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A novel antioxidant gene from Mycobacterium tuberculosis.

Ehrt S, Shiloh MU, Ruan J, Choi M, Gunzburg S, Nathan C, Xie Q, Riley LW - J. Exp. Med. (1997)

Bottom Line: Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI).Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages.These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of International Medicine and Infectious Disease, Department of Medicine, Cornell University Medical College, New York 10021, USA.

ABSTRACT
Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

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Expression of recombinant noxR1 in M. smegmatis and native noxR1 in M. tuberculosis. (A) Northern blot. Total  RNA (15 μg) from M. smegmatis  pOLYG (lanes 1 and 3) and M.  smegmatis pOLYG-NO14 (lanes  2 and 4) was probed with oligonucleotides specific for ORF1  (lanes 1 and 2) or ORF2 (lanes 3  and 4). (B) RT-PCR analysis.  Total RNA from recombinant  M. smegmatis strains (0.2 μg) and  wild-type M. tuberculosis (1.0 μg)  was analyzed by RT-PCR after  amplification of cDNA with random hexamer primers. Primer  sets I and II specific for noxR1-coding region are depicted. (C)  Ablation of phenotype by introduction of stop codon in ORF1.  E. coli HB101 were transformed  with pBS (open squares), pNO14.1  (solid squares), or pNO14.1-mut1  (solid triangles), and the latter was  mutated to introduce a stop at  codon 12 in ORF1 without affecting ORF2, and all three recombinants were subjected to  ASN.
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Figure 3: Expression of recombinant noxR1 in M. smegmatis and native noxR1 in M. tuberculosis. (A) Northern blot. Total RNA (15 μg) from M. smegmatis pOLYG (lanes 1 and 3) and M. smegmatis pOLYG-NO14 (lanes 2 and 4) was probed with oligonucleotides specific for ORF1 (lanes 1 and 2) or ORF2 (lanes 3 and 4). (B) RT-PCR analysis. Total RNA from recombinant M. smegmatis strains (0.2 μg) and wild-type M. tuberculosis (1.0 μg) was analyzed by RT-PCR after amplification of cDNA with random hexamer primers. Primer sets I and II specific for noxR1-coding region are depicted. (C) Ablation of phenotype by introduction of stop codon in ORF1. E. coli HB101 were transformed with pBS (open squares), pNO14.1 (solid squares), or pNO14.1-mut1 (solid triangles), and the latter was mutated to introduce a stop at codon 12 in ORF1 without affecting ORF2, and all three recombinants were subjected to ASN.

Mentions: To find out which ORF was responsible for the observed phenotype, we determined which was transcribed in recombinant M. smegmatis. In Northern blots with an oligonucleotide specific for ORF1, an ∼400-bp transcript was detected in RNA purified from logarithmically growing M. smegmatis pOLYG–NO14 but not from M. smegmatis pOLYG (Fig. 3 A). In contrast, neither RNA preparation hybridized with an oligonucleotide specific for ORF2. Exposure of M. smegmatis pOLYG–NO14 and M. smegmatis pOLYG to ASN before extraction of their RNA did not alter the Northern blot results (data not shown), suggesting that ORF1 but not ORF2 was expressed during manifestation of the phenotype conferred by transformation with pOLYG–NO14. The gene corresponding to ORF1 was designated noxR1.


A novel antioxidant gene from Mycobacterium tuberculosis.

Ehrt S, Shiloh MU, Ruan J, Choi M, Gunzburg S, Nathan C, Xie Q, Riley LW - J. Exp. Med. (1997)

Expression of recombinant noxR1 in M. smegmatis and native noxR1 in M. tuberculosis. (A) Northern blot. Total  RNA (15 μg) from M. smegmatis  pOLYG (lanes 1 and 3) and M.  smegmatis pOLYG-NO14 (lanes  2 and 4) was probed with oligonucleotides specific for ORF1  (lanes 1 and 2) or ORF2 (lanes 3  and 4). (B) RT-PCR analysis.  Total RNA from recombinant  M. smegmatis strains (0.2 μg) and  wild-type M. tuberculosis (1.0 μg)  was analyzed by RT-PCR after  amplification of cDNA with random hexamer primers. Primer  sets I and II specific for noxR1-coding region are depicted. (C)  Ablation of phenotype by introduction of stop codon in ORF1.  E. coli HB101 were transformed  with pBS (open squares), pNO14.1  (solid squares), or pNO14.1-mut1  (solid triangles), and the latter was  mutated to introduce a stop at  codon 12 in ORF1 without affecting ORF2, and all three recombinants were subjected to  ASN.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199150&req=5

Figure 3: Expression of recombinant noxR1 in M. smegmatis and native noxR1 in M. tuberculosis. (A) Northern blot. Total RNA (15 μg) from M. smegmatis pOLYG (lanes 1 and 3) and M. smegmatis pOLYG-NO14 (lanes 2 and 4) was probed with oligonucleotides specific for ORF1 (lanes 1 and 2) or ORF2 (lanes 3 and 4). (B) RT-PCR analysis. Total RNA from recombinant M. smegmatis strains (0.2 μg) and wild-type M. tuberculosis (1.0 μg) was analyzed by RT-PCR after amplification of cDNA with random hexamer primers. Primer sets I and II specific for noxR1-coding region are depicted. (C) Ablation of phenotype by introduction of stop codon in ORF1. E. coli HB101 were transformed with pBS (open squares), pNO14.1 (solid squares), or pNO14.1-mut1 (solid triangles), and the latter was mutated to introduce a stop at codon 12 in ORF1 without affecting ORF2, and all three recombinants were subjected to ASN.
Mentions: To find out which ORF was responsible for the observed phenotype, we determined which was transcribed in recombinant M. smegmatis. In Northern blots with an oligonucleotide specific for ORF1, an ∼400-bp transcript was detected in RNA purified from logarithmically growing M. smegmatis pOLYG–NO14 but not from M. smegmatis pOLYG (Fig. 3 A). In contrast, neither RNA preparation hybridized with an oligonucleotide specific for ORF2. Exposure of M. smegmatis pOLYG–NO14 and M. smegmatis pOLYG to ASN before extraction of their RNA did not alter the Northern blot results (data not shown), suggesting that ORF1 but not ORF2 was expressed during manifestation of the phenotype conferred by transformation with pOLYG–NO14. The gene corresponding to ORF1 was designated noxR1.

Bottom Line: Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI).Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages.These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of International Medicine and Infectious Disease, Department of Medicine, Cornell University Medical College, New York 10021, USA.

ABSTRACT
Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

Show MeSH
Related in: MedlinePlus