Correspondence: Lisa Y. Stein, Department of Biological Sciences, CW 405 Biological Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2E9. Tel.: +780 492 4782; fax: +780 492 9234; e-mail: firstname.lastname@example.org
Nitrite is the highly toxic end product of ammonia oxidation that accumulates in the absence of a nitrite-consuming process and is inhibitory to nitrifying and other bacteria. The effects of nitrite on ammonia oxidation rates and regulation of a common gene set were compared in three ammonia-oxidizing bacteria (AOB) to determine whether responses to this toxic metabolite were uniform. Mid-exponential-phase cells of Nitrosomonas europaea ATCC 19718, Nitrosospira multiformis ATCC 25196, and Nitrosomonas eutropha C-91 were incubated for 6 h in mineral medium supplemented with 0, 10, or 20 mM NaNO2. The rates of ammonia oxidation (nitrite production) decreased significantly only in NaNO2-supplemented incubations of N. eutropha; no significant effect on the rates was observed for N. europaea or N. multiformis. The levels of norB (nitric oxide reductases), cytL (cytochrome P460), and cytS (cytochrome c′-β) mRNA were unaffected by nitrite in all strains. The levels of nirK (nitrite reductase) mRNA increased only in N. europaea in response to nitrite (10 and 20 mM). Nitrite (20 mM) significantly reduced the mRNA levels of amoA (ammonia monooxygenase) in N. multiformis and norS (nitric oxide reductase) in the two Nitrosomonas spp. Differences in response to nitrite indicated nonuniform adaptive and regulatory strategies of AOB, even between closely related species.
Nitrification, the oxidative conversion of ammonium to nitrate via nitrite, is carried out by chemolithoautotrophic ammonia- and nitrite-oxidizing bacteria (AOB and NOB, respectively); ammonia-oxidizing thaumarchaea also convert ammonia to nitrite. Environments like wastewater treatment systems (van Dongen et al., 2001) and axenic cultures of AOB (Stein & Arp, 1998) can accumulate very high concentrations of nitrite, often in the range of 25–30 mM. Yet, the physiological mechanisms that AOB use to adapt to and resist high nitrite concentrations have not been broadly investigated and are limited to a single AOB strain, Nitrosomonas europaea ATCC 19718, and enrichment cultures (Tan et al., 2008). These studies show that nitrite and free nitrous acid have toxic effects on AOB (Tan et al., 2008) and specifically and irreversibly inactivate ammonia monooxygenase enzymes of N. europaea (Stein & Arp, 1998).
In N. europaea, the gene cluster, ncgABC-nirK, which encodes a copper-containing nitrite reductase and three functionally related proteins (Beaumont et al., 2004a, 2005), is under direct regulation by nitrite via a NsrR repressor protein (Beaumont et al., 2004a). No other genes in N. europaea have been identified as part of a nitrite regulon, although norB, encoding nitric oxide reductase, was shown to be more highly expressed in batch cultures of N. europaea in the presence of supplemental nitrite (Yu & Chandran, 2010). Furthermore, both nirK and norB genes were found to be essential for the anaerobic growth of N. europaea in which nitrite acts as the terminal electron acceptor (Schmidt et al., 2004). The irreversible inactivation of ammonia monooxygenase enzymes by nitrite in N. europaea was found to be under post-translational, but not transcriptional control (Stein & Arp, 1998).
The present study investigated the effect of moderately high nitrite concentrations on three genome-sequenced AOB strains: N. europaea ATCC 19718, the long-standing model strain that provided foundational knowledge of AOB physiology, biochemistry, and genetics (Chain et al., 2003); Nitrosomonas eutropha strain C-91, a close taxonomic relative of N. europaea that is apparently restricted to environments with very high ammonium loads like wastewater treatment plants (Stein et al., 2007); and Nitrosospira multiformis strain ATCC 25196, a representative of the most common AOB genus found in soils (Norton et al., 2008). The effects of nitrite on the ability of these three AOB to further convert ammonia to nitrite and on the expression of a common gene set were compared to determine whether the strains had similar or different responses to this toxic end product of their metabolism. Uniform responses would indicate that prior studies of nitrite effects on N. europaea could be universalized to other AOB strains. Different responses would indicate that each strain has evolved its own set of genetic and physiological adaptations to high-nitrite environments that must be explored independently.
Materials and methods
Nitrosospira multiformis strain ATCC 25196, N. europaea strain ATCC 19718, and N. eutropha strain C-91 were grown in a mineral medium containing per liter: 10 mM (NH4)2SO4, 0.4 mM KH2PO4, 0.2 mM MgSO4·7H2O, 1 mM CaCl2·2H2O, 1 mM KCl, 0.05% Phenol red, 1 mL of trace element solution (per liter distilled water: 11.5 mM Na2-EDTA, 10 mM FeCl2·4H2O, 0.5 mM MnCl2·2H2O, 0.1 mM NiCl2·6H2O, 0.1 mM CoCl2·6H2O, 0.1 mM CuCl2·2H2O, 0.5 mM ZnCl2, 0.1 mM Na2MoO4·2H2O, 1 mM H3BO3), and 15 mM HEPES buffer pH 7.5. The pH was maintained at c. 7.5 using 5% sodium bicarbonate, added daily following 48 h of growth. Nitrosomonas europaea and N. eutropha were also grown in the same medium buffered with 43 mM phosphate (per liter: 5.47 g KH2PO4 and 0.47 g NaH2PO4, pH 8) in place of HEPES. Nitrosospira multiformis was incapable of consistent growth in phosphate-buffered medium. Cultures were grown with shaking (180 r.p.m.) at 28 °C in the dark.
The maximum doubling times were similar at 24 h (± 1.90) for N. europaea, 20.6 h (± 1.73) for N. eutropha, and 22.1 h (± 1.71) for N. multiformis (Supporting Information, Fig. S1). Nitrosospira multiformis cultures produced half the cell numbers, but biomass equivalent to that of Nitrosomonas cultures. All cultures produced 13–15 mM nitrite by the late exponential phase. The maximum doubling times were significantly shorter at 7.1 (± 0.68) and 9.2 (± 1.38) h for N. europaea and N. eutropha, respectively, when grown in phosphate- instead of HEPES-buffered medium (Fig. S1) and produced c. 18 mM nitrite (± 0.04) by the late exponential phase.
Cells were harvested in the mid-exponential growth phase as determined by the levels of nitrite accumulation (c. 10 mM ± 0.76 for N. multiformis and c. 13 mM ± 0.23 for Nitrosomonas cultures). Cells were collected by centrifugation (15 000 g, 10 min), washed three times in HEPES buffer (15 mM, pH 7.5) or sodium phosphate buffer (50 mM NaH2PO4, 2 mM MgSO4, pH 8) for HEPES or phosphate-grown cells, respectively, and resuspended in 10 mL of a fresh medium to a concentration of 109 cells mL−1 as determined by a Petroff–Hausser counting chamber and phase-contrast light microscopy. The medium was amended with 0, 10, or 20 mM NaNO2. Flasks were incubated with shaking (180 r.p.m.) at 28 °C in the dark. Samples (2 mL) were taken at t=0, 0.5, 2, 4, and 6 h and cells were collected by centrifugation (21 000 g, 2 min). The supernatant was used for pH and nitrite measurements (Hageman & Hucklesby, 1971), and cell pellets were immediately treated with 500 μL RNAprotect (Qiagen, Valencia, CA) for storage at −80 °C. Three to seven replicates of each incubation condition using batches of cells grown on separate days were compared.
Cross-comparisons of nucleotide and predicted protein sequences were performed using genome sequences and blast functions available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). GenBank accession numbers for genome sequences are N. europaea, AL954747; N. eutropha, CP000450; and N. multiformis, CP000103.
Nucleotide sequences of each targeted gene from each strain were used to design specific primers using primer3 input 0.4.0 software (Rozen & Skaletsky, 2000) (Table S1). PCR reactions included standard reagents and concentrations for Taq polymerase (Sambrook & Russell, 2001) and isolated genomic DNA (AquaPure Genomic DNA Isolation Kit, Bio-Rad, Hercules, CA) as a template. Amplification conditions for all primer sets were: 95 °C for 5 min; 30 cycles of 95 °C for 40 s, 55 °C for 40 s and 72 °C for 50 s; and 72 °C for 7 min (iCycler, Bio-Rad). Single PCR products of appropriate size were verified by agarose gel and purified (QIAquick PCR Purification kit, Qiagen). Purified PCR products were labeled (Prime-a-Gene, Promega, Madison, WI) with [α-32P]-dCTP (3000 Ci mmol−1; Perkin-Elmer, Waltham, MA) and random hexamers. The dynamic range of detection for each probe was tested with 0.25–3 μg mRNA from control incubations of each culture. The r2 values for the slope of hybridization intensity vs. microgram of RNA concentration were between 0.92 and 1.00 for all probes and all strains (data not shown).
Total RNA was extracted from cell pellets using the Aurum Total RNA Mini kit as per the manufacturer's instructions (Bio-Rad). Nucleic acid concentrations were determined spectrophotometrically (NanoDrop Technologies, Wilmington, DE). Two micrograms of total RNA from each sample were blotted onto Zeta-Probe GT nylon membranes (Bio-Rad) using a Minifold filtration manifold (Schleicher & Schuell, Keene, NH). RNA from exponential-phase cells harvested directly from culture (and not resuspended into a fresh medium) was blotted onto the same membrane as RNA from cells subjected to short-term incubations to ensure comparability of the hybridization signals. Membranes were dried overnight and UV-crosslinked (FB-UVXL-1000, Fisher Scientific, Pittsburgh, PA). Prehybridization, hybridization, and washing of membranes were performed according to the manufacturer's instructions (Bio-Rad) at 30 °C. To allow reprobing, membranes were stripped of radioactivity by washing twice in a 0.1 × SSC/0.5% SDS solution at 95–100 °C for 20 min. Hybridization intensity was analyzed using a typhoon phosphorimager and imagequant software (Amersham, Piscataway, NJ).
Background from nonspecific binding of the probes to the membrane was subtracted from the hybridization signals. The relative hybridization intensity was normalized by dividing the gene-specific signals by 16S rRNA gene probes. The fold difference in the levels of mRNA for each gene, time point, and organism resulting from NaNO2 exposure was determined by dividing hybridization intensities from dot blots of RNA extracted from NaNO2 amended to those from unamended incubations. A twofold change in transcript level was considered a significant effect of nitrite on gene expression. Student's t-tests (P<0.05) were performed to verify significant differences between treatments for both activity and gene expression levels.
Effects of NaNO2 on the rates of ammonia oxidation to nitrite
Short-term (6 h) incubations were used to determine the effects of NaNO2 on the ability of the AOB to oxidize ammonia to nitrite. Concentrations of NaNO2 similar to that applied in previous studies of nitrite effects on N. europaea were used (Stein & Arp, 1998; Beaumont et al., 2004a, b). The final pH was significantly higher in NaNO2 amended than in unamended incubations for all three AOB, indicating less acidification and thus reduced rates of ammonia oxidation (Table 1). However, among the three AOB, only N. eutropha showed significantly slower rates of and less net nitrite production when incubated in the NaNO2-amended medium, although this strain also had the fastest maximum nitrite production rate among the three strains (Table 1). Similar results were observed for N. eutropha and N. europaea cells incubated in phosphate-buffered, rather than HEPES-buffered, medium (data not shown). Thus, among the three AOB, the ammonia-oxidizing activity of N. eutropha was the most negatively affected by the presence of high nitrite concentrations.
Table 1. pH and nitrite production measurements for incubations of AOB strains in HEPES-buffered medium with or without NaNO2 supplementation
pH at 6 h
Net NO2− (mM) produced at 6 h
Maximum rate of NO2− production (mM h−1)
10 mM NO2−
20 mM NO2−
10 mM NO2−
20 mM NO2−
10 mM NO2−
20 mM NO2−
Values in parentheses indicate the SE (n=3–7). Bold values indicate a significant difference between measurements for the same organism incubated in medium with NaNO2 supplementation relative to medium without added NaNO2. Superscript letters indicate measurements within the same statistical grouping for an individual treatment among the three AOB. Significant differences were determined using Student's t-test at P<0.05. Initial pH: 7.58 ± 0.02 for all incubations. The rates of NO2− production were measured over the maximum linear range for each incubation.
N. eutropha C91
Effect of nitrite on levels of select gene expression
Genes selected for this study included those with demonstrated involvement in the ammonia oxidation and/or the nitrite reduction pathways of N. europaea (Klotz & Stein, 2011). The genes were amoA, encoding the α-subunit of ammonia monooxygenase; nirK, encoding copper-containing nitrite reductase; norB and norS, both encoding cytochrome c-dependent nitric oxide reductases; cytS, encoding cytochrome c′-β; and cytL, encoding cytochrome P460. NirK and NorB have demonstrated activity in reducing nitrite to nitrous oxide via nitric oxide in N. europaea (Beaumont et al., 2002, 2004b; Schmidt et al., 2004). The norS gene has been identified only in AOB and a few other bacteria (Stein et al., 2007; Norton et al., 2008) and encodes a nitric oxide reductase with high similarity to NorB (J. Hemp, pers. commun.). Cytochrome c′-β has a putative function in nitrogen oxide detoxification, while the evolutionarily related cytochrome P460 was shown to oxidize hydroxylamine to nitrite in N. europaea (Elmore et al., 2007). Comparisons of similarity between nucleotide and translated protein sequences of genes in N. eutropha and N. multiformis to orthologues in N. europaea are shown in Table 2. Nitrosospira multiformis lacks cytochrome P460, and as it belongs to a different genus, there was less sequence similarity between N. multiformis and N. europaea than between the two Nitrosomonas strains for all genes.
Table 2. Similarity of nucleotide and predicted protein sequences of genes in Nitrosomonas eutropha C91 and Nitrosospira multiformis compared with related sequences in Nitrosomonas europaea
% Sequence identity relative to similar sequences in Nitrosomonas europaea (nucleotide/translated protein sequence)
Nitrosomonas eutropha C91
Two and three copies of nearly identical amoA genes reside in the two Nitrosomonas spp. and in N. multiformis, respectively; thus, the percent identity for the multiple copies is the same when comparing between species.
nirK genes are part of a four-member operon in the two Nitrosomonas spp. and is a singlet in N. multiformis (Cantera & Stein, 2007a).
norS genes are annotated as cytochrome c oxidase subunit II due to conservation among the heme-copper oxidase superfamily. Biochemical evidence shows NorS to be a functional nitric oxide reductase (J. Hemp, pers. commun.).
NS, no significant sequence similarity at a cut-off value of 1e−05.
Incubations supplemented with NaNO2 only caused significant changes in the expression levels of three of the six functional genes examined. No significant change was detected in the levels of norB, cytL, or cytS mRNA of any AOB, suggesting no regulation of these genes by nitrite (data not shown). The levels of amoA mRNA of N. multiformis were significantly reduced in incubations supplemented with 20 mM NaNO2, but not with 10 mM NaNO2 (Fig. 1). Similarly, the levels of norS mRNA of N. europaea and N. eutropha were also significantly reduced in incubations supplemented with 20 mM NaNO2, but not 10 mM NaNO2 (Fig. 2). The levels of nirK mRNA were only significantly increased in N. europaea with either 10 or 20 mM NaNO2, although the increase was short lived (Fig. 3). Similar trends in gene expression were observed for N. europaea and N. eutropha grown in phosphate-buffered medium, although norS mRNA levels decreased less than twofold relative to the no nitrite control (data not shown). No significant differences were found in the hybridization intensities of mRNA extracted from cells immediately harvested from culture vs. those taken at t=0 from the short-term incubations, indicating no immediate effects from resuspending cells into a fresh medium with or without NaNO2 amendment (data not shown).
The nonuniformity of the physiological and transcriptional responses of these three AOB to relatively high nitrite concentrations demonstrates that each strain, even those as closely related as N. europaea and N. eutropha, has a different ability and mechanism to tolerate the major end product of their metabolism. Therefore, the effects of nitrite on N. europaea found in this and prior studies cannot be universalized to other AOB.
Previous studies of N. europaea have shown that the expression of amoA is regulated primarily by the availability of NH3 (Sayavedra-Soto et al., 1996) and O2 (Yu & Chandran, 2010). However, exponential-phase N. europaea showed decreased amoA mRNA levels when grown in batch cultures supplemented with nitrite (Yu & Chandran, 2010), although this particular study involved a longer time course and supplementation of media with nitrite before inoculating cells for growth experiments, likely exposing them to a higher overall nitrite load than in the present study. In the present study, there was no acute effect of nitrite on amoA mRNA levels in either Nitrosomonas strain, only in N. multiformis (Fig. 1). The decrease in amoA mRNA did not translate to a significant decrease in the nitrite production rate of N. multiformis (Table 1). Similarly, the unchanged amoA mRNA levels in N. eutropha did not correlate with its decreased nitrite production rate. Thus, the expression of amoA did not correlate to ammonia-oxidizing activity in any of the AOB, at least in these short-term incubations. These observations indicate that caution must be exercised when using absolute amoA gene expression as a proxy for acute rates of ammonia-oxidizing activity.
Of the two genes encoding nitric oxide reductase, norB and norS, only the levels of norS mRNA in the two Nitrosomonas spp. were significantly decreased in incubations with nitrite supplementation (Fig. 2). A prior study showed upregulation of norS in NirK-deficient N. europaea under conditions where hydroxylamine conversion to nitrous oxide was highly favored (Cho et al., 2006; Cantera & Stein, 2007b). Thus, the decrease of norS mRNA levels could have been in response to less favorable conditions for ammonia oxidation, and hence hydroxylamine formation, if NorS indeed plays a specific role in reducing nitric oxide arising from incomplete hydroxylamine oxidation to nitrite.
Studies in N. europaea have linked the expression of nirK and norB genes with the reduction of nitrite to nitrous oxide via nitric oxide (Beaumont et al., 2002, 2004b; Schmidt et al., 2004). Similarly, the ability of Nitrosospira spp. to produce nitrous oxide has been suggested to involve orthologous genes (Shaw et al., 2006; Garbeva et al., 2007), although a direct linkage between this activity and nirK or norB expression has not yet been demonstrated in any Nitrosospira spp. The present study showed no effect of nitrite on the expression of either nirK (Fig. 2) or norB (data not shown) in N. multiformis, which is understandable at the molecular level as neither gene has a recognizable nitrite-responsive regulatory protein-binding motif in its promoter region (Norton et al., 2008). The more surprising result was the lack of increased nirK mRNA levels in N. eutropha from exposure to nitrite (Fig. 2) as the ncgABC-nirK operon, promoter-proximal NsrR-binding motif, and NsrR repressor share high sequence identity between N. europaea and N. eutropha (Cantera & Stein, 2007a; Stein et al., 2007). Together, the data suggest that while the expression of the NirK enzyme is vital to nitrite reduction (Schmidt et al., 2004) and tolerance (Beaumont et al., 2005; Cantera & Stein, 2007b) in N. europaea, it may play a lesser role in N. eutropha and N. multiformis or is constitutively expressed to perform these functions.
mRNA levels of the three remaining genes, norB, cytL (encoding cytochrome P460), and cytS (encoding cytochrome c′-β) were not affected by nitrite in any of the AOB, suggesting constitutive expression in the presence of this toxic metabolite (data not shown). In N. europaea, it was suggested that norB is constitutively expressed during aerobic metabolism (Beaumont et al., 2004b), but is induced during anaerobic metabolism (Beyer et al., 2009) and during growth in the presence of NaNO2 (Yu & Chandran, 2010). We were unable to confirm induction of norB expression by NaNO2, but did indicate a constant presence of norB mRNA (i.e. 0.03–0.08% of the 16S rRNA gene pool) for all three AOB in all incubations.
Although the present study examined only a small subset of shared genetic inventory among three AOB strains, the data revealed that the regulation of these genes was not predictable based on sequence or regulatory motif similarities. This observation was particularly surprising for the nirK genes of the two Nitrosomonas strains. Thus, nitrite and probably other metabolites of AOB are certain to have physiological and genetic effects that vary from strain to strain. This variability must be recognized when building predictive models of how environmental factors, like transiently high nitrite loads, affect AOB physiology, gene expression, and nitrification rates.
Funding was provided from grant #2001–04 to L.Y.S. from the Kearney Foundation of Soil Science and the faculties of UCR and UofA. The authors gratefully acknowledge M.G. Klotz, B.D. Lanoil, and anonymous reviewers for critical comments on this and previous versions of the manuscript.