Expression in Long-Term Continuous Cultures of Nitrosomonas
eutropha C91
Boran Kartal,aHans J. C. T. Wessels,bErwin van der Biezen,a* Kees-Jan Francoijs,cMike S. M. Jetten,a,dMartin G. Klotz,eand
Lisa Y. Steinf
Institute of Water and Wetland Research, Department of Microbiology, Radboud University Nijmegen, Nijmegen, The Netherlandsa; Radboud University Nijmegen Medical Centre, Nijmegen Centre for Mitochondrial Disorders, Nijmegen Proteomics Facility, Department of Laboratory Medicine, Nijmegen, The Netherlandsb; Radboud University, Department of Molecular Biology, NCMLS, Nijmegen, The Netherlandsc; Delft University of Technology, Department of Biotechnology, Delft, The Netherlandsd; Department of Biology, University of North Carolina, Charlotte, North Carolina, USAe; and Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canadaf
Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO2on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO2, and anoxic-plus-NO2culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO2under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO2resulted in increased representation of proteins and tran-scripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO2and anoxic-plus-NO2conditions. The results indicate that exposure to NO2results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO2as an oxidant.
N
itrosomonas eutropha is a betaproteobacterialammonia-oxi-dizing nitrifier with a niche preference for environments with a high ammonium flux, concentration, or load, such as wastewa-ter treatment plants (WWTPs) (13). Although chemolithotrophic ammonia-oxidizing bacteria (AOB) are considered obligate aer-obes, N. eutropha strain N904 was capable of anaerobic ammonia oxidation using nitrite as a terminal electron acceptor with exter-nally provided nitrogen dioxide gas (NO2) as an oxidant (18).
Anaerobic incubation of Nitrosomonas europaea ATCC 19718 with ammonia, NO2, and nitrite significantly increased levels of
transcripts encoding copper-containing nitrite reductase (nirK), cytochrome c-dependent nitric oxide reductase (norB), and the red copper protein nitrosocyanin (ncyA) relative to transcript lev-els in aerobically growing cultures without NO2(4). Although
dinitrogen gas (N2), not nitrous oxide (N2O), was considered
to be the main product of anaerobic metabolism in N. eutropha N904 (18,23), recognizable N2O reductase genes have never
been identified in genomes of ammonia-oxidizing bacteria, in-cluding N. eutropha C91.
Aside from anaerobic respiration, N. eutropha N904 generates and utilizes NO for aerobic growth; removal of NO by intensive aeration, chemical chelation, or consumption by a cocultivated denitrifier significantly reduced ammonia-oxidizing activity by N.
eutropha N904 (29). Furthermore, addition of NO2to N. eutropha
N904 cultures transitioning from anaerobic to aerobic cultivation (24) and addition of nitrite to ammonia-starved Nitrosomonas
europaea strain ATCC 19718 cultures (14) greatly increased the rate of metabolic recovery, indicating an important role for
nitro-gen oxides (NOx) in stimulating aerobic metabolism of
Nitro-somonas spp. Together, these observations led to the hypothesis of
the “NOx cycle,” whereby NO (initially generated from nitrite reduction by NirK) is oxidized to N2O4by O2and reduced back to
NO by ammonia monooxygenase (AMO), while ammonia is ox-idized to hydroxylamine (21). Based on the experiments with N.
eutropha N904, this model postulated that NO2rather than O2is
the primary oxidant for ammonia activation by AMO during aer-obic respiration; however, the mechanism of NO2(or N2O4)
re-duction by AMO has not been reconstructed. The anoxic oxida-tion of ammonia by AMO with externally provided NO2was not
inhibited by acetylene, whereas complete inhibition of AMO by acetylene was observed when O2was the sole oxidant in cultures of N. eutropha N904 (19). Only supplemented NO2, and not NO,
could replace O2during anoxic ammonia oxidation (19). Thus, it
appears that AMO can use either O2or NO2as an oxidant for
ammonia oxidation, and, based on the differential inhibition of
Received 4 March 2012 Accepted 26 April 2012 Published ahead of print 4 May 2012
Address correspondence to Lisa Y. Stein, lisa.stein@ualberta.ca. * Present address: Erwin van der Biezen, Utrecht University Medical Center, Department of Pathology, Utrecht, The Netherlands.
Supplemental material for this article may be found athttp://aem.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved.
AMO by acetylene, the enzyme is considered to have different binding sites for the oxidants. While both O2and NO2can be used
simultaneously by N. eutropha N904, resulting in simultaneous nitrification and denitrification under reduced aeration (5), it is not clear whether the postulated “NOx cycle” is operational dur-ing aerobic ammonia oxidation.
Addition of NO2to mixed cultures of betaproteobacterial
am-monia oxidizers and anammox bacteria stimulated both aerobic and anaerobic ammonia oxidation (20,30), and addition of NO stimulated activity of an anammox enrichment (10). From these studies it was concluded that anammox bacteria could be sup-ported either by externally added NO or by NO and nitrite pro-duced by aerobic ammonia oxidizers. Indeed, NO has been iden-tified as a central metabolite of anammox metabolism (9). As a whole, these studies suggest that N-oxides are important metabo-lites that support lithotrophic N metabolism, particularly in low-oxygen to anoxic environments. The current study investigated the global transcriptional and proteomic responses of axenic N.
eutropha C91 cultures to long-term exposure to NO2under
both aerobic and anaerobic conditions to better define the un-derlying physiology and significance of NO2to the metabolism
of N. eutropha.
MATERIALS AND METHODS
Reactor setup. A 7-liter glass vessel with a working volume of 4 liters was used for growing N. eutropha strain C91. The reactor was fed continuously at a flow rate of 1.4 ml · min⫺1(2 liters · day⫺1). The influent mineral medium (18) was modified and contained 50 mM NH4Cl and the follow-ing nutrients (per 1 liter of medium): 585 mg NaCl, 147 mg CaCl2· 2H2O, 74 mg KCl, 54 mg KH2PO4, 49 mg MgSO4· 7H2O, 12 g HEPES, and 1 ml trace element solution containing 0.02 M HCl, 973 mg FeSO4· 7H2O, 49 mg H3BO3, 43 mg ZnSO4· 7H2O, 37 mg (NH4)6Mo7O24· 4H2O, 34 mg MnSO4· H2O, and 16 mg CuSO4. The pH of the medium was adjusted to 7.0. A pH controller unit was used to supply a solution of Na2CO3(100 g · liter⫺1) when necessary to keep the pH stable at 7.0. For the homoge-neous distribution of substrates, the vessel was stirred at 200 rpm. A heat-ing blanket was used to keep the temperature at 30°C. A gas mixture of air-N2(90:10%) with a flow of 500 ml · min⫺1was sparged through the
reactor for continuous O2supply. On day 82 the gas flow was changed to
450 ml · min⫺1of air and 50 ml · min⫺1of 4,000 ppm NO2. On day 189,
the reactor was converted into an anaerobic batch reactor with a gas flow of 200 ml · min⫺1N2and 2.5 ml · min⫺1of 4,000 ppm NO2. At the start of
the anoxic-plus-NO2period, there was approximately 50 mM NO2⫺and
NH4⫹in the reactor as determined using standard assays (7). The biomass
remained unchanged throughout the 244 days of the bioreactor run as determined by measurement of stable protein levels and a continuous optical density at 600 nm (OD600) of 0.2. Samples for RNA isolation and
proteomic analyses were collected at the end of each period at days 82, 189, and 244.
RNA isolation and analysis. Culture (20 ml for each of the two oxic phases and 100 ml for the anoxic phase) was harvested from the reactor. RNA was isolated using the RiboPure Bacteria kit according to the man-ufacturer’s instructions (Ambion, Foster City, CA). At the final step, RNA was resuspended in 50l diethyl pyrocarbonate (DEPC)-treated water. First-strand cDNA was synthesized with random primers using the Re-vertAid H Minus first-strand cDNA synthesis kit, and the second strand was synthesized using DNA polymerase according to the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). mRNA was sequenced and analyzed as described elsewhere (9). The quality scores of the obtained Solexa reads (3.5 million) were converted to PHRED format, initially mapped with Maq (http://maq.sourceforge.net) to the genome of the N.
eutropha C91 chromosome and two plasmids (accession numbers
NC_008344, NC_008341, and NC_008342), and verified with CLCBIO
software using various settings. From the aligned reads, the per-position coverage was calculated for each contig and used to calculate the coverage for each open reading frame (ORF), intergenic region, and predicted RNA element.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data processing. For each phase, 4 liters of effluent was collected. Cells were centrifuged and resuspended in 5 ml 20 mM potas-sium phosphate buffer, pH 7.5. Cells were disrupted using a French press (3 times at 1,200 PSI) and centrifuged at 4°C. In-solution digestion of proteins was performed as described in detail elsewhere (28). Analyses were performed using a nanoflow liquid chromatograph (Proxeon Easy nLC) coupled online via a nanoflow electrospray ionization source to a 7T linear ion trap Fourier transform ion cyclotron resonance mass spectrom-eter (LTQ FT Ultra; Thermo Fisher Scientific). Samples were loaded directly onto the analytical column with 0.5% acetic acid and 5% aceto-nitrile at a pressure of 240⫻ 105Pa. The analytical column was a
10-cm-long, 100-m-inner-diameter electrospray emitter (PicoTip emitter FS360-100-8-N-5-C15; New Objective), which was packed in-house with 3m (120-Å-pore-size) C18-AQ ReproSil-Pur reversed-phase material
(Dr. Maisch GmbH). Peptides were eluted from the column using a linear gradient of 10 to 40% acetonitrile at a flow rate of 300 nl/min. The mass spectrometer was programmed to acquire a precursor scan by the ion cyclotron resonance cell (mass range, m/z 350 to 1600; resolution [R], 100.000; 1E6 ions; 1 microscan), from which the 4 most abundant ions were selected for fragmentation experiments in the linear ion trap (1E4 ions, 3-threshold [Th] isolation width, 30-ms activation time, 30% nor-malized collision energy, activation Q of 0.250) each cycle. Ions with charge state z⫽ 2⫹ or z ⫽ 3⫹ were exclusively selected for fragmentation analysis. Dynamic exclusion was enabled (exclusion duration time⫽ 300 s with early expiration enabled, count⫽ 10, S/N threshold ⫽ 2) to prevent oversampling.
Database searches were performed using the Mascot search engine (Matrix Science) in combination with the Refseq N. eutropha C91 data-base. Search parameters included a 10-ppm precursor mass tolerance, a 0.8-Da fragment ion mass tolerance, ESI-TRAP instrument settings for fragment ions, tryptic specificity with a maximum of 1 missed cleavage, carbamidomethylation (Cys) as fixed modification, and oxidation (Met) and acetylation (protein N term) as variable modifications. Peptide and protein identifications were validated using the in-house-developed PROTON (Proteomics Tools Nijmegen) software (unpublished). Valida-tion criteria required a minimum Mascot peptide identificaValida-tion score of 30 for peptides originating from proteins identified with multiple unique peptide sequences. Single-peptide matches required a minimum Mascot score of 49 and a modified delta score of 10 or better. Exponentially modified protein abundance index (emPAI) values were calculated by PROTON and were used to identify proteins with significantly changed expression levels between culturing conditions. The protein expression ratios between samples were calculated from the protein emPAI values and normalized via median centering. Only proteins identified by at least 3 unique peptides were considered for further analysis. A one-sided Stu-dent t test with a critical P value of⬍0.01 was used to identify proteins with significantly changed expression between culturing conditions.
Categorization. Assignment of proteins and transcripts into func-tional groups was based on the N. eutropha C91 genome annotation (http: //genome.jgi-psf.org/nitec/nitec.annotation.html).
RESULTS
Effect of NO2and anoxia on ammonia-oxidizing activity of Ni-trosomonas eutropha C91 under long-term continuous cultiva-tion. Continuous cultures of N. eutropha C91 were grown in
min-eral medium with 50 mM ammonium at the same dilution factor and under two aeration phases: oxic and oxic plus 4,000 ppm NO2
(Fig. 1). Total biomass remained statistically unchanged over the course of the experiment as measured by a stable OD600of 0.2
and nitrite levels averaged 45⫾ 4.2 mM in the absence of NO2.
Upon addition of NO2, ammonium levels were significantly
higher (P⬍ 0.01 by Student’s t test) at 6.5 ⫾ 4, while average nitrite levels were significantly lower (P⬍ 0.01 by Student’s t test) at 40⫾ 3.6 mM. Following these two aerobic growth phases, the culture was switched to an anoxic-plus-NO2(4,000 ppm)
atmo-sphere and from continuous to batch mode, as N. eutropha was incapable of growth under these conditions as measured by the absence of increasing biomass in batch mode. Ammonium levels averaged 42⫾ 7.5 mM and nitrite concentrations averaged 46.5 ⫾ 1.2 mM over the course of anaerobic incubation (Fig. 1). Approx-imately 4 mM ammonium was consumed over the course of an-oxic-plus-NO2 incubation without complete conversion to
ni-trite, indicating possible N loss through gas production as reported previously (18).
General proteome. A total of 713 unique proteins out of 2,443
predicted coding sequences (CDSs) (26) were detected in N. eutropha C91 over the course of the experiment (see the proteome data in the supplemental material). Of these, 652, 555, and 352 proteins from cells cultivated under oxic, oxic-plus-NO2, and anoxic-plus-NO2conditions,
respectively, were detected in at least one of three experimental replica-tions. Proteins representing CDSs within the ammonia monooxygenase (amoCAB [YP_748501-499 and YP_748266-64]/orf5 [YP_748497 and YP_748262]/copC [YP_748496]), hydroxylamine oxidoreductase (haoAB-cycAB [YP_748517-14, YP_747992-89, and YP_747878-76]), and nitrite reductase (ncgABC-nirK [YP_747619-16]) gene clusters (26) were expressed under all three conditions. Although none of the proteins encodingtheNorCBnitricoxidereductaseweredetectedunderanycon-dition, the NorY nitric oxide reductase (sNOR; YP_748072) (26) protein was expressed under oxic-plus-NO2and anoxic-plus-NO2conditions.
The N-oxide transformation proteins cytochrome c=-beta (CtyS; YP_747560) and cytochrome P460 (CytL; YP_746385) were expressed under all three conditions, as was nitrosocyanin (NcyA; YP_748360). Subunit II of the low-affinity cytochrome aa3 terminal oxidase
(YP_748574)wasexpressedunderallthreeconditions,whereasthehigh-affinity cbb3heme-copper oxidase (YP_747791-93) was expressed under
oxic-plus-NO2and anoxic-plus-NO2conditions only.
Effect of NO2on proteins and transcripts of N. eutropha C91.
The classes of proteins responsive to NO2exposure indicate that N. eutropha C91 was experiencing a state of energetic deficiency in
the presence of NO2, although differences in specific protein ratios
were more pronounced in the absence than in the presence of O2
(Table 1). Greater representation of proteins for ammonia mono-oxygenase, terminal heme-copper oxidase, and proton-translo-cating pyrophosphatase suggested a need for higher electron flow and proton motive force to maintain biomass. Increased represen-tation of HflK-C proteins indicated protease activation. However, levels of the Hsp20 chaperone, which is often associated with a stress response, were lower in NO2-exposed cells. Decreased levels
of key enzymes for arginine and leucine biosynthesis indicated a decrease in N assimilation and/or N storage activity. Similarly, decreased levels of proteins involved in the tricarboxylic acid (TCA) cycle and gluconeogenesis indicated a decline in central pathway activity and C assimilation and/or storage.
In general, transcript levels for each of the NO2-responsive
proteins changed insignificantly (⬍2-fold) and not in correlation with protein levels under oxic (without NO2) and anoxic (with
NO2) conditions (Table 1). The exception was
fructose-1,6-bis-phosphatase, a key enzyme in gluconeogenesis, which was the only CDS with a significant decrease in both transcript and protein levels after transition to anoxic-plus-NO2conditions. Transcript
levels from the orphan amoC gene (Neut_1520) were increased under anoxic-plus-NO2conditions relative to levels of the two
operonic amoC genes, although a protein corresponding to Neut_1520 was not identified in the proteome. Transcript levels of
norC (Neut_0521) were 2.2-fold higher in cells from oxic
condi-tions than in those from anoxic-plus-NO2conditions, whereas
transcript levels of norBQD (Neut_0520-18) remained unchanged
FIG 1 Concentrations of ammonium and nitrite measured during continuous cultivation of N. eutropha. The first arrow indicates the transition when NO2 (4,000 ppm) was added to the air mix. The second arrow indicates the transition from continuous to batch culture along with the cessation of O2addition to the air mix. During the anoxic-plus-NO2phase, complete biomass retention was required to collect material for transcriptome and proteome analysis; hence, the cells were not actively growing during the anoxic-plus-NO2phase.
(see the transcriptome data in the supplemental material). Tran-script levels for the norSY-senC nitric oxide reductase gene cluster (Neut_1874-76) also remained unchanged.
Effect of anoxia plus NO2on proteins and transcripts of N. eutropha C91. Proteins specifically and positively affected by
an-oxia plus NO2that were not similarly affected by oxic-plus-NO2
conditions indicated responsive alterations in membrane func-tions and carbon fixation, whereas most negatively affected
pro-teins were implicated in biosynthesis, protein processing, and in-formation processing (Table 2andFig. 2). Only 8 of 28 transcripts with significant changes in response to anoxia plus NO2relative to
oxic conditions were also represented in the proteome (Table 3). The most positively responsive transcripts to anoxia plus NO2
with a predicted cellular function were those for metal resistance genes (Fig. 3). The majority of transcripts assigned to biosynthesis, carbon fixation, central carbon pathways, and cell envelope and
TABLE 1 Proteins regulated by exposure to NO2independently of oxygen
Functional category Protein name
NCBI accession
no. Genome locus tag
Proteome expression ratio
Transcriptome expression ratio, anoxic plus NO2/oxic Oxic plus
NO2/oxic
Anoxic plus NO2/oxic
Energy metabolism Ammonia monooxygenase subunit C gi|114332279 Neut_2319 2.17 4.23 2.1a
and⫺1.4
Ammonia monooxygenase subunit A gi|114332278 Neut_2318 2.59 3.58 ⫺2.6
Ammonia monooxygenase subunit B gi|114332277 Neut_2317 3.05 4.68 ⫺1.9
Cytochrome c oxidase subunit II gi|114332352 Neut_2395 3.46 4.32 1.0
Multicopper oxidase, type 3 gi|114331847 Neut_1872 2.22 4.07 1.2
Membrane-bound proton-translocating pyrophosphatase gi|114330799 Neut_0786 4.48 4.91 ⫺1.6 NADH:flavin oxidoreductase/NADH oxidase gi|114332147 Neut_2182 ⫺1.87 ⫺2.23 ⫺1.3
Carboxysome Microcompartment protein gi|114330820 Neut_0810 2.22 4.07 ⫺1.3
Protein folding, processing, and turnover
HflK protein gi|114330966 Neut_0963 4.92 4.61 1.4
HflC protein gi|114330967 Neut_0964 4.60 5.36 1.0
Heat shock protein Hsp20 gi|114331583 Neut_1596 ⫺2.06 ⫺1.75 1.9
Redoxin domain-containing protein gi|114331049 Neut_1050 ⫺1.60 ⫺2.80 ⫺1.2
Amino acid metabolism Acetylglutamate kinase gi|114332341 gi|114331240
Neut_2384 Neut_1244 Neut_1261
⫺2.84 ⫺2.53 ⫺1.5
2-Isopropylmalate synthase 124 gi|114331256 Neut_1176 ⫺1.50 ⫺2.78 ⫺2.0
Acetolactate synthase 3 regulatory subunit
gi|114331174 Neut_1633 ⫺2.37 ⫺2.84 1.1
Carbohydrate metabolism Fructose-1,6-bisphosphatase gi|114331619 Neut_0832 ⫺2.00 ⫺2.45 ⫺5.4
Malate dehydrogenase gi|114330839 Neut_1271 ⫺2.05 ⫺2.32 1.4
Function unknown Hypothetical protein Neut_0832 gi|114331266 4.23 5.10 ⫺1.3
Hypothetical protein Neut_1271 2.72 3.95 ⫺1.2
aExpression of the orphan amoC (Neut_1520) gene was increased, whereas expression of the two operonic amoC gene copies were decreased, under anoxic-plus-NO
2relative to
oxic conditions.
TABLE 2 Proteins most responsive to anoxiaa
Change and functional class Protein Locus tag Ratio, anoxic/oxic
Increase
Energy FoF1ATP synthase subunit B Neut_0273 6.1
Cytochrome c1 Neut_1112 5.0
Nitrosocyanin Neut_2173 3.5
Carbon fixation Carboxysome shell protein CsoS2 Neut_0806 5.1
Von Willebrand factor, type A Neut_0816 5.1
Protein processing/secretion PpiC-type peptidyl-prolyl cis-trans isomerase Neut_0662 3.4
Transport RND efflux membrane fusion protein
subunit
Neut_2159 3.5
Decrease
Central carbon Phosphoenolpyruvate synthase Neut_0868 ⫺3.1
Protein processing/secretion Protein export chaperone Neut_0204 ⫺3.5
Replication DNA gyrase subunit A Neut_1573 ⫺3.3
Transcription Rho termination factor Neut_2479 ⫺3.1
Translation Initiation factor IF-3 Neut_2330 ⫺3.0
Hypothetical NAb Neut_2349 ⫺3.0
a
Protein levels are reported for anoxic-plus-NO2cultures with aⱖ3-fold difference from oxic cultures.
transport functions were found to decrease in response to anoxia plus NO2(Fig. 3), suggesting a pronounced response to energetic
deficiency beyond that observed under the oxic-plus-NO2growth
condition.
DISCUSSION
Aside from a focus on N. eutropha C91 rather than N. europaea ATCC 19718 as in prior studies (4,22), the present study is also unique in that no adjustments were made to the dilution rate during oxic growth in response to turbidity changes, and each culture condition continued for several weeks rather than a few days prior to RNA and protein extraction. Therefore, long-term physiological adaptations to NO2and anoxia plus NO2were
cap-tured in the transcriptome and proteome, rather than short-term stress responses. Although based on a single experiment, the data clearly show that NO2as a sole oxidant for anaerobic growth (18)
or a vital cooxidant with O2(19) was not validated for N. eutropha
C91. The lower rate of ammonia oxidation to nitrite under the oxic-plus-NO2culturing condition (Fig. 1) and lack of biomass
gain (data not shown) under the anoxic-plus-NO2atmosphere
indicated that both conditions resulted in energy and reductant deficiency. However, anaerobic ammonia oxidation in the pres-ence of NO2did support biomass retention and viability (as
de-termined by the ability of the cells to continue ammonia
oxida-FIG 2 Ratio of specific protein abundances in anoxic-plus-NO2versus oxic cultures based on functional category (Kyoto Encyclopedia of Genes and Ge-nomes [KEGG] and Clusters of Orthologous Groups [COG]). Ratios are re-ported only for proteins with greater than a 2-fold difference between treat-ments. Actual numbers of proteins for each category are printed below or above each bar. Error bars represent standard deviations inclusive of all ex-pression ratios within a category.
TABLE 3 Transcripts most responsive to anoxiaa
Change and functional class Protein Locus tag Ratio, anoxic/oxic
Increase
Energy CcdA, cytochrome c biogenesis protein Neut_0054 7.5
Envelope PilQ, pilus assembly protein Neut_1804 11.2
Metals CopC, copper homeostasis Neut_0023 22.4
MerA, mercuric reductase Neut_0030 7.5
IucA/IucC aerobactin synthasec Neut_1745c 6.2c
Mobile elements Mutator type transposase Neut_1318 11.2
Motility FlaG, flagellar protein Neut_1822 5.2
Protein secretion GTP-binding signal recognition particle Neut_2445 8.4
Stress CRISPR-associated protein, Csm2 fam. Neut_2217 9.0
Cytochrome c peroxidasec Neut_1521c 7.1c
Translation tRNA-Arg-TCT Neut_R0027 7.5
tRNA-Thr-CGT Neut_R0014 5.6
Transport Cation diffusion facilitator Neut_0043 9.0
Hypothetical NAb Neut_1464 22.4
NA Neut_1477 22.4
NA Neut_1812 22.4
Decrease
Biosynthesis Biotin-acetyl-coenzyme A carboxylase ligase Neut_0232 ⫺5.2
Carbon fixation Microcompartments proteinc Neut_0817c ⫺5.1c
Central carbon Glutathione synthetasec Neut_0953c ⫺6.2c
Chromosome Fructose 1,6-bisphosphatasec Neut_1176c ⫺5.4c
Excinuclease ABC, A subunitc Neut_2459c ⫺7.6c
2-Oxoglutarate-Fe(II) oxygenase Neut_1349 ⫺7.1
Mobile elements Transposase, inactivated derivatives Neut_1748 ⫺5.9
Protein processing/secretion Type II secretion proteinc Neut_0507c ⫺8.7c
Fructosamine kinase Neut_1270 ⫺5.3
Transcription Two-component, sigma 53, regulator Neut_1416 ⫺5.1
Translation Cysteinyl-tRNA synthetasec Neut_0191c ⫺8.6c
tRNA-Ala-TGC Neut_R0007 ⫺5.7
a
Transcript levels of predicted genes with known function are reported for anoxic-plus-NO2cultures with aⱖ5-fold difference from the oxic control. Hypothetical CDSs with
a⬎10-fold difference in transcript level between anoxic and oxic cultures are reported.
b
NA, information not available.
tion) but not growth. Although the function of NO2as an oxidant
capable of supporting anaerobic growth of N. eutropha C91 was not observed, it remains possible that short-term NO2exposure
could briefly stimulate ammonia-oxidizing activity. In addition, comparative studies of AOB genomes indicated that genome con-tent varies significantly between closely related AOB (12), which could be responsible for the observed physiological differences between N. eutropha N904 and the type strain C91 (Nm57).
The translation of three proteins, i.e., proton-translocating py-rophosphatase (Neut_0786/NE1935), a hypothetical protein (Neut_0832/NE1907), and cytochrome c1(Neut_1112/NE0811),
was commonly increased in NO2-exposed or anoxic-plus-NO2
incubations of N. eutropha C91 and ammonia-starved
Nitrosomo-nas europaea ATCC 19718 (16). Few indicators of nitrosative stress were identified in the proteome or the transcriptome (6,11). Elevated levels of transcript and presence of protein for aerobactin synthase (siderophore) and cytochrome c peroxidase along with elevated transcription of metal homeostasis genes were observed in anoxic-plus-NO2incubations of N. eutropha C91 (Table 3;Fig. 3), indicating some overlap with typical oxidative or heavy metal stress responses (1,17). Furthermore, increased transcription of the orphan amoC gene has been implicated in the recovery from starvation and general stress response of N. europaea ATCC 19718 (3).
The increase in AmoCAB and other redox-active proteins along with diminished biosynthetic and nutrient storage activities (Table 1) could explain a short-term enhancement of ammonia oxidation rates observed in batch cultures of N. eutropha N904 (19) and in WWTP sludges amended with NO2(31), as more
energy would be diverted to essential metabolism than to nutrient storage. While all proteins in the nitrite reductase gene cluster were expressed, there was no visible change in protein or tran-script levels of nirK, nor was there any detectable expression of nitric oxide reductase (NorCB) proteins or significant changes in
norBQD transcripts under any condition. Expression of NorY in
NO2-exposed cells under both oxic and anoxic conditions
indi-cates that this alternative nitric oxide reductase is perhaps more important than NorB for NOx metabolism by N. eutropha C91 (26). Nitrosocyanin protein levels increased with anoxia plus NO2, but transcription of the ncyA gene did not change
signifi-cantly between oxic (no NO2) and anoxic (with NO2) conditions.
These results are contradictory to a prior study of N. europaea ATCC 19718 incubated with ammonia, nitrite, and NO2in which nirK, norB, and ncyA transcription significantly increased relative
to that in aerobically grown cells (4); however, the prior study examined mRNA levels at 24 to 48 h after switching from aerobic to anaerobic conditions. The present results suggest that steady-state N-oxide metabolism by N. eutropha is likely controlled by a combination of proteins such as hydroxylamine oxidoreductase (HaoAB), cytochromes c554 (CycA) and cM552 (CycB), nitrite
reductase and related proteins (NcgABC to NirK), nitrosocyanin (NcyA), cytochrome c=-beta (CytS), and cytochrome P460 (CytL) (2,8,25,27), all of which were expressed and unchanged from condition to condition. Prior measurements of changes in expres-sion of these genes and proteins in nitrosomonads likely reflected the transient environmental conditions experienced in much-shorter-term batch and continuous culture incubations, includ-ing expression of norB in N. europaea ATCC 19718 (4).
Disagreement between specific transcript and protein abun-dances or presence (Tables 1 to 3) indicates independent and dy-namic control at transcriptional, translational, and posttransla-tional levels. While it is not yet feasible to integrate discrete levels of mRNA to protein content or protein content to activity, it is logical to presume that the presence of an enzyme, assuming that it is not inhibited, is indicative of a function. Levels of neither mRNA nor protein can predict in vivo activity levels; in bacteria, mRNA levels do not even correlate well to the absolute abun-dances of the proteins they encode (ca. 47% correlation in
Esche-richia coli) (15). Given the ambiguity of relationships between transcripts, enzymes, and activities, conclusions that can be drawn from this study are as follows: (i) NO2 exposure results in
de-creased steady-state levels of ammonia oxidation in aerobically growing N. eutropha C91; (ii) anaerobic ammonia oxidation with NO2and nitrite can maintain viability of N. eutropha but does not
provide enough energy for growth, which suggests that NO2is
likely not produced as an internal oxidant from NO and could also negatively impact WWTPs that seek to bolster ammonia oxida-tion rates with NO2amendments; (iii) the transcriptome and
pro-teome of N. eutropha suggest a general stress response to NO2and
anoxia, including a shift from nutrient storage and biosynthesis toward more efficient energy conservation and utilization; and (iv) steady-state levels of proteins involved in N-oxide metabo-lism are likely functionally important under all growth conditions, with NorY nitric oxide reductase playing perhaps a more signifi-cant role than NorB in response to NO2and anoxia (26).
ACKNOWLEDGMENTS
B.K. was supported by KRW (European Framework Directive on Water, grant number 09035) and the Netherlands Organization for Scientific Research (VENI grant 863.11.003), H.J.C.T.W. was supported by KRW (European Framework Directive on Water, grant number 09035), M.G.K. was supported by the U.S. National Science Foundation (EF-0541797, MCB-0948202) and incentive funds from UNC Charlotte, M.S.M.J was FIG 3 Ratio of specific transcript abundances in anoxic-plus-NO2versus oxic
cultures based on functional category (KEGG and COG). Ratios are reported only for transcript levels having greater than a 2-fold difference between treat-ments. Black bars represent categories of transcripts with higher levels under anoxic-plus-NO2than under oxic conditions; gray bars represent categories of transcripts with lower levels under anoxic-plus-NO2than under oxic condi-tions. The actual numbers of coding sequences (CDSs) for each category are printed below the bars for increased transcript levels or above the bars for decreased transcript levels. Error bars represent standard deviations inclusive of all CDSs within a category.
supported by ERC 232937, and L.Y.S. was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. We thank Nico G. C. Tan for initial experiments with enrichment cultures.
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