Download The Mycobacterium tuberculosis katG promoter region contains a

Microbiology (1999), 145, 2507–2518
Printed in Great Britain
The Mycobacterium tuberculosis katG promoter
region contains a novel upstream activator
Michelle A. Mulder, Harold Zappe and Lafras M. Steyn
Author for correspondence : Lafras M. Steyn. Tel : j27 21 406 6363. Fax : j27 21 448 8153.
e-mail : lsteyn!medmicro.uct.ac.za
Department of Medical
Microbiology, University of
Cape Town and Groote
Schuur Hospital, Medical
School, Observatory, 7925
Cape Town, South Africa
An Escherichia coli–mycobacterial shuttle vector, pJCluc, containing a
luciferase reporter gene, was constructed and used to analyse the
Mycobacterium tuberculosis katG promoter. A 19 kb region immediately
upstream of katG promoted expression of the luciferase gene in E. coli and
Mycobacterium smegmatis . A smaller promoter fragment (559 bp) promoted
expression with equal efficiency, and was used in all further studies. Two
transcription start sites were mapped by primer extension analysis to 47 and
56 bp upstream of the GTG initiation codon. Putative promoters associated
with these show similarity to previously identified mycobacterial promoters.
Deletions in the promoter fragment, introduced with BAL-31 nuclease and
restriction endonucleases, revealed that a region between 559 and 448 bp
upstream of the translation initiation codon, designated the upstream
activator region (UAR), is essential for promoter activity in E. coli, and is
required for optimal activity in M. smegmatis . The katG UAR was also able to
increase expression from the Mycobacterium paratuberculosis PAN promoter
15-fold in E. coli and 12-fold in M. smegmatis . An alternative promoter is active
in deletion constructs in which either the UAR or the katG promoters identified
here are absent. Expression from the katG promoter peaks during late
exponential phase, and declines during stationary phase. The promoter is
induced by ascorbic acid, and is repressed by oxygen limitation and growth at
elevated temperatures. The promoter constructs exhibited similar activities in
Mycobacterium bovis BCG as they did in M. smegmatis .
Keywords : Mycobacterium tuberculosis, katG, promoter, regulation
INTRODUCTION
The genus Mycobacterium is one of the largest bacterial
genera and includes the pathogenic species Mycobacterium leprae and Mycobacterium tuberculosis. At
present little is known about the control of gene
expression in the mycobacteria. A number of mycobacterial promoters have been studied (reviewed by
Mulder et al., 1997). Consensus promoter sequences
have been proposed (Ramesh & Gopinathan, 1995 ;
Bashyam et al., 1996) ; however, many of these
promoters are specifically regulated in vivo and may not
be typical. Some of the mycobacterial promoters resemble the typical Escherichia coli σ(! consensus promoter and function in this organism, but most have a
higher GjC content and differ from the E. coli
.................................................................................................................................................
Abbreviations : ADC, albumin dextrose catalase ; RLU, relative light units ;
tss, transcription start site ; UAR, upstream activator region.
consensus. These function more efficiently in Streptomyces (Kieser et al., 1986). As yet, too few mycobacterial
promoters have been studied to make accurate predictions on consensus sequences and promoter structures.
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.
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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.
METHODS
Bacterial strains and plasmids. All cloning steps were per-
formed in E. coli LKIII (Zabeau & Stanley, 1982) grown in
2YT medium (Miller, 1992). Expression studies were performed in M. smegmatis LR222, a high-frequency transforming strain (Beggs et al., 1995), and M. bovis BCG Tokyo
vaccine strain 172 (supplied by the State Vaccine Institute,
Pinelands, South Africa). M. tuberculosis H37Rv was obtained
from the American Type Culture Collection. All mycobacterial strains were cultivated in Middlebrook 7H9 medium
(Difco) supplemented with catalase-free ADC (albumin and
dextrose) and 0n05 % (v\v) Tween 80, or on MiddlebrookADC agar plates. pGEM-luc (Promega) and pJC86 (J. T.
Crawford, Centers for Disease Control and Prevention,
Atlanta, GA, USA) were used for construction of the plasmids.
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 5h end
of the luciferase gene, 5h-CTTTATGTTTTTGGCGTC-3h.
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 (0n1 µg) as the template with the primers 5h-CTGGTAAGCttGGCCGCAAAACAGC-3h and 5h-CACAGggaTCCTTCCAGGAGTTGGT-3h (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 HindIII–StuI 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
SmaI–BamHI 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 HindIII–BamHI 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 HindIII–SphI 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.
Nuclease BAL-31 deletions. pK20 DNA (30 µg) was digested
to completion with HindIII, extracted with phenol\
chloroform\isoamyl alcohol and precipitated with ethanol.
The linear fragment was digested with nuclease BAL-31
according to a standard protocol (Ausubel et al., 1987). The
digested DNA was then precipitated with ethanol, religated
and used to transform competent E. coli LKIII cells.
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 0n1 cm gap
Gene Pulser cuvettes (Bio-Rad) at 1n8 kV, 25 µF and 1000 Ω,
using a Gene Pulser apparatus (Bio-Rad). After electroporation, 900 µl Middlebrook-ADC was added, and the cells
were incubated at 37 mC for 3–4 h before plating. Plates were
incubated at 37 mC for 4–5 d. Electrocompetent M. bovis BCG
cells were prepared and electroporated using the method of
Jacobs et al. (1991), with minor modifications. A stationaryphase culture of M. bovis BCG grown in Middlebrook-ADCTween was diluted 100-fold in freshly prepared MiddlebrookADC-Tween, and grown to mid-exponential phase (8 d) in a
roller culture at 37 mC. Cells were harvested by centrifugation
at 4 mC 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. MiddlebrookADC (700 µl) was added and the cells were incubated for 3–4 h
at 37 mC. The cells were plated on Middlebrook-ADC agar
plates containing 30 µg kanamycin ml−" and 0n01 % (w\v)
cycloheximide (Fluka Biochemicals), and incubated at 37 mC
for 3–4 weeks.
Preparation of cell extracts and luciferase assays. For the
luciferase activity assays, 5 ml cultures were grown to late
exponential phase at 37 mC with aeration (Orbital shaker,
Ha$ gar 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 1i Cell Culture
Lysis Reagent (Luciferase Assay System, Promega). The cells
were lysed by sonication in ice water (three bursts of 40 s each)
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Table 1. Plasmids used in this study
Plasmid
pJCluc
pK10
pK20
pK20∆EN
pK20∆HA
pK20∆AS
pK20∆SE
pK21
pK22
pK23
pK24
pK25
pK20∆SB
pK20∆HS
pLPan
pLPuar
Size (bp)
Description
7427
9370
7956
7943
7845
7801
7850
7656
7588
7733
7521
7402
7689
7727
7646
7913
Promoterless firefly luciferase gene in pJC86
1943 kb katG upstream region in pJCluc (HindIII–BamHI)
559 bp katG upstream region in pJCluc (SmaI–BamHI)
pK20 with the 13 bp EcoRV–NruI fragment deleted
pK20 with the 111 bp HindIII–AluI fragment deleted
pK20 with the 155 bp AluI–SphI fragment deleted
pK20 with the 106 bp SalI–EcoRV fragment deleted
pK20 with 300 bp vector upstream of insert resected*
pK20 with 18 bp insert resected*
pK20 with 123 bp insert resected*
pK20 with 167 bp insert resected*
pK20 with 283 bp insert resected*
262 bp HindIII–SphI katG UAR in pJCluc
300 bp SphI–BamHI katG promoter fragment in pJCluc
218 bp PAN promoter in pJCluc
218 bp PAN promoter downstream of the katG UAR in pJCluc
* BAL-31 nuclease derivatives of pK20.
.....................................................................................................
Fig. 1. Schematic representation of the
construction of the luciferase reporter
plasmid pJCluc.
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
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Fig. 2. Schematic representation of
promoter deletion derivatives of pK20.
The 559 bp M. tuberculosis katG promoter
region was deleted as described in the text.
The HindIII site represents that upstream of
the luciferase gene in pJCluc. The positions
of the UAR, the 24 bp AT-rich region and
the transcription start sites (tss) are
indicated. The relative decrease in luciferase
activity of the derivatives of pK20 in M.
smegmatis and M. bovis BCG host cells is
shown. The relative decreases were calculated from the means of three independent
experiments.
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 (5h-GCAGTTGCTCTCCAGCGG-3h) was designed to
anneal 58 bp downstream of the ATG initiation codon of the
luciferase gene. Approximately 100 ng primer was endlabelled with [γ-$#P] dATP using polynucleotide kinase, in a
30 µl reaction with the supplied buffer. After incubation at
30 mC 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 mC for 10 min, and annealed overnight at
50 mC. 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 mC for 60 min, using 200 units of MMLV reverse transcriptase (Promega), in a reaction containing
40 units RNase inhibitor, 1i 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 mC 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 1n4 % (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 7n75) and 622 µl
distilled water. The ATP assays were performed according to
the manufacturer’s 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 mC with
shaking. Growth was monitored using OD
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
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Analysis of the M. tuberculosis katG promoter
sodium acetate (pH 7n0) ; and (4) 200 µM of the iron chelator
2,2h-dipyridyl. In the fifth culture, the pH was adjusted to 4n0
with glacial acetic acid. The remaining three cultures were
resuspended in medium with no additions. One of these was
heat-shocked at 45 mC. 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 mC 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.
RESULTS
M
P
T
A
Construction of pJCluc
The E. coli–mycobacterial shuttle vector pJCluc was
constructed as shown in Fig. 1. The plasmid pJC86
contains the EcoRV–HpaI pAL5000 backbone for replication in mycobacteria (Snapper et al., 1990), the lacZ
gene and oriE from pUC18, and the kanamycinresistance gene from Tn5. pJCluc contains the
promoterless luc gene in the opposite orientation to the
lacZ gene, and has no Shine–Dalgarno sequence. pJCluc
and pJC86 were assayed for luciferase activity in E. coli
and M. smegmatis. Cells containing pJC86 had no
detectable luciferase activity, indicating no endogenous
luminescence in either E. coli or M. smegmatis. E.
coli\pJCluc exhibited a low level of activity
[45p18 RLU (mg protein)−"] but a higher level of
activity was detected in M. smegmatis\pJCluc
[143p28 RLU (mg protein)−"]. This activity may be due
to read-through from an upstream promoter which
appears to be more active in M. smegmatis and,
therefore, may be located in the pAL5000 fragment of
the plasmid.
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 41 800p2400 and
38 500p10 700 RLU (mg protein)−", respectively. Those
for E. coli\pK10 and E. coli\pK20 were 381p100 and
474p13 RLU (mg protein)−", 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 SmaI–
BamHI 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
G
A
T
C
E
P
G
A
C
A
C
T
T
C
G
C
G
A
T
C
A
C
A
T
C
C
.................................................................................................................................................
Fig. 3. Determination of the transcription start sites of M.
tuberculosis katG. Primer extension analysis was carried out as
described in Methods, using a primer complementary to
sequences downstream of the luciferase ATG and RNA isolated
during late exponential phase from E. coli and M. smegmatis
cells harbouring pK20. Primer extension products were run on a
sequencing gel adjacent to sequencing reactions primed with
the same primer. The experiment was repeated three times and
one representative autoradiograph is shown. Two potential
transcription start sites were identified, 47 and 56 bp upstream
of the GTG translation initiation codon, and are indicated with
arrows. Lanes G, A, T, C show the sequencing reaction. M, M.
smegmatis RNA ; E, E. coli RNA ; P, primer extension products.
The sequence surrounding the transcription start sites is shown.
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 5h 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
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(a)
(b)
(c)
.................................................................................................................................................
Fig. 4. Nucleotide sequence of the M. tuberculosis katG
promoter region including the first three codons, labelled with
respect to the published sequence (GenBank accession no.
X68081). The transcription start sites (tss) are indicated with
arrows. The putative k10 and k35 hexamers associated with
each transcription start site are underlined, and the putative
ribosome-binding site (RBS) is shown in bold. The lower-case
nucleotides represent the restriction endonuclease sites EcoRV
and NruI. (a) Promoters associated with tssA and tssB. (b)
Promoters associated with tssC, which becomes functional in
deletion derivatives pK20∆HS, pK20∆EN and pK20∆SE. (c)
Summary of the potential katG promoter sequences.
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 k10 and
k35 sequences relative to these transcription start sites
are represented in Fig. 4. The putative k10 hexamer for
tssA, GACACT, lies 8 bp upstream of the transcription
start site and shows a high degree of similarity to other
mycobacterial k10 sequences. The first G of the
hexamer is the least conserved. Associated with this
k10 hexamer are three possible k35 sequences :
TCTATG 19 bp upstream, TGTCCT 15 bp upstream,
and TCCTGA 13 bp upstream. The putative k10
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 k10 region
are two possible k35 sequences, TCTACT 22 bp
upstream, and TACTGG 20 bp upstream.
Deletion analysis of the promoter region
A number of deletions were made, using the restriction
sites present on the insert of pK20, in order to determine
whether other regions of the M. tuberculosis katG
upstream region are required for promoter activity. The
constructs were tested for luciferase activity in E. coli
and M. smegmatis. The regions present in the various
deletions and the resulting decreases in luciferase activity
in M. smegmatis are summarized in Fig. 2. Removal of
111 bp from the 5h end of the insert (pK20∆HA) resulted
in an 82- and 55-fold reduction in activity in E. coli and
M. smegmatis, respectively, suggesting that this region
is essential for optimal promoter activity. Removal of
the 13 bp between the EcoRV and NruI restriction sites
(pK20∆EN) in the promoter region resulted in a threefold decrease in promoter activity in E. coli (results not
shown). This deletion removes the putative k10
hexamer associated with tssA, as well as tssB and a
region of its putative k10 hexamer. The deletion
should, therefore, abolish promoter activity completely.
In M. smegmatis, deletion of this region had little effect
(1n3-fold reduction) on expression. Removal of
sequences upstream of the EcoRV site (pK20∆SE) gave a
similar result, i.e. a threefold decrease in activity in E.
coli, and no significant change in M. smegmatis.
Deletion of the SalI–EcoRV fragment results in the
removal of half of the k10 hexamer of tssB and the k35
sequences of both tssA and tssB. Transcription initiates
at a different promoter in both pK20∆EN and pK20∆SE
(see below). The absence of the 155 bp AluI–SphI
fragment had no significant effect on promoter activity
in either E. coli or M. smegmatis. Thus, based on these
deletion constructs, the sequences necessary for maximal
promoter activity lie upstream of the AluI site. This
region may act as a regulatory region which enhances
transcription initiation and it was designated an upstream activator region (UAR). The results also indicate
that the distance between the UAR and the promoters is
not critical.
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 5h 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 SphI–BamHI
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 5h 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 5h end of the insert is
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Analysis of the M. tuberculosis katG promoter
(a) Px G A T C
(b)
Px G A T C
(c)
Px G A T C
.....................................................................................................
C
C
C
required for maximal promoter activity in both hosts.
The region between 123 and 283 bp from the 5h 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 HindIII–SphI 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
Fig. 5. Primer extension analysis of the
M. tuberculosis katG upstream region using
a primer complementary to sequences
downstream of the luciferase ATG and RNA
isolated during late exponential phase from
M. smegmatis cells harbouring (a) pK20∆HS,
(b) pK20∆EN, and (c) pK20∆SE. The potential transcription start site, tssC, is indicated
with an arrow. This corresponds to a C
residue, 23 bp upstream of the GTG translation initiation codon. Lanes G, A, T, C show
the sequencing reaction. Px, primer extension
products.
promoter sequences, a third region, designated PC, is
recognized as a promoter. Located 7 bp upstream of
transcription start site C is the putative k10 hexamer,
CACAGC (Fig. 4b). Associated with this are two
putative k35 regions : TTCGCG, 14 bp upstream, and
TCCGAC, 22 bp upstream.
Enhancement of the pAN promoter by the katG UAR
The functionality of the katG UAR with other mycobacterial promoters was tested by cloning of the 262 bp
HindIII–SphI fragment from pK20 upstream of the M.
paratuberculosis PAN promoter in pJCluc (pLPuar). The
PAN promoter was also cloned into pJCluc on its own
(pLPan) as a control. The constructs were electroporated
into M. smegmatis and cell extracts from the resulting
transformants were tested for luciferase activity. The
presence of the katG UAR resulted in a 15-fold increase
in expression of the PAN promoter in E. coli [278p72 to
4100p1400 RLU (mg protein)−"] and a 12-fold increase
in M. smegmatis (9900p1900 to 115 600p24 200 RLU
(mg protein)−"]. This result confirms that the region acts
as an enhancer of transcription initiation.
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
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M. A. M U L D ER, H. Z A P P E a n d L. M. S T E Y N
Table 2. Luciferase activities of the pJCluc constructs in
M. bovis BCG
Relative activity†
12p3
25 600p2400
300p53
46p8
3 300p1800
4 800p1300
21 420p600
0n001
1n000
0n012
0n002
0n129
0n188
0n837
Growth-phase-dependent expression from the katG
promoter in M. smegmatis
Expression of the katG promoter in M. smegmatis was
monitored over a 68 h period of growth by measurement
of luciferase activity. The values used to plot the curves
in Fig. 6 are taken from one experiment, but are
representative of three independent experiments. The
growth curves generated by optical density readings and
ATP concentration showed similar trends (Fig. 6). The
cells reach late exponential phase at approximately 30 h.
Expression of luciferase from the katG promoter is low
in early exponential phase, but increases steadily and
reaches a transient peak in the late exponential phase,
followed by a rapid decline as the cells enter stationary
phase.
Expression from the katG promoter in cells exposed
to various stresses
Some of the factors that are known to modulate katG
expression in other bacteria were tested for their ability
to influence expression from the M. tuberculosis katG
promoter. Preliminary experiments revealed that a 4 h
period was suitable for measurable response to stress in
M. smegmatis. The experiments were performed in
triplicate, and the luciferase activities of one represen-
10–1
10–9
10–2
10–10
10–11
* Mean of three independent experimentspthe standard error of
the mean.
† Activity normalized to that of the construct pK20.
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 HindIII–SphI 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.
10–8
OD600
Luciferase activity
RLU (mg protein)−1*
1
10–7
ATP concn (M)
pJCluc
pK20
pK20∆HS
pK20∆SB
pK24
pLPan
pLPuar
(a)
Luciferase activity [RLU (mg protein)–1]
Construct
10–6
10–3
120
(b)
100
80
60
40
20
0
0
10 20
30 40 50 60
Time (h)
.................................................................................................................................................
Fig. 6. Growth-phase-dependent expression of the M.
tuberculosis katG promoter in M. smegmatis. The results
are from one experiment but are representative of
three independent experiments. (a) Growth curve of M.
smegmatis/pK20 plotted as a measure of ATP concentration ($)
and OD600 (#). (b) Luciferase activity of the M. tuberculosis
katG promoter monitored over a 68 h growth period in M.
smegmatis.
Table 3. Luciferase activities of M. smegmatis/pK20
exposed to various stresses
Stress
Luciferase activity
RLU (µg protein)−1 Relative activity*
Control
Hydrogen peroxide
Sodium acetate
Ascorbic acid
Oxygen limitation
Low iron
pH 4
45 mC
88n60
87n82
80n02
164n48
8n25
78n26
79n52
2n12
1n00
0n99
0n90
1n86
0n09
0n89
0n90
0n02
* Activity normalized to that of the control.
tative experiment are given in Table 3. Treatment with
10 mM ascorbic acid resulted in an almost twofold
(86 %) increase in luciferase activity. Growth under
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Analysis of the M. tuberculosis katG promoter
oxygen-limiting conditions and at 45 mC resulted in an
11- and 42-fold decrease in luciferase activity, respectively. The other changes in the culture conditions did
not significantly affect luciferase activity.
DISCUSSION
In this study, the sequences required for expression from
the M. tuberculosis katG promoter have been identified.
The 1943 bp and 559 bp M. tuberculosis katG promoter
fragments promoted expression of the luciferase gene
with equal efficiency under the conditions used in this
study, showing that sequences upstream of the SmaI site
are not essential for optimal promoter efficiency during
normal growth. Deletion analysis revealed that the first
111 bp at the 5h end of the SmaI–BamHI M. tuberculosis
katG promoter fragment are required for optimal
expression in both E. coli and M. smegmatis. The
absence of the 155 bp AluI–SphI fragment, on the other
hand, had no significant effect on promoter activity in
either E. coli or M. smegmatis, indicating that this
region is not important for expression. The sequences
necessary for maximal promoter activity, therefore, lie
upstream of the AluI site. This region does not contain
a functional promoter but may act as a regulatory region
which enhances transcription initiation. The distance
between the UAR and the promoter sequences does not
appear to be critical. Within the UAR lies a 24 bp
sequence (k523 to k499 with respect to the GTG
translation initiation codon), with an AjT content of
66n67 mol %. This AjT content is high in comparison
to normal M. tuberculosis promoter regions (Bashyam
et al., 1996), and may be analogous to the UP elements
found upstream of some E. coli promoters. These
elements are usually AT-rich and can increase promoter
activity up to 30-fold (Ross et al., 1993). They are also a
frequent feature of Gram-positive promoters (Moran et
al., 1982 ; Graves & Rabinowitz, 1986). Movahedzadeh
et al. (1997) have recently identified a similar region,
269–310 bp upstream of the M. tuberculosis recA gene,
which is essential for expression from the promoter. A
database search with the M. tuberculosis katG UAR
sequence revealed that it is not dispersed in the M.
tuberculosis genome, but that it is similar to the region
upstream of the M. fortuitum katG gene. The 24 bp ATrich sequence within the katG UAR, described above, is
79n2 % homologous to a region located 489 bp upstream
of the M. fortuitum katG ATG translation initiation
codon. This suggests possible similarities in the mode of
regulation of the gene in the two mycobacterial species.
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 45–50 % 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 k10 and k35 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 Eσ(! in E. coli. The
spacing between the k10 and k35 regions, however,
differs from that found in most E. coli promoters (17p1)
(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 k10
and k35 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 k10 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 k10 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
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M. A. M U L D ER, H. Z A P P E a n d L. M. S T E Y N
to sites at various distances upstream of the k35 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 σ 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 5n7 mM ascorbic acid results in an
eightfold increase in catalase activity (Richter &
Loewen, 1981), while treatment with 0–80 mM sodium
acetate (pH 7n0) 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.
ACKNOWLEDGEMENTS
This work was supported by the University of Cape Town and
MRC and was conducted as part of the Glaxo-Wellcome
Action TB initiative.
REFERENCES
Aldovini, A. & Young, R. A. (1991). Humoral and cell-mediated
immune responses to live recombinant BCG-HIV vaccines.
Nature 351, 479–482.
2516
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 13:22:45
Analysis of the M. tuberculosis katG promoter
Altuvia, S., Almiron, M., Huisman, G., Kolter, R. & Storz, G. (1994).
The dps promoter is activated by OxyR during growth and by
IHF and sigma S in stationary phase. Mol Microbiol 13, 265–272.
Jacobs, W. R., Jr, Kalpana, G. V., Cirillo, J. D., Pascopella, L.,
Snapper, S. B., Udani, R. A., Jones, W., Barletta, R. G. & Bloom,
B. R. (1991). Genetic systems for mycobacteria. Methods
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G., Smith, J. A. & Struhl, K. (1987). Current Protocols in
Kieser, S., Moss, M. T., Dale, J. W. & Hopwood, D. A. (1986).
Molecular Biology. Cambridge, MA : Wiley Interscience.
Barletta, R. G., Kim, D. D., Snapper, S. B., Bloom, B. R. & Jacobs,
W. R., Jr (1992). Identification of expression signals of the
mycobacteriophages Bxb1, L1 and TM4 using the Escherichia–
Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed
to create translational fusions to the lacZ gene. J Gen Microbiol
138, 23–30.
Enzymol 204, 537–555.
Cloning and expression of Mycobacterium bovis BCG DNA in
Streptomyces lividans. J Bacteriol 168, 72–80.
Kremer, L., Baulard, A., Estaquier, J., Content, J., Capron, A. &
Locht, C. (1995). Analysis of the Mycobacterium tuberculosis 85A
antigen promoter region. J Bacteriol 177, 642–653.
Labidi, A., David, H. L. & Roulland-Dussoix, D. (1985). Restriction
Dellagostin, O. A., Esposito, G., Eales, L. J., Dale, J. W. &
McFadden, J. (1995). Activity of mycobacterial promoters during
endonuclease mapping and cloning of Mycobacterium fortuitum
var. fortuitum plasmid pAL5000. Ann Inst Pasteur Microbiol
136B, 209–215.
Levin, M. E. & Hatfull, G. F. (1993). Mycobacterium smegmatis
RNA polymerase : DNA supercoiling, action of rifampicin and
mechanism of rifampicin resistance. Mol Microbiol 8, 277–285.
Loewen, P. C., Switala, J. & Triggs-Raine, B. L. (1985). Catalases
HPI and HPII in Escherichia coli are induced independently. Arch
Biochem Biophys 243, 144–149.
Middlebrook, G. & Cohn, M. L. (1953). Some observations on the
pathogenicity of isoniazid-resistant varients of tubercle bacilli.
Science 118, 297–299.
Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold
Spring Harbor, NY : Cold Spring Harbor Laboratory.
intracellular and extracellular growth. Microbiology 141,
1785–1792.
Mitchison, D. A., Wallace, J. G., Bhatia, A. L., Selkon, J. B.,
Subbaiah, J. & Lancaster, M. C. (1960). A comparison of the
Deretic, V., Philipp, W., Dhandayuthapani, S., Mudd, M. H.,
Curcic, R., Garbe, T., Heym, B., Via, L. E. & Cole, S. T. (1995).
virulence in guinea pigs of South India and British tubercle bacilli.
Tubercle 41, 1–22.
Mycobacterium tuberculosis is a natural mutant with an
inactivated oxidative-stress regulatory gene : implications for
sensitivity to isoniazid. Mol Microbiol 17, 889–900.
Dhandayuthapani, S., Mudd, M. & Deretic, V. (1997). Interactions
of OxyR with the promoter region of the oxyR and ahpC genes
from Mycobacterium leprae and Mycobacterium tuberculosis. J
Bacteriol 179, 2401–2409.
Dussurget, O., Rodriguez, M. & Smith, I. (1996). An ideR mutant
of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative-stress response. Mol Microbiol
22, 535–544.
Farr, S. B. & Kogoma, T. (1991). Oxidative stress responses in
Escherichia coli and Salmonella typhimurium. Microbiol Rev 55,
561–585.
Graves, M. C. & Rabinowitz, J. C. (1986). In vivo and in vitro
transcription of the Clostridium pasteurianum ferredoxin gene.
Evidence for ‘ extended ’ promoter elements in gram-positive
organisms. J Biol Chem 261, 11409–11415.
Harley, C. B. & Reynolds, R. P. (1987). Analysis of E. coli promoter
sequences. Nucleic Acids Res 15, 2343–2361.
Moran, C. P., Jr, Lang, N., LeGrice, S. F., Lee, G., Stephens, M.,
Sonenshein, A. L., Pero, J. & Losick, R. (1982). Nucleotide
Bashyam, M. D., Kaushal, D., Dasgupta, S. K. & Tyagi, A. K.
(1996). A study of mycobacterial transcriptional apparatus :
identification of novel features in promoter elements. J Bacteriol
178, 4847–4853.
Beggs, M. L., Crawford, J. T. & Eisenach, K. D. (1995). Isolation
and sequencing of the replication region of Mycobacterium
avium plasmid pLR7. J Bacteriol 177, 4836–4840.
Christman, M. F., Morgan, R. W., Jacobson, F. S. & Ames, B. N.
(1985). Positive control of a regulon for defenses against oxidative
stress and some heat-shock proteins in Salmonella typhimurium.
Cell 41, 753–762.
Heym, B., Zhang, Y., Poulet, S., Young, D. & Cole, S. T. (1993).
Characterization of the katG gene encoding a catalase-peroxidase
required for the isoniazid susceptibility of Mycobacterium
tuberculosis. J Bacteriol 175, 4255–4259.
Heym, B., Stavropoulos, E., Honore, N., Domenech, P., SaintJoanis, B., Wilson, T. M., Collins, D. M., Colston, M. J. & Cole,
S. T. (1997). Effects of overexpression of the alkyl hydroperoxide
reductase AhpC on the virulence and isoniazid resistance of
Mycobacterium tuberculosis. Infect Immun 65, 1395–1401.
Jackett, P. S., Aber, V. R. & Lowrie, D. B. (1978). Virulence and
resistance to superoxide, low pH and hydrogen peroxide among
strains of Mycobacterium tuberculosis. J Gen Microbiol 104,
37–45.
sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 186, 339–346.
Morgan, R. W., Christman, M. F., Jacobson, F. S., Storz, G. &
Ames, B. N. (1986). Hydrogen peroxide-inducible proteins in
Salmonella typhimurium overlap with heat shock and other stress
proteins. Proc Natl Acad Sci USA 83, 8059–8063.
Movahedzadeh, F., Colston, M. J. & Davis, E. O. (1997). Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNAdamaging agents suggests that two modes of regulation exist. J
Bacteriol 179, 3509–3518.
Mukhopadhyay, S. & Schellhorn, H. E. (1994). Induction of
Escherichia coli hydroperoxidase I by acetate and other weak
acids. J Bacteriol 176, 2300–2307.
Mulder, M. A., Zappe, H. & Steyn, L. M. (1997). Mycobacterial
promoters. Tubercle Lung Dis 78, 211–223.
Mulvey, M. R., Sorby, P. A., Triggs-Raine, B. L. & Loewen, P. C.
(1988). Cloning and physical characterization of katE and katF
required for catalase HPII expression in Escherichia coli. Gene 73,
337–345.
Murray, A., Winter, N., Lagranderie, M., Hill, D. F., Rauzier, J.,
Timm, J., Leclerc, C., Morlarty, K. M. & Gicquel, B. (1992).
Expression of Escherichia coli β-galactosidase in Mycobacterium
bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular
immune responses. Mol Microbiol 6, 3331–3342.
Nesbit, C. E., Levin, M. E., Donnelly-Wu, M. K. & Hatfull, G. F.
(1995). Transcriptional regulation of repressor synthesis in
mycobacteriophage L5. Mol Microbiol 17, 1045–1056.
Pagan-Ramos, E., Song, J., McFalone, M., Mudd, M. H. & Deretic,
V. (1998). Oxidative stress response and characterization of the
2517
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 13:22:45
M. A. M U L D ER, H. Z A P P E a n d L. M. S T E Y N
oxyR–ahpC and furA–katG loci in Mycobacterium marinum. J
Bacteriol 180, 4856–4864.
Predich, M., Doukhan, L., Nair, G. & Smith, I. (1995). Characterization of RNA polymerase and two sigma-factor genes from
Mycobacterium smegmatis. Mol Microbiol 15, 355–366.
Prioli, R. P., Tanna, A. & Brown, I. N. (1985). Rapid methods for
counting mycobacteria – comparison of methods for extraction
of mycobacterial adenosine triphosphate (ATP) determined by
firefly luciferase assay. Tubercle 66, 99–108.
Raibaud, O. & Schwartz, M. (1984). Positive control of transcription initiation in bacteria. Annu Rev Genet 18, 173–206.
Ramesh, G. & Gopinathan, K. P. (1995). Cloning and characterization of mycobacteriophage I3 promoters. Indian J Biochem
Biophys 32, 361–367.
Richter, H. E. & Loewen, P. C. (1981). Induction of catalase in
Escherichia coli by ascorbic acid involves hydrogen peroxide.
Biochem Biophys Res Commun 100, 1039–1046.
Rocha, E. R. & Smith, C. J. (1995). Biochemical and genetic analyses
of a catalase from the anaerobic bacterium Bacteroides fragilis. J
Bacteriol 177, 3111–3119.
Ross, W., Gosink, K. K., Salomon, J., Igarashi, K., Zou, C.,
Ishihama, A., Severinov, K. & Gourse, R. L. (1993). A third
recognition element in bacterial promoters : DNA binding by the
alpha subunit of RNA polymerase. Science 262, 1407–1413.
Rouse, D. A. & Morris, S. L. (1995). Molecular mechanisms of
isoniazid resistance in Mycobacterium tuberculosis and Mycobacterium bovis. Infect Immun 63, 1427–1433.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular
Cloning : a Laboratory Manual, 2nd edn. Cold Spring Harbor,
NY : Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing
with chain-terminating inhibitors. Proc Natl Acad Sci USA 74,
5463–5467.
Schnell, S. & Steinman, H. M. (1995). Function and stationaryphase induction of periplasmic copper-zinc superoxide dismutase
and catalase\peroxidase in Caulobacter crescentus. J Bacteriol
177, 5924–5929.
Sherman, D. R., Mdluli, K., Hickey, M. J., Arain, T. M., Morris,
S. L., Barry, C. E., 3rd & Stover, C. K. (1996). Compensatory ahpC
gene expression in isoniazid-resistant Mycobacterium tuberculosis. Science 272, 1641–1643.
Snapper, S. B., Lugosi, L., Jekkel, A., Melton, R. E., Kieser, T.,
Bloom, B. R. & Jacobs, W. R., Jr (1988). Lysogeny and trans-
formation in mycobacteria : stable expression of foreign genes.
Proc Natl Acad Sci USA 85, 6987–6991.
Snapper, S. B., Melton, R. E., Kieser, T., Mustafa, S. & Jacobs,
W. R., Jr (1990). Isolation and characterization of efficient plasmid
transformation mutants of Mycobacterium smegmatis. Mol
Microbiol 4, 1911–1919.
Stanley, P. E., McCarthy, B. J. & Smither, R. (1989). Rapid Methods
in Microbiology. Oxford : Blackwell Scientific.
Stolt, P. & Stoker, N. G. (1996). Functional definition of regions
necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000. Microbiology 142,
2795–2802.
Storz, G., Tartaglia, L. A. & Ames, B. N. (1990). Transcriptional
regulator of oxidative stress-inducible genes : direct activation by
oxidation. Science 248, 189–194.
Stover, C. K., de la Cruz, V. F., Fuerst, T. R. & 11 other authors
(1991). New use of BCG for recombinant vaccines. Nature 351,
456–460.
Suzuki, Y., Nagata, A. & Yamada, T. (1991). Analysis of the
promoter region in the rRNA operon from Mycobacterium bovis
BCG. Antonie Leeuwenhoek 60, 7–11.
Tartaglia, L. A., Storz, G. & Ames, B. N. (1989). Identification and
molecular analysis of oxyR-regulated promoters important for
the bacterial adaptation to oxidative stress. J Mol Biol 210,
709–719.
Toledano, M. B., Kullik, I., Trinh, F., Baird, P. T., Schneider, T. D. &
Storz, G. (1994). Redox-dependent shift of OxyR-DNA contacts
along an extended DNA-binding site : a mechanism for differential
promoter selection. Cell 78, 897–909.
VanBogelen, R. A., Kelley, P. M. & Neidhardt, F. C. (1987).
Differential induction of heat shock, SOS, and oxidation stress
regulons and accumulation of nucleotides in Escherichia coli. J
Bacteriol 169, 26–32.
Wayne, L. G. & Sramek, H. A. (1994). Metronidazole is bactericidal
to dormant cells of Mycobacterium tuberculosis. Antimicrob
Agents Chemother 38, 2054–2058.
Wilson, T. M., de Lisle, G. W. & Collins, D. M. (1995). Effect of
inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis. Mol Microbiol 15, 1009–1015.
Winder, F. G. (1960). Catalase and peroxidase in mycobacteria :
possible relationship to the mode of action of isoniazid. Am Rev
Resp Dis 81, 68–78.
Zabeau, M. & Stanley, K. K. (1982). Enhanced expression of cro–βgalactosidase fusion proteins under the control of the PR promoter
of bacteriophage λ. EMBO J 1, 1217–1224.
Zhang, Y., Heym, B., Allen, B., Young, D. & Cole, S. (1992). The
catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358, 591–593.
Zhou, Y., Merkel, T. J. & Ebright, R. H. (1994a). Characterization
of the activating region of Escherichia coli catabolite gene
activator protein (CAP). II. Role of class I and class II CAPdependent promoters. J Mol Biol 243, 603–610.
Zhou, Y., Pendergrast, P. S., Bell, A., Williams, R., Busby, S. &
Ebright, R. H. (1994b). The functional subunit of a dimeric
transcription activator protein depends on promoter architecture.
EMBO J 13, 4549–4557.
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Received 22 February 1999 ; revised 29 April 1999 ; accepted 7 May 1999.
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