Genetics and Molecular Biology

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii

Christine Martineau, Florian Mauffrey, Richard Villemur
V. Müller, Editor
DOI: 10.1128/AEM.00848-15

ABSTRACT

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.

INTRODUCTION

Hyphomicrobium spp. are restricted facultative methylotrophs that reproduce by budding at the tip of a polar prostheca. They are ubiquitous in water and soil but can also be found in sewage treatment plants (1). Some strains have been characterized by their denitrification capacities (2–5). They have often been identified as major players in denitrification systems supplemented with methanol (4, 6–8), and their presence has been associated with high denitrification rates (9, 10).

Denitrification takes place in bacterial cells where N-oxides and/or N-oxyanions serve as the terminal electron acceptor instead of oxygen (O2) for energy production when oxygen depletion occurs, leading to the production of gaseous nitrogen (N2) (11). Four sequential reactions are essential for the reduction of nitrate to gaseous nitrogen via nitrite, nitric oxide, and nitrous oxide intermediates, and each of these reactions is catalyzed by different enzymes, namely, nitrate reductases (Nar and Nap), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos) (12–14).

Previous studies using DNA hybridization with probes targeting genes encoding the catalytic subunit of denitrification reductases (narG, nirK, nirS, and nosZ) detected denitrification genes in several members of the genus Hyphomicrobium, including H. denitrificans and H. zavarzinii (2, 3). H. denitrificans (5) is commonly found in soils, freshwater environments, and activated sludge. This bacterium has the capacity to denitrify (2), and its nitrite and nitrous oxide reductases were previously characterized (15–18). H. zavarzinii is a soil bacterium that has been studied mainly for the biotechnological potential of the exceptional formaldehyde dehydrogenase found in strain ZV580 (19, 20). Recently, we described a new denitrifying species of the genus Hyphomicrobium, H. nitrativorans NL23T (21, 22). This bacterium was isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome in Canada. H. nitrativorans was one of the most abundant bacterial species in the denitrifying biofilm, along with Methylophaga nitratireducenticrescens JAM1T, which was also isolated from this biofilm and which can reduce nitrate into nitrite (23, 24). Given that pure cultures of H. nitrativorans grow poorly in seawater (21), we believe that M. nitratireducenticrescens JAM1 and H. nitrativorans NL23 work together in syntrophy in the biofilm to achieve the complete denitrification pathway.

Despite the presence of denitrifying Hyphomicrobium spp. in many environments or denitrification processes, genetic and physiological information regarding their denitrification capacities is poorly documented. This could be related to the fact that Hyphomicrobium spp. can be hard to culture, especially under denitrifying conditions. In this study, three denitrifying Hyphomicrobium species, H. nitrativorans, H. denitrificans, and H. zavarzinii, were compared at the genetic and physiological levels. Because H. nitrativorans was isolated from a marine environment, we focused on the effect of salt on denitrification. Among the major differences found between their genomes was the type of nitrate reductase that they encode. Our results showed that H. nitrativorans sustained better denitrifying activity than the other two species and had a better tolerance to higher nitrate and NaCl concentrations. The effect of variations in the nitrate and NaCl concentrations on the relative expression levels of the nitrate and nitrite reductase genes was also assessed.

MATERIALS AND METHODS

Genome sequence analyses.Hyphomicrobium nitrativorans strain NL23T (ATCC BAA-2476), Hyphomicrobium denitrificans strain XT (ATCC 51888), and Hyphomicrobium zavarzinii strain ZV622T (ATCC 27496) were used in this study. The general characteristics of the three bacterial species are presented in Table S1 in the supplemental material. Publicly available genome sequences of H. nitrativorans NL23 (GenBank accession number NC_022997) (22), H. denitrificans ATCC 51888 (GenBank accession number NC_014313) (25), and H. zavarzinii ATCC 27496 (GenBank accession numbers KB911255 to KB911269 for 15 scaffolds) were retrieved from GenBank, imported into RAST (Rapid Annotation Using Subsystem Technology) (26) for annotation, and processed in SEED viewer (27) to identify denitrification gene clusters and associated regulatory genes. Orthologous genes were determined with the “sequence-based comparison” tool in SEED viewer and refined by hand. Briefly, the deduced amino acid sequences for all the annotated genes of one genome were compared to those of the other two genomes by BLAST (automated process in SEED viewer). This comparison was performed three times with a different genome set as the reference each time. Only genes that resulted from reciprocal analysis were considered orthologous. The three genomes were also annotated by the Integrated Microbial Genomes (IMG) comparative analysis system of the Joint Genome Institute (http://img.jgi.doe.gov/) (28). The majority of the differences in annotations between RAST and IMG were in genes encoding hypothetical proteins with unknown functions. However, both annotation systems provided similar results for orthologous genes. The results were derived from RAST because supplemental options were not available in IMG. A genome comparison of the three species was further performed with MAUVE (29). The average nucleotide identity (ANI) was determined for each pair of species (http://enve-omics.ce.gatech.edu/ani/). Deduced amino acid sequences derived from the napA, narG, nirK, cnorB, and nosZ genes that were retrieved from the genomes of the three Hyphomicrobium species, as well as sequences of their closest relatives and representative sequences of the Proteobacteria, were aligned by using ClustalW, and phylogenetic trees were constructed by using the neighbor-joining algorithm (30) with the Poisson correction method in MEGA version 6.06 (31).

Bacterial strains and growth conditions.Bacteria were cultured in modified Hyphomicrobium medium 337a (32) [composition (per liter), 1.3 g KH2PO4, 1.13 g Na2HPO4, 0.5 g (NH4)2SO4, 0.2 g MgSO4·7H2O, 3.09 mg CaCl2·2H2O, 2.0 mg FeSO4·7H2O, 1.0 mg Na2MoO4·2H2O, 0.88 mg MnSO4·4H2O, and 10 μg CuCl2·2H2O (pH 7.5)] at 30°C. The NaCl concentrations were variable depending on the assay and are specified below. When required, nitrate was added to the growth medium as sodium nitrate (NaNO3) at a concentration of 14.3 mM (200 mg NO3-N liter−1, unless otherwise specified). Nitrate was added to the precultures for all of the assays conducted under denitrifying conditions (unless otherwise specified). Methanol was added to the growth medium as a carbon source at a concentration of 0.3% (vol/vol) prior to inoculation. Vitamin B12 was also added at a concentration of 0.1 mg liter−1 prior to inoculation to stimulate bacterial growth on solid medium and in liquid medium to start cultures from bacterial colonies. The inoculum was made of fresh aerobic liquid cultures. Bacterial cells were pelleted, dispersed in a 0.85% NaCl solution, and homogenized prior to inoculation. The initial optical density of the cultures was adjusted to reach an optical density at 600 nm (OD600) close to 0.1. In order to assess the capacity of the three Hyphomicrobium species to achieve complete denitrification, the N2O concentrations in the headspace of cultures incubated with and without 10% (vol/vol) acetylene, an inhibitor of the nitrous oxide reductase, were compared (33).

Aerobic cultures used for RNA extraction were incubated with constant shaking (250 rpm) in 200-ml Erlenmeyer flasks containing 30 ml of growth medium. For cultures under denitrifying conditions, 30 ml of growth medium supplemented with nitrate was dispensed into 70-ml serum vials. The vials were sealed with rubber stoppers and metal seals, flushed for 10 min with N2 to remove oxygen from the headspace, and autoclaved. Substrate addition, inoculation, and sampling of these vials were performed through the rubber stopper using a syringe and a needle. All cultures were incubated in the dark. Bacterial growth was monitored by spectrophotometry (OD600). The nitrate and nitrite concentrations in the cultures were determined by ion chromatography using an 850 Professional IC instrument (Metrohm, Herisau, Switzerland) with a Metrosep A Supp 5 analytical column (250 by 4.0 mm), while N2O was analyzed by injecting 250 μl of the headspace into an Agilent 6890 gas chromatograph equipped with a microelectron capture detector.

RNA extraction.RNA was extracted from 15 to 30 ml of bacterial culture in the mid-exponential growth phase. Bacterial cells were centrifuged, transferred into 2-ml tubes containing 500 mg of 0.1-mm zirconia-silica beads (Biospec Products, Bartlesville, OK, USA), and dispersed in 700 μl of a solution containing precooled TPM buffer (50 mM Tris-HCl [pH 7.0], 1.7% [wt/vol] polyvinylpyrrolidone K25, 20 mM MgCl2), 35 μl of 20% SDS, and 500 μl of phenol. The samples were flash frozen with liquid nitrogen and stored at −80°C until RNA extraction was performed. After thawing, RNA was extracted by bead beating twice with a FastPrep-24 instrument (MP Biomedicals, Solon, OH, USA), with the power set at 6.5 for 45 s. The tubes were then centrifuged at 20,000 × g for 5 min at 4°C, and the upper phase was transferred into a new tube. The bead beating and centrifugation steps were repeated following the addition of 700 μl of PBB buffer (5 mM Tris-HCl [pH 7.0], 5 mM Na2 EDTA, 1% [wt/vol] SDS, 6% [vol/vol] water-saturated phenol) to the tubes containing the bacterial cells. Both upper phases were further extracted with phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol/vol]) and chloroform-isoamyl alcohol (24:1 [vol/vol]). For each extraction step, centrifugation was performed at 20,000 × g for 5 min at 4°C. RNA was recovered by precipitation with a 1/10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at −20°C overnight. After centrifugation at 20,000 × g for 45 min at 4°C, the supernatant was discarded, and the pellets were washed with 70% ethanol (13,000 × g for 10 min at 4°C), dried at room temperature for 15 min, dissolved in diethyl pyrocarbonate (DEPC)-treated water, and treated with Turbo DNase (Ambion; Life Technologies Inc., Burlington, ON, Canada) for 30 min to remove all contaminating DNA. Finally, RNA extracts were purified by using the RNeasy MinElute Cleanup kit according to the manufacturer's instructions (Qiagen, Toronto, ON, Canada). RNA integrity was verified by gel electrophoresis, and RNA quantification was performed by using a NanoDrop ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE, USA). The absence of DNA was confirmed by endpoint PCR analysis using 16S rRNA gene universal primers.

Reverse transcription and quantitative PCR assays.cDNAs were generated from RNA by using hexameric primers and the reverse transcription system from Promega (Madison, WI, USA) with 1 μg of extracted RNA, according to the manufacturer's protocols. Real-time quantitative PCR (qPCR) assays were performed with PerfeCTa SYBR green SuperMix ROX (Quanta; VWR International, Ville Mont-Royal, QC, Canada). The final volume of all of the reaction mixtures was 20 μl, and the amplification mix was composed of 10 μl of PerfeCTa SYBR green SuperMix, 0.4 μl of each primer (40 pmol), 4.2 μl of RNA-free water, and 5 μl of cDNA (50 ng). All of the reactions were performed with a Corbett Rotor-Gene 6000 real-time PCR machine (Qiagen). PCR started with an initial denaturation step for 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C. The fluorescence signal was acquired at the end of the 72°C step and at the end of a 15-s reading step at 80°C that was added to the end of each cycle to avoid fluorescence from primer dimers. Primers were designed with Primer3web version 4.0.0 (34, 35) to target the napA, narG, nirK, cnorB, and nosZ denitrification genes and the rpoB and dnaG reference genes of the three Hyphomicrobium species (see Table S2 in the supplemental material). Each of the primer sets was tested for specificity and efficiency of amplification by qPCR with serial dilutions of purified PCR fragments (efficiencies of between 0.92 and 1.05).

Calculations and statistical analyses.Growth and denitrification rates were obtained by calculating the slope of the linear portion of the growth and denitrification curves, respectively. Relative gene expression was calculated according to the ΔΔCT method (36). For each calculation, only one housekeeping gene was used as a control. For each Hyphomicrobium species, the control gene was chosen from among the two genes tested (rpoB and dnaG) by using RefFinder (http://www.leonxie.com/referencegene.php), an online tool that allows the selection of the appropriate control gene based on the lowest variability according to geNorm (37), Normfinder (38), BestKeeper (39), and the comparative ΔΔCT method (40). Statistical analyses were performed with R (version 2.9.0; R Foundation for Statistical Computing). The effects of nitrate and NaCl concentrations on growth and denitrification rates were tested by using factorial analysis of variance (ANOVA). Prior to factorial ANOVA, normality was tested by using the “shapiro.test” function. When necessary, data were log transformed to meet parametric ANOVA assumptions. ANOVA and post hoc Tukey honestly significant difference (HSD) tests were then carried out by using the “aov” and “TukeyHSD” functions, respectively. If significant interactions were detected between the two factors, ANOVA was performed on a subset of data that were generated for each factor. Normality was tested again on each data subset prior to ANOVA. The statistical significance of the ΔΔCT values was tested by Student's t test (two tailed, heteroscedastic).

RESULTS

Genome comparison.The general characteristics of the three genomes are presented in Tables 1 and 2 and Fig. 1. A total of 1,961 orthologous genes are shared among the three species (Fig. 1), with 2,150 orthologous genes shared between H. nitrativorans and H. denitrificans, 2,687 shared between H. nitrativorans and H. zavarzinii, and 2,355 shared between H. denitrificans and H. zavarzinii (Table 2). The number of unique genes (found in one species but not in the other two) constitutes between 16.3 and 25.7% of the total genes, with the highest number of unique genes being found in H. denitrificans (Fig. 1). However, a large number (>75%) of these unique genes are annotated as hypothetical proteins with unknown functions (HP). When the genomes were compared for the average deduced amino acid sequence identity between orthologous genes and for the average nucleotide identity (ANI) between whole-genome sequences, H. nitrativorans and H. zavarzinii showed a higher level of identity with each other than with H. denitrificans (Table 2). These results are consistent with the results of phylogenetic analysis of the 16S rRNA gene sequences (21).

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TABLE 1

General characteristics of the genomes of the three Hyphomicrobium speciesa

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TABLE 2

Comparative analysis of the genomes of the Hyphomicrobium species

FIG 1
FIG 1

Venn diagram illustrating the number of orthologs found across the three species. Numbers in the nonsharing zones represent unique genes with no homology to sequences in the other two species. Percentages of unique genes that were identified as encoding a HP (hypothetical protein with unknown function) are illustrated. In the shared zones, only genes that resulted from reciprocal analysis (e.g., H. nitrativorans versus H. denitrificans and H. denitrificans versus H. nitrativorans) were considered.

Putative unique pathways were found in each species or in two out of three species (see Table S3 in the supplemental material). A long cluster of genes associated with photosynthesis is present in H. zavarzinii, although there is no indication in the literature of photosynthesis-related activities in this bacterial species. The urea decomposition pathways differ among the three species. Urea is degraded into CO2 and NH3 by urease in H. denitrificans and by urea carboxylase and allophanate hydrolase in H. zavarzinii. No urea decomposition pathway was found in H. nitrativorans. Genes encoding the Na+/H+ multisubunit antiporter were found in H. nitrativorans, and NhaA-type and NhaD-type Na+/H+ antiporter genes were found in the other 2 species. H. nitrativorans and H. zavarzinii carry genes encoding methylamine dehydrogenase and associated proteins. These genes were not found in H. denitrificans, which is surprising because this bacterium can utilize monomethylamine, dimethylamine, and trimethylamine as sole carbon sources (5). Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated proteins were found in H. nitrativorans, whereas only CRISPR-associated proteins were found in H. zavarzinii. However, the draft genome showed an ∼3,000-bp gap flanking the chromosomal region of the genome, suggesting a repeated region that could not be assembled properly.

Genes associated with the denitrification pathway.All three Hyphomicrobium species studied here possess gene clusters encoding the full set of enzymes necessary to achieve complete denitrification, although they differ greatly in their nitrate reductases (Table 3). The genome of H. nitrativorans contains a gene cluster encoding the periplasmic nitrate reductase Nap, the genome of H. denitrificans contains a gene cluster encoding the membrane-bound nitrate reductase Nar, and the genome of H. zavarzinii contains gene clusters encoding one Nap and two Nar enzymes. The three species also have a nirK gene encoding the copper-containing nitrite reductase, along with the nirV gene encoding an accessory protein in H. nitrativorans and H. zavarzinii. Gene clusters encoding the nitric oxide (Nor) and nitrous oxide (Nos) reductases and with similar gene organizations were also detected in the three genomes. The only exception is norE, which is located upstream in a direction opposite that of cnorCBQD in H. denitrificans (Table 3). Genes associated with nitrate transport were detected upstream of the nar gene clusters in H. denitrificans and H. zavarzinii. Two distinct genes encoding NarK transporters, a NarK1-type symporter and a NarK2-type nitrate/nitrite antiporter, are located upstream of the nar-2 gene cluster in H. zavarzinii. One gene encoding a fused NarK1-NarK2-type transporter (here named narK12f) (41) is located upstream of the nar-1 gene cluster in H. zavarzinii and the nar gene cluster in H. denitrificans.

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TABLE 3

Gene clusters associated with the dissimilatory pathway of denitrification

Genes commonly associated with the regulation of denitrification gene expression, such as nnrS and nrrR, were detected in the three genomes. Most of these genes belong to the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulation) family; some of them are located close to the denitrification gene clusters (Table 3). The narXL two-component transcription regulatory system was found in H. denitrificans and H. zavarzinii. However, it is not located in the vicinity of the denitrification gene clusters in either species.

Phylogenetic analyses were performed on the deduced amino acid sequences of genes encoding the catalytic subunits of the different reductases (NapA, NarG, NirK, cNorB, and NosZ) found in the three Hyphomicrobium species. All these reductases are phylogenetically related to known denitrification reductases found in other Alphaproteobacteria, with sequence identities generally above 80%, except for NarG2 and NosZ of H. zavarzinii (maximum of 69% identity in both cases) (see Fig. S1 in the supplemental material).

Effect of nitrate and NaCl on bacterial growth and denitrification activity.The three Hyphomicrobium species cultivated under aerobic conditions with or without the addition of 14.3 mM NaNO3 showed similar growth curves. No nitrate reduction or nitrite production was detected under these conditions (data not shown). When incubated under denitrifying conditions at the same nitrate concentration, the three Hyphomicrobium species had the capacity to grow and to completely eliminate nitrate without nitrite accumulation, although growth was much slower than that under aerobic conditions (Fig. 2). The addition of acetylene led to the accumulation of N2O in the headspace of the vials, while no (H. nitrativorans) or less (H. denitrificans and H. zavarzinii) N2O accumulation was observed in cultures incubated without acetylene (Fig. 3), indicating that the three species can achieve complete denitrification.

FIG 2
FIG 2

Effect of nitrate concentration on growth and denitrification. H. nitrativorans (A and D), H. denitrificans (B and E), and H. zavarzinii (C and F) were cultured in triplicate under denitrifying conditions with an initial concentration of 14.3 mM (diamonds), 42.8 mM (squares), 71.4 mM (triangles), or 107 mM (crosses) NaNO3. At regular intervals, growth (A to C) and nitrate and nitrite concentrations (D to F) were measured. In panels D to F, full lines indicate nitrate concentrations, and dotted lines indicate nitrite (NOx) concentrations. Assays were performed with an NaCl concentration of 0.5% in the growth medium. The standard deviations of the means for each time point are presented.

FIG 3
FIG 3

Effect of acetylene on the production of N2O. H. nitrativorans (triangles), H. denitrificans (squares), and H. zavarzinii (circles) were cultured in triplicate under denitrifying conditions with (full lines) or without (dotted lines) acetylene at an initial concentration of 14.3 mM NaNO3. At regular intervals, the nitrous oxide concentration in the headspace was measured.

The effect of increasing nitrate concentrations on the growth and denitrification kinetics of the three Hyphomicrobium species was assessed under denitrifying conditions, with the NaNO3 concentration ranging from 14.3 to 107 mM (200 to 1,500 mg NO3-N/liter) (Fig. 2). H. nitrativorans had the capacity to grow (Fig. 2A) and denitrify without significant nitrite accumulation (Fig. 2D) at all of the concentrations of NaNO3 tested, with optimal growth at 71.4 mM NaNO3. In contrast, growth and denitrification by H. denitrificans and H. zavarzinii were almost completely inhibited at 107 mM NaNO3 (Fig. 2B, C, E, and F). For H. denitrificans and H. zavarzinii, optimal growth and denitrification were observed at 42.8 mM NaNO3, although higher levels of nitrite were constantly detected in cultures of H. denitrificans. While these higher levels of nitrite were transient at initial concentrations of 14.3 and 42.8 mM NaNO3 in H. denitrificans, nitrite accumulation leading to incomplete denitrification was detected at 71.4 mM NaNO3 (Fig. 2E).

Because H. nitrativorans was isolated from a marine environment, tolerance to increasing NaCl concentrations ranging from 0 to 2% NaCl was assessed under denitrifying conditions for the three species. Growth of and denitrification by H. nitrativorans were detected at all of the NaCl concentrations tested, without nitrite accumulation, although a concentration of 2% led to slower growth and denitrification (Fig. 4A and D). Increasing NaCl concentrations ranging from 0 to 1% led to delayed growth and denitrification in H. denitrificans (Fig. 4B and E) and slower growth and denitrification in H. zavarzinii (Fig. 4C and F). A transient accumulation of nitrite was also observed in H. denitrificans cultures. At 2% NaCl, these two species showed poor or no growth and no denitrifying activity.

FIG 4
FIG 4

Effect of NaCl concentration on growth and denitrification. H. nitrativorans (A and D), H. denitrificans (B and E), and H. zavarzinii (C and F) were cultured in triplicate under denitrifying conditions with 0% (diamonds), 0.5% (squares), 1% (triangles), and 2% (crosses) NaCl. At regular intervals, growth (A to C) and nitrate and nitrite (NOx) concentrations (D to F) were measured. In panels D to F, full lines indicate nitrate concentrations, and dotted lines indicate nitrite concentrations. Standard deviations of the means for each time point are presented.

To further evaluate whether the interaction between nitrate and NaCl could lead to significant differences in growth and denitrification rates in the three Hyphomicrobium species, we carried out a factorial experiment using two different concentrations of each of the two factors in question. Growth and denitrification were assessed at 14.3 and 71.4 mM NaNO3 and at 0 and 0.5% NaCl (Table 4). The results showed that the three species responded differently to variations of nitrate and NaCl levels. While no significant interactions between the nitrate and NaCl concentrations were detected for H. nitrativorans and H. denitrificans, significant interactions between these two factors were detected for the growth rate and the denitrification rate in H. zavarzinii (Table 4). Single-factor analysis of the data subsets showed that growth and denitrification rates in H. zavarzinii were negatively affected by higher nitrate and NaCl concentrations, with the lowest rates being detected at 71.4 mM NaNO3 and 0.5% NaCl (Tables 5 and 6). In H. nitrativorans, while nitrate and NaCl had no significant effect on the growth rate, a significant effect of both factors on the denitrification rate was detected, with higher values being observed at higher nitrate and NaCl concentrations (Tables 5 and 6). In contrast, neither of the two factors had a significant effect on the denitrification rate in H. denitrificans (Table 6), but the nitrate concentration had a significant effect on the growth rate, with a considerably lower value at 71.4 mM NaNO3 than at 14.3 mM NaNO3 (Table 5).

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TABLE 4

Factorial ANOVA of the effects of nitrate and NaCl concentrations on growth and denitrification ratesa

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TABLE 5

Effect of nitrate and NaCl concentrations on growth rates of three Hyphomicrobium speciesa

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TABLE 6

Effect of nitrate and NaCl concentrations on denitrification rates of three Hyphomicrobium speciesa

Effect of nitrate, NaCl, and oxygen on relative gene expression levels of the nitrate and nitrite reductase genes.Our results showed the influence of nitrate and NaCl on the growth and denitrification of the three Hyphomicrobium species. The effects of these parameters were also assessed at the nitrate and nitrite reductase gene expression levels. We focused our analysis on these genes because the three species differ greatly in their nitrate reductases; H. denitrificans also harbors a very distinct nitrite reductase, while NO and N2O reductases found in the three species are more similar.

The three Hyphomicrobium species were cultured under the following denitrifying conditions: (i) 14.3 mM NaNO3 and 0% NaCl, (ii) 71.4 mM NaNO3 and 0% NaCl, (iii) 14.3 mM NaNO3 and 0.5% NaCl, and (iv) 71.4 mM NaNO3 and 0.5% NaCl. Relative expression levels of the napA, narG, and nirK genes were measured during the mid-exponential growth phase, with cultures grown in 14.3 mM NaNO3 and 0% NaCl as the reference culture (Fig. 5A to C). In H. nitrativorans, higher nitrate and NaCl concentrations led to a small (1.4- to 3.4-fold) but significant increase in the relative gene expression levels of napA and nirK (Fig. 5A). In H. denitrificans, a significant effect was observed exclusively for relative gene expression levels of nirK in cultures grown with 71.4 mM NaNO3 and 0% NaCl (5.8-fold increase) (Fig. 5B). In H. zavarzinii, the nitrate and NaCl concentrations affected the three nitrate reductase genes to different degrees. The relative gene expression level of napA did not change under any of the conditions tested. Both factors had a negative effect on narG1 gene expression but a positive effect on narG2 gene expression (Fig. 5C). Similar to narG1, the relative gene expression level of nirK in H. zavarzinii was negatively affected by higher nitrate and NaCl concentrations.

FIG 5
FIG 5

Relative expression levels of the nitrate and nitrite reductase genes in the three species cultured under different conditions. (A to C) Effects of nitrate and NaCl concentrations. The three species were cultured in triplicate under denitrifying conditions with 14.3 mM NaNO3 and 0% NaCl (reference cultures are represented by the dashed line set at 1 in each panel), with 71.4 mM NaNO3 and 0% NaCl, with 14.3 mM NaNO3 and 0.5% NaCl, or with 71.4 mM NaNO3 and 0.5% NaCl. (D and E) Effects of oxygen. The three species were cultured in triplicate under aerobic conditions (reference cultures are represented by the dashed line set at 1 in each panel), aerobic conditions with 14.3 mM NaNO3, or denitrifying conditions with 14.3 mM NaNO3. Aerobic precultures for this assay were prepared without adding nitrate to the growth medium. ***, P < 0.001; **, 0.01 > P > 0.001; *, 0.05 > P > 0.01.

Oxygen is also known to have a major effect on denitrification and the denitrification reductases. The three Hyphomicrobium species were cultured under aerobic conditions, under aerobic conditions with 14.3 mM NaNO3, or under denitrifying conditions with 14.3 mM NaNO3. The relative expression levels of the napA, narG, and nirK genes were measured as described above, with the culture grown aerobic conditions set as the reference culture (Fig. 5D and E). In H. nitrativorans, the addition of nitrate to aerobic cultures led to small but significant increases in the relative gene expression levels of napA and nirK (1.8- and 3.2-fold, respectively), while incubation under denitrifying conditions led to much higher relative gene expression levels for both genes (7.1- and 35.1-fold, respectively) (Fig. 5D). In H. denitrificans, the only difference observed was for the relative gene expression level of narG, which was significantly higher in the presence of nitrate under aerobic conditions (Fig. 5E). In H. zavarzinii, none of the genes tested was significantly affected by the addition of nitrate to aerobic cultures (Fig. 5F). However, the incubation of this bacterial species under denitrifying conditions led to a strong increase in the relative gene expression level of narG1 (36.4-fold), a slight but significant increase in the level of narG2 (1.8-fold), and a significant decrease in the level of napA (−8.3-fold) (Fig. 5F).

DISCUSSION

H. nitrativorans, H. denitrificans, and H. zavarzinii possess genes encoding the full set of enzymes required to perform complete denitrification, and they were able to achieve this process. Under the culture conditions tested, H. nitrativorans was found to be the most efficient of the three Hyphomicrobium species evaluated. Compared to the two other species, it has the capacity to denitrify at high nitrate concentrations, without nitrite accumulation. Consistent with this observation, an increase in the nitrate concentration in the growth medium induced significant increases in narG and nirK relative gene expression levels in H. nitrativorans. In contrast, an increase of the initial nitrate concentration in the growth medium led to nitrite accumulation in H. denitrificans cultures despite a significant increase in the nirK relative gene expression level at a higher nitrate concentration. Nitrate inhibition of nitrite reduction was previously reported for several bacteria (42–44) and has been associated with a phenomenon of competition between nitrate and nitrite reductases for electron donors. These results suggest that in H. denitrificans, the nitrate reductase might have a higher affinity for electrons than the nitrite reductase. H. denitrificans is characterized by its unique nitrite reductase, which is clearly distinct from the nitrite reductases found in other Hyphomicrobium species. It is characterized by the presence of two type I Cu sites, as opposed to the single type I Cu site found in the nitrite reductases of most bacteria (16, 17, 45). The role of this second type I Cu site has not been elucidated yet. Currently, the role of this distinct nitrite reductase in the denitrifying capacity of H. denitrificans is unknown, but it might be linked to the nitrite accumulation observed in H. denitrificans cultures. Growth of H. denitrificans and H. zavarzinii was totally inhibited at the highest nitrate concentration tested. Because nitrate was supplied as NaNO3, it is possible that this growth inhibition was exacerbated by the input of sodium in the growth medium, as previously observed for Shewanella oneidensis cultures (46).

As expected, H. nitrativorans also demonstrated better tolerance to NaCl than the two other Hyphomicrobium species tested and showed small but significant increases in the napA and nirK relative gene expression levels when NaCl was added to the growth medium. More surprisingly, H. nitrativorans showed better tolerance to NaCl when cultured under denitrifying conditions than previously reported for aerobic growth. Indeed, while total growth inhibition at NaCl concentrations of >1% was previously reported for H. nitrativorans under aerobic conditions (21), our results demonstrate growth and complete denitrification by this bacterium at NaCl concentrations of up to 2%. The mechanisms of this enhanced tolerance to NaCl could be associated with the multisubunit Na+/H+ antiporter. A gene cluster encoding this type of antiporter was found in the H. nitrativorans genome, whereas genes encoding an NhaA-type Na+/H+ antiporter were found in the genomes of the two other species. The multisubunit Na+/H+ antiporter has been associated with high Na+ extrusion activity in halotolerant Staphylococcus aureus (47). Despite having better NaCl tolerance than the other Hyphomicrobium species, H. nitrativorans remains quite sensitive to NaCl compared to other bacteria isolated from denitrifying biofilms, such as Methylophaga nitratireducenticrescens JAM1, which can tolerate NaCl concentrations of up to 8% (24). We hypothesize that H. nitrativorans benefits from the osmoprotection of other bacteria, such as M. nitratireducenticrescens JAM1, to thrive in the artificial seawater of the Biodome aquarium (NaCl concentration of 2.4%). This hypothesis is currently being studied in our laboratory.

The detailed comparison of the H. nitrativorans, H. denitrificans, and H. zavarzinii genomes has highlighted the facts that a large number of orthologous genes are shared among these three species and that very few genes with known functions are unique to a single species. From this perspective, the fact that the three species differ greatly in their nitrate reductases is noteworthy, and it can be hypothesized that the differences observed in their denitrification capacities reflect these variations observed at the genetic level. Indeed, while H. nitrativorans and H. denitrificans are characterized by the presence of a single Nap or Nar gene cluster, respectively, one Nap and two Nar gene clusters have been identified in the genome of H. zavarzinii. Such an occurrence of multiple operons encoding nitrate reductases has previously been reported for other microorganisms. One well-characterized example is E. coli K-12, which also expresses two different cytoplasmic membrane nitrate reductases (NRA and NRZ), encoded by narGHJI and narZYWV, respectively, and one periplasmic nitrate reductase, encoded by napFDAGHBC (48–50). Gene deletions in E. coli K-12 indicate that Nap can support anaerobic growth when it is the only nitrate reductase available, although NRA is clearly the main enzyme responsible for nitrate reduction when all enzymes are present (48).

While Nap in E. coli is expressed under anaerobic conditions (50) and is specifically involved in nitrate reduction at low nitrate concentrations (48, 51), different functions have been proposed for the Nap system in other bacteria. In Paracoccus spp., in which both the Nar and Nap systems are present, Nar is expressed under anaerobic conditions, while Nap is expressed during aerobic growth, in the presence or absence of nitrate (13, 52, 53). This expression pattern is similar to our results for H. zavarzinii, with growth under denitrifying conditions leading to the repression of napA and the overexpression of narG1 and, to a lesser extent, narG2. In Paracoccus spp., Nap is responsible for aerobic nitrate reduction, and it has been suggested that this activity might help dissipate excess reductant, which is further supported by the fact that Nap is induced by growth on highly reduced carbon substrates (13, 52). Despite higher levels of Nap expression under aerobic conditions than under denitrifying conditions, we did not detect significant nitrate reduction in aerobic liquid cultures of H. zavarzinii. Similarly, the expression level of the Nap-α operon in Rhodobacter sphaeroides 2.4.3 was higher in aerobic liquid cultures than in anaerobic cultures, without any detectable nitrate reduction (54). However, aerobic reduction of nitrate into nitrite occurred when R. sphaeroides 2.4.3 was cultivated on solid medium, indicating a role of Nap-α in redox homeostasis specific for these culture conditions. Preliminary assays performed in our laboratory indicated that this is not the case for H. zavarzinii, but more extensive work will have to be performed to rule out this hypothesis.

While it is not uncommon to find multiple nitrate reductases in a single bacterium, the presence of multiple nitrate reductases of the same type has been reported only rarely in previous studies. Rhodobacter sphaeroides 2.4.3 possesses two Nap operons, one, identified as Nap-α, being similar to Nap operons found in other Alphaproteobacteria (napKEFDABC) and the other, identified as Nap-β, being similar to the Nap operons found in Gammaproteobacteria (napFDAGHBC) such as Shewanella spp. and E. coli (54). These two distinct Nap isoforms have also been reported for several strains of Shewanella (55, 56). Recently, we also described the new bacterial species Methylophaga nitratireducenticrescens JAM1 (Gammaproteobacteria), which expresses two distinct Nar nitrate reductases (Nar1 and Nar2) encoded by two narGHJI operons (23, 24, 57). For this bacterium, phylogenetic analyses revealed that Nar1 is more closely related to the Nar system of Betaproteobacteria, whereas Nar2 is more closely related to the Nar system found in other Gammaproteobacteria, suggesting that the Nar1 system was acquired by horizontal gene transfer (23). In H. zavarzinii, while narG1 is closely related to narG sequences found in other Alphaproteobacteria, narG2 is only distantly related to any known narG sequence. The expression patterns of these two genes were also slightly different, with a much stronger effect of anaerobic conditions on the expression of narG1 than on the expression of narG2. Further work, including the production of gene knockout mutants for the different nitrate reductases in H. zavarzinii, is necessary to completely elucidate the physiological roles played by these enzymes.

Although the napA gene product in H. nitrativorans is more related to Nap-α than to Nap-β of Rhodobacter sphaeroides 2.4.3, a distinct napA expression pattern was observed. In H. nitrativorans, napA gene expression levels were significantly higher in the presence of nitrate and under denitrifying conditions than in the absence of nitrate and under aerobic conditions. Similar results have been reported for Bradyrhizobium japonicum, which also possesses a single periplasmic nitrate reductase of the α isoform (58). Although results from studies on Paracoccus pantotrophus and Rhodobacter sphaeroides point toward a specific role of the Nap-α isoform in aerobic denitrification and redox balancing, both the α and β isoforms have been associated with anaerobic nitrate respiration in Shewanella spp. (55, 56). It is important to note that the highest denitrification efficiencies were observed for a Shewanella strain lacking the nap-β operon (55) and for a nap-β mutant Shewanella strain (56), indicating that the α isoform plays a major role in anaerobic respiration in this bacterial genus. Taken together, these results further illustrate that there is no clear association between the isoform and the physiological role of Nap and that other factors, such as the presence of distinct enzymes with similar functions, might also play an important role.

Denitrifying conditions were shown to increase the relative gene expression level of nirK in H. nitrativorans. However, such an effect was not observed for H. denitrificans and H. zavarzinii. No previous studies assessed quantitatively the expression of nirK under different culture conditions in Hyphomicrobium spp. Microaerobic or anaerobic conditions are known to regulate nirK expression in many bacterial species (59–61). In Rhodobacter sphaeroides 2.4.3, nirK expression is regulated by NnrR, a transcriptional activator of the CRP/FNR family (60, 62). Gene annotation of the H. denitrificans genome did not reveal sequences closely related to nnrR, which could explain the absence of regulation of nirK expression. The lack of an effect of denitrifying conditions on H. zavarzinii is surprising, as nnrR was found downstream of nirK. In fact, H. zavarzinii and H. nitrativorans have the same gene arrangement in the nirK locus (Table 3), and both showed a potential NnrR binding site (data not shown). Further work will be required in order to elucidate this phenomenon.

ACKNOWLEDGMENTS

We thank Karla Vasquez for her technical assistance.

This project was financially supported by a grant to R.V. from the Natural Sciences and Engineering Research Council of Canada and by scholarships to C.M. from the Fondation Universitaire Armand-Frappier and the Fonds de Recherche du Québec Nature et Technologies.

FOOTNOTES

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