Diel
794 Oscillations of Particulate Metabolites Reflect Synchronized Microbial Activity in the North
795 Pacific Subtropical Gyre. bioRxiv 2020; doi: https://doi.org/10.1101/2020.05.09.086173.
796 72. Bayer B, Hansman RL, Bittner MJ, Noriega Ortega BE, Niggemann J, Dittmar T, et al.
797 Ammonia oxidizing archaea release a suite of organic compounds potentially fueling
798 prokaryotic heterotrophy in the ocean. Environ Microbiol 2019; 21: 4062–4075.
799 73. Sañudo-Wilhelmy SA, Gómez-Consarnau L, Suffridge C, Webb EA. The Role of B
800 Vitamins in Marine Biogeochemistry. Ann Rev Mar Sci 2014; 6: 339–367.
801 74. Graham RM, Deery E, Warren MJ. Vitamin B12: Biosynthesis of the Corrin Ring.
802 Tetrapyrroles: Birth, Life and Death. 2009. Springer New York, New York, NY, pp 286–
803 299.
804 75. Murphy MJ, Siegel LM. Siroheme and sirohydrochlorin. The basis for a new type of
805 porphyrin-related prosthetic group common to both assimilatory and dissimilatory sulfite
806 reductases. J Biol Chem 1973; 248: 6911—6919.
807 76. Shelton AN, Seth EC, Mok KC, Han AW, Jackson SN, Haft DR, et al. Uneven distribution
808 of cobamide biosynthesis and dependence in bacteria predicted by comparative
809 genomics. ISME J 2019; 13: 789–804.
810 77. Saito MA, Rocap G, Moffett JW. Production of cobalt binding ligands in a Synechococcus
811 feature at the Costa Rica upwelling dome. Limnol Oceanogr 2005; 50: 279–290.
812 78. Noble AE, Ohnemus DC, Hawco NJ, Lam PJ, Saito MA. Coastal sources, sinks and
33 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.
813 strong organic complexation of dissolved cobalt within the US North Atlantic
814 GEOTRACES transect GA03. Biogeosciences 2017; 14: 2715–2739.
815 79. Hawco NJ, McIlvin MM, Bundy RM, Tagliabue A, Goepfert TJ, Moran DM, et al. Minimal
816 cobalt metabolism in the marine cyanobacterium Prochlorococcus. Proc Natl Acad Sci U
817 S A 2020; 12: doi: 10.1073/pnas.2001393117.
818 80. Ducklow HW, Oliver JL, Jr WOS. The Role of Iron as a Limiting Nutrient for Marine
819 Plankton Processes. Hydrosphere 2000; 2: 295–310.
820 81. Martin JH. Iron as a Limiting Factor in Oceanic Productivity. Prim Product Biogeochem
821 Cycles Sea 1992; 123–137.
822 82. Santoro AE, Sakamoto CM, Smith JM, Plant JN, Gehman AL, Worden AZ, et al.
823 Measurements of nitrite production in and around the primary nitrite maximum in the
824 central California Current. Biogeosciences 2013; 10: 7395–7410.
825 83. Schalk IJ, Guillon L. Fate of ferrisiderophores after import across bacterial outer
826 membranes: Different iron release strategies are observed in the cytoplasm or periplasm
827 depending on the siderophore pathways. Amino Acids 2013; 44: 1267–1277.
828 84. Füssel J, Lam P, Lavik G, Jensen MM, Holtappels M, Günter M, et al. Nitrite oxidation in
829 the Namibian oxygen minimum zone. ISME J 2012; 6: 1200–1209.
830 85. Sun X, Kop LFM, Lau MCY, Frank J, Jayakumar A, Lücker S, et al. Uncultured
831 Nitrospina-like species are major nitrite oxidizing bacteria in oxygen minimum zones.
832 ISME J 2019; 13: 2391–2402.
833 86. D’Hondt S, Inagaki F, Zarikian CA, Abrams LJ, Dubois N, Engelhardt T, et al. Presence of
834 oxygen and aerobic communities from sea floor to basement in deep-sea sediments. Nat
835 Geosci 2015; 8: 299–304.
836 87. Karstensen J, Stramma L, Visbeck M. Oxygen minimum zones in the eastern tropical
837 Atlantic and Pacific oceans. Prog Oceanogr 2008; 77: 331–350.
838 88. Vollmer AC, Bark SJ. Twenty-Five Years of Investigating the Universal Stress Protein:
34 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.
839 Function, Structure, and Applications. Advances in Applied Microbiology, 1st ed. 2018.
840 Elsevier Inc., pp 1–36.
841 89. Nyström T, Neidhardt FC. Expression and role of the universal stress protein, UspA, of
842 Escherichia coli during growth arrest. Mol Microbiol 1994; 11: 537–544.
843 90. O’Toole R, Smeulders MJ, Blokpoel MC, Kay EJ, Lougheed K, Williams HD. A two-
844 component regulator of universal stress protein expression and adaptation to oxygen
845 starvation in Mycobacterium smegmatis. J Bacteriol 2003; 185: 1543–1554.
846 91. Schreiber K, Boes N, Eschbach M, Jaensch L, Wehland J, Bjarnsholt T, et al. Anaerobic
847 Survival of of Pseudomonas aeruginosa by Pyruvate Fermentation Requires an Usp-
848 Type Stress Protein. J Bacteriol 2006; 188: 659–668.
849 92. Coulthurst S. The Type VI secretion system: A versatile bacterial weapon. Microbiology
850 2019; 165: 503–515.
851 93. Gallique M, Bouteiller M, Merieau A. The type VI secretion system: A dynamic system for
852 bacterial communication? Front Microbiol 2017; 8: 1454.
853 94. Mellbye BL, Spieck E, Bottomley PJ, Sayavedra-Soto LA. Acyl-Homoserine Lactone
854 Production in Nitrifying Bacteria of the Genera. Appl Environ Microbiol 2017; 83: e01540-
855 17.
856 95. Erb TJ. Carboxylases in natural and synthetic microbial pathways. Appl Environ Microbiol
857 2011; 77: 8466–8477.
858 96. Yamamoto M, Arai H, Ishii M, Igarashi Y. Role of two 2-oxoglutarate:ferredoxin
859 oxidoreductases in Hydrogenobacter thermophilus under aerobic and anaerobic
860 conditions. FEMS Microbiol Lett 2006; 263: 189–193.
861 97. Ikeda T, Ochiai T, Morita S, Nishiyama A, Yamada E, Arai H, et al. Anabolic five subunit-
862 type pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6.
863 Biochem Biophys Res Commun 2006; 340: 76–82.
864 98. Yun NR, Yamamoto M, Arai H, Ishii M, Igarashi Y. A novel five-subunit-type 2-
35 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.
865 oxoglutalate:ferredoxin oxidoreductases from hydrogenobacter thermophilus TK-6.
866 Biochem Biophys Res Commun 2002; 292: 280–286.
867 99. Kletzin A, Adams MWW. Molecular and phylogenetic characterization of pyruvate and 2-
868 ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate
869 ferredoxin oxidoreductase from Thermotoga maritima. J Bacteriol 1996; 178: 248–257.
870 100. Berg IA. Ecological aspects of the distribution of different autotrophic CO2 fixation
871 pathways. Appl Environ Microbiol 2011; 77: 1925–1936.
872 101. Pineda E, Encalada R, Rodríguez-Zavala JS, Olivos-García A, Moreno-Sánchez R,
873 Saavedra E. Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde-alcohol
874 dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba
875 histolytica. FEBS J 2010; 277: 3382–3395.
876 102. Preisig O, Zufferey R, Thöny-Meyer L, Appleby CA, Hennecke H. A high-affinity cbb3-
877 type cytochrome oxidase terminates the symbiosis-specific respiratory chain of
878 Bradyrhizobium japonicum. J Bacteriol 1996; 178: 1532–1538.
879 103. Han H, Hemp J, Pace LA, Ouyang H, Ganesan K, Roh JH, et al. Adaptation of aerobic
880 respiration to low O2 environments. Proc Natl Acad Sci U S A 2011; 108: 14109–14114.
881 104. Ngugi DK, Blom J, Stepanauskas R, Stingl U. Diversification and niche adaptations of
882 Nitrospina-like bacteria in the polyextreme interfaces of Red Sea brines. ISME J 2016;
883 10: 1383–1399.
884 105. Mueller AJ, Jung M-Y, Strachan CR, Herbold CW, Kirkegaard RH, Wagner M, et al.
885 Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting. bioRxiv 2020;
886 doi: https://doi.org/10.1101/2020.06.09.141952.
887 106. Shao MF, Zhang T, Fang HHP. Sulfur-driven autotrophic denitrification: Diversity,
888 biochemistry, and engineering applications. Appl Microbiol Biotechnol 2010; 88: 1027–
889 1042.
890 107. Mulkidjanian AY, Dibrov P, Galperin MY. The past and present of the sodium energetics:
36 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.
891 May the sodium-motive force be with you. Biochim Biophys Acta 2008; 1777: 985–992.
892 108. Watson SW, Waterbury JB. Characteristics of two marine nitrite oxidizing bacteria,
893 Nitrospina gracilis nov. gen. nov. sp. and Nitrococcus mobilis nov. gen. nov. sp. Arch
894 Mikrobiol 1971; 77: 203–230.
895
37 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.
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−ligase 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.