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Applied and Environmental Microbiology, April 1999, p. 1652-1657, Vol. 65, No. 4
Department of Microbiology,
Received 25 September 1998/Accepted 15 January 1999
Using consensus regions in gene sequences encoding the two forms of
nitrite reductase (Nir), a key enzyme in the denitrification pathway,
we designed two sets of PCR primers to amplify
cd1- and Cu-nir. The primers
were evaluated by screening defined denitrifying strains,
denitrifying isolates from wastewater treatment plants, and
extracts from activated sludge. Sequence relationships of nir genes were also established. The
cd1 primers were designed to amplify a 778 to
799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp
region in seven of the eight published Cu-nir sequences.
Together, the two sets of PCR primers amplified nir genes
in nine species within four genera, as well as in four of the seven
sludge isolates. The primers did not amplify genes of nondenitrifying
strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only
obtained in five samples. PCR products of the expected
sizes were verified as nir genes after hybridization to DNA
probes, except in one case. The sequenced nir fragments
were related to other nir sequences, demonstrating that the
primers amplified the correct gene. The selected primer sites
for Cu-nir were conserved, while broad-range primers
targeting conserved regions of cd1-nir seem to
be difficult to find. We also report on the existence of
Cu-nir in Paracoccus denitrificans Pd1222.
Biological denitrification is a
respiratory process defined as the enzymatic, stepwise reduction of
nitrogen oxides associated with electron transport phosphorylation and
evolution of the gases nitric oxide (NO), nitrous oxide
(N2O), and molecular itrogen (N2). The
ability to denitrify is a facultative trait spread among a wide variety
of physiological and taxonomic groups. Nearly 130 denitrifying
bacterial species are found within more than 50 genera (32). The denitrification pathway gives them a
competitive advantage in low-oxygen environments. However, some
also denitrify aerobically. Denitrifying bacteria prosper in
practically all habitats, and the reductive process is of global
concern. Denitrification causes nitrogen loss in agricultural soils,
and emitted N2O destroys the ozone layer and contributes to
global warming. Still, denitrification fills an important function in
waste treatment by removing excess nitrogen in local environments and
by anaerobically degrading organic pollutants.
The key step in the denitrification pathway is the reduction of nitrite
by nitrite reductase (Nir). This reaction distinguishes denitrifiers
from nitrate respirers. Denitrification includes two known and distinct
types of Nir enzymes: one with heme c and heme
d1 (cd1-Nir) and the
other containing copper (Cu-Nir) (2, 4, 12). The two Nir
types are functionally and physiologically equivalent, which is
indicated by the fact that the Cu-nir gene from
Pseudomonas aureofaciens can be expressed in a P. stutzeri mutant lacking the gene encoding
cd1-Nir (10). Whereas
cd1-Nir is the predominant reductase in
denitrifying bacteria, copper reductases show greater variation
in molecular weight and immunological reactions and are present
in more taxonomically unrelated strains (5). It has also
been demonstrated that the two reductases are mutually exclusive in any
given strain, although the Nir type may differ within the same
genera and even within the same species (5). This is
particularly true for Alcaligenes faecalis isolates.
The classic approach, using 16S rRNA gene sequences to detect and
analyze bacterial communities in environmental samples without isolation and cultivation, is not possible when studying denitrifying bacteria, except at the species or genus level. Instead, the
phylogenetic diversity of denitrifying bacteria suggests the use of
functional probes and PCR primers based on structural
nir genes to detect denitrifying bacteria in general. This
implies sufficient genetic homology of the structural gene. DNA probes
have been more or less successfully used to detect
cd1- and Cu-nir genes in culturable denitrifying bacteria, enrichment cultures, and a variety of
environmental samples (9, 15, 26, 29, 31). One attempt has
been made to PCR amplify nir fragments from
denitrifying bacteria (29). That study was, however,
limited to experiments with culturable strains, and the primers
used were based on three sequences of the
cd1-Nir-encoding gene derived from two species
(13, 25, 26).
Since then, the number of available nir sequences has
increased, facilitating the construction of more reliable and general primers for the study of denitrifying populations. We designed two
sets of PCR primers to amplify fragments of the gene coding for the
two Nir enzymes using consensus regions in published sequences for the
structural nir gene. Our primers were evaluated by
screening DNAs from denitrifying strains from culture collections and
unidentified denitrifying isolates from different wastewater treatment
plants, as well as DNA extracts from activated sludge. Sequence
relationships of nir genes were established by Southern
blotting and sequencing.
Bacterial strains and culture conditions.
Designations and
sources of all of the defined bacterial strains used in this study are
listed in Table 1. Isolates of
denitrifying bacteria from different activated-sludge plants in
Stockholm, Sweden, were kindly provided by Gunnel Dalhammar, Royal
Institute of Technology, Stockholm, Sweden (Table
2). Strains and isolates were cultivated
aerobically in nutrient broth (Oxoid) at 30°C and stored in 15%
glycerol at DNA extraction.
Bacteria were grown aerobically on an
orbital shaker (100 rpm). Cells were harvested after overnight growth
from 6 ml of a bacterial suspension by centrifugation for 10 min at
19,000 × g. The pellets were suspended in 2 ml of 0.15 M
NaCl-0.10 M EDTA-0.01 M Tris-HCl (pH 7.9) with lysozyme and RNase A
added to final concentrations of 1 mg ml
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
PCR Detection of Genes Encoding Nitrite
Reductase in Denitrifying Bacteria
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C.
TABLE 1.
Bacterial strains used in this study and results of PCR
amplification of cd1- and Cu-nir
genes and Southern blot hybridization of PCR products
TABLE 2.
Results of PCR amplification of
cd1- and Cu-nir genes in
bacterial isolates from activated sludge
1 and 50 µg
ml1, respectively. After the cells were incubated for
1 h at 37°C, 0.1 ml of a saturated solution of sodium dodecyl
sulfate (SDS) in 45% ethanol was added. After 5 min at 37°C, 0.65 ml
of 5 M NaClO4 was added. DNA was recovered in the water
phase after extraction with 3 ml of chloroform and 0.15 ml of isoamyl
alcohol. The DNA was precipitated by adding 2 volumes of ice-cold 95%
ethanol. The precipitate was washed once in 70% ice-cold ethanol, air
dried, and dissolved in 200 µl of water.
70°C for 1 min. The thawing-freezing procedure was repeated three times. The
supernatants were collected after centrifugation (19,000 × g, 10 min), and DNA originating from 3 ml of activated sludge was purified and concentrated by using an Elutip-d minicolumn (Schleicher & Schuell) attached to a 0.45-µm-pore-size cellulose acetate prefilter. DNA was concentrated by ethanol precipitation, dried, and suspended in 100 µl of water.
PCR amplification of nir genes. Primers for PCR were based on cd1- and Cu-nir genes from denitrifying bacteria and amplified nir fragments of approximately 800 and 473 bp, respectively (Fig. 1). The oligonucleotides were purchased from Pharmacia Biotech. PCR amplification of nir fragments was carried out with 50-µl reaction mixtures in 0.5-ml Eppendorf tubes containing 10 to 50 ng of template DNA, 1.25 U of Taq polymerase (Pharmacia Biotech) with the manufacturer's reaction buffer at 1.5 mM MgCl2, 10 nmol of each deoxynucleotide triphosphate, and 50 pmol of each primer. The reaction mixtures were covered with mineral oil and placed in a thermocycler (Perkin Elmer). The PCR was run with initial denaturation of the DNAs at 94°C for 3 min followed by 35 cycles of 30 s at 94°C, 1 min at 57°C, and 1 min at 73°C. The reaction was completed after 10 min at 75°C. We determined an optimal annealing temperature in the interval between 50 and 65°C.
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Nucleotide sequencing and computer analysis.
PCR products
recovered from an agarose gel and purified with GenElute Minus EtBr
Spin Columns (Supelco) were sequenced on both strands by using the
Thermo Sequenase dye terminator cycle sequencing premix kit
(Amersham Life Science) with 50 to 100 ng of template DNA. The
original PCR primers (Fig. 1) and two other oligonucleotides were
used as sequencing primers (Table 3).
An ABI PRISM 377 (Perkin-Elmer) automated DNA sequencer was used for
sequencing. DNA sequences were analyzed with the PCGene program package
(IntelliGenetics). Pairwise similarity was calculated with the NALIGN
module by the method of Myers and Miller (17), while
multiple-sequence alignments were determined with the Clustal V
software (11). Phylogenetic trees were constructed from the alignments by using the neighbor-joining method of Saitou and Nei
(22). Tree topology was evaluated by bootstrap analysis using 10,000 replicates.
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DNA probes for detection of nir genes.
DNA
probes for detection of nir genes were created by amplifying
nir fragments from P. stutzeri (ATCC 14405)
and A. faecalis (ATCC 8750) with internal primers
(Table 3). The cd1 probe was a 736-bp fragment
starting at 882 bp from the 5' end within the amplified 799-bp
region of the nir gene of P. stutzeri (ATCC
14405). The Cu probe was a 398-bp fragment within the 473-bp region
of the nir gene of A. faecalis (ATCC 8750).
After PCR amplification, the fragments were separated by agarose gel
electrophoresis, purified with GenElute Minus EtBr Spin Columns
(Supelco), and then labeled with [
-32P]CTP by random
priming using the Multiprime DNA labeling system kit (Amersham). The
labeled probes were separated from unincorporated nucleotides on a
Sepharose CL-6B (Pharmacia Biotech) spin column and used immediately.
Southern blotting and hybridization conditions.
The
PCR-amplified fragments were transferred to Hybond-N+ nylon
membranes (Amersham) with 20× SSC (3 mol of NaCl liter1
and 0.3 mol of Na citrate liter1) by vacuum blotting
(VacuGene XL; Pharmacia) and cross-linked by UV light in a UV oven
(Hoefer Pharmacia Biotech). The blots were prehybridized at 45°C for
2 h in tubes containing 50 ml of a hybridization solution
containing 6× SSC, 0.5% SDS, and 3× Denhardt's reagent (0.6 g each
of Ficoll, polyvinylpyrrolidone, and bovine serum albumin
liter1). The blots were hybridized for 17 h at
45°C in 15 ml of hybridization solution with the respective,
previously heat-denatured probe (4 × 105 cpm
ml1). After hybridization, the filters were washed twice
for 15 min each time in 2× SSC-0.1% SDS at room temperature and then
twice for 15 min each time in 0.1× SSC-0.1% SDS at 45, 50, 55, 60, and 65°C, respectively, in consecutive steps. The hybridization
signals were visualized on a PhosphorImager (Molecular Dynamics) after each washing step.
Nucleotide sequence accession numbers. The partial nir gene nucleotide sequences of A. faecalis ATCC 8750, Achromobacter cycloclastes ATCC 21921, Paracoccus denitrificans Pd1222, Ralstonia eutropha CCUG 13724, P. aeruginosa CCUG 241, P. aeruginosa Mi11, and P. fluorescens Mi32 have been submitted to GenBank under accession no. AF114786 to AF114792.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Selection of PCR primers and optimization of annealing temperature. We created PCR primers to amplify nir fragments coding for cd1- and Cu-Nir (Fig. 1). To do this, we used consensus regions in sequences for the structural genes encoding Nir. These sequences were retrieved from the GenBank, EMBL, and DDBJ databases. The primers were evaluated by searching the databases with the NCBI sequence similarity search tool BLAST. Single primers had similarity to other sequences, but for the pairwise primers, BLAST predicted only genes encoding Nir. The cd1 primers were designed to amplify a 778 to 799-bp region of the nir gene in the six published sequences, i.e., P. stutzeri ATCC 14405 (13; X56813), P. stutzeri JM300 (26; M80653), Alcaligenes eutrophus H16 (21; X91394), P. aeruginosa NCTC 6750 (25; X16452), P. denitrificans Pd1222 (6; U05002), and P. denitrificans LMD 92.63 (U75413). Likewise, the Cu primers amplified a 473-bp region in seven Cu-nir sequences, Achromobacter cycloclastes (Z48635), A. faecalis S-6 (18; D13155), Pseudomonas sp. strain G-179 (31; M97294), Rhodobacter sphaeroides ATCC 17025 (28; U62291), Alcaligenes xylosoxidans NCIB 11015 (AF051831), Bradyrhizobium japonicum USDA 110 (AJ002516), and Rhizobium "hedysari" HCNT1 (27; U65658). The nucleotide sequence of the gene encoding Cu-Nir from P. aureofaciens ATCC 13985 (10; Z21945) did not share consensus regions of suitable sizes with the other seven Cu-nir sequences and was excluded when we designed the Cu primers. The Cu primers have 100% homology with the Cu-nir sequence of R. "hedysari" HCNT1, but nitrite reduction is not coupled to energy conservation in this strain (27). All four PCR primers include wobbling at one to three positions to cover some of the variation found in the published sequences.
Annealing temperatures in the interval between 50 and 65°C were evaluated, and the optimum was determined to be 57°C. At temperatures below 57°C, nonspecific binding of the primers resulted in a noticeable number of amplification products from all strains. Although fewer bands were observed at higher temperatures, some of the fragments of the expected size were lost. We decided to allow some nonspecific binding in order to obtain as many different fragments of the expected size as possible. Nevertheless, it was not possible to amplify the cd1-nir fragment in A. eutrophus ATCC 17699 at an annealing temperature of 57°C. The 800-bp fragment was only visible when the annealing temperature was 50°C.Detection of cd1- and Cu-nir genes in different strains and sewage sludge. The two sets of PCR primers for cd1-nir and Cu-nir genes amplified nir genes in several of the defined strains (Table 1; Fig. 2), as well as in bacterial isolates from activated sludge (Table 2). The primers did not amplify genes of nondenitrifying strains, although cd1-nir from isolate 89, which cannot reduce nitrite, was amplified. The cd1 primers were not able to amplify 800-bp nir fragments from the two strains of P. denitrificans, even though the gene from P. denitrificans Pd1222 has been sequenced and includes the primer sites (6). However, the Cu-nir primers amplified a 473-bp fragment from P. denitrificans Pd1222.
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Similarity of amplified nir fragments. The DNA probes hybridized to almost every PCR product of the expected size, including the fragments amplified from the sludge samples (Fig. 2). The cd1-nir probe derived from P. stutzeri ATCC 14405 hybridized to all 800-bp fragments but one from P. fluorescens ATCC 33512. A second band of approximately 450 bp also hybridized to the probe. The hybridization signal from the smaller fragment was even stronger in the sludge samples. The Cu-nir probe derived from A. faecalis ATCC 8750 hybridized to all of the amplified 473-bp fragments. The probe hybridized to a 473-bp fragment in A. faecalis ATCC 19018 that was not detectable on an ethidium bromide-stained agarose gel. This probe also detected a second, smaller fragment of 400 bp in all of the strains that displayed the 473-bp fragment. This fragment was clearly visible in the control, A. faecalis ATCC 8750, with a short exposure time. The strong signals visualized after washing at 45°C were still detectable after washing at 65°C, but they were not as distinct as the hybridization signals seen in the controls.
A subset of the PCR fragments of the expected sizes (473 and 800 bp) was sequenced. The sequences were similar to those of other cd1- and Cu-nir genes, demonstrating that the primers amplified the correct genes (Fig. 3). The degree of pairwise similarity was the same within the cd1- and Cu-nir gene fragments, respectively (data not shown). The cd1-nir fragments divided into two major clusters (Fig. 3). One cluster contained only the two published sequences from P. stutzeri, and the other cluster included all of the other sequences. The three nir fragments from P. aeruginosa strains were closely related, and the new sequence from R. eutropha CCUG 13724, formerly known as A. eutrophus, was most similar to the A. eutrophus H16 sequence. The Cu-nir fragments were distributed in two heterogeneous clusters. The two strains of A. cycloclastes were identical, with 100% sequence similarity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our strategy was to design PCR primers general enough to amplify denitrifying bacteria regardless of their genus or species affiliation. General PCR primers permit the study of denitrifying bacteria without prior cultivation. Denitrifying bacteria have at least Nir in common, and we therefore based the primers on the structural nir genes. Because there are two structurally different reductases, no conserved regions for both genes were found. However, the use of two sets of PCR primers to distinguish between cd1 heme-type Nir and copper-dependent Nir will enhance resolution when analyzing denitrifying populations in environmental samples.
Both sets of primers were able to amplify the correct fragment of the nir gene in different strains and in a total DNA extract from sewage sludge as well. In some cases, such as the two P. denitrificans strains and A. eutrophus, the primers did not amplify the cd1-nir gene under the conditions used in this study, although cd1-Nir exists in these strains. This might be explained by a tendency for the target DNA to form secondary structures, which makes it difficult for the primers to anneal. Amplification of a Cu-nir fragment in P. denitrificans Pd1222 surprised us. To our knowledge, the existence of Cu-nir in P. denitrificans Pd1222 has not been reported before. This strain has been shown to carry a nir gene of the cd1 type (6). A Cu-nir fragment of the expected size in this strain hybridized with a strong signal to the DNA probe derived from A. faecalis ATCC 8750, and the sequence of that fragment was similar to the other sequences of copper-containing nir genes. The second band visualized in all Southern blot hybridizations could be a mutated copy of the cd1- or Cu-nir gene, but there is no published evidence that several copies exist.
Researchers have used nir genes to construct DNA probes that identify denitrifying bacteria in general. The detection of cd1-nir genes has been largely based on sequences from different P. stutzeri strains. One DNA probe, constructed from P. stutzeri JM300 for the detection of heme-type denitrifiers, identified a great number of defined denitrifying strains and found nir genes in bioreactors, aquifer microcosms, and toluene-degrading enrichment cultures (9, 26). Nevertheless, this particular probe did not hybridize to DNA extracted from soil or wetland sediments. The distribution of denitrifying bacteria in soil was investigated by Linne von Berg and Bothe (15). The cd1-nir probe used in that study was fairly large and contained additional sequences not encoding the structural gene, which caused nonspecific hybridization. Marine denitrifying isolates were detected with cd1-nir probes derived from P. stutzeri ATCC 14405 (29). Ye et al. (31) constructed a Cu-nir probe from Pseudomonas sp. which was then used to detect Cu-nir in denitrifying strains and enrichment cultures (9). DNA probes based on genes encoding cd1-Nir in P. aeruginosa and Cu-Nir in A. xylosoxidans were used to screen Hyphomicrobium isolates to separate denitrifiers from nondenitrifiers (7, 14).
Ward (29) suggested that PCR amplification is a more specific test than DNA hybridization, since PCR primers are dependent on a high degree of similarity in two separate 15 to 25-bp regions. The regions we selected for use as primer sites for the Cu-nir genes appear well conserved. The amplified fragment is exactly 473 bp in the available sequences and the sequenced fragments presented in this report, which further indicates that these regions are conserved in the Cu-nir genes. Ward (29) concluded that the functional gene encoding cd1 heme-type Nir was variable enough to support species-specific identification with DNA probes or primers and that a general probe for denitrifiers requires a different approach. Her conclusions were based on information from three sequences in two different species. The primer sites used by Ward et al. (30) are less conserved than the regions suggested in this report, although we agree that true broad-range primers for amplification of cd1-nir are more difficult to construct than Cu-nir primers.
By using an annealing temperature of 57°C, we allowed some nonspecific binding of the primers. This was done to obtain as many different nir fragments of the expected sizes as possible. If the next step is to separate and analyze the PCR products, it is important not to lose potential nir genes in crude DNA extracts. Although the primers amplified the correct gene segment in many different strains, the primers were constructed on the basis of nir sequences in culturable denitrifying bacteria. Since less than 1% of all bacteria are considered culturable (1), the amplified cd1- and Cu-nir fragments may not reflect the dominant denitrifiers present in the environment. Even though current research results are still fragmentary, evidence from rRNA gene sequencing of clone libraries generated from DNA extracted from soil indicates the existence of great unexplored bacterial diversity (3).
It is uncertain whether nir gene diversity is related to the phylogenetic diversity of denitrifying bacteria. Ohkubo et al. (20) showed that the grouping in a phylogenetic tree based on 5S rRNA sequences was almost compatible with the type of nitrite reductase in denitrifying bacteria. Otherwise, the relationship among denitrifying isolates derived from hybridization to a DNA probe for a nir gene was not correlated with restriction fragment length polymorphism patterns of ribosomal genes (29). Even if the diversity of the structural gene is not correlated with phylogenetic relationships, the nir gene still reveals the diversity of the gene encoding the key enzyme in denitrifying bacteria.
Primers capable of amplifying genes from all denitrifiers may prove to be impossible to design. Perhaps several pairs of primers will better detect more denitrifiers, in particular, those carrying cd1-Nir. New molecular approaches, such as denaturing gradient gel electrophoresis of PCR-amplified DNA fragments, can be used to examine the ecological importance of denitrifiers, as well as detect and identify new denitrifying bacteria. It might also be possible to quantify denitrifying bacteria with a competitive PCR (8). The use of the primers reported here will further our understanding of denitrifier complexity and diversity in the environment.
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ADDENDUM |
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After submission of the manuscript, a similar report by Braker et al. (3a) was published. We acknowledge their work.
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ACKNOWLEDGMENTS |
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Financial support for this work was partially provided by the National Board for Industrial and Technical Development in Sweden, the Swedish Environmental Protection Agency, and the Swedish Council for Forestry and Agricultural Research.
We thank Lars Frykberg for constructive discussions. Thanks are also extended to Martin Nilsson for assistance with vacuum blotting, as well as to Marianne Boysen, Nora Ausmees, and Karin Jacobsson for help with the sequencing.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden. Phone: 46 18 67 32 88. Fax: 46 18 67 33 92. E-mail: Sara.Hallin{at}mikrob.slu.se.
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Amann, R. I.,
W. Ludwig, and K. H. Schleifer.
1995.
Phylogenetic identification and in situ detection of individual microbial cells without cultivation.
Microbiol. Rev.
59:143-169 |
| 2. | Berks, B. C., S. J. Ferguson, J. W. B. Moir, and D. J. Richardson. 1995. Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim. Biophys. Acta 1232:97-173[Medline]. |
| 3. | Borneman, J., P. W. Skroch, K. M. O'Sullivan, J. A. Palus, N. G. Rumjanek, J. L. Jansen, J. Nienhuis, and E. W. Triplett. 1996. Molecular microbial diversity of an agricultural soil in Wisconsin. Appl. Environ. Microbiol. 62:1935-1943[Abstract]. |
| 3a. |
Braker, G.,
A. Fesefeldt, and K.-P. Witzel.
1998.
Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.
Appl. Environ. Microbiol.
64:3769-3775 |
| 4. | Brittain, T., R. Blackmore, C. Greenwood, and A. J. Thomson. 1992. Bacterial nitrite-reducing enzymes. Eur. J. Biochem. 209:793-802[Medline]. |
| 5. |
Coyne, M. S.,
A. Arunakumari,
B. A. Averill, and J. M. Tiedje.
1989.
Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria.
Appl. Environ. Microbiol.
55:2924-2931 |
| 6. | de Boer, A. P. N., W. N. M. Reijnders, J. G. Kuenen, A. H. Stouthamer, and R. J. M. van Spanning. 1994. Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans. Antonie van Leeuwenhoek 66:111-127[Medline]. |
| 7. | Fesefeldt, A., K. Kloos, H. Bothe, H. Lemmer, and C. G. Gliesche. 1998. Distribution of denitrification and nitrogen fixation genes in Hyphomicrobium spp. and other budding bacteria. Can. J. Microbiol. 44:181-186. |
| 8. | Förster, E. 1994. Rapid generation of internal primers for competitive PCR by low stringency primer annealing. BioTechniques 16:1006-1008[Medline]. |
| 9. |
Fries, M. R.,
J. Zhou,
J. Chee-Sandford, and J. M. Tiedje.
1994.
Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats.
Appl. Environ. Microbiol.
60:2802-2810 |
| 10. |
Glockner, A. B.,
A. Jüngst, and W. G. Zumft.
1993.
Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS) of Pseudomonas stutzeri.
Arch. Microbiol.
160:18-26[Medline].
|
| 11. |
Higgins, D. G.,
A. J. Bleasby, and R. Fuchs.
1991.
CLUSTAL V: improved software for multiple sequence alignment.
Comp. Appl. Biosci.
8:189-191 |
| 12. | Hochstein, L. I., and G. A. Tomlinson. 1988. The enzymes associated with denitrification. Annu. Rev. Microbiol. 42:231-261[Medline]. |
| 13. | Jüngst, A., S. Wakabayashi, H. Matsubara, and W. G. Zumft. 1991. The nirSTBM region coding for cytochrome cd1-dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di-, and tetraheme proteins. FEBS Lett. 279:205-209[Medline]. |
| 14. | Kloos, K., A. Fesefeldt, C. G. Gliesche, and H. Bothe. 1995. DNA-probing indicates the occurrence of denitrification and nitrogen fixation genes in Hypohomicrobium. Distribution of denitrifying and nitrogen fixing isolates of Hypohomicrobium in a sewage treatment plant. FEMS Microbiol. Ecol. 18:205-213. |
| 15. | Linne von Berg, K.-H., and H. Bothe. 1992. The distribution of denitrifying bacteria in soils monitored by DNA-probing. FEMS Microbiol. Ecol. 86:331-340. |
| 16. | Magnusson, G., H. Edin, and G. Dahlhammar. 1998. Characterisation of efficient denitrifying bacterial strains isolated from activated sludge by 16S-rDNA analysis. Water Sci. Technol. 38:63-68. |
| 17. |
Myers, E. W., and W. Miller.
1988.
Optimal alignments in linear space.
Comp. Appl. Biosci.
4:11-17 |
| 18. |
Nishiyama, M.,
J. Suzuki,
M. Kukimoto,
T. Ohnuki,
S. Horinouchi, and T. Beppu.
1993.
Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli.
J. Gen. Microbiol.
139:725-733 |
| 19. | Novick, R. P. 1967. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33:155-156[Medline]. |
| 20. |
Ohkubo, S.,
H. Iwasaki,
H. Hori, and S. Osawa.
1986.
Evolutionary relationship of denitrifying bacteria as deduced from 5S rRNA sequences.
J. Biochem.
100:1261-1267 |
| 21. | Rees, E., R. A. Siddiqui, F. Köster, B. Schneider, and B. Friedrich. 1997. Structural gene (nirS) for the cytochrome cd1 nitrite reductase of Alcaligenes eutrophus H16. Appl. Environ. Microbiol. 63:800-802[Abstract]. |
| 22. | Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract]. |
| 23. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 24. | Sann, R., S. Kostka, and B. Friedrich. 1994. A cytochrome cd1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus H16. Arch. Microbiol. 161:453-459[Medline]. |
| 25. | Silvestrini, M. C., C. L. Galeotti, M. Gervais, E. Schininà, D. Barra, F. Bossa, and M. Brunori. 1989. Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein. FEBS Lett. 254:33-38[Medline]. |
| 26. |
Smith, G. B., and J. M. Tiedje.
1992.
Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.
Appl. Environ. Microbiol.
58:376-384 |
| 27. | Toffanin, A., Q. Wu, M. Maskus, S. Caselia, H. D. Abruna, and J. P. Shapleigh. 1996. Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the nondenitrifying Rhizobium "hedysari" strain HCNT1. Appl. Environ. Microbiol. 62:4019-4025[Abstract]. |
| 28. |
Tosques, I. E.,
A. V. Kwiatkowski,
J. Shi, and J. P. Shapleigh.
1997.
Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3.
J. Bacteriol.
179:1090-1095 |
| 29. | Ward, B. B. 1995. Diversity of culturable denitrifying bacteria. Arch. Microbiol. 163:167-175. |
| 30. |
Ward, B. B.,
A. R. Cockcroft, and K. A. Kilpatrick.
1993.
Antibody and DNA probes for detection of nitrite reductase in seawater.
J. Gen. Microbiol.
139:2285-2293 |
| 31. |
Ye, R. W.,
M. R. Fries,
S. G. Bezborodnikov,
B. A. Averill, and J. M. Tiedje.
1993.
Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers.
Appl. Environ. Microbiol.
59:250-254 |
| 32. | Zumft, W. G. 1992. The denitrifying procaryotes, p. 554-582. In A. Balows, H. G. Trüper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The procaryotes. Springer Verlag, New York, N.Y. |
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