INTRODUCTION

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Despite the seriousness of tuberculosis in the world, efforts in prevention and control of this disease suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. A variety of mechanisms contribute to the survival of Mycobacterium tuberculosis in the host, and protection against oxidative stress is one of the primary defenses (1). However, little is known about regulation and expression of the genes involved in oxidative stress resistance. Most bacteria, exposed to toxic oxygen metabolites, exhibit an adaptive response, expressing several genes involved in detoxification of oxygen species (4). The pathogenic mycobacteria have a limited, nonprotective oxidative stress response, which in the case of M. tuberculosis is restricted to induction of a single gene, katG, encoding a catalase-peroxidase (14, 28). Consistently, the KatG protein is an important virulence factor in M. tuberculosis, since its expression is required for growth and persistence in mouse and guinea pig model systems (17). The importance of a better understanding of the elements involved in katG regulation is increased by its implication in the innate susceptibility and acquired resistance to the front-line antituberculosis drug isoniazid (isonicotinic acid hydrazide) (9, 33). Indeed, inactivation of katG is frequently found among isoniazid-resistant M. tuberculosis strains (32).

Response to oxidative stress in mycobacteria differs from the OxyR-dependent induction observed in gram-negative bacteria (25). Indeed, both M. tuberculosis and Mycobacterium smegmatis lack a functional oxyR gene (6, 8, 31). In all the mycobacterial species studied, the DNA region immediately upstream of the katG genes is highly conserved and encodes the furA gene, suggesting a putative involvement of the FurA protein in katG regulation. Recently, Zahrt et al. (31) demonstrated that in M. smegmatis a knockout furA mutant overexpressed the catalase-peroxidase KatG and that this altered phenotype is complemented by expression of FurA; thus, FurA appears to be a negative regulator of katG.

Fur-like proteins are transcriptional repressors that exhibit a Fe²⁺-dependent DNA binding activity and regulate several genes involved in iron metabolism (10). There is an intimate relationship between iron metabolism and oxidative stress: indeed, the cytotoxic effects of reactive oxygen species are largely mediated by iron, and it has been shown elsewhere that regulators of Escherichia coli involved in oxidative stress response, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric iron uptake (34). E. coli fur mutants are more sensitive to hydrogen peroxide than are Fur-proficient strains (30). In M. smegmatis, hydrogen peroxide sensitivity is increased by iron starvation (18); in Staphylococcus aureus, Fur is necessary for oxidative stress resistance (15); and in Streptomyces spp., Fur-like proteins regulate catalase-peroxidase genes in an iron-dependent manner (13, 35).

In this work, using M. smegmatis as a model system, we investigated transcription of furA and katG after oxidative stress. Specific transcripts and promoters were identified for furA and katG, respectively. Although transcription of both genes was induced upon oxidative stress, an inducible promoter could be identified only upstream of furA. A similar transcriptional regulation was found in the furA-katG region of M. tuberculosis.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. E. coli XL1-Blue (3) was grown in LD medium (26), supplemented when necessary with kanamycin (50 µg/ml). M. smegmatis mc²155 was grown in LD medium containing 0.2% (vol/vol) glycerol and 0.05% (vol/vol) Tween 80 and supplemented when necessary with kanamycin (20 µg/ml) or spectinomycin (100 µg/ml).

Cloning and sequencing of M. smegmatis furA gene. A cosmid library of M. smegmatis mc²155 was produced by standard procedures in the vector Tropist4 (7). The library was screened using a 380-bp DNA internal to the katG coding region, obtained by PCR amplification with a couple of primers (218 and 219 [see Fig. 1]) based on the published sequence (GenBank accession no. U46844).

Construction of plasmids. The pSG10ter vector was derived from pSG10 (11) by inserting the E. coli Rho-independent  terminator (24) upstream of the reporter gene luxAB, in order to reduce basal luciferase expression. Plasmid pSM128 is an integrative vector carrying the lacZ reporter gene (29). Plasmid pSG10ter and pSM128 derivatives, containing DNA fragments from either M. smegmatis (MS3, MYS42, MYS44, MYS48, MYS65, and MYS66) or M. tuberculosis (MT3, MYT43, and MYT45), were obtained by PCR amplification of the regions (the oligonucleotide sequences will be provided upon request). All the inserted fragments were sequenced.

RNA extraction, Northern blotting, and primer extension analysis. Fifteen milliliters of M. smegmatis mc²155 cultures (optical density at 600 nm = 0.8), untreated or treated with H₂O₂ for 1 h at 37°C at the concentrations indicated in Fig. 1, was pelleted and resuspended in 100 µl of Tris-EDTA buffer; 75 µl of RNA lysis buffer (4 M guanidinium thiocyanate, 0.01 M Tris [pH 7.5], 0.97% -mercaptoethanol) was added; and the suspension was sonicated (20 s at 40 W). The RNA was then purified using the SV Total RNA Isolation System (Promega). Twenty micrograms of each RNA sample was fractionated, blotted, and hybridized as described previously (5). The ³²P-labeled riboprobes furA and katG were prepared by SP6 polymerase transcription of pGEMT-easy (Promega) derivatives, carrying the M. smegmatis 349-509 (oligonucleotides 240 and 256) and 724-1113 (oligonucleotides 218 and 219) DNA regions, respectively (see Fig. 1). The oligonucleotide probes (indicated in Fig. 1) were 5' end labeled with T4 polynucleotide kinase as described by Sambrook et al. (27). Potential transcription start sites in the M. smegmatis furA-katG region were identified by primer extension analysis (27), using the oligonucleotides indicated.

Reverse transcriptase PCR (RT-PCR). Two micrograms of RNA extracted from M. smegmatis mc²155 mock treated and treated for 1 h with either 0.6 or 20 mM H₂O₂ was retrotranscribed with Moloney murine leukemia virus reverse transcriptase (Promega). Samples (50 ng) were amplified with oligonucleotides 254 and 219 and, as a control, with oligonucleotides 218 and 219 and oligonucleotides 240 and 271. The presence of an amplified DNA was evaluated both by ethidium bromide staining and by Southern blot hybridization.

Luciferase assay. Independent cultures of M. smegmatis mc²155 carrying the plasmids indicated in Table 1 were grown in LD medium at 37°C. The turbidity of the cultures at 600 nm was adjusted to 0.03, and the cultures were sampled and either mock treated or treated with H₂O₂ (final concentration, 0.125 mM) for 1 h at 37°C. One hundred microliters of 1% (vol/vol) decyl aldehyde solution (Sigma-Aldrich) in ethanol was then added to 1 ml of a 1:10 dilution of the culture, and luminescence was measured over 10 s, using a Stratec luminometer. At least three independent clones of each construct were tested.

-Galactosidase assay. The cultures were grown as described for the luciferase assay to an optical density at 600 nm of 1.7 and mock treated or treated for 1 h with 0.5 mM H₂O₂ at 37°C, and the cells were disrupted by sonication. -Galactosidase activity of the extracts was measured as described in reference 20. The enzyme activity was expressed as nanomoles of o-nitrophenol--D-galactopyranoside converted to o-nitrophenol minute¹ milligram of protein¹.

Nucleotide sequence accession number. The sequence upstream of katG was determined and deposited in the GenBank database under accession number AF196484.

	RESULTS

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Cloning and sequencing of M. smegmatis furA. The katG region of M. smegmatis was identified by screening a cosmid library with an internal katG PCR fragment as a probe (see Materials and Methods). Sequence inspection of this region revealed the presence of an open reading frame predicted to encode a polypeptide of 153 amino acids, with a calculated molecular mass of 17 kDa, homologous to Fur-like proteins. The gene was named furA, by analogy with other fur genes located upstream of katG in mycobacteria (23) (Fig. 1A). A Shine-Dalgarno sequence is present upstream of katG, while no typical Shine-Dalgarno sequence was identified upstream of furA.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Transcription of the furA-katG region in M. smegmatis. (A) Map of the region. The furA-katG map of M. smegmatis is drawn to scale according to the sequence in the GenBank database with accession no. AF196484 (coordinate 1 corresponds to nt 245 of the deposited sequence). The genes are indicated by boxes. The potential Rho-independent termination site, downstream of katG, is shown. The transcripts are indicated by arrows, and the 5' ends are indicated by vertical arrows below the map. The positions of the riboprobes and oligonucleotides used for Northern blotting, primer extension analysis, and RT-PCR are reported. (B to E) Northern blotting analysis of furA-katG transcription upon oxidative stress. RNAs were extracted from a culture of M. smegmatis mc2155 treated for 1 h with the millimolar concentration of hydrogen peroxide indicated on the top of the lanes, and equal amounts of each sample (20 µg) were separated by agarose (B, C, and D) or polyacrylamide (E) gel electrophoresis, blotted, and hybridized to the different probes. (B) Riboprobe katG (coordinates 724 to 1113); (C and E) riboprobe furA (coordinates 332 to 511); (D) the same filter used in panel C was hybridized successively to oligonucleotides 554 (coordinates 366 to 385), 553 (coordinates 715 to 742), and 244 (coordinates 803 to 822). Molecular size markers, run in the same gel, are indicated on the left. For panels C and D, probe 554 was exposed about three times longer. In agarose gels, two nonspecific bands (about 1.5 and 3 kb) were observed after prolonged exposure of the filters (particularly visible in panels C and D, oligonucleotide 554). These bands are likely to be furA-katG RNA entrapped by rRNA (5). (F) Primer extension of 5' ends of furA and katG transcripts. The primer extension experiments were performed with the 554 (furA) and 244 (katG) oligonucleotides. Sequence reactions, performed with the same oligonucleotides on MS3 DNA (Table 1), are reported in the first four lanes of the two panels. The coordinates of the 5' ends are reported on the left of each panel.

Transcription of furA-katG in M. smegmatis upon oxidative stress. Transcription of katG and furA in M. smegmatis in response to oxidative stress was analyzed by Northern blotting, and the transcripts were mapped by hybridization with different probes. The RNAs were extracted from mc²155 cultures after 1 h of treatment with different concentrations of hydrogen peroxide (Fig. 1B, C, D, and E). The katG coding sequence is 2,223 nucleotides (nt) long. A single transcript, about 2.3 kb long, hybridized with the katG riboprobe (Fig. 1B). The same transcript could be observed with the 244 probe, internal to katG, and with the 553 probe, encompassing the 3' end of the furA coding sequence (Fig. 1D). With the furA riboprobe and with the 554 oligonucleotide, both mapping in the 5' half of the furA coding sequence, the 2.3-kb transcript was not detected, even after prolonged exposure of the filter (Fig. 1C and D). Thus, the katG transcript extended to the intergenic region between furA and katG but did not cover the whole furA gene. Immediately downstream of katG, a potential Rho-independent termination site was identified by the TERMINATOR program of the GCG package (data not shown).

Identification of promoter regions upstream of katG and furA. The 5' ends of the transcripts were mapped by primer extension. Using an oligonucleotide internal to the furA coding sequence (oligonucleotide 554 in Fig. 1A), a unique 5' end was identified at nt 271 (Fig. 1F), 1 base upstream of the translation start codon of furA. The intensity of the signal increased 9- to 12-fold upon oxidative stress. No other signals could be identified further upstream, neither with this oligonucleotide nor with an oligonucleotide located upstream of furA (oligonucleotide 729 in Fig. 1A; data not shown).

View this table: [in this window] [in a new window]   TABLE 1.   Expression of luciferase and -galactosidase from M. smegmatis and M. tuberculosis DNA fragments

Promoters of furA and katG in M. tuberculosis. Fragments of M. tuberculosis DNA of the furA-katG region were cloned in pSG10ter. M. smegmatis mc²155 was transformed with the plasmids, and luciferase activity was determined (Table 1). The DNA regions cloned were homologous to the corresponding M. smegmatis fragments, except for the first 113 nt of MT3. The expression of luciferase activity from the different plasmids indicated that in M. tuberculosis furA-katG transcription is regulated in a way similar to that found in M. smegmatis: an inducible pfurA promoter is located immediately upstream of furA, where 35 (TTGACT) and 10 (TATTGT) sequences were identified. Deletion of the 35 region eliminated inducibility upon oxidative stress (compare MYT43 and MYT45), although a basal level of luciferase activity was still observed.

	DISCUSSION

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Expression of the furA-katG region in M. smegmatis. We have cloned and sequenced the M. smegmatis region upstream of katG. It contains the furA gene, which codes for FurA, one of the two Fur-like proteins identified in the M. tuberculosis genome. Zahrt et al. (31) have recently sequenced the same region of M. smegmatis (GenBank accession no. AF012631). The FurA proteins deduced from the two sequences differ for some residues, with FurA deduced from our sequence presenting a higher similarity to M. tuberculosis FurA. The M. smegmatis FurA protein conserves both the helix-turn-helix DNA binding domain (35) and the metal binding domain (2), suggesting that this protein might carry out regulatory functions in response to stress conditions such as iron depletion or oxidative stress.

Transcription of furA. Four transcripts covering furA were detected with a riboprobe encompassing the 5' half of furA. The two longest ones cover the entire furA coding sequence, whereas the two shorter terminate before the end of the gene. Thus, they may either represent stable intermediates of the processed furA transcript or be produced by premature transcription termination.

Transcription of katG. Two possible 5' ends were identified for the 2.3-kb katG transcript. This suggests the presence of two mRNAs, differing by 35 bp in length, that were not resolved by agarose gel electrophoresis. The transcript starting at nt 713 might be partially processed, generating the nt 748 5' end. Alternatively, transcription may start at both sites. Similarly, for M. tuberculosis Mulder et al. (21) identified three potential 5' ends for katG transcripts, two of them internal to the furA coding sequence and one in the intergenic region, whereas Master et al. (19) identified a single 5' end internal to furA, 54 nt upstream of the katG coding sequence.