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Journal of Bacteriology, July 1999, p. 4266-4274, Vol. 181, No. 14
Division of Infectious Diseases, Children's
Hospital, Harvard Medical School, Boston, Massachusetts 02115
Received 10 September 1998/Accepted 5 May 1999
Extracytoplasmic function (ECF) sigma factors are a heterogeneous
group of alternative sigma factors that regulate gene expression in
response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a
closely related sigma factor, sigH, was cloned from
Mycobacterium tuberculosis and Mycobacterium
smegmatis. A single copy of this gene is present in these and
other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode
highly similar proteins, there are multiple differences in adjacent
genes. The single in vivo transcriptional start site identified in
M. smegmatis and one of two identified in M. bovis BCG were found to have Extracytoplasmic function (ECF)
sigma factors constitute a diverse family of proteins within the
Because of the role of some ECF sigma factors in regulating the
interaction of bacteria with the extracellular environment and in the
adaptation of bacteria to stress, these proteins are of interest as
potential regulators of virulence factors in bacterial pathogens.
Examples of this role have been described for P. aeruginosa and Salmonella spp. (6, 11). Mycobacteria are
major pathogens of humans, yet little is known regarding determinants
of virulence in these organisms or the regulation of mycobacterial gene
expression during infection. As a step toward determining whether sigma
factor-regulated gene expression plays a role in mycobacterial
pathogenesis, we have begun to examine the role of ECF sigma factors in
the mycobacterial stress response.
We previously characterized a mycobacterial ECF sigma factor,
designated SigE, that plays a role in bacterial survival following a
variety of in vitro stresses (39). Examination of the
sequence of the genome of Mycobacterium tuberculosis H37Rv
demonstrated the presence of a gene closely related to sigE.
This report describes the cloning and initial characterization of this
mycobacterial ECF sigma factor designated SigH.
Bacterial strains, plasmids, and culture and stress
conditions.
Strains and plasmids are described in Table
1. E. coli cultures were grown
on L agar or in L broth. M. smegmatis liquid cultures were
grown in Middlebrook 7H9 broth supplemented with 0.2% glucose, 0.5%
albumin, 0.085% NaCl, and 0.05% Tween 80 (7H9-ADCTw). M. smegmatis was plated on Middlebrook 7H10 plates supplemented with 0.2% glucose or on L agar. Ampicillin (50 to 70 µg/ml), kanamycin (20 to 50 µg/ml), zeocin (30 µg/ml), and apramycin (30 µg/ml) were added to culture media as indicated.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Mycobacterial Extracytoplasmic Sigma Factor
Involved in Survival following Heat Shock and Oxidative
Stress
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
35 promoter sequences that match
the ECF-dependent 35 promoter consensus. Expression from these
promoters was strongly induced by 50°C heat shock. In comparison to
the wild type, an M. smegmatis sigH mutant was found to be
more susceptible to cumene hydroperoxide stress but to be similar in
logarithmic growth, stationary-phase survival, and survival following
several other stresses. Survival of an M. smegmatis sigH
sigE double mutant was found to be markedly decreased following
53°C heat shock and following exposure to cumene hydroperoxide.
Expression of the second gene in the sigH operon is
required for complementation of the sigH stress phenotypes. SigH is an alternative sigma factor that plays a role in the
mycobacterial stress response.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70 class of bacterial RNA polymerase sigma subunits.
This group was originally defined by conservation of sequence, and in
some cases of function, of these proteins among several bacterial
species (24). Many of these proteins, including those first
described for Escherichia coli and Pseudomonas
aeruginosa, have been shown to play a role in the regulation of
gene expression required for survival following exposure to stress
(6, 9, 13, 18, 31). With the rapid expansion of bacterial
genomic sequence data, it has become apparent that many bacterial
species have several genes that encode ECF-type sigma factors, although
in most cases the functions of these proteins have not been defined.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Cloning of mycobacterial sigH genes.
The
following primers, based on the M. tuberculosis H37Rv
sequence from cosmid MTCY7D11, were synthesized and used to amplify a
464-bp product: 5'-CTGTACGGCGGTGCGCTGCG-3' and
5'-AAGACCGCGCAACTGACGTCG-3'. This PCR product was used to
probe a M. tuberculosis genomic DNA library in 
gt11
(40). A clone containing a 3.5-kb insert was obtained and
verified to contain the M. tuberculosis sigH locus by
restriction analysis and limited DNA sequencing. The M. tuberculosis sigH clone was used to probe an M. smegmatis genomic DNA library in pUC19 to obtain a clone
containing a 7-kb insert that included the M. smegmatis sigH
locus (15).
DNA manipulation and sequencing. Lambda DNA was purified and plasmid DNA was isolated according to standard methods (34). Restriction and modifying enzymes were obtained from New England Biolabs or Boehringer Mannheim. Genomic DNA from M. smegmatis, M. fortuitum, M. bovis BCG, and M. avium was purified as previously described (17). M. tuberculosis genomic DNA was obtained from Patrick Brennan and John Belisle (Colorado State University). Southern blot analysis was performed with an NEBlot Phototope kit (New England Biolabs) or with an ECL kit (Amersham), using M. tuberculosis DNA from the sigH-coding region as a probe, according to the manufacturer's instructions.
Sequencing of the M. smegmatis sigH gene was performed on subcloned restriction fragments and by using oligonucleotide primers based on adjacent sequence. Sequencing was performed on automated DNA sequencers, using Taq dye terminator chemistry (Applied Biosystems, Foster City, Calif.), in the core sequencing facility of the Children's Hospital Mental Retardation Research Center. Sequence assembly and analysis were performed with Sequencher (Gene Codes Corp., Ann Arbor, Mich.), the Wisconsin Package (Genetics Computer Group, Madison, Wis.), and Macvector (Oxford Molecular, Oxford, England).Transcriptional analysis. Primer extension was performed as previously described (39). Total RNA was isolated from M. smegmatis and M. bovis BCG by the hot phenol method or by using a Fast RNA Prep kit (Bio 101) (34). Primers used to determine the sigH transcriptional start sites were 5'-GGCGTCTCGGGCTCGACCCGGTCGACGTCAG-3' and 5'-TCGCGTCGCGCTCGAAACGCGCGGTCAACTC-3' for M. smegmatis and M. bovis BCG, respectively. Quantification of primer extension bands was performed by scanning of the film and performing densitometry with NIH Image software.
Reverse transcription (RT) of 2 µg of total mycobacterial RNA, using the primer 5'-CGCGGATCCTGGTTGTCTTCTAGCCC-3' (underlined bases correspond to the end of the gene immediately 3' to sigH), was performed by using Superscript II reverse transcriptase (Life Technologies) incubated at 42°C, followed by removal of RNA by RNase H; 2 µl of this reaction mixture was used as the substrate for PCR (30 cycles of 95°C for 2 min, 56°C for 1 min, and 72°C for 1 min in a 50-µl reaction mixture with 2 mM MgCl2), using Amplitaq DNA polymerase (Perkin-Elmer). For PCR, the primer 5'-TGTTTCCCACGATGACTGACG-3', corresponding to the beginning of sigH, was used together with the primer used in the RT reaction. The expected size of the product of this reaction is 979 bp.Construction of a transcriptional fusion of the heat-inducible
sigH promoter to lacZ.
A transcriptional fusion
of the heat shock-inducible promoter region of M. tuberculosis (M. bovis BCG) sigH (MtP2) to a
promoterless 
-galactosidase gene was constructed in the E. coli-mycobacterial shuttle vector pJEM15 (38). This
promoter region was amplified by PCR using the primers
5'-CACCGGACCGCGGGACAGGC-3' and
5'-TGCAGGTACCGAACCAATC-3'. Restriction sites
incorporated into the primers are underlined. The resulting PCR
products were digested with SacII plus KpnI and
cloned into the corresponding sites in pJEM15 to generate pRH1313.
Correct promoter sequence was confirmed by DNA sequencing. The
resulting construct incorporated 64 bp 5' to the transcription start
sites of MtP2. pRH1313 was transformed into M. smegmatis mc2-155 and M. bovis BCG to assess the activity
of MtP2 in these mycobacterial species.
-Galactosidase assays were performed as previously described
(25), with the following modifications. M. smegmatis and M. bovis BCG were grown to log phase
(OD600 of 0.2 to 0.5), pelleted by centrifugation, and
resuspended in Z buffer (25). The cells were then lysed by
beating in a 2-ml microcentrifuge tube approximately one-fourth filled
with glass beads in a Bead-Beater (Biospec Products). The cell debris
was pelleted, and -galactosidase activity was determined in the
supernatant following addition of
o-nitrophenyl--D-galactopyranoside (final
concentration, 4 mg/ml) by measuring absorbance at 405 nm at 5-min
intervals for 1 h. Because of the potential for variable cell
lysis and less accurate correlation of OD600 with cell
number in mycobacteria, -galactosidase activity was calculated as
OD405 per minute per milligram of protein in the cleared
cell lysate, rather than in Miller units. Protein concentrations were
determined by using a NanoOrange kit (Molecular Probes).
-Galactosidase activity and protein concentration were measured in triplicate.
Construction and complementation of M. smegmatis sigH mutants. pRH1276 was digested with BamHI, and the 1.3-kb aph (kanamycin resistance) gene of Tn903 from pUC4KSac was inserted in the same transcriptional orientation as sigH, to generate pRH1301 (1). This insertion disrupts the sigH structural gene between codons 81 and 82 of the inferred SigH protein. This suicide construct was electroporated into mc2-155; transformants were selected on kanamycin plates and then screened by PCR to distinguish single from double homologous recombination events. Two independent clones in which a double crossover had occurred were identified. The occurrence of allelic exchange resulting in a single disrupted copy of sigH in the chromosome was confirmed by Southern blotting of chromosomal DNA, and these strains were designated RH280 and RH296.
RH244 was constructed by transforming mc2-155 with pRH1264Z followed by PCR screening and Southern analysis to document gene replacement of the wild-type sigE with the zeocin-disrupted sigE, as previously described (39). To generate a sigE sigH double mutation in M. smegmatis, RH244 was transformed with pRH1317. Cells were grown at 32°C, plated on LB plates containing kanamycin and sucrose, and incubated at 39°C as described elsewhere (28). DNA from individual colonies was screened as described above. The occurrence of allelic exchange resulting in gene replacement was confirmed for two independent isolates by Southern blotting of chromosomal DNA, and the strains were designated RH315 and RH328. For complementation experiments, a DNA fragment containing an intact copy of the M. smegmatis sigH coding and promoter regions was introduced into RH315 and RH328 by using pMH94A, a derivative of the integrating vector pMH94 in which the kanamycin resistance gene had been replaced with the apramycin resistance gene of pVK173T (23, 27). The same vector was used to introduce the sigH promoter and coding regions together with the coding sequence of the adjacent 3' gene into RH315 and RH328.Western blot analysis. Lysates of M. smegmatis RH244, RH280, RH315, and mc2-155 were made following growth to log phase and 50°C heat shock. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nylon membranes were performed according to standard methods. Blots were probed with monoclonal antibody IT-41 (HAT3) directed against DnaK (Hsp70) of M. tuberculosis and known to be cross-reactive with the M. smegmatis DnaK (21). Colorimetric detection of bound antibody was performed with the Protoblot II AP system (Promega) according to the manufacturer's instructions.
Nucleotide sequence accession number. The sequence of the sigH locus of M. smegmatis has been deposited in GenBank under accession no. AF144091.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning, Southern blotting, and sequence analysis of the mycobacterial sigE locus. A BLAST search of the M. tuberculosis H37Rv genomic DNA sequence with the M. tuberculosis sigE sequence identified an open reading frame in the cosmid MTCY7d11 related to but distinct from sigE (4). A PCR product based on this sequence was used as a probe to isolate clones containing this putative sigma factor gene, named sigH (Rv3223c), from genomic DNA libraries of M. smegmatis and M. tuberculosis (15, 40). Southern blot analysis using the coding region of the M. tuberculosis sigH gene as a probe showed that a single copy of this gene was present in M. smegmatis, M. fortuitum, M. avium, M. tuberculosis, and M. bovis BCG (not shown).
Analysis of the sigH locus in M. tuberculosis and M. smegmatis revealed that the M. smegmatis deduced amino acid sequence of 216 amino acids was 89% identical to the M. tuberculosis sequence of 217 amino acids. Additional potential translational start sites using GTG as the initiation codon can be identified 5' to those used in this analysis. However, the deduced amino acid sequences of these upstream regions in M. tuberculosis and M. smegmatis are not significantly similar. In contrast to the conserved organization of the mycobacterial sigE locus, the organization of the mycobacterial chromosome surrounding sigH differs substantially between M. tuberculosis and M. smegmatis (Fig. 1). Immediately 5' to M. smegmatis sigH is an open reading frame in the opposite transcriptional orientation that is highly similar to a gene of unknown function, designated ybaK, that is present in E. coli and several other species. Further 5' in M. smegmatis are two open reading frames that are highly similar to catechol dioxygenase (catA) and muconolactone isomerase (catC) genes of Acinetobacter lwoffii and other species. In contrast, immediately 5' to the M. tuberculosis sigH gene is a putative oxidoreductase gene. Further 5' is a fragment of ybaK, followed by an open reading frame with similarity to the carboxy-terminal region of aminoglycoside phosphotransferases.
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35 promoter regions in
ECF-dependent promoters, the greatest proportion of conserved amino
acid sequence among these proteins is in region 4.2, where M. tuberculosis SigH is 48% identical to M. tuberculosis
SigE and 43% identical to P. aeruginosa AlgU. As is the
case in many ECF sigma factors, SigH has a short region 1 compared to
the region 1 of the primary sigma factors of most bacteria.
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Characterization of sigH promoters in M. bovis BCG and M. smegmatis. Primer extension analysis was performed to identify the promoters of the sigH genes of M. bovis BCG and M. smegmatis (Fig. 3A). For both species, a strong band identifying the 5' end of an RNA transcript (MtP2 for M. bovis and MsP1 for M. smegmatis) was observed following heat shock. The band corresponding to this transcription start site was faintly visible in the absence of heat shock in both M. smegmatis and M. bovis BCG and was induced approximately fivefold following heat shock. In M. bovis BCG but not in M. smegmatis, an additional, less intense band was seen at 37°C that decreased in intensity following heat shock (MtP1). These transcription start sites are located 163 (MsP1), 296 (MtP1), and 198 (MtP2) bases 5' of the inferred initiation codon of sigH in M. smegmatis and M. bovis BCG.
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10 regions of these two promoters are identical in four of six
positions. In addition, the MtP1 promoter matches the rpsL
promoter sequence at all three positions in the extended 10 region, a
region that was demonstrated to be important for optimal expression of
rpsL. This extended 10 region has been observed to play an
important role in efficient transcription initiation from promoters
that lack 35 consensus regions (26). The MtP1 and
rpsL promoters show less similarity in the 35 region.
MtP2 and MsP1, the heat shock-inducible promoters of M. tuberculosis and M. smegmatis, both show substantial
similarity in the 35 region to the consensus sequence of ECF sigma
factor-dependent promoters (24), with seven of nine and six
of nine matches, respectively. MtP2 and MsP1 are identical to each
other in five of nine positions in this region. No highly conserved
consensus 10 region has been defined for ECF sigma factor-dependent
promoters; limited similarity of unknown significance was found in the
10 regions of MtP2 and MsP1.
Genes encoding ECF sigma factors of other species have been shown to be
autoregulated; i.e., RNA polymerase incorporating the ECF sigma factor
is required for transcription of the gene encoding that sigma factor.
To determine whether sigH was autoregulated, primer
extension analysis was performed on RNA isolated from
mc2-155 and RH280 following growth at 37°C or after
50°C heat shock (Fig. 4). No
sigH transcript was visible in either strain grown at
37°C. Following heat shock, however, a transcript was present in the
wild type but not in the sigH mutant. This result indicates that RNA polymerase incorporating SigH is required for transcription of
sigH from MsP1 following heat shock.
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-Galactosidase expression from the heat shock-inducible promoter
MtP2.
The heat shock-inducible sigH promoter MtP2
identified in M. bovis BCG was cloned upstream of the
promoterless -galactosidase gene in the shuttle vector pJEM15
(38). -Galactosidase activity from this construct was
measured in M. bovis BCG and in M. smegmatis. MtP2 showed substantial activity at 37°C (10-fold greater than vector
control activity) in M. bovis BCG but not in M. smegmatis. Following a 50°C heat shock, expression from MtP2
remained undetectable in M. smegmatis and declined slightly,
followed by recovery to baseline or slightly higher levels in M. bovis BCG.
Analysis and complementation of M. smegmatis sigH mutants. The sigH gene was disrupted by introducing the kanamycin resistance gene (aph) of Tn903 into the coding region of this gene on the chromosome of mc2-155 by allelic exchange. No differences in colony morphology were observed between the sigH mutants and the parental strain mc2-155 when plated on solid medium, and no difference in growth rate at 30 or 37°C in liquid medium was observed. No significant differences in survival were observed following several distinct stresses, including 42°C heat shock, 0°C cold shock, 50 mM citric acid stress, pH 4 HCl acid stress, exposure to 5 mM hydrogen peroxide, and exposure reactive nitrogen stress (20 mM NaNo2 incubated at pH 5.3). In addition, no difference was observed in survival during stationary phase for up to 10 days. The sigH mutant strain RH280 was substantially more susceptible to organic peroxide stress when measured by plating at serial time points following exposure, as was the sigE mutant strain RH244 (Fig. 6A). When measured by inhibition of growth around a paper disc impregnated with cumene hydroperoxide, RH280 but not RH244 was significantly more susceptible than mc2-155 (Fig. 6B). In multiple experiments, RH280 was no different from or slightly more susceptible to 53°C heat shock than the wild type, as was RH244 (Fig. 7).
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,
mc2-155/pMH94A; 
, RH244/pMH94A; 
, RH280/pMH94A; 
,
RH315/pMH94A; 
, RH315/pRH1335; 
, RH315/pRH1341.
Expression of DnaK in sigE and sigH mutant strains of M. smegmatis. Because of the apparent role of both SigE and SigH in the heat shock response, we determined whether expression of the heat shock chaperone DnaK was dependent on either of these sigma factors. Western blot analysis of lysates of mc2-155, RH244, RH280, and RH315 using the monoclonal antibody HAT3 (IT-41) (21), which recognizes an epitope present in M. tuberculosis and M. smegmatis DnaK, demonstrated substantial expression of this protein in all strains, whether grown at 37°C or after 50°C heat shock, indicating that its expression is not SigE or SigH dependent (Fig. 8).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The initial identification of the ECF subfamily of sigma factors
linked a relatively small number of alternative sigma factors in a
group based on sequence similarity, conservation of 35 region sequences of ECF sigma factor-dependent promoters, and a broad definition of conserved function (24). The large number of
ECF-type sigma factors identified in bacterial genomes that have been
recently sequenced suggests that members of this family are likely to
regulate many types of genes in response to a wide variety of conditions.
In M. tuberculosis, 10 of 13 putative sigma factors are members of the ECF subfamily (4). In addition to SigH, whose initial characterization we describe in this report, four mycobacterial sigma factors have been characterized to some extent. SigA appears to be the primary mycobacterial sigma factor (10, 30). SigB is highly similar to SigA but is nonessential and plays a role in the response to stress (10). SigE is widely distributed in mycobacterial species and plays a role in survival following stress (39). SigF is present only in species of the M. tuberculosis complex and is expressed in response to stress and starvation (5).
The results presented here indicate that SigH, like SigE, is present in fast- and slow-growing mycobacteria and plays a role in the bacterial stress response. The induction of transcription following heat shock suggests that genes regulated by SigH may be important in the heat shock response of mycobacteria. The absence of a difference in survival of the sigH mutant compared to wild-type M. smegmatis following 42°C heat shock and the small difference following 53°C heat shock is surprising in this regard. In the context of these phenotypes, the strong induction of sigH transcription suggests the presence of multiple mechanisms for the response to heat shock in mycobacteria.
This interpretation is supported by the expression of the heat shock chaperone DnaK in single- and double-mutant strains as well as in the wild type. The presence of multiple, possibly overlapping, responses to high temperature heat shock is also supported by the much greater susceptibility to heat shock of the sigE sigH double mutant compared to either single mutant. Overlap of gene regulation by ECF sigma factors has been found recently in B. subtilis, where for four different genes, RNA polymerase holoenzyme containing either SigW or SigX was shown to initiate transcription from the same promoter (13, 14).
The second stress for which SigH appears to be important in mycobacterial survival is oxidative stress from exposure to organic peroxide. Increased killing following exposure to cumene hydroperoxide of the sigH mutant, and to a lesser extent the sigE mutant, relative to the wild type, and greater susceptibility of the sigE sigH double mutant, suggests the presence of protective mechanisms mediated by each of these two sigma factors. The lack of altered susceptibility to hydrogen peroxide of the sigH mutant or of the sigE sigH double mutant relative to wild type indicates that the responses in M. smegmatis to these oxidative stresses differ. Differences in both phenotype and protein expression of mycobacteria following responses to these different peroxides have been noted previously (7, 32, 36). Greater susceptibility of mycobacteria to organic hydroperoxide than to hydrogen peroxide could result from the large lipid content of the mycobacterial cell wall that may be subject to peroxidation by these organic reagents. The lipid-rich mycobacterial cell wall may also act as a barrier to the effects of water-soluble hydrogen peroxide.
Both sigH and the adjacent 3' gene were found to be required for complementation of sigH mutant stress phenotypes in M. smegmatis. The expression of these genes as a single transcript from the autoregulated sigH promoter indicates that this gene, like sigH itself, is dependent on SigH for its expression. While it is possible that this gene encodes a protein that functions in a direct protective role against these stresses, the near-complete complementation of two distinct phenotypes is more consistent with the product of this gene functioning to regulate the expression of other gene products, either directly or as a positive regulator of SigH activity. The latter mechanism is well described for the regulation of alternative sigma factor activity in B. subtilis, where activities of the stress-responsive SigB and the sporulation-specific SigF are both regulated through interactions of positive and negative regulators (12, 35).
The sigH MtP1 promoter is of interest in the context of what
is currently known of mycobacterial promoters. Its 10 region is
highly similar to the well-characterized rpsL promoter of
M. smegmatis and to the consensus 10 region identified in
several putative promoters identified in M. smegmatis and
M. tuberculosis (2, 20). Of the two bases in the
MtP1 10 hexamer that diverge from this consensus, one is at the
second position, where C is present in the place of the highly (>90%)
conserved A. This divergence from the consensus may account for the
weak signal observed in the primer extension experiments. Like the
rpsL promoter, MtP1 lacks a 35 region that matches known
consensus 35 sequences, a finding typical of the majority of
mycobacterial promoters (2). MtP1 does have the extended
10 TGN motif that plays an important role mycobacterial
rpsL transcription as well as in E. coli and other species in promoters lacking consensus 35 elements (19, 29, 33). This extended 10 region appears to be relatively common among mycobacterial promoter sequences, occurring in more than
20% of those identified (3).
The 35 elements of MsP1 and MtP2 are highly similar and match closely
the ECF consensus. Like previously defined ECF-dependent promoters,
MsP1 and MtP2 lack consensus 10 elements. The similarity between
regions 4.2 of the mycobacterial SigH proteins and this region in AlgU
and other ECF sigma factors supports the importance of the 35 element
in transcription initiation. The nature of the two promoters identified
in M. bovis BCG suggests (i) transcription from MtP1 by RNA
polymerase containing the primary sigma factor SigA and (ii) expression
from MtP2 mediated by RNA polymerase incorporating SigH.
While SigH proteins of M. tuberculosis and M. smegmatis are highly similar and have similar 35 promoter
elements, several results suggest that they may have different
functions or regulation. The lack of activity of the
MtP2-lacZ transcriptional fusion in M. smegmatis,
both at 37°C and after heat shock is surprising given its activity at
37°C in M. bovis BCG. While differences in activity of
other promoters between these species has been noted, most
mycobacterial promoters that are active in one species are active in
the other (2, 38). The differences in the 35 regions of
these promoters may be sufficient to account for the difference in
activity of the two promoters in M. smegmatis and in
M. bovis BCG.
The second observation that suggests different functions for sigH in M. tuberculosis and M. smegmatis is the marked difference in the organization of the chromosome adjacent to sigH in these species. Of particular interest is the gene immediately 3' to sigH. Our data indicate that this gene plays an important role in the SigH-mediated stress response in M. smegmatis. The absence of sequence similarity in the corresponding gene in M. tuberculosis makes it unlikely that this gene has a similar role in this species.
Taken together, functional data for M. smegmatis and the sequence and transcriptional analyses of both species suggest a model in which sigH is nonessential and is expressed at low levels under nonstressed conditions. Following stress, e.g., heat shock, transcription of sigH and its adjacent 3' gene is markedly increased. Autoregulation of the inducible sigH promoter provides a positive feedback mechanism for rapidly increasing the amount of SigH present in the cell in response to stress. At least in M. smegmatis, the second gene in the sigH operon is essential for the sigH-mediated stress response.
Our data do not exclude the formal possibility that this second gene is solely responsible for the phenotypes of the mutant strains as well as for the autoregulation of the sigH promoter. However, a model in which this gene acts as a positive regulator of SigH function, for example, through direct interaction with SigH, is consistent with our data as well as with known mechanisms for the regulation of sigma factor activity. Alternatively, the product of this gene could directly regulate the expression of genes involved in the mycobacterial stress response. Whether the unrelated gene in the corresponding position in the M. tuberculosis has a similar function remains to be determined, as does the mechanism by which heat shock is transduced into activation of transcription from the inducible mycobacterial sigH promoters.
SigH, like SigE and SigF, plays a role in the mycobacterial stress response. The presence of multiple mechanisms for the regulation of stress responses is consistent with the need for the bacteria to adapt to a variety of stresses during extracellular growth and after uptake by macrophages during the course of infection. The major stresses with which M. tuberculosis must contend are those encountered during the course of infection. Thus, sigma factors that regulate gene expression in response to stress response are likely to play an important role in regulation of gene expression that is essential for M. tuberculosis pathogenesis.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant AI-37901 from the National Institute of Allergy and Infectious Diseases and by a grant from the World Health Organization Vaccine Research and Development Program. S.G. was supported by a student intern award from the Elizabeth Glaser Pediatric AIDS Foundation.
Monoclonal antibody IT-41 (HAT3) was obtained from the UNPD/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. We thank John Belisle and Patrick Brennan for M. tuberculosis genomic DNA and Eric Rubin for helpful discussions.
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ADDENDUM IN PROOF |
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During review of this article, we became aware of a report in which M. tuberculosis sigH transcription was shown to be induced following heat shock (R. Manganelli, E. Dubnau, S. Tyagi, F. R. Kramer, and I. Smith, Mol. Microbiol. 31:715-724, 1999).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, Children's Hospital, Enders 609, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 355-5151. Fax: (617) 355-8387. E-mail: husson{at}a1.tch.harvard.edu.
Present address: Dyax Corporation, Cambridge, MA 02139.
Present address: Department of Cardiology, Brigham and Women's
Hospital, Boston, MA 02115.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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