Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital, Medical School, Observatory, 7925 Cape Town, South Africa1
Author for correspondence: Lafras M. Steyn. Tel: +27 21 406 6363. Fax: +27 21 448 8153. e-mail: lsteyn{at}medmicro.uct.ac.za
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ABSTRACT |
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Keywords: Mycobacterium tuberculosis, katG, promoter, regulation
Abbreviations: ADC, albumin dextrose catalase; RLU, relative light units; tss, transcription start site; UAR, upstream activator region
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INTRODUCTION |
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The isolation of plasmids which replicate in the mycobacteria, for example pAL5000 from Mycobacterium fortuitum (Labidi et al., 1985 ), has facilitated the construction of vectors for studying mycobacterial gene expression in a homologous host (Snapper et al., 1988
; Aldovini & Young, 1991
; Stover et al., 1991
; Barletta et al., 1992
). The surrogate host of choice is Mycobacterium smegmatis because of its non-pathogenicity and rapid growth. The M. smegmatis RNA polymerase has been purified and used for transcription initiation in vitro (Levin & Hatfull, 1993
). The major form of this enzyme has marked conservation with that of E. coli (Predich et al., 1995
), which suggests some common mechanisms of transcription initiation.
The M. tuberculosis katG gene encodes a dual-function catalase-peroxidase enzyme which protects the cell against excess hydrogen peroxide and, therefore, contributes to its survival in macrophages (Middlebrook & Cohn, 1953 ; Mitchison et al., 1960
; Jackett et al., 1978
; Wilson et al., 1995
; Heym et al., 1997
). Another important feature of the M. tuberculosis katG gene is its well-documented association with susceptibility to isoniazid (e.g. Winder, 1960
; Zhang et al., 1992
; Heym et al., 1993
; Rouse & Morris, 1995
). Examination of the mechanisms of regulation of this gene may, therefore, contribute to our knowledge of both the interaction of the organism with its host and the conditions which lead to resistance to isoniazid. We report here on the characterization of the M. tuberculosis katG promoter region and examination of the conditions under which katG is expressed in an M. smegmatis host.
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METHODS |
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DNA manipulations.
Standard recombinant DNA methods were performed by previously described protocols (Ausubel et al., 1987 ; Sambrook et al., 1989
). DNA fragments were sequenced by the dideoxy sequencing protocol (Sanger et al., 1977
) using a T7 Polymerase Sequencing kit (Pharmacia), pUC19 forward and reverse sequencing primers (Promega), and a synthetic oligonucleotide complementary to the 5' end of the luciferase gene, 5'-CTTTATGTTTTTGGCGTC-3'.
Isolation of genomic DNA and PCR.
Genomic DNA was purified from M. tuberculosis H37Rv as described by Jacobs et al. (1991 ). PCR amplification of the katG upstream sequences was performed using M. tuberculosis H37Rv genomic DNA (0·1 µg) as the template with the primers 5'-CTGGTAAGCttGGCCGCAAAACAGC-3' and 5'-CACAGggaTCCTTCCAGGAGTTGGT-3' (based on the published sequence, GenBank accession no. X68081). The lower-case nucleotides indicate mismatches and the underlined nucleotides represent the restriction sites for HindIII and BamHI, respectively. The amplifications were performed using Taq polymerase according to the manufacturers specifications, except that DMSO was added to a final concentration of 10% (v/v).
Plasmid constructions.
The plasmids constructed in this study are listed in Table 1. Plasmid pJCluc was constructed by cloning the 1748 bp HindIIIStuI fragment from pGEM-luc (GenBank/EMBL accession no. X65316), containing the promoterless firefly luciferase gene, into the KpnI and HindIII sites of pJC86 (Fig. 1
). A 1943 bp PCR product containing the region upstream of the M. tuberculosis katG gene was digested with BamHI and HindIII and cloned into the corresponding restriction sites of pJCluc to yield plasmid pK10. A 559 bp SmaIBamHI fragment of the PCR product, immediately upstream of the katG gene, was cloned upstream of the luciferase gene in pJCluc to produce pK20. Various deletion derivatives of pK20 were constructed, making use of the restriction sites present in the insert. These are summarized in Table 1
and Fig. 2.
The construct pANIIIW (a gift from Karen Kempsall, Glaxo-Wellcome), consisting of a 170 bp PCR fragment containing the PAN promoter cloned into the T-tailed EcoRV restriction site of the vector pT7Blue (Novagen), was used as a source of the M. paratuberculosis promoter (Murray et al., 1992
). The 218 bp HindIIIBamHI fragment from pANIIIW was cloned into the corresponding restriction sites of pJCluc to form pLPan. In this construct, the PAN promoter is in the correct orientation to promote expression of the luciferase gene. The 262 bp HindIIISphI fragment of pK20 containing the katG upstream activator region (UAR) was cloned upstream of the PAN promoter in pLPan to form pLPuar. The structures of all the above constructs were confirmed by DNA sequencing, using the pUC19 forward sequencing primer (Promega) and the luciferase primer described above.
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Electroporation.
M. smegmatis cells were prepared for electroporation as described previously (Jacobs et al., 1991 ). For electroporation, 60 µl cells were combined with 140 µl 10% (v/v) glycerol and 1 µg DNA (5 µl), and placed on ice for 5 min. Electroporations were performed using 0·1 cm gap Gene Pulser cuvettes (Bio-Rad) at 1·8 kV, 25 µF and 1000
, using a Gene Pulser apparatus (Bio-Rad). After electroporation, 900 µl Middlebrook-ADC was added, and the cells were incubated at 37 °C for 34 h before plating. Plates were incubated at 37 °C for 45 d. Electrocompetent M. bovis BCG cells were prepared and electroporated using the method of Jacobs et al. (1991
), with minor modifications. A stationary-phase culture of M. bovis BCG grown in Middlebrook-ADC-Tween was diluted 100-fold in freshly prepared Middlebrook-ADC-Tween, and grown to mid-exponential phase (8 d) in a roller culture at 37 °C. Cells were harvested by centrifugation at 4 °C and washed five times in sterile deionized water to remove all salts and residual medium. Washed cells were resuspended in a final volume of 1 ml ice-cold 10% (v/v) glycerol per 100 ml cells (original volume). Electroporations were performed as described for M. smegmatis. The cells were kept on ice for 5 min post-electroporation. Middlebrook-ADC (700 µl) was added and the cells were incubated for 34 h at 37 °C. The cells were plated on Middlebrook-ADC agar plates containing 30 µg kanamycin ml-1 and 0·01% (w/v) cycloheximide (Fluka Biochemicals), and incubated at 37 °C for 34 weeks.
Preparation of cell extracts and luciferase assays.
For the luciferase activity assays, 5 ml cultures were grown to late exponential phase at 37 °C with aeration (Orbital shaker, Hägar Designs, at 150 r.p.m.). E. coli cell extracts were prepared by harvesting cells from 1 ml of culture and resuspension of the cells in 100 µl ice-cold 1x Cell Culture Lysis Reagent (Luciferase Assay System, Promega). The cells were lysed by sonication in ice water (three bursts of 40 s each) in a Branson 1200 ultrasonic cleaner. Cell debris was removed by centrifugation, and the supernatant equilibrated to room temperature before being assayed for luciferase activity. Luciferase activity assays were performed using the Promega Luciferase Assay System, according to the manufacturers instructions. Light output was measured in a Bio-Orbit 1253 luminometer. M. smegmatis cell extracts were prepared by resuspension of cell pellets in Cell Culture Lysis Reagent, followed by processing in a FastPrep FP120 instrument (Savant Instruments) for two 40 s cycles at a setting of 6. Unlysed cells and cell debris were removed by centrifugation. The supernatants were assayed for luciferase activity as described above. The protein concentrations of the cell extracts were measured using the DC Protein Assay kit (Bio-Rad), and the luciferase activities are quoted as relative light units (RLU) per mg protein used in each assay.
RNA extractions and primer extension analysis.
RNA was extracted from E. coli and M. smegmatis using the FastPrep system [FastPrep FP120 instrument (Savant Instruments) and FastRNA Kit-Blue (Bio101)], according to the manufacturers instructions. Potential transcription start sites of the M. tuberculosis katG gene were identified by primer extension analysis (Sambrook et al., 1989 ). A complementary 18-mer primer (5'-GCAGTTGCTCTCCAGCGG-3') was designed to anneal 58 bp downstream of the ATG initiation codon of the luciferase gene. Approximately 100 ng primer was end-labelled with [
-32P] dATP using polynucleotide kinase, in a 30 µl reaction with the supplied buffer. After incubation at 30 °C for 30 min, the unincorporated nucleotides were removed using a Promega G25 spin column. RNA samples (100 µg) were precipitated with sodium acetate and ethanol, resuspended in 30 µl RNA hybridization buffer (Ambion), and added to 25 ng labelled primer. The nucleic acids were denatured at 85 °C for 10 min, and annealed overnight at 50 °C. The annealed RNA was precipitated with sodium acetate and ethanol, and resuspended in diethyl pyrocarbonate (DEPC)-treated water. The primer was extended on the mRNA template at 37 °C for 60 min, using 200 units of M-MLV reverse transcriptase (Promega), in a reaction containing 40 units RNase inhibitor, 1x Reverse Transcriptase buffer (Promega), and an equal mixture of dNTPs (1 mM each) (Boehringer Mannheim). The primer extension products were precipitated and resuspended in the Stop buffer supplied in the Pharmacia T7 Sequencing kit. The products were denatured at 90 °C for 2 min and separated on a 6% (w/v) polyacrylamide sequencing gel adjacent to dideoxy sequencing reactions primed with the same 18-mer primer. Gels were dried and the bands visualized by autoradiography.
ATP assays.
ATP concentration was measured as an indication of cell mass using the Promega Enliten luciferase/luciferin bioluminescence detection reagent in a Bio-Orbit 1253 luminometer. The ATP levels were monitored by measuring the production of light when ATP, luciferin and oxygen were combined in the presence of luciferase. The assay system was calibrated using an ATP standard of known concentration (Prioli et al., 1985 ; Stanley et al., 1989
). The M. smegmatis cells were treated with 1·4% (w/v) (final concentration) trichloroacetic acid (TCA), and incubated on ice for 30 min. The precipitation was terminated by the addition of 50 µl neutralization buffer (1 M Tris/acetate, pH 7·75) and 622 µl distilled water. The ATP assays were performed according to the manufacturers instructions. At higher cell densities, the TCA-treated samples were diluted for the assay and the calculation adjusted accordingly.
Conditions of expression of katG.
Stationary-phase cultures of M. smegmatis/pK20 were diluted 100-fold into 500 ml Middlebrook-ADC-Tween, and incubated at 37 °C with shaking. Growth was monitored using OD600 readings to measure cell density and ATP concentration to measure cell mass. Samples were removed at various times and assayed for luciferase activity. To test the effect of various stresses on promoter activity, an M. smegmatis/pK20 culture (800 ml) was grown to mid-exponential phase (24 h), and divided into eight 100 ml cultures. The cells were harvested by centrifugation and resuspended in Middlebrook-ADC-Tween with the following additions per individual culture: (1) 10 mM hydrogen peroxide; (2) 10 mM ascorbic acid; (3) 60 mM sodium acetate (pH 7·0); and (4) 200 µM of the iron chelator 2,2'-dipyridyl. In the fifth culture, the pH was adjusted to 4·0 with glacial acetic acid. The remaining three cultures were resuspended in medium with no additions. One of these was heat-shocked at 45 °C. One was incubated in a 100 ml bottle with no head volume and without agitation, to simulate oxygen limitation (Wayne & Sramek, 1994 ), and the cells that settled to the bottom of the bottle were used as the anaerobic sample. The remaining culture served as the control. Unless otherwise stated, the cultures were incubated at 37 °C with shaking. After 4 h, samples were removed and assayed for luciferase activity. Preliminary experiments showed that a heat shock response occurs in M. smegmatis within the first hour, whereas it takes between 3 and 6 h for the cells to respond to cold shock (unpublished data, this laboratory). A time period of 4 h for exposure to the stresses in this experiment was, therefore, chosen to accommodate these differences in response time and to allow for lag phases.
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RESULTS |
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Amplification and expression analysis of the M. tuberculosis katG promoter
Plasmids pK10 and pK20, containing different-sized fragments of the katG upstream region (Table 1), were tested for luciferase activity in M. smegmatis and E. coli. For M. smegmatis/pK10 and M. smegmatis/pK20, the luciferase activities were 41800±2400 and 38500±10700 RLU (mg protein)-1, respectively. Those for E. coli/pK10 and E. coli/pK20 were 381±100 and 474±13 RLU (mg protein)-1, respectively. These results indicate that the katG promoter lies within the amplified fragment and is recognized by the E. coli and M. smegmatis transcription apparatus. They also suggest that the promoter region lies within the smaller SmaIBamHI fragment, since the luciferase activities of cells harbouring pK10 and pK20 do not differ significantly for either E. coli or M. smegmatis . Thus it appears that no sequences required for optimal expression are located in the 1374 bp region upstream of the SmaI restriction site. The smaller construct, pK20, was therefore used for all further studies.
Mapping of the transcription start site by primer extension analysis
Primer extension analysis was performed using an antisense strand primer complementary to sequences near the 5' end of the luciferase gene, and total RNA isolated from late-exponential-phase M. smegmatis/pK20 and E. coli/pK20 cells. In both E. coli and M. smegmatis, transcription was initiated at two sites located 47 bp (transcription start site A, tssA) and 56 bp (tssB) upstream of the translation initiation codon (Fig. 3). The possibility that the bands are due to a premature stop site of the reverse transcription reaction can be excluded, as sequencing of this region revealed no signs of band compressions, suggesting no tendency for secondary structure formation. The putative -10 and -35 sequences relative to these transcription start sites are represented in Fig. 4.
The putative -10 hexamer for tssA, GACACT, lies 8 bp upstream of the transcription start site and shows a high degree of similarity to other mycobacterial -10 sequences. The first G of the hexamer is the least conserved. Associated with this -10 hexamer are three possible -35 sequences: TCTATG 19 bp upstream, TGTCCT 15 bp upstream, and TCCTGA 13 bp upstream. The putative -10 hexamer for tssB, GATATC, lies 6 bp upstream of the transcription start site. Again, the first G of the hexamer is the least conserved. Associated with this -10 region are two possible -35 sequences, TCTACT 22 bp upstream, and TACTGG 20 bp upstream.
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Deletion analysis of the katG promoter fragment using nuclease BAL-31
In a further attempt to locate the sequences required for promoter activity, the pK20 insert was progressively deleted from the HindIII restriction site at the 5' end, using nuclease BAL-31. Five recombinants were selected and sequenced to determine the size of the deletions. Since the activity of BAL-31 is bidirectional, the construct pK20 HS, containing the 300 bp SphIBamHI katG promoter region in pJCluc, was used to determine whether the deletion of vector sequences was responsible for the observed differences in luciferase activity. The plasmid pK20
SB was constructed to determine whether the UAR on its own was able to promote expression of the luciferase gene. E. coli and M. smegmatis cells harbouring the deletion plasmids, as well as the undeleted and vector controls, pK20 and pJCluc, respectively, were tested for luciferase activity (Fig. 2
). Removal of the first 18 bp (pK22) of the insert resulted in an approximately 40% reduction in promoter activity, while the removal of 123 bp from the 5' end (pK23) resulted in a sixfold reduction in activity in M. smegmatis. Removal of a further 160 bp (pK25) resulted in an 11-fold reduction in activity in this host. Similar results were obtained for E. coli. These results show that the region up to 123 bp from the 5' end of the insert is required for maximal promoter activity in both hosts. The region between 123 and 283 bp from the 5' end is not additionally important. Deletion plasmid pK21 has no insert sequences deleted, but has 300 bp of vector sequence removed. The construct exhibited similar luciferase activities to the parent plasmid, pK20, suggesting that deletion of at least the first 300 bp of vector sequence upstream of the katG promoter fragment has no effect on promoter activity. In addition the construct pK20
HS, in which no vector sequences are deleted but 262 bp of insert are removed, exhibited a similar decrease in luciferase activity to pK23, pK24 and pK25. These results indicate that the decreases in luciferase activity are due to removal of essential insert fragments, rather than vector sequences. The background luciferase activity of cells harbouring pK20
SB suggests that the 262 bp HindIIISphI UAR contains no promoter sequences capable of promoting expression of the luciferase gene. The MIC of kanamycin for M. smegmatis and E. coli cells harbouring a selection of the restriction-endonuclease- and BAL-31-generated deletion constructs was determined as a measure of the relative copy number of the relevant plasmids (Stolt & Stoker, 1996
) (results not shown). There were no significant differences in the MICs of the cells harbouring different deletion constructs in either of the two species. Thus it is unlikely that the effects of the deletions on the luciferase activities observed were due to differences in the plasmid copy number.
Primer extension analysis of selected deletions
Primer extension analysis was performed on total RNA extracted from M. smegmatis/pK20 HS, M. smegmatis/pK20
EN and M. smegmatis/pK20
SE, in order to identify alternative promoter sequences that allowed expression of the luciferase gene. In all three cases, transcription initiated at a C residue located 23 bp upstream of the translation initiation codon (Fig.
5). Therefore, in the absence of either the UAR or the usual promoter sequences, a third region, designated PC, is recognized as a promoter. Located 7 bp upstream of transcription start site C is the putative -10 hexamer, CACAGC (Fig. 4b
). Associated with this are two putative -35 regions: TTCGCG, 14 bp upstream, and TCCGAC, 22 bp upstream.
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Transformation of M. bovis BCG with the pJCluc constructs and luciferase activity assays
The following constructs were electroporated into M. bovis BCG and tested for luciferase activity: pJCluc, pK20, pK20 HS, pK20
SB, pK24, pLPan and pLPuar (Table 2,
Fig. 2
). The background luciferase activity resulting from transcriptional readthrough was low in M. bovis BCG/pJCluc. The activity of the katG promoter in this host was, however, comparable to that in M. smegmatis. Removal of the katG UAR (pK20
HS) resulted in a 78-fold decrease in luciferase activity, indicating that the region also plays an important role in promoter activity in this host. The nuclease BAL-31 deletion plasmid, pK24, exhibited an eightfold decrease in luciferase activity relative to pK20. This plasmid contains an extra 93 bp of insert sequence relative to pK20
HS, which explains the smaller reduction in luciferase activity. The low background levels of luciferase activity resulting from pK20
SB indicates, as shown before, that the HindIIISphI UAR of katG contains no active promoter sequences. The activity of the M. paratuberculosis PAN promoter increased fourfold in the presence of the katG UAR. This further confirms the importance of the region in enhancing transcription in this host and indicates that similar sequences are required for expression of the promoter in a slow-growing host.
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DISCUSSION |
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Two transcription start sites were identified for the M. tuberculosis katG promoter in both E. coli/pK20 and M. smegmatis/pK20. This suggests that the gene is transcribed from two promoters and that the same promoter sequences are recognized by both hosts. The presence of two promoters upstream of mycobacterial genes is not uncommon and has been reported previously (Stover et al., 1991 ; Suzuki et al., 1991
; Dhandayuthapani et al., 1997
; Movahedzadeh et al., 1997
). It is possible that the promoters are recognized by different RNA polymerase holoenzymes, and that they are utilized to different extents during growth. Removal of the sequence between the ribosome-binding site and tssB of the katG promoter results in a 4550% reduction in luciferase activity (unpublished data, this laboratory). This suggests that the promoters contribute equally to katG expression under the conditions of the above experiment. In the absence of either the UAR, or the putative katG promoters, PA and PB, M. smegmatis utilizes a third promoter, PC. It is possible that a similar situation occurs in E. coli. This promoter functions as efficiently as the other two in M. smegmatis; however, the presence of the UAR is also essential for optimal activity. If promoter PC is used to promote expression of the luciferase gene in E. coli/pK20
EN and E. coli/pK20
SE, then it is evidently not as efficient as promoters PA and PB in this host, since these strains exhibit a threefold lower luciferase activity than E. coli/pK20.
The putative -10 and -35 sequences identified for the katG promoters show significant homology to many mycobacterial promoters characterized thus far, and may, therefore, be typical mycobacterial promoters (Mulder et al., 1997 ). These sequences also all match the consensus E. coli sequences in at least one of the three highly conserved positions. The promoters are therefore probably recognized and bound by E
70 in E. coli. The spacing between the -10 and -35 regions, however, differs from that found in most E. coli promoters (17±1) (Harley & Reynolds, 1987
). This, together with the divergence from the consensus sequences, may be responsible for the poor efficiency of the promoters in E. coli. It has been noted that the distance between the -10 and -35 region is not critical in mycobacterial promoters (Kremer et al., 1995
), and varies between 13 and 24 bp (Ramesh & Gopinathan, 1995
). All three of the katG -10 hexamers are least conserved in the first position, where the T is replaced by a G or C. It has previously been reported that less conserved bases in M. tuberculosis promoters tend towards G and C substitutions (Bashyam et al., 1996
).
Primer extension analysis using total RNA isolated from M. tuberculosis was unsuccessful, possibly due to low levels of the katG mRNA. The experiment was therefore performed using RNA isolated from M. smegmatis cells harbouring various katG promoter clones. The determination of transcription start sites for M. tuberculosis genes, using this heterologous host, has been shown to correlate well with results obtained from native mRNA transcripts (Levin & Hatfull, 1993 ; Dhandayuthapani et al., 1997
; Movahedzadeh et al., 1997
), and has successfully been used as a substitute in many cases (Murray et al., 1992
; Kremer et al., 1995
; Nesbit et al., 1995
).
As mentioned above, the first nucleotide of all three putative katG -10 hexamers deviates from the consensus E. coli sequence. E. coli promoters which deviate in this position generally require activators for efficient transcription initiation. These activators normally bind to sites at various distances upstream of the -35 region (Raibaud & Schwartz, 1984 ;Zhou et al., 1994a
, b
).
J. Song & V. Deretic (GenBank accession no. AF002194) and Pagan-Ramos et al. (1998 ) have identified an open reading frame with homology to the Fur protein upstream of the M. tuberculosis and M. marinum katG genes, respectively. The FurA translation start codon lies immediately downstream of the 24 bp AT-rich sequence described above, while the stop codon lies immediately downstream of the katG tssA. This overlap of the FurA ORF with the katG promoter region and UAR may have implications for the regulation of both genes and suggests a possible coupling of the regulation of oxidative stress and iron metabolism genes in this organism.
The growth-phase-dependent expression of the M. tuberculosis katG promoter in M. smegmatis is similar to that in other bacteria. Many bacterial catalases are produced at low levels during exponential growth and are induced either during late exponential phase or during the transition to stationary phase (Mukhopadhyay & Schellhorn, 1994 ; Rocha & Smith, 1995
; Schnell & Steinman, 1995
). In E. coli, the induction of katG as the cells enter stationary phase is dependent on the stationary-phase or starvation response
factor, KatF or RpoS (Loewen et al., 1985
; Mulvey et al., 1988
; Mukhopadhyay & Schellhorn, 1994
).
The effects of host cell stress on expression from the katG promoter were also investigated in this study. Mycobacterial cells were stressed during late exponential phase, at the time corresponding to maximum expression. The repression of the katG promoter under anaerobic conditions has been noted for other catalases (Morgan et al., 1986 ), and is probably due to a reduced requirement for the enzyme. The factor(s) responsible for this repression, and the repression of the promoter at elevated temperatures, have not been identified. In E. coli, treatment with 5·7 mM ascorbic acid results in an eightfold increase in catalase activity (Richter & Loewen, 1981
), while treatment with 080 mM sodium acetate (pH 7·0) induces catalase activity sevenfold (Mukhopadhyay & Schellhorn, 1994
). The treatment of M. smegmatis/pK20 with 10 mM ascorbic acid, however, resulted in less than twofold induction of the M. tuberculosis katG promoter, while no induction was observed in response to sodium acetate. These results suggest that there are differences in the regulation of the gene in the two organisms. The lack of induction of the M. tuberculosis katG promoter in the presence of hydrogen peroxide agrees with the results of Deretic et al. (1995
), but not those of Sherman et al. (1996
). The latter authors detected a sevenfold increase in expression from the M. tuberculosis katG promoter in an M. bovis BCG host in response to hydrogen peroxide. The differences in the response of the katG promoter to hydrogen peroxide may have been due to the use of different mycobacterial hosts. M. tuberculosis does not have a functional OxyR, which is responsible for the hydrogen-peroxide-dependent induction of the katG gene in most bacteria (Christman et al., 1985
; VanBogelen et al., 1987
; Tartaglia et al., 1989
; Storz et al., 1990
; Farr & Kogoma, 1991
; Altuvia et al., 1994
; Toledano et al., 1994
). In addition, no homologous oxyR sequences have been detected in M. smegmatis. These observations suggest that the katG gene is regulated differently in these organisms, and that induction of the gene in response to hydrogen peroxide is unlikely, unless it occurs through an alternative mechanism.
IdeR-deficient mutants of M. smegmatis exhibit reduced catalase and peroxidase activities, associated with KatG (Dussurget et al., 1996 ). This suggests that the protein is either directly or indirectly responsible for the regulation of the katG gene in response to iron limitation in this organism. In this study, no changes in the expression from the katG promoter were noted under iron-limiting conditions. It is possible that the M. tuberculosis katG promoter is not recognized by the M. smegmatis IdeR or the factor(s) induced by the protein, or that regulation in response to iron limitation occurs at a post-transcriptional level in this organism. Alternatively, the M. smegmatis IdeR may not be induced or active under the conditions used in this study.
That the pJCluc promoter constructs in this study exhibited similar luciferase activities in M. bovis BCG and M. smegmatis was not unexpected. There were no significant differences between the activities of the M. bovis BCG hsp60, M. leprae 28 kDa and M. leprae 18 kDa promoters in M. smegmatis and M. bovis BCG (Dellagostin et al., 1995 ). In addition, the efficiency and specificity of transcriptional recognition is conserved in M. tuberculosis, M. smegmatis and M. bovis BCG (Bashyam et al., 1996
). M. smegmatis is, therefore, a suitable host for the characterization of M. tuberculosis promoters.
A novel observation reported here is the identification of the UAR sequence upstream of the katG promoter. As mentioned earlier, these elements do occur in other bacteria, but there is a large degree of sequence divergence. No identical UAR sequences were found elsewhere in the M. tuberculosis genome and it would be intriguing to determine if other mycobacterial genes are controlled, in part, by divergent UAR elements. Furthermore, are these elements particularly common to the mycobacteria? Generally, the UAR or UP regions are only identified by functional analysis of the regions upstream of promoters, indicating the value of this kind of study.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 February 1999;
revised 29 April 1999;
accepted 7 May 1999.