JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.13.4033–4039.2001

July 2001, p. 4033–4039 Vol. 183, No. 13

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Mapping of Mycobacterium tuberculosis katG Promoters andTheir Differential Expression in Infected Macrophages

SHARON MASTER, THOMAS C. ZAHRT, JIAN SONG, AND VOJO DERETIC*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan

Received 29 December 2000/Accepted 9 April 2001

Intracellular pathogenic bacteria, including Mycobacterium tuberculosis, frequently have multitiered defensemechanisms ensuring their survival in host phagocytic cells. One such defense determinant in M. tuberculosisis the katG gene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG isconsidered to be important for protection against reactive oxygen and nitrogen intermediates produced byphagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence renderingM. tuberculosis exquisitely sensitive to this compound. In this context, katG expression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determinesensitivity to INH. Thus, it is important to delineate the regulation and expression of katG, as this not only canaid understanding of how M. tuberculosis survives and persists in the host but also may provide information ofrelevance for better management of INH therapy. Here, we report the first extensive analysis of the katGpromoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped thekatG mRNA 5* ends and demonstrated that two promoters, P1furA and P1katG, control transcription of katG.The furA and katG genes are cotranscribed from P1furA. Both P1furA and P1katG promoters show inductionupon challenge with hydrogen peroxide and cumene hydroperoxide. Studies carried out using the transcrip-tional fusions P1furA-gfp, P1katG-gfp, and P1furA-P1katG-gfp confirmed the existence of two katG promoters. Inaddition, we showed that both promoters are expressed in vivo during intracellular growth of virulent M.tuberculosis H37Rv. P1furA is induced early upon infection, and P1katG becomes active only upon extendedgrowth in macrophages. These studies delineate the transcriptional organization of the furA-katG region andindicate differential regulation in vivo of the two katG promoters. These phenomena most likely reflect thediffering demands at sequential stages of the infection cycle and may provide information for improvedunderstanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.

Mycobacterium tuberculosis is the most common cause ofdeath from a single infectious agent worldwide. It is a faculta-tive, intracellular pathogen capable of surviving and persistingin the highly oxidative environment of phagocytic cells (1, 7).M. tuberculosis can evade the host immune system by prevent-ing phagosome-lysosome fusion and resists killing by reactiveoxygen and reactive nitrogen intermediates (2, 5, 8, 16, 25, 31,33, 34, 37, 44, 45). As is the case with most pathogenic bacteria,M. tuberculosis has evolved mechanisms of protection againstoxidative stress by way of specific defenses and global re-sponses. Examples of such defense systems include katG (en-coding a catalase-peroxidase), and ahpC (encoding a homologof alkyl hydroperoxide reductase). The products of these twogenes are important in protection against oxidative stress, spe-cifically peroxides, and in macrophage parasitism of patho-genic mycobacteria (3, 6, 15, 20, 22, 24–26, 28, 43).

Unlike other mycobacteria with two catalases (14) and othermicroorganisms with multiple peroxide-dismutating or -reduc-ing enzymes (27) which are differentially regulated during thegrowth cycle (17), M. tuberculosis has a single catalase-perox-idase, encoded by the katG gene (21). KatG has also beenshown to have peroxynitritase activity (41). KatG activity isnecessary for growth and persistence in mice and guinea pigs

(24). KatG is also involved in the activation of isonicotinic acidhydrazide (INH) (4, 19, 42, 49) and hence has been implicatedin the exquisite sensitivity of M. tuberculosis to INH (9, 48). Inthis context, one of the mechanisms of M. tuberculosis resis-tance to INH is via the inactivation of the katG gene (30, 49).As KatG is an important virulence determinant in M. tubercu-losis and at the same time plays a critical role in rendering thetubercle bacilli sensitive to INH, its potentially varied levels ofexpression in vivo during infection and periods of antibiotictreatment may play opposing roles in the survival of the patho-gen. In this context, our present knowledge of katG regulationand expression is limited, and KatG levels have not been con-sidered in the majority of studies published to date.

In all mycobacteria, the katG locus is genetically linked tothe furA gene (Fig. 1A) (11, 32). In this study we presentcharacterization of the katG promoters and their expression.We show that katG is transcribed from two promoters, onetranscript encompassing both the furA and the katG genes andthe other corresponding to katG alone. We also show theinduction of both promoters by peroxides in vitro and theirdifferential expression during growth of M. tuberculosis in mac-rophages. These studies indicate the existence of a dual andstage-specific induction of katG promoters, a phenomenon ofpotential significance for the physiology of the tubercle bacil-lus. Due to the presence of only one catalase in M. tuberculosis,unlike in other bacteria where at least two enzymes allowbalanced H2O2 and peroxide homeostasis (17), the tuberclebacillus may have evolved ways of appropriately tuning katG

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Michigan Medical School, Med-ical Science Building II, Ann Arbor, MI 48109. Phone: (734) 763-1580.Fax: (734) 647-6243. E-mail: deretic@umich.edu.

4033

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

expression from two promoters to respond to varied environ-mental inputs and physiological demands.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and electrotransformation. Unless oth-erwise noted, Mycobacterium bovis BCG (strain Pasteur; ATCC 27291) and M.tuberculosis H37Rv (ATCC 27294) were grown in Middlebrook 7H9 medium oron 7H10 agar plates (Difco) supplemented with 0.05% Tween, 0.2% glycerol,and ADC (10% bovine serum albumin fraction V, dextrose, and catalase) en-richment for M. bovis BCG or OADC (which also contains oleic acid) forM. tuberculosis. All manipulations of live M. tuberculosis were carried out underbiosafety level 3 conditions. All transformations in Escherichia coli were per-formed with the strain DH5a. E. coli was grown in Luria broth (Difco) at 37°C.Wherever necessary, 25 mg of kanamycin/ml was added to the medium. Thepreparation and the transformation of electrocompetent mycobacteria were per-formed as previously described (23).

Construction of plasmids. The furA promoter was PCR amplified using prim-ers Mtbfur1 (59-GCTCATCGGAACATACGAAGG-39, located at positions2138 to 2117 upstream of the furA initiation codon) and katGS1-P22 (59-TGTGGATGCGCATTCACTGCT-39, located at 180 to 1102 relative to the furAinitiation codon). This fragment was cloned into pCR2.1, digested with EcoRI,and ligated to HindIII/EcoRI adapters. This was then digested with HindIII andcloned into pMYGFP2 to give pTZ175. The katG promoter was amplified usingprimers Mtbfur2 (59-ACCATCACATCGTCTGCCGGT-39, located at 2215 to2194 relative to the katG initiation codon) and katGS1 (59-TGGGTGGGTGTTGCTCGGGCACAGCA-39, located at 24 to 122 relative to the katG initiationcodon) and cloned as for pTZ175 to yield pTZ176. The furA and katG promoterswere amplified using primers Mtbfur1 and katGS1 and cloned as for pTZ175 toyield pTZ177. All constructs were sequenced to confirm the correct orientationand sequence of nucleotides. pMYGFP2 and pahpC-gfp were constructed else-where (10). All plasmids are kanamycin resistant.

RNA isolation and S1 nuclease protection analysis. Total cellular RNA wasisolated by centrifugation through a cushion of 5.7 M CsCl (13). Uniformly32P-labeled single-stranded hybridization probes were prepared using pJS121,containing a 1,724-bp SmaI-BamHI fragment of pYZ55 (49) that encompassesthe furA-katG loci, and primers katGS1, katGS1-P2 (59-TGGACTCGTAGCG

CGCGACGGAG-39), and katGS1-P22. Equal amounts of RNA (33 mg) werehybridized with aliquots of the radioactively labeled DNA probe. S1 nucleaseprotection analysis was carried out as described previously (13). S1 nucleasedigestion products were analyzed on sequencing gels (7.5% polyacrylamide–8 Murea–100 mM Tris–100 mM boric acid–2 mM EDTA, pH 8.3) along with thesequencing ladder. Because of the uniform labeling of single-stranded DNA,which dramatically improves the sensitivity of the assay, radioactive decay con-tributes to the presence of multiple bands corresponding to the 59 end of mRNA,as has been noted (13). Images were quantitated using densitometry (at leastthree samples). All bands corresponding to the promoter region were included inthe quantitation.

Fluorescence microscopy. M. tuberculosis H37Rv strains harboring promoter-gfp fusion plasmids were grown in static cultures until the cells reached mid-exponential phase. J774A (ATCC TIB-67) macrophage infections and fluores-cence microscopy analyses were carried out as described previously (46).Relative fluorescence units (RFU) were previously defined (46).

Flow cytometry. M. tuberculosis H37Rv, containing the various promoter-gfpfusion plasmids, were grown in static cultures (7H9 plus OADC or Sautonminimal medium, which contains 3.6 mM potassium dihydrogen phosphate, 6mM citric acid, 6% glycerol, 30 mM L-asparagine, 1 mM magnesium sulfateheptahydrate, 2 mM copper sulfate, 7 mM zinc sulfate heptahydrate, and 70 mMferric ammonium citrate) until the cells reached mid-exponential phase. Flowcytometric analysis was then carried out, as previously described (10, 38) using aFACStar Plus system (Becton Dickinson Immunocytometry Systems). Illumina-tion was with a 200-mW, 488-nm argon ion laser, and emission light was detectedthrough a 530/30-nm band pass filter. Data were collected for 20,0000 individualparticles per sample, with gating for size to eliminate interference due to poten-tial clumping, as published previously (12).

RESULTS

Mapping of the 5* end of transcripts in the furA-katG region.With the long-term goal of investigating expression and regu-lation of katG in M. tuberculosis, we mapped the katG mRNA59 ends using S1 nuclease protection analysis as described inMaterials and Methods. First, by using a primer located within

FIG. 1. (A) Genetic map of the furA-katG loci in mycobacteria. In all mycobacteria, furA and katG are linked. Both furA and katG areinactivated by multiple mutations in M. leprae (circles, insertions; triangles, deletions). (B) Promoter-gfp reporter fusions. The katG promoters(P1furA and P1katG) were cloned in front of the green fluorescent reporter gene (gfp) either individually or together as described in Materials andMethods. pTZ175 carries P1furA upstream of gfp; pTZ176 carries P1katG upstream of gfp; pTZ177 carries both P1furA and P1katG upstream ofkatG. pTZ177 also carries an intact furA gene. All three plasmids confer resistance to kanamycin.

4034 MASTER ET AL. J. BACTERIOL.

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

the katG coding sequence, a single-stranded DNA probe wasgenerated. This probe (probe I) (Fig. 2), when hybridized withRNA from M. bovis BCG, yielded two products that wereprotected from digestion by S1 nuclease. The pattern observedsuggested the presence of two katG transcripts (Fig. 2A), as themRNA corresponding to both the upstream and the down-stream bands encompassed katG coding sequences. The down-stream mRNA 59 end (termed P1katG) was located 54 nucle-otides upstream of the katG coding sequences. The signal fromP1katG was much stronger than the upstream signal and was

located within the 39-end portion of the furA gene (13 bpupstream of the furA stop codon). We next asked whether theupstream transcript was located within the coding region offurA or encompassed a complete FurA sequence. The mappingof the 59 end of the corresponding mRNA was carried outusing a series of primers at various positions downstream of thesecond transcription start site (Fig. 2B and C). The results ofthese studies indicate that the two detectable transcripts ofkatG have their 59 ends at positions 54 and 471 bp upstream ofthe initiation codon of katG. Significantly, the upstream tran-

FIG. 2. Mapping of the 59 ends of the katG mRNA by S1 nuclease protection analysis. RNA was isolated from M. bovis BCG. S1 nucleaseprotection assays were carried out as described in Materials and Methods. The protected products (lanes 1) were analyzed on standard sequencinggels alongside the undigested probe (lanes 2) and sequencing ladders (lanes G, A, T, and C) generated by the primer and template used tosynthesize the corresponding probes. (A) Mapping of P1katG and detection of a transcript containing katG sequences that initiates furtherupstream (a detailed mapping of the 59 end of this transcript is shown in panels B and C) using probe I, generated with the primer katGS1 (atpositions 24 to 122 relative to the katG initiation codon). (B) Mapping of the mRNA 59 end using probe II, generated with the primer katGS1-P2(at positions 1226 to 1250 relative to the furA initiation codon). (C) Mapping of the P1furA 59 end using probe III, generated with the primerkatGS1-P22 (at positions 180 to 1102 relative to the furA initiation codon). (D) Schematic representation of the probes and the protectedfragments in relationship to the furA and katG genes. P1katG and P1furA, mRNA 59 ends. The P1furA mRNA start site coincides with the furAtranslation initiation site (GenBank accession number AF002194). Arrows between the panels indicate the stepwise mapping of P1furA. Note thatone of the transcripts initiating from P1furA, shown at the bottom of panel D, encompasses both furA and katG coding regions. p, a transcriptinitiating further upstream from P1furA for which the 59 end has not been mapped.

VOL. 183, 2001 IN VIVO M. TUBERCULOSIS katG PROMOTER ACTIVITY 4035

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

script also coincided with the translational start site of FurA.Thus, we conclude that the katG transcript initiating furtherupstream also encompasses the FurA coding sequence, andhence, the upstream promoter was termed P1furA (Fig. 2D).

Induction of furA and katG transcription by hydrogen per-oxide and CHP. It has been shown previously that katG isinduced upon exposure of M. tuberculosis to oxidants (35, 36).We reasoned that if both mRNA 59 ends were involved inexpression of katG, then both transcripts may be induced uponstimulation with peroxides. Thus, the effects of peroxides onthe induction of P1katG and P1furA were tested. M. bovis BCGwas exposed to either 62.5 mM hydrogen peroxide (H2O2) or62.5 mM cumene hydroperoxide (CHP) for 2 h. After expo-sure, the RNA was isolated and S1 nuclease protection analysiswas carried out as described above. As can be seen in Fig. 3,the levels of both transcripts were significantly increased. Asdetermined by PhosphorImager analysis, a 5.4-fold increaseand a 3.7-fold increase in the proximal katG transcript levels(starting at P1katG) were detected upon exposure to H2O2 andCHP, respectively, compared to untreated controls (Fig. 3A).Similarly, a 7.1-fold increase and a 2.2-fold increase comparedto untreated controls were observed in the distal transcriptlevels (starting at P1furA) (Fig. 3B).

Two promoters control expression of M. tuberculosis katG.To rule out the possibility that the two katG mRNA 59 endswere processing products, we generated transcriptional fusionsbetween P1furA and P1katG with gfp (Fig. 1B) and subjectedthem to promoter activity analyses using previously establishedmethodologies (13, 38). The results of these experiments areshown in Table 1. After 7 days of growth, both P1furA andP1katG showed promoter activity. Furthermore, the effects ofthe two promoters were additive, as pTZ177 (with both P1furAand P1katG fused in tandem with gfp) showed the highesttranscriptional activity. Similar results were obtained when thestrains were tested using a different (Sauton minimal) medium(Table 1).

In vivo expression of P1furA and P1katG in infected macro-phages. Since our primary interest is in understanding katGregulation and expression in vivo, we used the gfp fusions to studykatG promoter activities in infected macrophages. For the invivo analyses, J774A macrophages were infected with M. tu-berculosis H37Rv containing the promoter-gfp fusion plasmidspTZ175, pTZ176, and pTZ177. The results, as visualized by

fluorescence microscopy at 2 h, 3 days, and 7 days postinfec-tion, are shown in Fig. 4A. As evidenced by gfp fluorescence,P1furA activity can be seen immediately (2 h) postinfection(Fig. 4A, panel I). In contrast, expression from P1katG was notdetectable until the later stages of infection, but by the thirdday postinfection a high level of activity was observed (Fig. 4A,panels IV and V). At 3 days, the amount of fluorescencedetected in the presence of both promoters was significantlygreater than the level for P1furA or P1katG alone (Fig. 4A,panels II, V, and VIII). At 7 days following phagocytosis, thelevels of expression from both promoters individually werevirtually identical (Fig. 4A, panels III and VI). These resultsshow that both katG promoters are active in vivo and reveal thephenomenon of a differential expression of the two katG pro-moters in vivo.

DISCUSSION

Protection against oxidative stress is one of the primarydefenses that enable microbial pathogens to survive in the host.Understanding the regulation and expression of virulence de-terminants is fundamental to deciphering how pathogenic my-cobacteria, e.g., M. tuberculosis, survive in host macrophages.The catalase-peroxidase KatG is an important virulence factorin M. tuberculosis, and it is thus significant to determine how itis regulated and expressed. Our results, obtained by using S1nuclease mapping of M. bovis BCG RNA, indicate that thereare two transcriptional start sites and two promoters of katG.One is located immediately upstream of the katG gene. Theother transcript starts from P1furA and corresponds to the furApromoter, from which both furA and katG are cotranscribed.We propose a model in which the two katG promoters aredifferentially expressed with potentially different outcomes, asthe transcription from the upstream promoter allows expres-sion of both furA and katG while the downstream promotersupports expression of katG only. Since mycobacterial furA is aregulatory gene (47), expression of katG alone and in combi-nation with furA may lead to different physiological outcomesin M. tuberculosis.

The furA gene and the katG gene are genetically linked in allspecies of mycobacteria including Mycobacterium smegmatis,M. tuberculosis complex, and Mycobacterium marinum, asshown in Fig. 1A. Interestingly, in Mycobacterium leprae these

FIG. 3. Activation of P1furA and P1katG in M. bovis BCG uponexposure to hydrogen peroxide and CHP. Lanes 1, untreated control;lanes 2, 62.5 mM H2O2; lanes 3, 62.5 mM CHP. (A) S1 nucleasemapping using probe I, showing induction from P1katG; (B) S1 nucle-ase mapping using probe III, showing induction from P1furA. Equalamounts of total RNA (33 mg) were used in each sample.

TABLE 1. Promoter activity of P1furA and P1katG inM. tuberculosis H37Rv

Medium

Mean relative fluorescencea (RFU)

pTZ175b pTZ176c pTZ177d Positivecontrole

Negativecontrolf

7H9 1 OADC 23.2 12.8 43.8 101.3 6.2Sauton 16.7 17 60 BDL

a Determined from 20,000 individual particles per sample. The bacteria weregrown for 7 days without inducing agents.

b P1furA-gfp.c P1katG-gfp.d Transcriptional fusion of both (P1furA and P1katG) promoters in tandem

with gfp.e pahpC-gfp with PahpC from M. bovis BCG upstream of gfp. The pahpC from

M. bovis BCG was used instead of the one from M. tuberculosis because it isconstitutively expressed, while the one from M. tuberculosis is not.

f pMYGFP2 containing promoterless gfp. BDL, below detection limit.

4036 MASTER ET AL. J. BACTERIOL.

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

genes are inactive due to the presence of insertions and dele-tions (Fig. 1A). A similar linkage of the furA and katG or-thologs is also seen in Streptomyces coelicolor (furA-catC) andStreptomyces reticuli (furS-cpeB) (18, 50). S. coelicolor furA ishighly homologous to the furA gene from M. tuberculosis. Stud-ies on the S. coelicolor furA-catC loci (18) have revealed pro-moter activities similar to that which we have reported here,with the existence of two transcripts. One transcript encom-passes the furA-catC genes and the other covers only the catCgene. The previously reported studies on the M. tuberculosiskatG promoter have suggested the existence of a number oftranscriptional start sites near the katG initiation codon (29)that do not coincide with the mRNA start sites reported in ourwork. However, such studies were performed with a plasmid-borne M. tuberculosis katG gene studied for expression in E.coli and M. smegmatis (29), which could explain the discrep-ancies between studies carried out in heterologous hosts andour findings derived in an autologous system.

By using a katG-lux construct, Sherman et al. (35) observeda sevenfold induction of katG expression upon exposing BCGto H2O2. Our results are in agreement with these findings andfurthermore show that peroxides induce both katG promoters.Both organic and hydrogen peroxides affect katG transcription.Most importantly, our green fluorescent protein fluorescencedata show that both promoters are expressed in synthetic me-dia and during infection of mouse macrophages. Hence, bothtranscriptional starts are utilized in vitro and in vivo. Theresults reported here also reveal a staged expression from thetwo promoters. The promoter corresponding to the largertranscript, starting at P1furA, is activated early in infection,

while the smaller transcript, starting at P1katG, is producedlater in infection. The effects of the two promoters are cumu-lative.

The genetic linkage of furA and katG in mycobacteria sug-gests that FurA may control katG. Indeed, recent studies in-cluding inactivation of mycobacterial furA have demonstratedthat it is a negative regulator of katG (47). A similar role forFurA has also been reported for S. coelicolor (18), albeit basedon indirect observations without inactivating the furA gene inthis organism. Interestingly, the S. coelicolor FurA is closelyrelated to FurA of M. tuberculosis. From our in vivo data, thereis no obvious repression of katG up to 7 days postinfection,although furA is expressed at corresponding time points basedon the P1furA-gfp data. This may reflect the possibility that thebacteria are in a highly oxidative environment in macrophagesfor prolonged periods of time or that additional signals con-tribute to the activation in vivo. Studies are currently underway to characterize FurA further and to reveal the molecularmechanisms by which it regulates one or both of the katGpromoters and possibly additional genes involved in oxidativestress.

The observation that katG expression continues for pro-longed periods of time in infected macrophages may explainthe exquisite sensitivity of the tubercle bacillus to INH in vivo.However, important variabilities in clinical responses to INHchemotherapy have been noted (39, 40). Often, such phenom-ena are not associated with mutations in katG or other genesimplicated in INH resistance or with host factors such as dif-ferences in INH inactivation in the body. We propose thatsome of these phenomena could be due to phenotypic differ-

FIG. 4. Differential induction of P1furA and P1katG in M. tuberculosis H37Rv in infected macrophages. (A) Fluorescence microscopy of J774Amurine macrophages infected with H37Rv carrying the transcriptional fusion constructs shown in panel B. Numbers are RFU, quantitated aspreviously described (47). Columns correspond to 2 h, 3 days, and 7 days postinfection from left to right, respectively. (B) Graphic representationof the promoter-gfp fusions used (see Fig. 1 for details).

VOL. 183, 2001 IN VIVO M. TUBERCULOSIS katG PROMOTER ACTIVITY 4037

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ences based on expression patterns of M. tuberculosis katGfrom its two promoters defined in this work, as their regulationmay vary depending upon different conditions in anatomicallydiverse infection sites.

ACKNOWLEDGMENTS

S.M., T.C.Z., and J.S. contributed equally to this paper.T.C.Z. was supported by an NRSA fellowship from NIH. This work

was supported by grant AI42999 from NIAID.

REFERENCES

1. Armstrong, J. A., and P. D. A. Hart. 1971. Response of cultured macro-phages to Mycobacterium tuberculosis, with observations on fusion of lyso-somes with phagosomes. J. Exp. Med. 134:713–740.

2. Chan, J., X. D. Fan, S. W. Hunter, P. J. Brennan, and B. R. Bloom. 1991.Lipoarabinomannan, a possible virulence factor involved in persistence ofMycobacterium tuberculosis within macrophages. Infect. Immun. 59:1755–1761.

3. Chen, L., Q. W. Xie, and C. Nathan. 1998. Alkyl hydroperoxide reductasesubunit C (AhpC) protects bacterial and human cells against reactive nitro-gen intermediates. Mol. Cell 1:795–805.

4. Chouchane, S., I. Lippai, and R. S. Magliozzo. 2000. Catalase-peroxidase(Mycobacterium tuberculosis KatG) catalysis and isoniazid activation. Bio-chemistry 39:9975–9983.

5. Clemens, D. L., and M. A. Horwitz. 1995. Characterization of the Mycobac-terium tuberculosis phagosome and evidence that phagosomal maturation isinhibited. J. Exp. Med. 181:257–270.

6. Cooper, A. M., B. H. Segal, A. A. Frank, S. M. Holland, and I. M. Orme.2000. Transient loss of resistance to pulmonary tuberculosis in p47phox2/2

mice. Infect. Immun. 68:1231–1234.7. Dannenberg, A. M., Jr., and G. A. Rook. 1994. Pathogenesis of pulmonary

tuberculosis: an interplay of tissue-damaging and macrophage-activating im-mune responses—dual mechanisms that control bacillary multiplication, p.459–483. In B. R. Bloom (ed.), Tuberculosis: pathogenesis, protection, andcontrol. American Society for Microbiology, Washington, D.C.

8. Deretic, V., and R. A. Fratti. 1999. Mycobacterium tuberculosis phagosome.Mol. Microbiol. 31:1603–1609.

9. Deretic, V., E. Pagan-Ramos, Y. Zhang, S. Dhandayuthapani, and L. E. Via.1996. The extreme sensitivity of Mycobacterium tuberculosis to the front-lineantituberculosis drug isoniazid. Nat. Biotechnol. 14:1557–1561.

10. Deretic, V., W. Philipp, S. Dhandayuthapani, M. H. Mudd, R. Curcic, T.Garbe, B. Heym, L. E. Via, and S. T. Cole. 1995. Mycobacterium tuberculosisis a natural mutant with an inactivated oxidative-stress regulatory gene:implications for sensitivity to isoniazid. Mol. Microbiol. 17:889–900.

11. Deretic, V., J. Song, and E. Pagan-Ramos. 1997. Loss of oxyR in Mycobac-terium tuberculosis. Trends Microbiol. 5:367–372.

12. Dhandayuthapani, S., L. E. Via, C. A. Thomas, P. M. Horowitz, D. Deretic,and V. Deretic. 1995. Green fluorescent protein as a marker for gene ex-pression and cell biology of mycobacterial interactions with macrophages.Mol. Microbiol. 17:901–912.

13. Dhandayuthapani, S., Y. Zhang, M. H. Mudd, and V. Deretic. 1996. Oxida-tive stress response and its role in sensitivity to isoniazid in mycobacteria:characterization and inducibility of ahpC by peroxides in Mycobacteriumsmegmatis and lack of expression in M. aurum and M. tuberculosis. J. Bacte-riol. 178:3641–3649.

14. Farr, S. B., and T. Kogoma. 1991. Oxidative stress responses in Escherichiacoli and Salmonella typhimurium. Microbiol. Rev. 55:561–585.

15. Fazal, N. 1997. Influence of Mycobacterium tuberculosis catalase gene(KatG) expression on nitric oxide production and the intracellular growth oftransfected Mycobacterium smegmatis strains within murine macrophages.Biochem. Mol. Biol. Int. 42:135–142.

16. Fratti, R. A., I. Vergne, J. Chua, J. Skidmore, and V. Deretic. 2000. Regu-lators of membrane trafficking and Mycobacterium tuberculosis phagosomematuration block. Electrophoresis 21:3378–3385.

17. Gonzalez-Flecha, B., and B. Demple. 1997. Homeostatic regulation of intra-cellular hydrogen peroxide concentration in aerobically growing Escherichiacoli. J. Bacteriol. 179:382–388.

18. Hahn, J. S., S. Y. Oh, and J. H. Roe. 2000. Regulation of the furA and catCoperon, encoding a ferric uptake regulator homologue and catalase-peroxi-dase, respectively, in Streptomyces coelicolor A3(2). J. Bacteriol. 182:3767–3774.

19. Heym, B., P. M. Alzari, N. Honore, and S. T. Cole. 1995. Missense mutationsin the catalase-peroxidase gene, katG, are associated with isoniazid resis-tance in Mycobacterium tuberculosis. Mol. Microbiol. 15:235–245.

20. Heym, B., E. Stavropoulos, N. Honore, P. Domenech, B. Saint-Joanis, T. M.Wilson, D. M. Collins, M. J. Colston, and S. T. Cole. 1997. Effects ofoverexpression of the alkyl hydroperoxide reductase AhpC on the virulenceand isoniazid resistance of Mycobacterium tuberculosis. Infect. Immun. 65:1395–1401.

21. Heym, B., Y. Zhang, S. Poulet, D. Young, and S. T. Cole. 1993. Character-ization of the katG gene encoding a catalase-peroxidase required for theisoniazid susceptibility of Mycobacterium tuberculosis. J. Bacteriol. 175:4255–4259.

22. Hillas, P. J., F. S. del Alba, J. Oyarzabal, A. Wilks, and P. R. Ortiz DeMontellano. 2000. The AhpC and AhpD antioxidant defense system ofMycobacterium tuberculosis. J. Biol. Chem. 275:18801–18809.

23. Jacobs, W. R., Jr., G. V. Kalpana, J. D. Cirillo, L. Pascopella, S. B. Snapper,R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom. 1991. Geneticsystems for mycobacteria. Methods Enzymol. 204:537–555.

24. Li, Z., C. Kelley, F. Collins, D. Rouse, and S. Morris. 1998. Expression ofkatG in Mycobacterium tuberculosis is associated with its growth and per-sistence in mice and guinea pigs. J. Infect. Dis. 177:1030–1035.

25. Manca, C., S. Paul, E. C. Barry III, V. H. Freedman, and G. Kaplan. 1999.Mycobacterium tuberculosis catalase and peroxidase activities and resistanceto oxidative killing in human monocytes in vitro. Infect. Immun. 67:74–79.

26. Middlebrook, G., and M. L. Kohn. 1953. Some observations on the patho-genicity of isoniazid resistant variants of the tubercle bacilli. Science 118:297–299.

27. Milano, A., E. De Rossi, L. Gusberti, B. Heym, P. Marone, and G. Riccardi.1996. The katE gene, which encodes the catalase HPII of Mycobacteriumavium. Mol. Microbiol. 19:113–123.

28. Mitchison, D. A., J. B. Selkon, and J. Lloyd. 1963. Virulence in the guineapig, susceptibility to hydrogen peroxide, and catalase activity of isoniazid-sensitive tubercle bacilli from South Indian and British patients. J. Pathol.Bacteriol. 86:377–386.

29. Mulder, M. A., H. Zappe, and L. M. Steyn. 1999. The Mycobacteriumtuberculosis katG promoter region contains a novel upstream activator.Microbiology 145:2507–2518.

30. Musser, J. M. 1995. Antimicrobial agent resistance in mycobacteria: molec-ular genetic insights. Clin. Microbiol. Rev. 8:496–514.

31. Mustafa, T., S. Phyu, R. Nilsen, G. Bjune, and R. Jonsson. 1999. Increasedexpression of Fas ligand on Mycobacterium tuberculosis infected macro-phages: a potential novel mechanism of immune evasion by Mycobacteriumtuberculosis? Inflammation 23:507–521.

32. Pagan-Ramos, E., J. Song, M. McFalone, M. H. Mudd, and V. Deretic. 1998.Oxidative stress response and characterization of the oxyR-ahpC and furA-katG loci in Mycobacterium marinum. J. Bacteriol. 180:4856–4864.

33. Pancholi, P., A. Mirza, N. Bhardwaj, and R. M. Steinman. 1993. Sequestra-tion from immune CD41 T cells of mycobacteria growing in human mac-rophages. Science 260:984–986.

34. Russell, D. G. 1995. Mycobacterium and Leishmania: stowaways in theendosomal network. Trends Cell Biol. 5:125–128.

35. Sherman, D. R., K. Mdluli, M. J. Hickey, T. M. Arain, S. L. Morris, C. E.Barry III, and C. K. Stover. 1996. Compensatory ahpC gene expression inisoniazid-resistant Mycobacterium tuberculosis. Science 272:1641–1643.

36. Sherman, D. R., P. J. Sabo, M. J. Hickey, T. M. Arain, G. G. Mahairas, Y.Yuan, C. E. Barry III, and C. K. Stover. 1995. Disparate responses tooxidative stress in saprophytic and pathogenic mycobacteria. Proc. Natl.Acad. Sci. USA 92:6625–6629.

37. Stenger, S., K. R. Niazi, and R. L. Modlin. 1998. Down-regulation of CD1 onantigen-presenting cells by infection with Mycobacterium tuberculosis. J. Im-munol. 161:3582–3588.

38. Via, L. E., S. Dhandayuthapani, D. Deretic, and V. Deretic. 1998. Greenfluorescent protein. A tool for gene expression and cell biology in mycobac-teria. Methods Mol. Biol. 101:245–260.

39. Wallis, R. S., S. Patil, S. H. Cheon, K. Edmonds, M. Phillips, M. D. Perkins,M. Joloba, A. Namale, J. L. Johnson, L. Teixeira, R. Dietze, S. Siddiqi, R. D.Mugerwa, K. Eisenach, and J. J. Ellner. 1999. Drug tolerance in Mycobac-terium tuberculosis. Antimicrob Agents Chemother. 43:2600–2606.

40. Wallis, R. S., M. D. Perkins, M. Phillips, M. Joloba, A. Namale, J. L.Johnson, C. C. Whalen, L. Teixeira, B. Demchuk, R. Dietze, R. D. Mugerwa,K. Eisenach, and J. J. Ellner. 2000. Predicting the outcome of therapy forpulmonary tuberculosis. Am. J. Respir. Crit. Care Med. 161:1076–1080.

41. Wengenack, N. L., M. P. Jensen, F. Rusnak, and M. K. Stern. 1999. Myco-bacterium tuberculosis KatG is a peroxynitritase. Biochem. Biophys. Res.Commun. 256:485–487.

42. Wengenack, N. L., H. Lopes, M. J. Kennedy, P. Tavares, A. S. Pereira, I.Moura, J. J. Moura, and F. Rusnak. 2000. Redox potential measurements ofthe Mycobacterium tuberculosis heme protein KatG and the isoniazid-resis-tant enzyme KatG(S315T): insights into isoniazid activation. Biochemistry39:11508–11513.

43. Wilson, T., G. W. de Lisle, J. A. Marcinkeviciene, J. S. Blanchard, and D. M.Collins. 1998. Antisense RNA to ahpC, an oxidative stress defence geneinvolved in isoniazid resistance, indicates that AhpC of Mycobacterium bovishas virulence properties. Microbiology 144:2687–2695.

44. Yu, K., C. Mitchell, Y. Xing, R. S. Magliozzo, B. R. Bloom, and J. Chan.1999. Toxicity of nitrogen oxides and related oxidants on mycobacteria: M.tuberculosis is resistant to peroxynitrite anion. Tuber. Lung Dis. 79:191–198.

45. Yuan, Y., R. E. Lee, G. S. Besra, J. T. Belisle, and C. E. Barry III. 1995.Identification of a gene involved in the biosynthesis of cyclopropanated

4038 MASTER ET AL. J. BACTERIOL.

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

mycolic acids in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA92:6630–6634.

46. Zahrt, T. C., and V. Deretic. 2000. An essential two-component signal trans-duction system in Mycobacterium tuberculosis. J. Bacteriol. 182:3832–3838.

47. Zahrt, T. C., J. Song, J. Siple, and V. Deretic. 2000. Mycobacterial FurA isa negative regulator of catalase-peroxidase gene katG. Mol. Microbiol. 39:1174–1185.

48. Zhang, Y., S. Dhandayuthapani, and V. Deretic. 1996. Molecular basis for

the exquisite sensitivity of Mycobacterium tuberculosis to isoniazid. Proc.Natl. Acad. Sci. USA 93:13212–13216.

49. Zhang, Y., B. Heym, B. Allen, D. Young, and S. Cole. 1992. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis.Nature 358:591–593.

50. Zou, P., I. Borovok, D. Ortiz de Orue Lucana, D. Muller, and H. Schrempf.1999. The mycelium-associated Streptomyces reticuli catalase-peroxidase, itsgene and regulation by FurS. Microbiology 145:549–559.

VOL. 183, 2001 IN VIVO M. TUBERCULOSIS katG PROMOTER ACTIVITY 4039

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from