Disruption of narH, narJ, and moaE Inhibits Heterotrophic Nitrification in Pseudomonas Strain M19

Interruptions in three nitrate reductase-related genes, narH,narJ, and moaE, inhibited heterotrophic nitrification in Pseudomonasstrain M19. No nitrate was detected in the medium, and nitrificationproceeded in the presence of a nitrate reductase inhibitor.Heterotrophic nitrification was greatly stimulated by the additionof nitrate.

INTRODUCTION

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Introduction

Nitrification is a key step in the global nitrogen cycle; italters both the mobility and utilization potential of nitrogenin soil (21). The chemoautotrophic bacteria Nitrosospira andNitrobacter are typically identified as the main agents of nitrification,but many heterotrophic bacteria and fungi can also nitrify (8,13). The physiological role of heterotrophic nitrification isunknown (8). In addition, because many microorganisms are notamenable to traditional culturing techniques (2), the phylogeneticdiversity and ecological significance of heterotrophic nitrifiersare not known. Knowledge of the molecular mechanisms of heterotrophicnitrification would facilitate cultivation-independent studiesof these organisms and provide information about the physiologicalrole of this process.

Reports of the molecular mechanisms of heterotrophic nitrificationhave presented either functional or sequence data, but not both.Moir et al. (14) used heterologous expression to show that theParacoccus denitrificans ammonia monooxygenase consists of twosubunits and is a quinol oxidase. Ono et al. (16) purified a115-kDa pyruvic oxime dioxygenase containing three 40-kDa subunitsfrom Alcaligenes faecalis. Other studies (11, 15) have reportedthe purification of hydroxylamine oxidase proteins from Paracoccusdenitrificans and Pseudomonas strain PB16. None of these reportshave provided corollary sequence data. Daum et al. (7) usedSouthern analysis and reported a weak hybridization of Pseudomonasputida genomic DNA with Nitrosomonas europaea amoA. They didnot provide any functional data to demonstrate that this geneproduct was involved in heterotrophic nitrification. This isthe first study that provides both functional and sequence analysesof genes involved in heterotrophic nitrification.

Bacterial strains and plasmids.

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Bacterial strains and plasmids.

Pseudomonas strain M19 was isolated with a glutamate enrichmentfrom a tundra dry meadow soil at the Niwot Ridge long-term ecologicalresearch site in the front range of the Colorado Rocky Mountains.It was determined that M19 was a heterotrophic nitrifier bytesting for the production of nitrite (see below) after growthin nitrification broth (22). A partial sequence of the 16S rRNAgene of Pseudomonas strain M19 was generated (accession numberAF539808), and BLAST (1) searches revealed that this sequenceis 99% identical to the 16S rRNA gene from Pseudomonas fluorescens.

Escherichia coli strain S17-1 carrying pJFF350 was kindly providedby Sven Valla. S17-1 has been described elsewhere (20) and featureschromosomally encoded tra genes for conjugation. pJFF350 (9)carries the Omegon-Km transposon containing the aphA kanamycinresistance gene and an E. coli origin of replication.

Conjugation.

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Conjugation.

Transposon mutagenesis was used to interrupt the genome of Pseudomonasstrain M19 to identify genes required for heterotrophic nitrification.M19 was conjugated with S171-1/pJFF350 to mobilize the Omegon-Kmtransposon into M19. Approximately 4 x 10⁸ of both the E. coliS171-1/pJFF350 donor cells and the M19 recipient cells weremixed on a 0.2-µm-pore-size filter. The cells were incubatedon Luria-Bertani (LB) agar medium for 16 h at room temperature.The filter was resuspended in 10 ml of 0.9% NaCl, and 100-µlaliquots were plated on Pseudomonas agar B (2% peptone, 1% glycerol,0.15% MgSO₄ · 7H₂O, 0.15% K₂HPO₄, 1.5% agar) containingampicillin (100 µg/ml) and chloramphenicol (5 µg/ml)(to select against S17-1/pJFF350) and kanamycin (100 µg/ml)(to select against M19). Approximately 9,500 recombinant colonieswere screened for the lack of nitrification activity. Cellswere grown in selective LB medium in deep 96-well plates atroom temperature for 20 h, and cultures were tested for nitrite.Nitrite analysis was performed by a modified version of themethod of Bundy and Meisinger (5). Briefly, 100 µl ofdiazotizing reagent (0.5 g of sulfanilamide in 100 ml of 600mM HCl) and 100 µl of coupling reagent [0.3 g of N-(1-naphthyl)-ethylenediaminedihydrochloride in 100 ml of 120 mM HCl] were added to 100-µlsamples of the cultures. A pink color was considered a positiveindication of the presence of nitrite. Three mutants that wereincapable of nitrification, mt54, mt97, and mt105, were obtained.

Molecular techniques.

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Molecular techniques.

Southern hybridization analysis was used to determine the numberof transposon insertions in the mutant strains. DNA was extractedfrom the three mutants using Genomic-tips (Qiagen, Valencia,Calif.). Genomic DNA (750 ng) was digested with EcoRI (Promega,Madison, Wis.) and used for Southern hybridization by standardprotocols (19). An aphA probe was generated using the PCR digoxigeninprobe synthesis kit (Roche Molecular Biochemicals, Mannheim,Germany) and the primers aphAF (5'-CAAGATGGATTGCACGCAGGT-3')and aphAR (5'-GAACTCGTCAAGAAGGCGAT-3') following the manufacturer'sdirections. For mutants mt54 and mt97, a single band of approximately10 kb hybridized to the probe, signifying a single transposoninsertion. For mutant mt105, two bands (approximately 12 and9 kb) hybridized to the probe, suggesting that two transposonshad integrated into the genome.

The Omegon Km-based aphA gene and E. coli origin of replicationwere used to generate plasmids containing the interrupted genes.Serial dilutions of EcoRI-digested genomic DNA were ligatedusing T4 DNA ligase (Promega) overnight at 16°C. For eachmutant, we pooled the ligation dilution series and transformedthe plasmids into TOP10 (Invitrogen, San Diego, Calif.) chemicallycompetent cells following the manufacturer's directions. Weplated the bacteria on LB containing kanamycin (100 µg/ml)to select for intramolecular ligations that contained the transposon-encodedorigin of replication and aphA gene. For each mutant, 10 transformantswere selected for analysis. Plasmids were extracted using theQIAprep Spin Miniprep kit (Qiagen) and were screened with EcoRI/BamHI,EcoRI/ApaI, and EcoRI/SmaI (Promega) restriction digestion.For both mt54 and mt97, all 10 plasmids generated identicalrestriction patterns. For mutant mt105, two restriction patternscorresponded to the 12-kb EcoRI restriction fragment and the9-kb EcoRI restriction fragment (data not shown).

Plasmids were sequenced using primers OmegonOri (5'-GTCAGAGGTTTTCACCGTCATC-3')and aphA2 (5'-GCTCACAGCCAAACTATC-3') with the BigDye TerminatorCycle Sequencing kit version 2.0 (PE Biosystems, Foster City,Calif.) following the manufacturer's directions. Sequencingproducts were analyzed at the Iowa State University DNA SequencingFacility. Sequences were edited with Sequencher 4.1 (Gene CodesCo., Ann Arbor, Mich.) and used to search GenBank with BLASTX(1). The transposons interrupted narH in mt54 and narJ in mt97.In mt105, one transposon interrupted a genomic region with nosignificant BLASTX matches and the other transposon interruptedmoaE (Fig. 1). Pseudomonas strain M19 is closely related toP. fluorescens, and the interrupted nar genes were highly identicalto P. fluorescens sequences (Fig. 1). No moaE P. fluorescenssequences are available in GenBank; therefore, the sequenceidentity for the closest BLAST match is not as high (Fig. 1).

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FIG. 1. Sites of transposon insertions in mt54, mt97, and mt105 mutants. The transposon insertion site (X) and sequenced areas (lines) are shown. The numbers below the lines are amino acid positions. Values for identities refer to amino acid sequences. The closest BLASTX match to the mutants and GenBank accession numbers are also shown.

All interrupted genes are required for the function of the molybdo-bis(molybdopteringuanine dinucleotide) (Mo-bisMGD) containing membrane-boundnitrate reductase NAR (for a recent review, see reference 18).NarH is an Fe-S-containing electron transfer subunit (18). NarJis a system-specific chaperone involved in the acquisition ofthe Mo-bisMGD cofactor by the NarGH complex (4). MoaE is requiredfor molybdopterin biosynthesis (17).

To rule out nitrate reduction as the source of nitrite, themedium was tested for the presence of nitrate. Nitrate analysiswas performed by a reduction to nitrite; 200 µl of themedium was mixed with 1,000 µl of 0.1 M NH₄Cl, and thesolution was passed over a copperized cadmium column. Nitritewas measured as described above. This assay is sensitive to20 µM concentrations of nitrate, and no nitrate was detectedin the medium. Pseudomonas strain M19 produced 100 µMconcentrations of nitrite after 20 h in this medium at roomtemperature (data not shown). Thus, trace amounts of nitratein the medium could not account for the production of nitrite.Furthermore, concentrations of sodium azide (3) that are inhibitoryto membrane-bound nitrate reductase did not inhibit nitriteproduction in M19 (data not shown). It is possible that nitrogenwas first oxidized to nitrate and then reduced to nitrite bythe membrane-bound nitrate reductase. If this were the case,then nitrate would accumulate in the mutant strains in whichthe NAR activity was blocked. However, no nitrate accumulatedin the mutant strains (data not shown). The periplasmic nitratereductase NAP is also found in strains of Pseudomonas and isinsensitive to sodium azide (18). However, this is also a Mo-bisMGD-containingenzyme that would be inactive in the moaE mutant, yet we detectedno nitrate accumulation by this strain (data not shown). Takentogether, these results imply that the nitrite production byM19 was not the result of nitrate reduction.

To further investigate the role of nitrate reductase genes inheterotrophic nitrification, nitrite accumulation was assayedafter growth on different nitrogen sources with and withoutnitrate (Table 1). Heterotrophic nitrification was not specificto organic nitrogen; Pseudomonas strain M19 also produced nitritewhen grown on ammonium, while mutants mt54, mt97, and mt105were incapable of this activity (Table 1). M19 was also ableto reduce nitrate; millimolar quantities of nitrite accumulatedin these cultures. No nitrite accumulated in cultures of themutants grown on nitrate supplemented with peptone. However,the nar mutants (mt54 and mt97) produced micromolar quantitiesof nitrite when grown on nitrate alone, suggesting that theNAP pathway was active. The moaE mutant was incapable of growthand nitrite production on nitrate alone (Table 1), implyingthat the NAR and NAP pathways were inactive. Nitrite productiondue to heterotrophic nitrification in cultures grown withoutnitrate was low; micromolar quantities accumulated in culturessupplemented with peptone or ammonium (Table 1). Interestingly,nitrate addition greatly enhanced heterotrophic nitrification,increasing nitrite accumulation from the micromolar to millimolarrange in cultures grown with peptone. The nitrite accumulationin these cultures was much greater than the sum of nitrite yieldfrom separate cultures supplemented with nitrate and peptone(Table 1). Because the yield of nitrite was greater than theamount of nitrate in the medium, the difference must be dueto an increase in heterotrophic nitrification rather than anincrease in nitrate reduction.

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TABLE 1. Nitrite accumulation in Pseudomonas strain M19 and mutant cells after growth for 68 h at room temperature

Conclusions.

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Conclusions.

Interruptions in the narH, narJ, and moaE genes all inhibitheterotrophic nitrification in Pseudomonas strain M19. It ispossible that these gene products are involved in both nitratereduction and nitrogen oxidation. Indeed, the processes of aerobicdenitrification and heterotrophic nitrification are linked inmany microbes (13). In addition, subunits of the Nitrobacterhamburgensis nitrite oxidoreductase, which catalyzes both nitratereduction and nitrite oxidation, share sequence similarity withNarH and NarG (12). Furthermore, Crossman et al. (6) found thatheterologous expression of nitrate reductase genes resultedin the production of nitrite even though no nitrate was addedto the medium. These researchers provided no data to demonstratethat the quantities of nitrite produced could be explained bytrace amounts of nitrate in the medium. These results suggesta role for nitrate reductase genes in heterotrophic nitrification.In addition, heterotrophic nitrification was stimulated in Pseudomonasstrain M19 with the presence of nitrate. Nitrate reductase genesare induced by nitrate, probably increasing both their reductiveand oxidative roles in M19. However, although narH, narJ, andmoaE are required for nitrification, they are not sufficientto catalyze this reaction. Indeed, several bacteria, includingE. coli, contain all of these genes and are not capable of heterotrophicnitrification. More work is needed to elucidate the mechanismand physiological role of heterotrophic nitrification.

ACKNOWLEDGMENTS

This work was supported in part by National Science Foundationgrants MCB-0084223 and IBN-9817164.

We thank Stephen Spiro for critically reading the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Environmental, Population and Organismic Biology, University of Colorado, Campus Box 334, Boulder, CO 80309. Phone: (303) 492-6248. Fax: (303) 492-8699. E-mail: schmidts{at}spot.colorado.edu.

REFERENCES

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