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Journal of Clinical Microbiology, July 2003, p. 3252-3259, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3252-3259.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis

Marion Stermann, Antje Bohrssen, Catharina Diephaus, Silvia Maass, and Franz-Christoph Bange*

Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, 30625 Hannover, Germany

Received 3 February 2003/ Returned for modification 17 March 2003/ Accepted 1 April 2003


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ABSTRACT
 
Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply "M. bovis"]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide -215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG.


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INTRODUCTION
 
Mycobacterium tuberculosis, the major pathogen of human tuberculosis, predominantly affects the respiratory tract, whereas M. bovis, the major pathogen of tuberculosis in cattle, is typically found in extrapulmonary tuberculosis (5, 32). Attenuated M. bovis BCG, the only currently available vaccine against tuberculosis, has been administered to more than 3 billion people worldwide (17). It may be isolated from immunocompromised individuals, who might develop disseminated disease with the vaccine strain after vaccination with M. bovis BCG. M. africanum and M. microti are rarely encountered members of the M. tuberculosis complex (MTBC) (11, 18, 22). M. africanum causes human tuberculosis in certain regions of tropical Africa. M. microti causes naturally acquired generalized tuberculosis in voles and produces local lesions in guinea pigs, rabbits, and calves and has been described very occasionally as a cause of infection in human immunodeficiency virus-positive patients.

Nitrate reductase activity is a widely used phenotypic trait to differentiate between M. tuberculosis, which rapidly accumulates nitrite from nitrate, and other members of the MTBC (18). In other pathogenic and environmental bacteria, at least three different nitrate reductases have been found, one of which is the respiratory nitrate reductase encoded by narGHJI (19). The membrane-bound complex consists of NarG, -H, and -I, with NarG being the catalytic subunit, whereas NarJ is required for the assembly of the enzyme. Expression of narGHJI is typically induced under anaerobic conditions (20). Previous studies on mycobacterial nitrate reduction were limited to its role in classification and identification of the genus Mycobacterium (2, 6, 28, 29). However, recent reports revived interest in a possible role for enzymes involved in nitrate metabolism during infection with M. tuberculosis or M. bovis BCG (4, 12, 30). We provided evidence that M. bovis BCG weakly accumulates nitrite from nitrate under strictly anaerobic conditions, that this activity is mediated by narGHJI, and that a mutant of M. bovis BCG with a partial deletion of the narG gene was attenuated in mice, linking anaerobic nitrate reduction to mycobacterial pathogenesis (7, 31).

In the present study, we generated targeted deletion of narG to compare the role of narGHJI in nitrite accumulation by M. tuberculosis, M. bovis (non-BCG [referred to here as simply "M. bovis"]), and M. bovis BCG. Analysis of the promoter region of narGHJI from either species revealed a single nucleotide polymorphism that separated M. tuberculosis from the bovine mycobacteria. We tested a variety of different strains within the MTBC by using LightCycler technology and showed that this single nucleotide polymorphism was specific for M. tuberculosis, thus allowing rapid identification of M. tuberculosis. For further differentiation within the MTBC based on LightCycler technology, we adapted analysis of the oxyR polymorphism, which is specific for the bovine mycobacteria, and the region of differences 1 (RD1) polymorphism, which is specific for M. bovis BCG, to a LightCycler format.


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MATERIALS AND METHODS
 
Strains and cultures. To test nitrate reductase activity and the generation of narG mutants, we used M. tuberculosis H37Rv ATCC 25618, a clinical strain of M. bovis, and M. bovis BCG Pasteur ATCC 35734. All strains were cultured in 7H9 broth or on 7H10 plates (Difco Laboratories, Inc., Detroit, Mich.) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADS (0.5% bovine albumin fraction V, 0.2% glucose, 140 mM NaCl) unless indicated otherwise.

For LightCycler-based analysis, strains were grown in the MGIT 960 automated culture system (Becton Dickinson, Sparks, Md.). A pellet from 0.5 ml of a MGIT culture was resuspended in 10 mM Tris (pH 8.0) and 1 mM EDTA, supplemented with glass beads (106 µm in diameter), and vortexed for 2 min, and the supernatant was used for the LightCycler reaction. As indicated in Table 1, 62 strains were tested. Eight strains were obtained from the American Tissue Culture Collection (ATCC). A total of 54 strains were recovered from clinical sources and either identified in our laboratory between 2000 and 2002 (n = 41, including 33 strains of M. tuberculosis, 1 strain of M. africanum, 3 strains of M. bovis, and 4 strains of M. bovis BCG), or they were provided by the National Reference Center for Mycobacteria in Borstel, Germany (n = 4, including 2 strains of M. africanum, and 2 strains of M. microti) or by the Federal Research Centre for Virus Diseases of Animals in Jena, Germany (n = 9, all of which were M. bovis strains). Clinical isolates from the Medical School of Hannover were identified by partial sequencing of 16S rRNA gene (13), phenotypic testing of niacin/nitrate (18), and analysis of RD1 as described by Talbot et al. (27). Strains rendering doubtful results were sent to the National Reference Center for Mycobacteria in Borstel for further testing. Patients from whom mycobacteria were isolated at the Medical School of Hannover originated from Germany, from other European countries, and from Africa and Asia (8, 9). Human and animal isolates of M. bovis strains provided by the Federal Research Centre for Virus Diseases of Animals in Jena were recovered from sources in Germany and other European countries. ATCC strains originated from sources worldwide.


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  		TABLE 1. Melting temperatures for Mycobacteriumstrains in this study

Accumulation of nitrite under anaerobic conditions. The presence of nitrite can be demonstrated by naphthylamide and sulfanilic acid reagents, which form a red diazonium dye when they react with nitrite (18). Prior to testing for accumulation of nitrite, bacteria were grown under aerobic conditions in 7H9 broth to an optical density at 600 nm of between 0.7 and 1.0. For the experiment shown in Fig. 1, a dense culture was washed three times with MB medium without added nitrate. MB medium is a minimal medium based on various salts, buffers, and trace elements. Fully supplemented MB medium contains albumin, dextrose, and saline (i.e., ADS) and also nitrate. The addition of Tween 80 is necessary to prevent mycobacteria from clumping in liquid culture. The exact formula of 1 liter fully supplemented MB medium is as follows: 1 g of KH2PO4, 2.5 g of Na2HPO4, 2.0 g of K2SO4,0.5 mM MgCl2, 0.5 mM CaCl2, 0.2% glycerol, 0.05% Tween 80, 10% ADS, 10 mM nitrate, and 2 ml of trace elements. One liter of trace elements contained 40 mg of ZnCl2, 200 mg of FeCl3 · 6H2O, 10 mg of CuCl2 · 4H2O, 10 mg of MnCl2 · 4H2O, 10 mg of Na2B4O7 · 10H2O, and 10 mg of (NH4)6Mo7O24 · 4H2O. After the culture was washed three times, the pellet was resuspended in fully supplemented MB medium reaching an optical density at 600 nm of ca. 0.2 corresponding to ca. 107 bacilli/ml. An anaerobic environment was achieved with the AnaeroGen anaerobic system (Oxoid, Ltd., Bastingstoke, Hamphsire, England) in a standard anaerobic jar. As recommended by the manufacturer, an indicator strip was used to confirm anaerobic conditions. At the indicated time points, sulfanilic acid and N,N-dimethyl-1-naphthylamine (both reagents were taken from the API system [bioMerieux, Marcy l'Étoile, France]) were added to aliquots of culture, followed by centrifugation at 15,000 x g for 15 min at room temperature. The absorbency of supernatant was measured at 530 nm and compared to a known standard of nitrite (ranging from 10-6 to 10-3 mol/liter). Testing was always performed in duplicates.



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  		FIG. 1. Anaerobic nitrate reductase activity of wild-type and narG mutant strains of M. tuberculosis, M. bovis, and M. bovis BCG. A total of 107 wild-type (closed symbols) and narG mutant (open symbols) M. tuberculosis (squares), M. bovis (triangles), and M. bovis BCG (circles) bacteria/ml were cultured in MB medium supplemented with 10 mM nitrate, and aliquots were tested for production of nitrite after 1, 2, 3, 5, and 7 days.

Diagnostic nitrate reductase activity. According to the recommendations of the American Society for Microbiology, the diagnostic nitrate reductase activity test must be performed with actively growing cultures that are inoculated directly into phosphate buffer supplemented with nitrate, followed by incubation for 2 h at 37°C. Culture prior to testing should be done on solid medium (15, 18). Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco) supplemented with 0.2% glycerol and 10% ADS. For the experiment shown in Fig. 3, bacilli were inoculated into phosphate buffer supplemented with 10 mM nitrate. After 2 h of incubation at 37°C, naphthylamide and sulfanilic acid reagents were added, and a photograph was obtained.



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  		FIG. 3. Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis. Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on 7H10 agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.

Generation of narG mutants in M. tuberculosis, M. bovis, and M. bovis BCG. The vector pYUB657 does not replicate or integrate in mycobacteria and contains a hygromycin resistance cassette for positive selection and sacB for negative selection, thus allowing generation of unmarked mutants by targeting gene deletion (24). To generate narG mutants of M. bovis and M. bovis BCG, a 736-bp ClaI fragment within the narG gene was deleted. To generate a narG mutant of M. tuberculosis, a 684-bp BsmI/MscI fragment within the narG gene was deleted. The mutated fragments were cloned into the PacI site of pYUB657. M. tuberculosis, M. bovis, and M. bovis BCG were transformed and screened for clones resistant to hygromycin. We performed PCR to screen for the presence of the mutated narG gene with ACT GGT TCC ACA GCG ATG as a forward primer, targeting a region just upstream of the first ClaI site and the MscI site, respectively. AGG GAT GGA CGG TAT ATC was used as a reverse primers, targeting a region just downstream of the second ClaI site and the BsmI site, respectively. A 438-bp fragment was amplified in the presence of the mutated narG in M. tuberculosis, and a 488 bp in the presence of the mutated narG fragment in M. bovis and M. bovis BCG. A 1,122-bp fragment was amplified in the presence of wild-type narG in all three species. Cointegration of the plasmid carrying the mutated narG gene next to the narG wild-type locus within the mycobacterial genome was confirmed by using Southern blot analysis. Genomic DNAs from potential clones were digested with BssHII, and a DNA probe, targeting a 527-bp region downstream from the narG deletion, was constructed by using TCG GAC TTT GAC GCA TTC GC as a forward primer and GTA TCG GCG TAG GTG ATG CG as a reverse primer. A 4,723-bp fragment indicated wild-type M. tuberculosis, M. bovis, or M. bovis BCG, whereas a 17,458-bp fragment indicated cointegration in M. tuberculosis and a 17,406-bp fragment indicated cointegration in M. bovis and M. bovis BCG.

By using hygromycin for selection and sucrose for counterselection as described previously (24), clones were chosen in which the wild-type narG was replaced by the mutated narG. Chromosomal DNA was digested with SmaI restriction enzyme, since the deleted fragment of the narG site contained a SmaI site. Wild-type M. tuberculosis, M. bovis, and M. bovis BCG showed a 1,114-bp fragment. The narG mutant of M. tuberculosis carried a 2,302-bp fragment, and the narG mutants of M. bovis and M. bovis BCG contained a 2,250-bp fragment. Eventually, clones were chosen and named MS1 for the narG mutant of M. tuberculosis, SM118 for the narG mutant of M. bovis, and SR106 for the narG mutant of M. bovis BCG.

Standard LightCycler protocol. Using LightCycler model II (Roche, Mannheim, Germany), a commercially available ready-to-use hot-start reaction mixture (LightCycler FastStart DNA master hybridization probes; catalog no. 239272; Roche) containing FastStart Taq polymerase, reaction buffer, deoxynucleoside triphosphates, and 1 mM MgCl2 was supplemented with 2 mM MgCl2. Primers were supplied with 18 pmol (1.1 µM final concentration) per reaction and DNA probes with 2 pmol (100 nM final concentration). The amplification program began with denaturation at 95°C for 10 min, followed by 50 cycles of 95°C for 3 s (denaturation), a temperature range of 62 to 68°C for 2 s ("touch-down" annealing), and 72°C for 40 s (extension). For the first five cycles, annealing was performed at 68°C (step delay) and then reduced to 62°C with 1°C per cycle (step size). The temperature transition rate for all cycling steps was 20°C per s. The amplification program was followed by a melting program of 95°C for 30 s (denaturation), 38°C for 30 s (annealing), and then 38 to 80°C at a transition rate of 0.2°C/s with continuing monitoring of fluorescence. The 3.5.3 version of LightCycler profile software automatically adjusted the gain of the F2/F3 channel photometric detector.

Detection of T/C transition within the promoter of narGHJI. For DNA amplification, LC66 (AAC CGA CGG TGT GGT TGA C) was used as a forward primer, and LC67 (ATC TCG ATG GAT GGG CGT C) was used as a reverse primer. The polymorphic nucleotide was targeted by using LC63 (GTC GCC ACG CGT CCA GAA AAC C; antisense) as an anchor probe and LC64 (CGT GAT CGC TAC GGG CAT; antisense, where the underlined nucleotide is the polymorphic nucleotide within the promoter of narGHJI as a sensor probe. The anchor probe LC63 was labeled with fluorescein as a donor for fluorescence resonance energy transfer (FRET), and the sensor probe LC64 was labeled with LightCycler Red 640 as an acceptor for FRET.

Detection of A/G transition of oxyR. For DNA amplification, LC90 (CGG GTG CCG CTG ACC GCG) was used as a forward primer, and LC91 (CCA GCC GGC TTC GCG TGG) was used as a reverse primer. The polymorphic nucleotide was targeted by using LC92 (GCC GGT CAC GCA CTG CAC GAC G; antisense) as an anchor probe and LC94 (GGC CAG CCA CAC CGC; antisense) as a sensor probe. The anchor probe LC92 was labeled with fluorescein as a donor for FRET, and the sensor probe LC94 was labeled with LightCycler Red 705 as an acceptor for FRET.

Detection of RD 1 region. The 3' end of RD1 was detected by using LC73 (CTA GAC GAG GCC GCT CAA G) and LC74 (AAG ACG TGG CCT TTC TGC TG) as forward primers and LC75 (ACG GGT TAC TGC GAA TAC CG) as a reverse primer. The amplification product was targeted by using LC78 (GCT ATG CCA GAC AGA TGC TGG ATC; antisense) as an anchor probe and LC79 (GGC GGC TGG GTG GAA TGC C; antisense) as a sensor probe. The anchor probe LC78 was labeled with fluorescein as a donor for FRET, and the sensor probe LC79 was labeled with LightCycler Red 640 as an acceptor for FRET.


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RESULTS
 
Anaerobic nitrate reductase activity of wild-type and narG mutant strains of M. tuberculosis, M. bovis, and M. bovis BCG. In other bacteria narGHJI encodes a nitrate reductase that is typically upregulated in the absence of oxygen. We tested nitrite accumulation in anaerobic cultures containing M. tuberculosis and 10 mM nitrate. Peaking at ca. 1 mmol/liter after 2 days, the concentration of nitrite slowly decreased over the next 5 days (Fig. 1). In contrast to M. tuberculosis, the amount of accumulated nitrite in cultures inoculated with M. bovis was considerably lower, and M. bovis BCG produced even less nitrite, showing minimal accumulation over 7 days (Fig. 1). Next, narG mutants of M. tuberculosis, M. bovis, and M. bovis BCG were generated. Since a mutant strain without an antibiotic resistance marker is clearly preferable, we used a suicide plasmid with a counterselectable marker system as described by Pavelka and Jacobs (24) to generate an unmarked deletion of narG in all three species. Disruption of narG deleted a SmaI restriction site within the narG gene. Therefore, Southern hybridization of SmaI-digested DNA showed a larger fragment for the mutant strains compared to the wild-type strains (Fig. 2). Nitrite accumulation activity of the three narG mutant strains was compared under anaerobic conditions. All strains were cultured in fully supplemented MB medium containing 10 mM nitrate, and aliquots were analyzed at various time points. The narG mutants of M. tuberculosis, M. bovis, and M. bovis BCG were unable to accumulate nitrite (Fig. 1). Thus, in all three species nitrite accumulation under anaerobic conditions was exclusively mediated by narGHJI.



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  		FIG. 2. Genomic locus of M. tuberculosis encompassing narGHJI and Southern blot analysis of narG mutants of M. tuberculosis, M. bovis, and M. bovis BCG. An EcoRV fragment of M. tuberculosis contains the entire narGHJI gene cluster, including 0.9-kb prior to the translation start of narG. MscI/BsmI restriction sites for the construction of the narG deletion in M. tuberculosis, and ClaI restriction sites for construction of narG deletions in M. bovis and M. bovis BCG are depicted. The positions of the deleted fragments are shown ({Delta}M. tuberculosis and {Delta}M. bovis, BCG). The positions of the forward primer and the reverse primer that were used for screening narG mutants are depicted as arrows. A Southern blot analysis showing wild-type (lanes 1 to 3) and mutant (lanes 4 to 6) strains is shown below. Genomic DNAs from M. tuberculosis (lane 1), M. bovis (lane 2), M. bovis BCG (lane 3), {Delta}narG M. tuberculosis (lane 4), {Delta}narG M. bovis (lane 5), and {Delta}narG M. bovis BCG (lane 6) were digested with SmaI. The position of the DNA probe for Southern blot analysis is shown. The loss of a SmaI site due to deletion within the narG gene results in an "upshift" of the specific band in all three mutant strains.

Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis. Nitrate reductase activity has been used for many years in mycobacterial diagnostics. In the experiments described above, cultures were exposed to nitrate for 7 days; during this time continuous nitrite accumulation was measured at various time points. In diagnostic microbiology, however, mycobacterial nitrite accumulation is typically tested after just 2 h of incubation of bacilli in buffer containing nitrate (15, 18). Thus, diagnostic nitrate reductase activity is really more accurately described as the rapid accumulation of nitrite by the bacilli within 2 h. To test for diagnostic nitrate reductase activity, M. tuberculosis and the narG mutant of M. tuberculosis were inoculated from solid medium into phosphate buffer, and production of nitrite was measured after incubation at 37°C for 2 h. Nitrite accumulation by M. tuberculosis became visible and correlated with the amount of inoculated bacteria, whereas the narG mutant of M. tuberculosis showed no discernible production of nitrite (Fig. 3). As expected, neither M. bovis nor M. bovis BCG accumulated detectable amounts of nitrite from nitrate under these conditions within 2 h (data not shown). These results clearly indicate that the molecular mechanism of the nitrate reductase activity test, which is used in diagnostic microbiology for identification of M. tuberculosis, is also based on narGHJI.

Identification of M. tuberculosis by analyzing T/C polymorphism within the promoter of narGHJI by means of LightCycler technology. M. tuberculosis, M. bovis, and M. bovis BCG all possess the narGHJI operon. However, NarGHJI-dependent accumulation of nitrite appeared to be strikingly different. One explanation for this could be a variation within the promoter of narGHJI. Therefore, we sequenced a 500-bp region preceding the translation start of narG from M. tuberculosis, M. bovis, and M. bovis BCG. At position -215 prior to the GTG-start codon, a thymine residue was detected in M. tuberculosis, and a cytosine residue was found in M. bovis and M. bovis BCG. Next, we analyzed the nucleotide polymorphism of the narG promoter at position -215 in 35 strains of M. tuberculosis, 13 strains of M. bovis, and 9 strains of M. bovis BCG, and we also included 2 strains of M. microti and 3 strains of M. africanum. LightCycler technology provides rapid DNA amplification and sequence-specific detection of DNA using oligonucleotides labeled with two different fluorescence dyes. Typically, so-called sensor probes, which cover a polymorphic region, allow detection of as little as one nucleotide difference by melting off at a lower temperature. We constructed an oligonucleotide targeting position -215 with a perfect match to M. bovis and M. bovis BCG. Amplification of a 155-bp fragment of the narGHJI promoter region encompassing position -215 was followed by melting peak analysis. The probe dissociated from the target at 63°C when genomic DNA from M. bovis, M. bovis BCG, M. africanum, or M. microti was used as a template, indicating a perfect match between the target DNA and the hybridization probe. When M. tuberculosis was used as a template, the probe dissociated at 57°C, indicating a mismatch between the target DNA and the hybridization probe (Fig. 4 and Table 1). These results suggested that the thymine residue at position -215 prior to the GTG start codon of narG is specific for M. tuberculosis. In contrast, M. bovis, M. bovis BCG, M. africanum, and M. microti appear to carry a cytosine at this position of the narGHJI promoter.



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  		FIG. 4. LightCycler analysis of the polymorphic nucleotide at position -215 within the promoter region of narGHJI of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. Strains were cultured in the MGIT automated culture system, and DNA was prepared from an aliquot of positive culture by mechanical disruption. Melting peak analysis of the amplification product was done after the last amplification cycle. The melting curve analysis is displayed as the first negative derivative of the fluorescence (-dF/dT) versus temperature. F2 refers to channel 2, which is used by the LightCycler's optical unit to measure signals from LightCycler Red 640 at 640 nm.

Identification of M. bovis and M. bovis BCG by analysis of oxyR and RD1 polymorphisms using LightCycler technology. Next, we sought to include LightCycler-based identification and differentiation of M. bovis and M. bovis BCG. We generated FRET probes targeting either A/G polymorphism of oxyR, which is specific for bovine mycobacteria (26), or RD1, a 9,650-bp deletion that is specific for M. bovis BCG (27). M. bovis and M. bovis BCG carry an adenine residue at nucleotide 285 of the oxyR, whereas M. tuberculosis, M. africanum and M. microti carry a guanine residue. The FRET probe targeting A/G polymorphism of oxyR had a perfect match to M. tuberculosis. Amplification of a 200-bp fragment was followed by melting peak analysis of the amplification product. As shown in Table 1, the probe dissociated from the target at 59°C for M. bovis and M. bovis BCG and at 68°C for M. tuberculosis, M. africanum, and M. microti.

RD1, a DNA fragment comprising 9,650 bp, is absent in M. bovis BCG but present in M. bovis, M. tuberculosis, M. africanum, and M. microti. PCR identification of M. bovis BCG, as described by Talbot et al., is based on three primers (27). Two primers are complementary to regions flanking the RD1 sequence. In M. bovis BCG, which lack RD1, these primers bind and amplify a 200-bp fragment. In strains from other species of the MTBC, these primers bind but the 9,650-bp sequence is too large to efficiently amplify. However, a third primer, which is complementary to the 3' end within the RD1 sequence, results in amplification of a 150-bp product, which is thus specific for M. tuberculosis, M. bovis, M. africanum, and M. microti. The 200- and 150-bp products, respectively, are then visualized on an agarose gel. We adapted this multiplex PCR method to the LightCycler system by generating a FRET probe complementary to 12 bp of genomic DNA outside of, but adjacent to, the 3' end of RD1 (Fig. 5). An additional 7 bp extending toward RD1 varied in their homology to M. bovis BCG and the other members of the MTBC, respectively. Three primers were used for amplification, two of which were complementary to regions flanking the RD1 sequence, whereas the third primer targeted the 3' end within RD1. Primers LC73 and LC75 amplified a 320-bp fragment from M. bovis BCG, and primers LC74 and LC75 amplified a 290-bp fragment from M. tuberculosis, M. bovis, M. africanum, or M. microti. Differentiation between these two different amplification products was achieved by subsequent melting peak analysis. As shown in Fig. 6 and Table 1, a melting peak of 49°C was specific for M. bovis BCG, and a melting peak of 62°C was specific for M. tuberculosis, M. bovis, M. africanum, and M. microti.



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  		FIG. 5. Multiplex PCR design for LightCycler-based analysis of RD1. The RD1, which comprises 9,560 bp, is deleted in M. bovis BCG (II) but present in M. tuberculosis, M. bovis, M. africanum, and M. microti. (I). The PCR primers LC73, LC74, and LC75 are shown as arrows and oriented in the direction of amplification. The sensor probe LC79 is aligned to the target specific for MTBC except for BCG (I) and to the target specific for BCG (II). Mismatches between the sensor probe and the targets are shown as boldface, lowercase letters.



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  		FIG. 6. LightCycler analysis of RD1 of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. Strains were cultured in the MGIT automated culture system, and DNA was prepared from an aliquot of positive culture by mechanical disruption. Melting peak analysis of the amplification product was done following the last amplification cycle. The melting curve analysis is displayed as the first negative derivative of the fluorescence (-dF/dT) versus temperature. F2 refers to channel 2, which is used by the LightCycler's optical unit to measure signals from LightCycler Red 640 at 640 nm.


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DISCUSSION
 
In other bacteria, several nitrate reductases have been identified. NarGHJI is only one of at least three different nitrate reductases, and its presence has been demonstrated in both obligate aerobes such as Bacillus spp. and facultative anaerobes such as Escherichia coli (1, 10). Apart from NarGHJI, a second nitrate reductase, often referred to as Nas, mediates in concert with an assimilatory nitrite reductase the utilization of nitrate as a nitrogen source via ammonia. A third nitrate reductase, called Nap, may play a role in the disposal of excess reducing equivalents (19). By generating narG deletion mutants in M. tuberculosis, M. bovis, and M. bovis BCG, we ruled out that nitrate reductases other than NarGHJI contributed to nitrite accumulation under anaerobic conditions in the three species.

In comparison to M. bovis and M. bovis BCG, M. tuberculosis strongly accumulates nitrite from nitrate. At present the American Society for Microbiology recommends testing nitrite accumulation in mycobacteria by incubating bacilli at 37°C for 2 h in buffer containing nitrate (15, 18). We tested nitrite accumulation of the narG mutant of M. tuberculosis under these conditions and demonstrated that the molecular mechanism for this "diagnostic nitrate reductase activity" of M. tuberculosis, which could be more precisely described as rapid nitrite accumulation within 2 h by M. tuberculosis, is also mediated by narGHJI.

Single nucleotide polymorphism, as well as the absence or presence of entire DNA regions ranging in size from 2 to 12 kb, has been described as a variable between different tubercle bacilli. At present, we do not know whether the polymorphic nucleotide detected at position -215 prior to the start codon of narG is functional and plays a role in the different nitrite accumulation that is observed between M. tuberculosis and other members of the MTBC. By comparing the sequence data we obtained from sequencing 500 bp ustream of the narG gene with the genomic sequence data available for M. tuberculosis strains H37Rv/CDC1551 and M. bovis, the single nulceotide polymorphism at position -215 was confirmed. Within this 500-bp region, no additional nucleotide difference between M. tuberculosis and M. bovis was found. Previous studies showed that a single nucleotide polymorphism in oxyR separates the bovine mycobacteria from other members of the MTBC, and the absence of an DNA region encompassing 9,650 bp, called RD1, is specific for M. bovis BCG (3, 26). Whereas oxyR is nonfunctional in mycobacteria, the absence of RD1 is not only specific for M. bovis BCG but also contributes to attenuation of the vaccine strain (16, 25, 27).

We recently used LightCycler technology for the diagnosis of mycobacteria based on amplification and detection of the 16S rRNA gene (14). Since all members of the MTBC share identical 16S rRNA genes, further procedures were required for unambiguous identification of M. tuberculosis. Two recent molecular approaches greatly advanced differentiation within the MTBC, one is based on gyrB polymorphism, and the other is based on genomic deletion analysis (3, 21, 23). However, both methods require further manipulations after DNA amplification such as gel electrophoresis and DNA hybridization procedures. LightCycler allows DNA amplification and subsequent DNA analysis by hybridization in a one-tube format. In the present study, LightCycler technology was used to evaluate the single nucleotide polymorphism at position -215 of narGHJI for its role in diagnostic mycobacteriology. At the same time we subjected an adaptation of the analysis of the single nucleotide polymorphism of oxyR, as well as RD1, to the LightCycler system. We generated FRET probes targeting the single nucleotide polymorphism of the narGHJI promoter, and the single nucleotide polymorphism within oxyR. Detection of the presence or absence of the 9.6-kbp RD1 was achieved by constructing a FRET probe that was complementary to neither of the two possible target sequences, which resulted in a reduction of the melting peak specific for M. bovis BCG by 13°C in comparison to the melting peak specific for M. tuberculosis, M. bovis, M. africanum, and M. microti. Thus, the combination of the three FRET probes allowed rapid identification and differentiation of M. tuberculosis, M. bovis, and M. bovis BCG. This approach is currently being applied in our laboratory to DNA extracted from positive MGIT automated culture system specimens.

Diagnostic laboratories that receive predominantly specimens from human sources may use M. tuberculosis-specific FRET probe for initial diagnostic procedures, since M. tuberculosis will comprise almost all clinical isolates. In our previous study, we used a FRET probe specific for mycobacteria and a FRET probe specific for MTBC for identification of all mycobacteria and separation of MTBC from nontuberculous mycobacteria (14). Meanwhile, we submitted all specimens revealing MTBC to a second round of LightCycler analysis by using the M. tuberculosis-specific FRET probe, since in our laboratory the vast majority of these specimens contain M. tuberculosis. A thymine residue at position -215 of the narGHJI promoter establishes the unambiguous diagnosis, and no further processing, such as phenotypic testing for nitrate reductase activity or additional molecular analysis, is required. The LightCycler's optical unit is capable of measuring fluorescence from FRET probes in two separate channels simultaneously. Channel 2 (F2; 640 nm) is used to measure signals from LightCycler Red 640. Channel 3 (F3; 705 nm) is designed for using LightCycler Red 705. To utilize this technique, the FRET probe targeting RD1 was labeled with LightCycler Red 640, and the FRET probe targeting oxyR was labeled with LightCycler Red 705. Thus, the FRET probe specific for bovine mycobacteria combined with the FRET probe specific for M. bovis BCG allows simultaneous identification and differentiation of M. bovis and M. bovis BCG. Only definitive diagnosis of M. africanum and M. microti will require further testing.


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ACKNOWLEDGMENTS
 
We thank E. Richter and S. Rüsch-Gerdes from the National Reference Center for Mycobacteria in Borstel, Germany, as well as I. Moser from the Federal Research Centre for Virus Diseases of Animals in Jena, Germany, for providing various strains that were used in this study. We thank D. Bitter-Suermann for support. We also thank W. R. Jabobs, Jr., and M. S. Pavelka for providing pYUB657 and mc2155 and for advice on allelelic exchange in mycobacteria.

This study was supported by the Deutsche Forschungsgemeinschaft and the Niedersächsische Verein zur Bekämpfung der Tuberkulose, Lungen, und Bronchialerkrankungen.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Phone: 49-511-532-4359. Fax: 49-511-532-4366. E-mail: bange{at}mikrobio.mh-hannover.de. Back


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Journal of Clinical Microbiology, July 2003, p. 3252-3259, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3252-3259.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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