Báo cáo khoa học: The sigma factors of Mycobacterium tuberculosis: regulation of the regulators potx

REVIEW ARTICLE
The sigma factors of Mycobacterium tuberculosis:
regulation of the regulators
Preeti Sachdeva
1
, Richa Misra
1
, Anil K. Tyagi
2
and Yogendra Singh
1
1 Institute of Genomics and Integrative Biology (CSIR), Mall Road, Near Jubilee Hall, Delhi, India
2 Department of Biochemistry, University of Delhi South Campus, New Delhi, India
Introduction
The causative agent of tuberculosis (TB), the intracel-
lular bacterial pathogen Mycobacterium tuberculosis,
latently infects about one-third of the world’s popula-
tion and claims one life every 15 s. Although the
World Health Organization-recommended treatment
strategy for the detection and cure of TB, the ‘Directly
Observed Treatment, Short-course’ (DOTS), has
reduced the burden of TB to a great extent, the inci-
dence of TB has shown an increase in recent years as a
result of the emergence of drug-resistant TB and
co-infection with the human immunodeficiency virus
( Despite global efforts, no
new drugs for the treatment of TB have been devel-
oped successfully for more than four decades.
The success of M. tuberculosis as a highly adapted
pathogen rests upon its ability to establish a persistent
infection in the hostile environment of the host cell

through mechanisms involving transcriptional repro-
gramming, ensuring metabolic slowdown and the
upregulation of virulence and stress-response pathways
[1]. In the course of a successful infection, the bacte-
rium copes with numerous stresses (reviewed previ-
ously [2]) and modulates host responses through
coordinated regulation of its gene expression in
response to signals encountered in the host body. Gene
expression in bacteria is regulated primarily at the level
of transcription initiation, which is mediated by
the RNA polymerase (RNAP) holoenzyme. The
Keywords
Mycobacterium; post-translational
regulation; sigma factor; tuberculosis
Correspondence
Y. Singh, Institute of Genomics and
Integrative Biology, Mall Road, Delhi
110007, India
Fax: +11 2766 7471
Tel: +11 2766 6156
E-mail:
(Received 8 September 2009, revised
27 October 2009, accepted 4 November
2009)
doi:10.1111/j.1742-4658.2009.07479.x
One of the important determinants of virulence of Mycobacterium
tuberculosis is adaptation to adverse conditions encountered in the host
cells. The ability of Mycobacterium to successfully adapt to stress condi-
tions is brought about by the expression of specific regulons effected by a
repertoire of r factors. The induction and availability of r factors in

response to specific stimuli is governed by a complex regulatory network
comprising a number of proteins, including r factors themselves. A serine–
threonine protein kinase-mediated signaling pathway adds another dimen-
sion to the mycobacterial r factor regulatory network. This review high-
lights the recent advances in understanding mycobacterial r factors, their
regulation and contribution to bacterial pathogenesis.
Abbreviations
ECF, extracytoplasmic function; imp, immunopathology; M. bovis BCG, Mycobacterium bovis Bacille Calmette–Gue
´
rin; PPE, proline-
proline-glutamate; RNAP, RNA polymerase; STPK, serine–threonine protein kinase; Tat, twin-arginine translocation; TB, tuberculosis; TCS,
two-component system; TSP, transcription start point; ZAS, zinc-associated anti-r factor.
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 605
holoenzyme comprises a five-subunit core RNAP (sub-
unit composition, a
2
bb¢x) and a dissociable subunit,
sigma (r). The r factor contains many, if not all, of
the promoter recognition determinants and confers
promoter specificity to the RNAP holoenzyme. The
association and dissociation of distinct r factors with
core RNAP mediates specific cellular responses
through redirection of transcription initiation at vari-
ous regulons [3,4]. The temporal expression of specific
regulons controlled by the induction or availability of
one or more r factors allows M. tuberculosis to sustain
multiple stages of host–pathogen interactions, includ-
ing adhesion, invasion, intracellular replication and
dissemination to other sites [5]. In this review, we sum-
marize the current knowledge about mycobacterial r

factors and their regulation, and discuss their role and
importance in the pathogenesis of mycobacteria.
General overview of r factors
The r factors have been divided into two main fami-
lies, namely the sigma 70 (r
70
) family and the sigma
54 (r
54
) family, named after the 70 kDa primary r
factor and the 54 kDa nitrogen-regulation r factor
both from Escherichia coli, respectively. r
54
-type r fac-
tors are structurally different from the r
70
family
members and utilize a distinct mechanism of open
complex formation. There are no known representa-
tives of the r
54
family in any GC-rich, Gram-positive
bacteria and cyanobacteria; however, r
70
-related r fac-
tors are encoded in all bacterial genomes [4,6]. The r
70
family of proteins contain up to four conserved regions
(regions 1, 2, 3 and 4) and have been divided into four
groups (groups 1–4) based on their phylogenetic rela-

tionships and modular structure [4,7] (Fig. 1). Group 1
comprises essential housekeeping r factors and these r
factors contain all the four conserved regions. Group 2
r factors are most closely related to group 1 r factors,
but are not essential and lack the subregion 1.1
(Fig. 1). Most group 3 r factors contain conserved
regions 2–4. Group 4 includes r factors containing
only regions 2 and 4 and accommodates the highly
diverged extracytoplasmic function (ECF) subfamily,
members of which respond to signals from the extra-
cytoplasmic environment [4,8,9].
A r
70
-dependent promoter sequence may comprise
the following potential elements that mediate its recog-
nition by the RNAP holoenzyme: (a) the -10 element, a
hexameric sequence, centered about 10 bp upstream of
the transcription start point (TSP) and recognized by
the 2.4 subregion; (b) usually an extended -10 motif
(TGN motif), situated immediately upstream of the -10
sequence and recognized by an a-helix in the region 3.0;
(c) the -35 element, a hexameric sequence centered
about 35 bp upstream of the TSP and recognized by
the 4.2 subregion [3,10,11]; and (d) an AT-rich UP
element, generally located between 61 and 41 bp up-
stream of the TSP [12,13]. The upstream (UP) element
binds the C-terminal domain of the a subunit of the
RNAP and enhances the promoter activity by two-fold
to as much as 90-fold [12,14]. Besides, the presence of
GC-rich sequences in the spacer region has been shown

to drastically influence the promoter strength [15].
All bacteria have at least one essential r factor that
transcribes the genes required for cell viability, and
most bacteria harbor alternative r factors that tran-
scribe regulons in response to specific stimuli. The
number of alternative r factors correlates generally
with the variability of the environments encountered
by a given bacterial species. The number may range
from two alternative r factors in Streptococcus pyoge-
nes, whose primary niche is limited to the human oro-
pharynx, to more than 60 encoded by the soil
actinomycete, Streptomyces coelicolor, whose natural
habitat is highly variable in terms of nutrients, stresses
and competing microbial flora [16]. Also, while the
number of r factors is generally found to increase with
the genome size, microorganisms that have developed
differentiation programmes (e.g. sporulation) tend to
have higher alternative r factor ⁄ genome size ratios
than most obligate pathogens or commensal bacteria.
Interestingly, M. tuberculosis has the highest alterna-
tive r factor ⁄ genome size ratio amongst the obligate
pathogens, suggesting a highly complex regulatory
mechanism for its transcription [17]. Notably, a recent
report suggested endospore formation by mycobacteria
and speculated sporulation as a possible mechanism
for dormancy [18].
M. tuberculosis encodes a repertoire of 13 r factors
[19,20], of which r
A
,

r
B
and r
F
are representatives of
groups 1, 2 and 3 of the r
70
family, respectively, while
the remaining 10 r factors belong to group 4.
Amongst the ECF r factors, r
G
, r
I
and r
J
possess an
additional carboxy-terminal extension in their struc-
ture, which is presumed to provide a surface for inter-
action with other regulatory molecules [17]. The gene
loci for the principal r factor in M. tuberculosis, sigA,
and for the principal factor-like r factor, sigB, are well
conserved across all sequenced mycobacterial genomes.
The alternative r factors of M. tuberculosis have var-
ied representation, in terms of number and functional-
ity, across the Mycobacterium genus. sigC orthologs
have been found to be present in all pathogenic Myco-
bacterium spp. sequenced to date, including Myco-
bacterium leprae, but absent in nonpathogenic
Mycobacterium spp. sequenced so far. By contrast,
The r-factors of M. tuberculosis P. Sachdeva et al.

606 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
sigE is the only ECF r factor conserved across the
Mycobacterium genus. Five r factors (sigD, sigF, sigG,
sigH and sigJ) are present in all genomes, except for
that of M. leprae, where these r factors are present as
pseudogenes. The remaining ECF r factors have
mixed representation across pathogenic and nonpatho-
genic species. The details of the r factor content and
the paralogous members of a r subfamily in various
sequenced Mycobacterium spp. have already been
reviewed [17]. Interestingly, among all bacterial genus
types, Mycobacterium exhibits the maximum variation
in the number of r factors among its species [21] with
the saprophytic species, Mycobacterium smegmatis,
possessing 28 r factors and the obligate pathogen,
M. leprae, having only four functional r factors [17].
Physiological roles of mycobacterial r
factors
SigA (r
A
)
r
A
, the primary or principal r factor, is indispensable
for growth both in M. tuberculosis [2] and in
M. smegmatis [22]. The M. tuberculosis H
37
Rv sigA
transcript is maintained at a constant level under vari-
ous stress conditions; however, there have been a few

reports of its upregulation during infection in human
macrophages [23] and downregulation during low aera-
tion and stationary-phase growth [24]. During condi-
tions of low aeration and in the stationary phase, the
energy available to the bacteria may be quite low and
Fig. 1. Promoter recognition by the RNA
polymerase-r
70
holoenzyme. [Correction
added on 8 December 2009 after original
online publication: in Fig. 1 ‘transcription
atrat site’ was changed to ‘transcription
start site’, and ‘is acidic an nature’ was
changed to ‘is acidic in nature’.]
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 607
the decreased levels of the sigA transcript might reflect
a decrease in the mRNA pool relative to the total
RNA of the cell [24]. The sigA transcript continues to
be widely used as an internal standard for normaliza-
tion in quantitative RT-PCR experiments using
M. tuberculosis RNA isolated under all conditions
[24,25]. The unusual stability of sigA mRNA (half
life > 40 min) [26] makes it a preferred standard for
this purpose.
A point mutation in the 4.2 domain of r
A
(R515H)
resulted in attenuation of virulence of the M. bovis
strain ATCC 35721 in a guinea pig model of infection

[27]. The mutated r
A
was reported to be incapable of
interaction with an accessory transcription factor,
WhiB3. Inactivation of the whiB3 gene in another
M. bovis strain, ATCC 35723, resulted in the attenua-
tion of pathological changes in lungs, as reported for
the M. bovis sigA mutant strain. However, in a differ-
ent genetic background, such as M. tuberculosis
H
37
Rv, whiB3 deletion resulted in only partial attenua-
tion of virulence in both mice and guinea pig models
[28]. A number of such results have been reported in
the last few years, establishing the role of r
A
in host–
pathogen interactions, in addition to its housekeeping
function. In a clinical strain (the M. tuberculosis 210
isolate, TB294, known for its higher intracellular
growth rate compared with other strains), sigA is natu-
rally upregulated. Overexpression of sigA in M. tuber-
culosis H
37
Rv enhanced its growth in human
macrophages and in the lungs of mice after aerosol
infection, further suggesting its role in virulence [29].
The same group recently reported that this effect of
r
A

is mediated, in part, by upregulation of one of its
target genes, eis (enhanced intracellular survival),
which contributes to the enhanced capacity of
M. tuberculosis strain 210 to grow in monocytes [30].
The role of r
A
in the expression of virulence genes is,
however, a host-specific feature, because complementa-
tion with a functional r
A
is sufficient for the restora-
tion of virulence of M. bovis ATCC 35721 in a
subcutaneous guinea pig model but not in an intratrac-
heal Australian brushtail possum model of experimen-
tal TB [31]. Furthermore, the consensus sequence for a
r
A
-dependent promoter has been predicted upstream
of an operon, Rv3134c-devR-devS [32], which encodes
proteins involved in establishing and maintaining TB
latency under hypoxic conditions [33]. Recently,
PE_PGRS33, a cell-surface molecule that plays an
important role in TB pathogenesis [34], has also been
shown to be transcribed from a r
A
-dependent pro-
moter [35]. For a very long time, genes upregulated in
phagocytosed bacteria were considered as potential
candidates for virulence over constitutively expressed
genes. This perception, however, has been challenged

by a study which proposed that the majority of
M. tuberculosis genes required for intracellular survival
are constitutively expressed rather than regulated by
macrophages [36]. Interestingly, the role of r
A
in viru-
lence gene expression goes very well with this report.
SigB (r
B
)
r
B
, the principal factor-like r factor of M. tuberculosis,
is very similar to the C-terminal portion of r
A
[17,37].
But, unlike r
A
, it has been found to be dispensable for
growth in both M. smegmatis [22] and M. tuberculosis
[38]. Moreover, the r
A
and r
B
regulons do not overlap
much except for a few genes such as those belonging to
the PE_PGRS family [35,39]. A comparison of amino
acid residues from the 2.4 and 4.2 subregions of r
A
and

r
B
shows that these r factors differ at four of 23 posi-
tions in the 2.4 subregion. More significantly, the 4.2
subregion of r
A
and r
B
differs at 15 of 39 positions
and nine of these changes are nonconserved. While
these differences would definitely reflect on the pro-
moter sequences recognized by these two r factors,
other aspects that contribute to the nonoverlapping
repertoire of genes transcribed by the two r factors
remain to be elucidated [40]. Besides, unlike sigA,
expression of the M. tuberculosis sigB gene increases
upon exposure to various environmental stresses such
as low aeration [24], treatment with hydrogen peroxide
and heat shock, with a more pronounced effect seen in
stationary phase than in logarithmic phase [26].
A recent report demonstrated that inactivation of
the sigB gene did not affect the survival of M. tubercu-
losis during infection in human macrophages or in
mouse and guinea pig models [38]. However, deletion
of sigB in M. tuberculosis results in its higher sensitiv-
ity to SDS-induced surface stress, heat shock, oxidative
stress, exposure to vancomycin and hypoxic conditions
[17,38]. Further evidence for the role of r
B
in adapta-

tion to stationary phase and nutritionally poor condi-
tions came from a report by Mukherjee et al. [41] This
group reported that upon overexpression of M. tuber-
culosis sigB in M. smegmatis, the cell-surface glycopep-
tidolipids found in the outer layers of M. smegmatis
become hyperglycosylated, similarly to what is
observed during carbon starvation. Certain metabolic
enzymes, namely succinyl-coenzyme A synthetase,
glycosyltransferases (encoded by genes in the glycopep-
tidolipid locus), b-ketoacyl coenzyme A synthetase,
rhamnosyltransferase and acetylcoenzyme A acetyl-
transferase, were also found to be induced upon over-
expression of r
B
in M. smegmatis [42]. Overexpression
of sigB in M. tuberculosis resulted in a significant
The r-factors of M. tuberculosis P. Sachdeva et al.
608 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
upregulation of genes encoding proteins involved in
cell wall-related processes, several early culture filtrate
antigens (ESAT-6-like proteins), 50S ribosomal pro-
teins, PE-PGRS proteins, keto-acyl synthase, KasA
and the regulatory proteins, WhiB2, IdeR and r
B
itself
[39]. Also, sigB is transcribed from a predicted r
B
-
dependent promoter in an in vitro assay, further sug-
gesting its autoregulation [39]. Moreover, expression

profiling of the M. tuberculosis sigB mutant strain
revealed regulation of ideR, furA, katG, ppe19 and
hsp20 by r
B
[38].
Previous studies have suggested that there are two
more promoters upstream of r
B
: one is recognized by
RNAP containing any of the three r factors, r
E
[43],
r
H
[44] and r
L
; while the other is recognized by RNAP
containing r
F
[45]. The transcription of sigB under stan-
dard physiological growth conditions and its induction
upon exposure to surface stress is dependent on r
E
[43],
while its induction during heat shock or oxidative stress
is r
H
-dependent [44]. In contrast to the r
F
-dependent

transcription of sigB observed in an in vitro transcrip-
tion assay [45], the upregulation of sigB upon overex-
pression of sigF was not observed [46]. The possibility
of certain r
F
-dependent genes being missed in the study
by Williams et al. cannot be ruled out in view of induc-
tion of the anti-r
F
protein, UsfX, which is expected to
significantly reduce the effective concentration of active
r
F
in the cell. The conditions for r
F
- and r
L
-dependent
transcription of sigB are yet to be identified.
r
B
seems to operate as a downstream response regu-
lator in the hierarchy of the r factor regulation net-
work, the levels of which are adjusted in response to
different environmental conditions brought about by
an ensemble of five different r factors, including itself
(Fig. 2). The self-regulation of r
B
is expected to result
in its autoamplification and therefore a pronounced

effect on its level, even in the presence of minimal
changes in the levels of its upstream regulators. Inter-
estingly, other than r factors, a two-component system
(TCS) response regulator, MprA, also regulates the
in vivo expression of sigB in M. tuberculosis under
SDS-induced surface stress and exponential growth via
its binding to conserved motifs in the upstream region
of the gene [47].
SigC (r
C
)
sigC is conserved across all pathogenic mycobacterial
species, including M. leprae [48], and is absent in all
nonpathogenic species sequenced to date, such as
M. smegmatis [21], Mycobacterium gilvum, Mycobacte-
rium vanbaalenii, Mycobacterium sp. MCS, Mycobacte-
rium sp. KMS and Mycobacterium sp. JLS. Despite the
sigC transcript being the most abundant transcript of
all r factors, most of the core RNAP during exponen-
tial phase has been found to be associated with either
r
A
or r
B
. Therefore, it was speculated that M. tubercu-
losis sigC is either translated at a very low efficiency or
has a low affinity for RNAP [24]. M. tuberculosis sigC
is downregulated during stationary phase and in
response to heat shock and SDS-induced surface stress
[24]. Interestingly, sigC was also found to be downregu-

lated in the M. tuberculosis CDC1551 sigF mutant
strain [49], adding another r factor in the hierarchical
regulatory network of mycobacterial r factors (Fig. 2).
r
C
is not required for the survival of Mycobacterium
in murine bone marrow-derived macrophages or in
activated J774A.1 macrophages [50]. In a mouse model
of infection, the sigC mutant in both the CDC1551
and H
37
Rv backgrounds grows and persists in lungs
but shows attenuated disease progression and fails to
elicit the same degree of lethal immunopathology as
the wild-type strain [50,51]. This phenotype of bacterial
persistence at high colony counts with reduced host
mortality is designated as the immunopathology (imp)
defect and is associated with a significant reduction in
the number of infiltrating neutrophils and with the
production of pro-inflammatory cytokines such as
tumor necrosis factor- a-a, interleukin-1b, interleukin-6
and interferon-c in the lungs of the infected animal
[51]. The attenuation of the sigC mutant may result
from dysregulation of expression of several key viru-
lence-associated genes, such as hspX (encoding an
a-crystallin homolog), senX3 (a sensor kinase), mtrA (a
response regulator), polyketide synthases and fbpC
(antigen 85C) [50]. Most importantly, in a guinea pig
model of infection that best mimics the granuloma for-
mation and disease progression in humans, the

M. tuberculosis sigC mutant displayed delayed growth
with fewer caseating lesions compared with the wild-
type strain [51]. The two promoter-recognition
domains of r
C
(r
C
2
and r
C
4
) have been crystallized [52]
and interact in vitro involving occlusion of the Pribnow
box recognition region. This interdomain interaction is
suggestive of an alternate mechanism of regulation of
r
C
activity via autoinhibition in the absence of a cog-
nate anti-r factor [53].
SigD (r
D
)
r
D
is expressed at a moderately high and constitutive
level during exponential and stationary growth phases
and declines significantly, following hypoxia, in a pat-
tern very similar to that of r
A
in an in vitro culture

[54,55]. The stringent response is modulated by the relA
gene product via the synthesis of a key signaling
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 609
molecule, guanidine tetraphosphate (ppGpp) [56]. relA
deletion in Mycobacterium results in a loss of virulence
accompanied with a significant decrease in sigD expres-
sion during the logarithmic phase [57]. sigD is upregu-
lated during nutrient starvation [58] (a condition that is
at least partly Rel-dependent), further suggesting the
role of r
D
in physiological adaptations such as stringent
response and starvation. The intracellular r
D
levels
decrease, following infection, in both quiescent and acti-
vated macrophages [59] and the loss of sigD does not
affect the ability of M. tuberculosis CDC1551 to survive
in J774A.1 macrophages. However, the mutant strain
induced a lower level of tumor necrosis factor- a produc-
tion by macrophages relative to the wild-type strain.
The M. tuberculosis H
37
Rv sigD mutant showed a mod-
erate loss of virulence with less extensive inflammation
and histopathological changes in BALB ⁄ c mice [54],
while deletion of sigD in the CDC1551 background
resulted in significant attenuation of lethality in a
C3H:HeJ mouse model of infection [60].

Some of the important r
D
-regulated genes include
those encoding proteins involved in lipid metabolism,
cell wall-related processes, stress response and DNA
binding and repair. Several genes, such as rpfC (impli-
cated in the revival of dormant mycobacteria), mce1
(associated with the entry of Mycobacterium into non-
phagocytic cells), pks10 (a polyketide-like chalcone
synthase), recR and those encoding several chaperones,
ribosomal proteins, elongation factors and ATP syn-
thase subunits were also reported to be downregulated
in the M. tuberculosis sigD mutant in the late station-
ary phase [54,60]. Notably, several r
D
-regulated genes
were reported to be highly expressed in the MprAB
TCS mutant strain under SDS stress, indicating that
many of the r
D
-regulated genes are under the repres-
sive effect of MprA [61].
SigE (r
E
)
r
E
is one of the two ECF r factors encoded by the
M. leprae genome [48]. sigE is upregulated in myco-
bacteria grown within human macrophages compared

with those grown in an in vitro culture [23,62]. Its
expression also increases following exposure to heat
Fig. 2. Regulatory network of Mycobacterium tuberculosis r factors. Color coding and symbols for the various regulators are mentioned in
the key. The red horizontal line indicates blockade; (?) indicates that the significance of the phosphorylation is not known.
The r-factors of M. tuberculosis P. Sachdeva et al.
610 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
shock, SDS-mediated surface stress [24], isoniazid [63]
and vancomycin [64]. Although sigE is not essential
for growth of M. smegmatis, its deletion results in
increased susceptibility to oxidative stress and acidic
pH [65,66]. The M. tuberculosis CDC1551 sigE mutant
exhibits the imp phenotype in the C3H ⁄ HeJ mouse
model of lung infection [67]. The M. tuberculosis
H
37
Rv sigE mutant showed decreased viability in mac-
rophages [43] and is strongly attenuated for virulence
in both BALB ⁄ c and severe combined immunodeficient
(SCID) mice. Moreover, the sigE mutant manifested
the formation of granulomas with characteristics dif-
ferent from those induced by the wild-type strain [68]
and a reduced ability to grow at 4 days postinfection,
as well as impaired CXCL10 expression in monocyte-
derived dendritic cells. The impaired CXCL10 expres-
sion is thought to inhibit the recruitment of activated
effector cells involved in the formation of granulomas
[69]. Also, the global transcription profile of macro-
phages infected with the sigE mutant showed upregula-
tion of a number of components of the host defense
system, such as CCL4 chemokine, prostaglandin E,

toll-like receptor-2 and defensins, indicating the role of
r
E
in suppressing the immune system and the antibac-
terial response of the host [70].
In M. tuberculosis, sigE is transcribed from three dif-
ferent promoters: promoter P1, utilized during growth
under normal physiological conditions [71]; promoter
P2, regulated by MprAB TCS, induced under surface
stress and alkaline pH [47]; and a third, r
H
-dependent
promoter, P3, induced under the conditions of oxida-
tive stress and heat shock. The r
H
-dependent promoter
is also considered to be responsible for increased tran-
scription of sigE in macrophages [72]. The lack of a
functional sigH gene in M. leprae has in fact been
implicated in the defective response of the organism to
heat stress, despite the presence of a functional r
E
pro-
tein [73]. Various different sigE start codons have also
been characterized, which may give rise to different r
E
isoforms, depending on which promoter is used for
transcription [71]. Moreover, in both M. smegmatis
and M. bovis Bacille Calmette–Gue
´

rin (BCG), sigE is
transcribed from two TSPs, each preferred under dif-
ferent temperature conditions [65]. However, despite
the presence of identical upstream sequences, these
TSPs could not be detected in M. tuberculosis [71].
r
E
-dependent genes encode proteins belonging to dif-
ferent classes, such as transcriptional regulators (includ-
ing r
B
, Rv3050c, MprA and MprB), enzymes involved
in fatty acid metabolism (most importantly isocitrate
lyase) and the classical heat shock proteins [43,70]. As
stated above, the MprAB TCS encoded by the r
E
-
dependent genes mprA and mprB regulates the in vivo
expression of sigE as well as another stress-responsive r
factor, sigB,inM. tuberculosis. This regulation is medi-
ated by binding of the response regulator, MprA, to
conserved motifs in the upstream regions of r
E
and r
B
[47]. A number of stress-responsive genes have been
reported to be downregulated in the mprA mutant
strain, some of which may actually be targets of these r
factors [61]. In addition, the induction of mprA follow-
ing exposure to stress also suggests a direct role of this

regulatory system in the stress-response pathways in
M. tuberculosis [47]. Surprisingly, some of the r
E
-depen-
dent genes, such as Rv1129c, Rv1130 and Rv1131 (cit-
rate synthase; gltA), are also under an indirect repressive
effect of MprA, suggesting the MprAB–r
E
regulation
network to be highly complex [43,61].
Under conditions of stress (such as ATP depletion),
inorganic polyphosphate polyP (synthesized by the
enzyme PPK1) serves as a preferred donor for the
MprB-mediated phosphorylation of MprA. This results
in MprA-regulated transcription of the mprAB operon,
which thereby facilitates r
E
-mediated transcription of a
stringent response gene, rel [74]. As sigD is suggested to
be part of the RelA regulon [57], its expression is also
likely to be under the indirect control of r
E
. The positive
regulation of r
E
by MprAB, which results in bimodal rel
gene expression and thereby a phenotypic heterogeneity
in the bacterial population, may play a role in the devel-
opment of persistence in Mycobacterium [75]. The bind-
ing affinity of MprA for its promoter increases upon

phosphorylation and is required for the upregulation of
the mprA gene in vivo [76]. By contrast, the binding of
MprA to sigB and sigE upstream regions can occur,
even in the absence of phosphorylation [47]. In this view,
the downregulation of sigE and sigB and their corre-
sponding regulons in the ppk1 deletion mutant [74] is
likely to be an indirect consequence of diminished poly-
phosphate levels. The direct effect of phosphorylation of
MprA on its binding to sigB and sigE upstream regions
remains to be studied. The co-dependent transcriptional
regulation of r
E
and MprA [43,61], as well as the autore-
gulation of MprA [76], may result in the maintenance of
the levels of r
E
and MprA in a cyclic manner during
stress. r
E
, in addition to a complex transcriptional regu-
lation, is also subjected to translational regulation as
well as to post-translational regulation by a zinc-associ-
ated anti-r factor (ZAS) family protein [71].
SigF (r
F
)
M. tuberculosis r
F
, the only group 3 r factor represented
in the Mycobacterium genus, bears significant homology

to the stress response r factors in Bacillus subtilis, Staph-
ylococcus aureus and Listeria monocytogenes and to the
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 611
sporulation r factors in B. subtilis and S. coelicolor [77].
A study elucidating alignment of the sigF orthologs
across sequences obtained from various mycobacterial
species revealed a clustering pattern that differentiates
slow-growing and fast-growing species [78].
When introduced into M. bovis, the expression of
M. tuberculosis sigF is induced under a variety of stress
conditions, most notably antibiotic stress (rifampin,
ethambutol, streptomycin and cycloserine), nutrient
depletion, oxidative stress, cold shock and anaerobic
metabolism, particularly in the presence of metronida-
zole and during stationary-phase growth [79]. How-
ever, no such marked change in the transcript level of
sigF was seen in M. tuberculosis H
37
Rv following
exposure to cold shock, hypoxia, oxidative stress and
entry into stationary phase [24], suggesting a
differential regulation pattern of sigF expression in
M. tuberculosis and M. bovis BCG. Similarly, the
M. tuberculosis CDC1551 sigF mutant strain showed
no significant difference in the in vitro survival rate in
response to temperature shift, oxidative stress and
long-term stationary phase growth. However, the
mutant displayed increased susceptibility to rifampin
and rifapentine, as well as reduced uptake of a hydro-

phobic solute, chenodeoxycholate, suggesting that
the sigF deletion produces structural alterations in
the mycobacterial cell envelope [80]. In contrast to the
findings of Chen et al. [80] and their speculation for
the role of r
F
in bacterial growth during stationary
phase and under stress conditions, as well as suscepti-
bility to antibiotics, the conditional overexpression of
sigF during the early exponential growth phase neither
resulted in any growth arrest nor reduced the suscepti-
bility of the strain to rifampin and isonaizid [46]. Also,
M. tuberculosis H
37
Rv sigF was found to be upregulat-
ed in a nutrient starvation model of M. tuberculosis
[58] and during infection of cultured human macro-
phages [62]. However, a study by Williams et al.
revealed that M. tuberculosis CDC1551 r
F
is not
required for bacillary survival under nutrient starva-
tion conditions and within activated murine macro-
phages or for extracellular persistence in an in vivo
granuloma model of latent TB infection [46]. The sigF
mutant exhibits the imp phenotype in mice [49], as well
as reduced lethality in mouse and guinea pig infection
models [80,81], relative to the wild-type strain.
Unlike M. tuberculosis, M. smegmatis sigF is exp-
ressed throughout growth at levels almost comparable

to those of sigA. Expression profiling using a recombi-
nant M. smegmatis reporter strain revealed significant
induction of sigF upon treatment with ethambutol,
isoniazid, SDS and cold shock, whereas nutrient deple-
tion and salt stress stimulated a lower level of sigF
induction compared with the untreated control [78].
Another study with a M. smegmatis mc
2
155 sigF
mutant strain demonstrated that r
F
is required for
resistance to heat shock and acid stress, but not for the
survival of bacillus in human neutrophils. M. smegma-
tis r
F
also mediates resistance to oxidative stress, prob-
ably in a KatG-independent and AhpC-independent
manner [82]. As reported for M. tuberculosis [80],
M. smegmatis r
F
is also implicated in the regulation of
genes involved in cell wall permeability, as evident by
the decreased transformation efficiency in the presence
of a functional sigF gene. Also, r
F
is required for the
biosynthesis of carotenoids, complex lipids that act as
free-radical scavengers and protect the cells against
photodynamic injury in M. smegmatis [83].

In order to identify r
F
-dependent genes, transcrip-
tional profiling of an M. tuberculosis CDC1551 sigF
mutant strain at different growth stages and of a sigF-
overexpressing strain were carried out independently
[46,49]. [Correction added on 8 December 2009 after ori-
ginal online publication: in the preceding sentence
‘CDC551’ was changed to ‘CDC1551’.] Disruption of
the sigF gene resulted in the downregulation of a signifi-
cantly larger number of genes in the late-stationary
phase compared with the exponential phase. r
F
regu-
lates the expression of genes involved in the biosynthesis
and structure of the mycobacterial cell envelope, includ-
ing complex polysaccharides and lipids, particularly vir-
ulence-related sulfolipids. In addition to genes involved
in energy metabolism, nucleotide synthesis, intermediary
metabolism and information pathways, r
F
regulates
genes encoding several transcriptional regulators, for
example, MarR, GntR and TetR family regulators,
PhoY1 and Rv2884, as well as an ECF r factor, r
C
[46,49]. Conditional overexpression of sigF during the
early exponential phase resulted in the upregulation of
several genes encoding cell wall-associated proteins,
such as proline-glutamate (PE) and proline-proline-glu-

tamate (PPE) family proteins and mmpL family trans-
porters (mmpL2, mmpL5 and mmpL11) [46], known to
be involved in virulence [84,85].
sigF expression is regulated at the transcriptional
level via autoregulation of its promoter. Besides, r
F
activity is under the control of a complex post-transla-
tional regulatory network comprising an array of pro-
teins such as anti-r factors, anti-anti-r factors, as well
as certain proteins responsible for the modification of
these factors [86–88].
SigG (r
G
)
sigG is one of the most highly induced genes in
M. tuberculosis during macrophage infection [23,89]
The r-factors of M. tuberculosis P. Sachdeva et al.
612 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
and has been shown, in a macrophage infection model,
to be required for survival of the bacterium [90]. How-
ever, its expression is downregulated upon exposure to
various stress conditions, such as mild cold shock, heat
shock, low aeration and SDS-mediated surface stress.
sigG is one of the least represented mRNAs among all
r factors under normal in vitro growth conditions [24].
The sequence upstream of the M. tuberculosis sigG
gene showed similarity with the P1 promoter of a SOS
response gene, recA. Also, lexA, the gene encoding a
repressor for SOS genes, is regulated by r
G

. The
reduced lexA levels in the sigG mutant strain could
account for the upregulation of several SOS genes,
including recA. The sigG mutant is resistant to the
SOS response inducer, mitomycin C, further support-
ing the role of r
G
in the SOS stress response [90].
Microarray analysis of the mutant strain revealed
the downregulation of several genes encoding proteins
involved in fatty acid metabolism such as AceA (isoci-
trate lyase), FadE5 (acyl-coenzyme A dehydrogenase)
and ScoA (succinyl-coenzyme A). Interestingly, several
genes reported to be under the control of r
H
, such as
clpB, dnaK and trxB2, were also found to be
down-regulated in this study. Moreover, two other
r
D
-regulated genes, Rv1815 and rpfC (one of the five
resuscitation-promoting factor-like genes), were found
to be upregulated in the sigG mutant. The aforemen-
tioned result is expected in view of the downregulation
of sigH and the upregulation of sigD upon the deletion
of sigG in M. tuberculosis [90]. This finding further
corroborates the complex interplay of r factors in
M. tuberculosis (Fig. 2).
SigH (r
H

)
The role of r
H
as a central regulator of oxidative and
heat stress responses has been described for M. tuber-
culosis as well as for certain other mycobacterial spe-
cies, such as M. smegmatis and Mycobacterium avium
ssp. paratuberculosis [44,66,72,91]. As mentioned
earlier, the lack of a functional sigH plays a role in
the unresponsiveness of sigE during heat stress in
M. leprae [73]. Although the expression of M. tubercu-
losis sigH was found to be induced during macrophage
infection [62], the sigH mutant was not attenuated for
growth in human macrophages [44]. In a murine
model, the M. tuberculosis CDC1551 sigH mutant
strain demonstrated a distinctive imp phenotype [92].
To identify r
H
-regulated genes, microarray experi-
ments were carried out at different phases of growth
[92] and following diamide stress [44]. The r
H
regulon
includes its own structural gene and genes encoding
r
B
, r
E
, Rv0142 (putative transcriptional regulator)
DnaK, ClpB (heat shock proteins), TrxB and TrxC

(thioredoxin reductase ⁄ thioredoxin) [44,92]. r
H
also
induces enzymes involved in cysteine biosynthesis and
in the metabolism of ribose and glucose, indicating an
increased need for the synthesis of mycothiol precur-
sors [44] (mycothiols are known to be involved in
cellular protection during oxidative stress in actinomy-
cetes [93]). In a recent report on the long-term effects
of r
H
induction following diamide stress, it was
observed that in response to oxidative damage, certain
virulence ⁄ detoxification genes were induced, while
many lipid metabolism genes were repressed, as a part
of the stress-defense mechanism in M. tuberculosis.As
the effect of stress diminished with time, the expression
of lipid metabolism and of cell wall-associated genes
resumed, demonstrating a remarkable plasticity in gene
expression brought about by a mycobacterial r factor
[94]. r
H
activity is regulated at the transcriptional level
via autoregulation of the sigH promoter and post-
translationally via interaction with its cognate anti-r
factor, RshA [95]. The latter branch of regulation is
intersected by PknB, a serine–threonine protein kinase
(STPK), which further fine-tunes the stress response
regulon controlled by r
H

[96].
SigI (r
I
) and SigJ (r
J
)
sigI, and to a larger extent, sigJ, genes are expressed at
high levels in late stationary phase dormant cultures of
M. tuberculosis. The transcription of these two r fac-
tors continues following rifampicin treatment of these
cultures in an in vitro drug-persistent model of
M. tuberculosis [55]. However, no difference in viability
was observed between the sigJ mutant and the wild-
type strain in a late stationary phase culture following
rifampicin treatment, or in an immune stasis murine
model. The sigJ mutant is more susceptible to killing
by H
2
O
2
than its parental strain. As katG mRNA
levels remain unchanged upon deletion of sigJ, r
J
possibly mediates resistance to H
2
O
2
via a KatG-
independent pathway [97]. r
J

may also contribute to
the survival of M. tuberculosis in the host organism, as
suggested by an increase in sigJ expression in human
macrophages [89].
Using the E. coli two-plasmid system, it was found
that the expression of M. tuberculosis sigI is regulated
by r
J
[98], suggesting a possible relationship between
the two r factors. It is likely that r
I
may be involved in
regulating stress responses similar to those regulated by
r
J
. Based on the fact that the sigI transcript level
increases after mild cold shock (room temperature), the
r
I
regulon has been speculated to be involved in the sur-
vival of M. tuberculosis in aerosol particles, where the
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 613
ambient temperature is usually lower than 37 °C [24].
The role of sigI in cold shock adaptation has also been
suggested in the case of M. avium ssp. paratuberculosis,
where it was found to be one of the genes significantly
upregulated in cow fecal samples [91].
SigK (r
K

)
The sigK gene is present in all species of M. tuberculosis
complex (MTC, a group of pathogenic organisms in-
cluding Mycobacterium tuberculosis, Mycobacterium
bovis, Mycobacterium pinnipedii, Mycobacterium microti
and Mycobacterium caprae [100]) and in Mycobacte-
rium marinum, while it is absent in certain other myco-
bacterial species (e.g. M. avium ssp. paratuberculosis
and M. smegmatis) [2]. r
K
positively regulates the
expression of two antigenic proteins, MPB70 and
MPB83, in M. bovis BCG. A mutation in the start
codon of sigK, and certain mutations in the coding
region of its downstream regulator, rskA, account for
the variable production of these antigenic proteins in
the members of the M. tuberculosis complex [99,100].
The basal expression of mpt70 and mpt83 in M. tuber-
culosis is low and induced only during infection of
macrophages, while its M. bovis homologs are constitu-
tively expressed at high levels. The presence of
mutations in RskA, the negative regulator of r
K
, may
have led to a state of constitutive activity of r
K
and
therefore constantly high levels of expression of its reg-
ulon observed in the study [100]. Evolutionary analysis
indicates that the core regulatory system, r

K
⁄ RskA, is
conserved across the Mycobacterium genus, whereas the
regulon under its control varies considerably across
species. Gene alignments indicated insertion, deletion,
or re-arrangements within the r
K
regulon across differ-
ent mycobacteria (e.g. mpt83, dipZ and mpt70). It has
been suggested that from a minimal module of
mpt83 ⁄ rskA ⁄ sigK, a gene-duplication event resulted in
two MPT70 ⁄ 83 paralogs. Possibly during evolution,
this locus became bifurcated into two regions: the
sigK ⁄ rskA locus and the mpt70 ⁄ 83 locus. In slow-grow-
ing pathogenic mycobacteria, an additional gene, dipZ,
is inserted between the two mpt83 paralogs. The r
K
regulon is atypically small; however, the number and
identity of r
K
-regulated genes varies across different
mycobacterial species [101]. The role and trigger of this
regulon in M. tuberculosis pathogenicity merits investi-
gation.
SigL (r
L
)
sigL is constitutively expressed at a very low level from
a weak r
L

-independent promoter and is also transcribed
from a r
L
-dependent promoter [102]. In a murine
infection model, the sigL mutant exhibited marked
attenuation compared with the parental strain, suggest-
ing a role of r
L
in virulence; however, there were no
significant differences in the growth rate or in the size
and extent of lesions in the infected organs [45,102].
Microarray analysis was carried out by two groups
independently in order to characterize the r
L
regulon
[45,102]. In one of the approaches, sigL overexpres-
sion from an acetamide-inducible promoter led to the
strong upregulation of four small operons: sigL
(Rv0735)-rslA (Rv0736); mpt53 (Rv2878c)-Rv2877;
pks10 (Rv1660)-pks7 (Rv1661); and Rv1139c-Rv1138c
[102]. Mpt53, a DsbE-like protein, possibly acts as an
extracellular oxidant required for proper folding of
reduced unfolded secreted proteins [103]. While pks10
and pks7 are polyketide synthase genes, the other
r
L
-regulated gene pair, Rv1139c-Rv1138c, encodes a
membrane protein containing an isoprenyl cysteine
carboxy methyltransferase motif and a putative oxido-
reductase, respectively [102]. In another study, a

mutant strain lacking both sigL and rslA was comple-
mented by integrating a single wild-type copy of sigL
into its chromosome, resulting in its constitutive
expression [45]. Some of the genes (pks10, pks7,
Rv1138c, mpt53 and Rv2877c) were identified using
both approaches. Other genes identified using the lat-
ter method included the remaining genes from the
pks10 operon and the ppsA gene, involved in the
biosynthesis of dimycocerosyl phthiocerol (a cell wall-
associated lipid found only in pathogenic mycobacte-
ria [104]), mmpL13a and mmpL13b, involved in fatty
acid transport [105], another r factor-encoding gene,
sigB and two genes thought to be involved in host
cell invasion [45].
r
L
, like r
E
and r
H
, is post-translationally regulated
by a ZAS family protein with its gene located down-
stream of the sigL gene. However, unlike sigE and
sigH, sigL does not play a role in the oxidative or
nitrosative stress responses [45].
SigM (r
M
)
sigM expression was found to be induced at high tem-
perature and in the stationary phase during in vitro

growth of M. smegmatis, M. bovis BCG as well as
M. tuberculosis CDC1551 [106,107]. Its induced levels,
however, were markedly lower than that of sigA in the
late stationary phase in M. tuberculosis [107]. By
contrast, Raman et al. did not observe a significant
change in sigM expression at any growth stage in
M. tuberculosis H
37
Rv [108]. The M. smegmatis sigM
mutant is more susceptible to oxidative stress than the
The r-factors of M. tuberculosis P. Sachdeva et al.
614 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
parental strain [106]. However, M. tuberculosis
CDC1551 r
M
is dispensable for survival in the pres-
ence of oxidative and detergent stresses, suggesting dif-
ferent roles of the r
M
orthologs in M. tuberculosis and
M. smegmatis. The mutant does not display any defect
in survival in the activated macrophages and in a
mouse model of infection [107].
Microarray profiling of a sigM-overexpressing strain
revealed upregulation of four putative esat-6 homologs
(esxT, esxU, esxE and esxF) and of a few other genes,
such as Rv0035 (fadD34) and Rv0685 (tuf, encoding a
probable iron-regulated elongation factor) [107].
Raman et al. also carried out transcription profiling
experiments to characterize the M. tuberculosis r

M
reg-
ulon by comparison of a sigM deletion strain with the
wild-type strain as well as with the sigM overexpres-
sion strain. In addition to genes encoding two pairs of
Esx secreted proteins, a multisubunit nonribosomal
peptide synthetase operon and two PPE family
members – PPE1 and PPE19 – were found to be up-
regulated in the sigM overexpression strain compared
with the sigM mutant. Positive regulation of Esx
family secretory proteins by r
M
suggests its role in
long-term adaptation to specific host environments
[108]. Comparison of the sigM deletion strain with the
wild-type strain revealed the negative regulation of
several genes by r
M
, such as the devR-devS-Rv3134c
operon; another PPE gene (ppe60); a type I fatty acid
synthase gene (fas); and two polyketide synthases
(pks2 and pks3). Certain other genes involved in
surface lipid synthesis that were also found to be
upregulated in the sigM mutant strain include most of
the genes from the first operon of the phthiocerol
dimycocerosate biosynthetic and transport locus, the
independently transcribed mas gene from the same
locus and the kasA-kasB operon, required for mycolic
acid synthesis [108]. As phthiocerol dimycocerosates
have been shown to be important virulence determi-

nants required for efficient replication in the lung
during the short-term infection of mice [109,110], the
negative regulation of their synthesis by r
M
negates
the possibility of its role in virulence during the early
course of infection [108]. Consistent with this
inference, in a guinea pig aerosol model of infection,
the sigM mutant strain appeared to be hypervirulent
at early time-points, with greater numbers of granulo-
mas and increased necrosis compared with the
wild-type strain [81]. Despite the presence of certain
mutations in the 4.2 regions of r
M
, M. tuberculosis
H
37
Rv and M. bovis AF2122 ⁄ 97 are fully virulent. It is
possible that these mutations do not affect the function
of r
M
, or that r
M
may not be required for virulence
[17].
Promoter recognition by mycobacterial
r factors
During the last few years of work on mycobacterial r
factors, a major focus has been the identification of
genes whose expression lies under the control of differ-

ent r factors. The promoters recognized by different r
factors have been delineated principally by compara-
tive expression profiling using microarray platforms.
The comparison between wild-type and r factor over-
expressing or mutant strains, followed by analysis of
upstream regions of differentially expressed genes, led
to the selection of putative promoter sequences that
were validated using in vitro assays. Despite the accu-
mulation of extensive data obtained by employing this
strategy, the success in defining unambiguous arche-
typal promoter consensus sequences recognized by
most of the mycobacterial r factors has been quite
limited. A comprehensive analysis of the literature sug-
gests a number of factors that may account for the
poor sequence conservation in promoter consensus and
also for certain inconsistencies in different promoter
sequences reported for some of the mycobacterial r
factors.
A substantial body of research work on the constitu-
tively expressed mycobacterial promoters, under the
transcriptional control of r
A
, has demonstrated that
the -10 position of these promoters have sequences
similar to their counterparts in E. coli [40,111]. How-
ever, a majority of these mycobacterial promoters do
not work efficiently in E. coli. This could be attributed
to the heterogeneity in the -35 regions and variability
in the spacer distance between the -10 and -35 regions
in mycobacterial promoters [112]. It has also been

speculated that the presence of an AT-rich sequence at
the -15 region of E. coli promoters, which drastically
influences the promoter strength [15], and its absence
in the GC-rich mycobacterial promoters, may be
responsible, at least partially, for the reduced activity
of mycobacterial promoters in E. coli [111].
Another class of promoters, termed ‘extended -10
promoters’, is more crucially dependent on the -10
region. In these promoters, the -35 element plays a sec-
ondary role in promoter recognition [113]. However,
the -35 consensus sequences in promoters recognized
by various ECF r factors, such as r
D
, r
E
, r
H
, r
L
and
r
M
, are very similar [54,72,102,108,114], often with
only single base differences, whereas their -10 consen-
sus sequences are significantly divergent. In the
absence of reports of genes commonly regulated by
any of these r factors, it was suggested that the core
-35 region is required for efficient promoter binding
and ⁄ or transcription initiation, while binding specificity
P. Sachdeva et al. The r-factors of M. tuberculosis

FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 615
arises from differences in the nucleotides present at
either end of the -35 element and those in the -10
region sequences [108]. This notion supports the fact
that in contrast to region 3.0 containing group 1 and 2
r factors, for ECF r factors, the -35 sequence is
particularly important and should be indispensable in
the absence of an extended -10 motif. The consensus
promoter sequences of M. tuberculosis r factors are
listed in Table 1.
Promoters recognized by certain mycobacterial r
factors have been identified by using basic templates
adapted from promoter consensus sequence derived for
other unrelated organisms, including nonactino-
mycetes. M. tuberculosis r
F
bears significant homology
with B. subtilis r
B
in terms of its function as well as
conservation of a network of regulatory proteins for
modulation of its activity [77]. Use of the B. subtilis
r
B
promoter sequence as a template, in combination
with biochemical analysis, facilitated the identification
of the Mycobacterium r
F
-dependent promoter [86].
Similarly, the consensus sequences for both M. tuber-

culosis r
D
- and r
M
-dependent promoters are based on
the sequence recognized by r
W
of B. subtilis [60,107].
However, in these cases, the consensus derived using
the Bacillus sp. template differs from the consensus
sequences derived from in vivo promoter analysis using
methods such as TSP identification and chromatin
immunoprecipitation [54,108,115]. Hence, the criterion
of B. subtilis r
W
as a functional counterpart of both
r
M
and r
D
needs to be further established. In view of
the significantly higher GC content, higher variability
in spacing between the -10 and -35 regions and a low
level of general homology of mycobacterial promoters
to those of other prokaryotes [112], the use of a con-
sensus template from other unrelated prokaryotes may
bias the search for a promoter sequence.
Mycobacterial r factors widely recognize promoters
of various transcription factors; for example, r
F

affects
the expression of genes encoding EmbR, PhoY1, TetR,
MarR and LysR family regulators [46,49], while r
E
induces MprA and certain other putative transcription
regulators [43,70]. Such r factors are likely to influence
the global transcriptional profile via indirect regulation
of a number of genes, whose upstream regions may
actually not have binding sites for the r factor in ques-
tion. Furthermore, one r factor mediating transcrip-
tion of another r factor is a very common
phenomenon in the mycobacterial r factor network,
for example, r
H
is responsible for the induction of
both sigB [44] and sigE under oxidative stress and heat
shock conditions [72], while sigC is a r
F
-dependent
gene [49] (Fig. 2). Further intricacy in mycobacterial
transcriptional regulatory mechanisms is brought
about by the presence of multiple promoters and TSPs
for a single gene as well as binding of more than one
r factor to a single promoter sequence. Mycobacterium
recA, glnA, rrn operon, Rv1364c, sigB and sigE repre-
sent examples of genes with multiple promoters, whose
expression is differentially regulated in response to dif-
ferent environmental conditions [45,71,112,116]. More-
over, the sigB gene promoter can also be recognized
by multiple r factors [45]. Such diverse regulatory

strategies adopted by a limited number of r factors
with distinct promoter consensus sequences and recog-
Table 1. Proposed consensus promoter sequence of Mycobacterium tuberculosis r factors. Allowed spacing ranges are indicated by the
numbers in subscript. Appropriate references are shown for each r factor.
Sigma
factor
Promoter consensus sequence*
Reference(s)-35 -10
SigA TTGCGA –N
18
– TANNNT [111]
SigB NGTGG –N
14-18
– NNGNNG [39]
SigC SSSAAT –N
16-20
– CGTSSS [50]
SigD GTAACGct AT-rich region [54]
AGAAAG –N
16-20
– CGTTAA [60]
SigE gGGAACYa –N
15-16
– cGTT [114]
SigF GGWWT –N
16-17
– GGGTAY [115]
SigG GCGNGT –N
15-18
– CGANCA [90]

SigH gGGAAYA –N
16-17
– cGTT [72]
SigI unknown
SigJ (sigIp) GTCACA –N
16
– CGTCCT [98]
SigK CCATCC –N
15
– CCGAAT [101]
SigL TGAACC –N
16
– CGTgtc [102]
SigM GGAAC –N
16-18
– CGTCR [107]
GGGAACC –N
17
– gtCcgA [108]
*W=A⁄ T; M = A ⁄ C; S = C ⁄ G; Y = C ⁄ T; R = A ⁄ G.
The r-factors of M. tuberculosis P. Sachdeva et al.
616 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
nition properties might represent a key to the remark-
able transcriptional flexibility of this highly adaptable
pathogen. Hence, transcriptional profiling of r factor
mutant strains at different stages of growth, and fol-
lowing exposure to various stress conditions, could be
one of the approaches used to evaluate global as well
as specific contributions made by each r factor.
In addition to modulation of expression level, the

activity of an alternative r factor may also be
regulated post-translationally in response to different
physiological conditions. The latter aspect is essentially
governed by proteins such as anti-r factors, which
serve to negatively regulate the activity of r factors.
Generally, the anti-r factors are co-transcribed and
induced in the presence of their cognate r factors. The
overexpression of sigL in a sigL-rslA gene pair knock-
out strain led to the identification of a number of
r
L
-regulated genes [45], whose expression was not
found to be altered in a sigL overexpressing strain with
an intact rslA gene [102]. Because the r factor levels
attained on its overexpression in the presence of its
functional anti-r factor, as carried out in most of the
studies, may not completely reflect the state of activa-
tion, certain genes of the regulon might remain unex-
plored. Certain r factors, such as r
F
, possess a very
complex post-translational regulatory network com-
prising multiple factors. This could also be one of the
reasons for a major discordance observed in the
expression data obtained for the M. tuberculosis
CDC1551 sigF deletion mutant [49] and the sigF over-
expressing strain [46]. Hence, regulatory aspects of a r
factor need to be explored and understood well before
one can unambiguously interpret promoter consensus
from expression data obtained from overexpression

models.
Post-translational regulation of r
factors
As detailed in the previous sections, r factors play a
critical role in the modulation of the transcription pro-
file of M. tuberculosis under various environmental
challenges. Table 2 summarizes our present knowledge
of the M. tuberculosis r factors, obtained from both
experimental and bioinformatic analyses. Effective tun-
ing of bacterial gene expression is based on the correct
modulation of the level and activity of its r factors by
a robust regulatory mechanism. The bacterium
responds to environmental cues by recruitment of
appropriate r factor(s) to the core RNAP to carry out
the coordinate expression of a specific subset of genes.
The availability of the alternative r factors is
controlled at the transcriptional, translational and
post-translational levels. Almost all r factors are
constitutively expressed at a basal level and the levels
are upregulated through intricate regulatory mecha-
nisms involving various transcription factors, including
other r factors. The activity of certain r factors is
post-translationally regulated by antagonist proteins
termed anti-r factors, which sequester the r factor
thereby preventing its interaction with the core RNAP.
These anti-r factors operate via a partner-switching
system, in which key protein–protein interactions are
controlled by mechanisms such as phosphorylation or
intracellular redox conditions. Anti-r factors sense
external stimuli and release the r factor under stress

conditions facilitating the transcription of the stress-
specific regulons. As most of the r factors exhibit
autotranscription, the activation of r factor via this
mechanism also results in its transcriptional upregula-
tion. Adding to the complexity is the regulation of the
anti-r factors themselves, which can be negatively reg-
ulated by specific molecules termed anti-anti-r factors
[117]. Such anti-r factors belong to the gyrase, Hsp90,
histidine kinase, MutL (GHKL) family of kinases,
which inactivate their cognate anti-anti-r factor by
phosphorylation [118]. The other family of anti-r fac-
tors, called the ZAS family, is characterized by the
presence of a conserved HX
3
CX
2
C motif. Under
reducing conditions, a ZAS family protein binds and
inhibits r factor, while under oxidizing conditions, its
redox-sensitive cysteines form an intramolecular disul-
fide bridge, resulting in the reversal of inhibition
[119,120].
The regulation of r factors and their regulators in
M. tuberculosis has not been as extensively studied as
that for organisms such as E. coli and B. subtilis. Five
anti-r factors have been identified in M. tuberculosis
to date, of which three, namely regulator of sigmaE A
(RseA), regulator of sigmaH A (RshA) and regulator
of sigmaL A (RslA) belong to the ZAS family of pro-
teins. RseA (Rv1222) maps downstream of r

E
and
inhibits the transcription activity of a r
E
isoform in a
dose-dependent manner. Furthermore, RseA (Rv1222)
is known to interact directly with the two isoforms of
r
E
in a temperature-dependent manner [71]. Two
co-transcribed genes exist downstream of M. tubercu-
losis rseA; the first encodes a membrane serine protease
(HtrA), and the second encodes a twin-arginine trans-
location (Tat) system protein, TatB. Interestingly, the
RseA N-terminus region has two arginine residues,
suggesting that this protein could be targeted to the
bacterial membrane through the Tat system to allow
its degradation by HtrA [71]. However, earlier
attempts to demonstrate interactions between these
proteins were unsuccessful [17].
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 617
r
H
, similarly to its homolog, r
R
in S. coelicolor,is
negatively regulated by its cognate anti-r factor, RshA
(Rv3221A), encoded by a gene in the sigH operon [95].
RshA interacts with r

H
, and this interaction is dis-
rupted under oxidative stress as well as by elevated
temperature. RshA acts as a stress sensor and inhibits
r
H
-dependent transcription only under reducing condi-
tions. During oxidative stress conditions, r
H
-depen-
dent self-regulation of the sigH promoter results in a
rapid and substantial induction of the r
H
-dependent
genes, which serve to re-establish redox conditions
under which RshA can bind and inhibit r
H
-dependent
transcription [95]. Interestingly, the r
H
-RshA interac-
tion can also be modulated by signaling mediated
through an essential STPK, PknB. In a recent study, it
was observed that the phosphorylation of RshA by
PknB decreases its interaction with r
H
. This is consis-
tent with the observation that inhibition of r
H
activity

Table 2. Summary of Mycobacterium tuberculosis r factors.
Name ORF* Group
Auto-
regulation PTR
M. leprae
homolog Important genes in regulon
Proposed regulatory
role Reference(s)
SigA Rv2703 1 + + rrsP1, rrsP2, eis, other
housekeeping genes
Housekeeping function,
host–pathogen
interaction
[30,115]
SigB Rv2710 2 + + ideR, furA, katG, hsp20,
ppe19, kasA, whiB2
Adaptation to cell
envelope stress,
hypoxia, oxidative
stress
[38,39]
SigC Rv2069 4 + hspX, senX3, fbpC, mtrA Virulence,
immunopathology
phenotype
[50,51]
SigD Rv3414c 4 + P rpfC, recR, pks10, mce1 Stationary-phase
survival,
immunopathology
phenotype, stringent
response, starvation

[54,60]
SigE Rv1221 4 + + rseA, sigB, mprA, mprB, rel Surface stress and
heat shock response,
immunopathology
phenotype, host
immune-response
modulation,
virulence
[43,67,68,70,71]
SigF Rv3286c 3 + + P usfX, sigC, phoY1, mmpL,
marR, gntR, tetR
Biosynthesis of
mycobacterial
cell envelope,
immunopathology
phenotype
[46,49,86]
SigG Rv0182c 4 P sigH, lexA, aceA, clpB,
scoA, dnaK, trxB2, fadE5
SOS response,
survival during
macrophage
infection
[89,90]
SigH Rv3223c 4 + + P rshA, sigB, sigE, dnaK,
lpB, trxB, trxC
Oxidative and
heat stress,
immunopathology
phenotype

[44,72,92,95,96]
SigI Rv1189 4 P Unknown Unknown
SigJ Rv3328c 4 P sigI Oxidative stress [97,98]
SigK Rv0445c 4 + + P rskA, mpt83, dipZ, mpt70 Unknown [99–101]
SigL Rv0735 4 + + – rslA, sigB, mpt53, pks10,
pks7, ppsA, mmpL13a
Virulence, PDIM
biosythesis
[45,102]
SigM Rv3911 4 + P esxT, esxU, esxE, esxF,
tuf, fadD34
Long-term
in vivo adaptation
[107,108]
* M. tuberculosis H
37
Rv ORF designation. ORF, open-reading frame; P, pseudogene; PDIM, phthiocerol dimycocerosate; PTR, post-transla-
tional regulation.
The r-factors of M. tuberculosis P. Sachdeva et al.
618 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
by RshA is partially relieved in a strain overexpressing
pknB, resulting in an increased resistance to oxida-
tive stress. This STPK-mediated modulation of the
r
H
–RshA interaction may provide a means for partial
activation of the r
H
regulon in response to oxidative
or nitrosative stresses that are not potent enough to

inactivate RshA [96]. In an independent study, RshA
was also reported to be phosphorylated in vitro by
another STPK, PknE. The in vivo significance of this
observation is not yet known [88].
Rv0736 situated adjacent to sigL is co-transcribed
with sigL and encodes a transmembrane protein. The
intracellular domain of Rv0736 specifically interacts
with r
L
in a bacterial two-hybrid system and was desig-
nated as RslA [102]. Another group demonstrated nega-
tive regulation of r
L
activity by RslA in a dose-
dependent manner in an in vitro transcription assay [45].
rskA (Rv0444c), located immediately downstream of
sigK, is co-transcribed with sigK. It encodes a trans-
membrane protein, with an extracellular C-terminal
domain, possibly involved in sensing an in vivo stimulus
for the release of r
K
and an intracellular N-terminal
domain involved in interaction with r
K
. Certain muta-
tions in M. bovis rskA abolish its inhibitory effect on
r
K
, resulting in the high-level expression of the
r

K
-dependent genes, mpb70 and mpb83 [100]. [Correc-
tion added on 8 December 2009 after original online
publication: in the preceding sentence ‘mbp83’ was chan-
ged to ‘mpb83’.] However, none of these mutations map
to the region required for the interaction of RskA with
r
K
. Possibly, these mutations result in major changes in
the protein structure and folding [16], thereby making it
nonfunctional. The stimulus and the mechanism that
lead to the release of r
K
from RskA remain unknown.
The mode of regulation of M. tuberculosis r
F
and
its B. subtilis homolog, r
B
, is significantly similar [77].
B. subtilis r
B
activity is negatively regulated by
the anti-r factor, RsbW, and its antagonist, RsbV.
The anti-anti-r action of RsbV is counteracted by the
kinase activity of RsbW and is re-established by the
phosphatase activity of another protein, RsbU [117].
Similarly, M. tuberculosis r
F
seems to be regulated by

a complex signal-transduction cascade comprising a
number of regulatory molecules, including anti-r fac-
tors, anti-anti-r factors, kinases and phosphatases.
Located upstream of sigF in M. tuberculosis, M. bovis
and M. smegmatis is an anti-r factor gene, usfX (rsbW
homolog) [77,78]. sigF and usfX are known to be
co-transcribed from a r
F
-dependent promoter in
M. tuberculosis [77,86]. UsfX directly interacts with r
F
and inhibits r
F
-dependent transcription. The activity
of UsfX is, in turn, negatively regulated by two anti-
anti-r factors: regulator of sigmaF A (RsfA) and regu-
lator of sigmaF B (RsfB). RsfA (Rv1365c) exerts its
antagonistic effect on UsfX only under reducing condi-
tions, with its two critical cysteine residues acting as a
redox sensor. RsfB (Rv3687c) may be regulated by
phosphorylation as it is rendered nonfunctional by the
introduction of a phosphomimetic mutation. However,
UsfX, despite being an RsbW homolog, does not
phosphorylate RsfB [86]. Several other proteins have
also been shown to interact with r
F
and UsfX and are
thought to exert some control on the activity of r
F
[87]. Interestingly, one of the putative anti-anti-r fac-

tors, Rv0516c, is modulated by an apparently indepen-
dent signaling pathway mediated by an STPK, PknD.
The in vitro phosphorylation of Rv0516c by PknD
blocks its interaction with another putative r
F
regula-
tor, Rv2638. M. tuberculosis PknD phosphorylates
Rv0516c in vivo, and PknD overexpression strongly
induces the expression of Rv0516c and simultaneously
alters the expression of various r
F
-dependent genes.
Besides, the STPKs PknB and PknE also phosphory-
late Rv0516c and few other r
F
regulators (Rv1904 and
RsfA) in vitro; however, the role of these phosphoryla-
tion events remains to be determined [88]. There is a
paucity in the number of TCS histidine kinases [19]
and functional r regulatory GHKL family kinases in
the Mycobacterium genus compared with other bacte-
rial genomes (e.g. B. subtilis and E. coli). In view of
the abundance of eukaryotic-like STPKs and a signifi-
cant number of physiological processes, including regu-
lation of r
F
, being governed by them, the STPKs may
compensate for the lacunae created by the dearth of
other kinases in Mycobacterium.
Recently, we identified a pathogenic species-specific

putative multidomain regulator of r
F
(MursiF;
Rv1364c), in which the entire r
F
regulatory cascade
akin to the RsbU-RsbW-RsbV system appears to be
encoded in a single polypeptide with an additional Per-
Arnt-Sim (PAS) sensor domain. Its putative anti-r
factor domain interacts specifically with r
F
as well as
with the putative anti-anti-r factor domain. Although
we were unable to detect any kinase activity in full-
length MursiF or its isolated anti-r factor domain in an
in vitro assay [121], a recent study reported phosphory-
lation of the kinase–substrate fusion protein in an intra-
molecular reaction. The anti-r factor domain activates
the phosphatase domain through direct interaction,
while its kinase activity in turn is antagonized by the
phosphatase domain. This entire module has been pro-
posed to regulate the environmental phosphatase,
RsbU, of Rv1364c [122] akin to the RsbU-RsbT-RsbS
system of B. subtilis [117]. However, the signal govern-
ing this cascade, and other possible regulators playing a
role in this pathway, still needs to be determined.
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 619
Therapeutic potential of mycobacterial
r factors

Certain r factor deletion strains manifest attenuated
disease progression and prolonged survival of the
immunocompetent host, despite showing no growth
defect under in vivo conditions. Such mutants, called
imp phenotype mutants, which persist in the host at a
high level of infection and stimulate the immune
response without causing detrimental pathological
changes in the host tissues, may serve as good vaccine
candidates [123]. The mycobacterial r factor-deficient
strains exhibiting the imp phenotype are sigF, sigH,
sigE and sigD mutants and, most importantly, the sigC
mutant [51]. The sigF mutant is partially attenuated in
mice as well as guinea pig models despite its ability to
persist in lung tissues [49,81]. Geiman et al. [49]
reported that intradermal vaccination of rabbits with
the M. tuberculosis sigF mutant strain confers a higher
degree of protection against tubercle formation in the
aerosol challenge model than vaccination with
M. bovis BCG. However, a recent study by Williams
et al. [46] provided evidence against the role of r
F
in
extracellular persistence in an in vivo granuloma model
of latent TB infection. The potential of the M. tubercu-
losis sigF mutant as a likely therapeutic vaccine candi-
date therefore needs to be further explored.
Remarkably, the M. tuberculosis CDC1551 sigC
mutant is just as persistent as the parental strain, but is
significantly attenuated in the DBA ⁄ 2 mice model, caus-
ing no death of mice compared with 100% mortality

observed in mice infected with the wild-type strain [50].
In view of the downregulation of sigC in the sigF mutant
strain [49], Karls et al. [81] proposed r
C
to be a direct
regulator of genes that mediate adaptive survival of the
Mycobacterium and also the typical pathological
changes observed in TB. Most importantly, the role of
M. tuberculosis H
37
Rv r
C
has also been demonstrated
to be crucial for pathogenesis in the lung and spleen in a
guinea pig TB model, which bears a close resemblance
to human M. tuberculosis infection in terms of lung
granuloma formation and disease progression. There-
fore, in addition to the establishment of infection in the
lungs, r
C
is also required for successful dissemination of
bacteria in the host [81]. Such dissemination mutants
would be of particular utility in the development of safer
vaccines for immunocompromised individuals [123].
The M. tuberculosis sigC mutant seems to fulfill both
the essential features of a successful vaccine, namely,
safety and efficacy. The safety of imp mutant-based vac-
cine strains can be further enhanced by deleting more
than one r factor in a mycobacterial strain (e.g. deletion
of sigE or sigH genes in combination with sigC might

give an even more pronounced imp phenotype). Very
recently, Sadagopal et al. [124] reported that the sup-
pression of mycobacterial antioxidant mechanisms via
deletion of sigH paralogs in M. bovis BCG, in combina-
tion with reduced secretion and activity of superoxide
dismutase, leads to greater activation of innate immu-
nity, stronger antigen-specific T-cell responses and bet-
ter memory immunity than observed in the case of the
parent BCG vaccine.
The indispensable role of r
C
in immunopathology
and disease progression of TB also suggests it to be a
potential drug target. The availability of a r
C
crystal
structure [52] can act as a catalyst in targeting this r
factor for the development of antimycobacterial drugs.
Concluding remarks
The exceptional capacity of M. tuberculosis to adapt
to changing environmental conditions during infection
is mainly achieved through its transcriptional flexibility
brought about by a pool of r factors. Recent years
have seen several important developments in under-
standing mycobacterial r factors, especially their
in vivo role, the regulons, derivation of the promoter
consensus sequences and post-translational regulation.
For a long time, r
A
was largely considered as a r fac-

tor required for the maintenance of physiology of
M. tuberculosis but not virulence. However, the role of
r
A
in the regulation of virulence genes has added a
new dimension to its function [27,29].
Assigning a discrete role to each r factor has been
hindered by discordant results obtained with bacilli in
culture, infected macrophages and animal models. For
example, strains mutated for certain r factors control-
ling virulence and stress-associated regulons fail to
show any loss in virulence in the animal models. While
this suggests functional redundancy among certain r
factors, the lack of overlap between the regulons
controlled by different r factors with similar pro-
moter-recognition sequences, weakens the argument.
However, we cannot formally rule out the possibility
that the complete repertoire of genes regulated by each
r factor is yet to be discovered. Expression profiling
of a r factor deletion ⁄ overexpression strain under dif-
ferent stages of growth and various stress conditions
might help to clarify the role of r factors. In the light
of the regulatory network of r and anti-r factors
highlighted in the earlier sections, we speculate that
the overexpression of r factor, along with knockout of
anti-r factors, would be effective in unraveling the
entire regulon of an alternate r factor.
The r-factors of M. tuberculosis P. Sachdeva et al.
620 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
The complex interplay of r factors shown in Fig. 2

highlights the convergence of the stress-associated reg-
ulatory pathways at r
B
. Stitched in to this network is
also a two-component response regulator, MprA,
which directly regulates sigE and sigB [47]. In addition
to regulation of r factors by anti-r factors belonging
to the redox-sensitive ZAS family and the GHKL fam-
ily of kinases, fine-tuning of the r–anti-r interaction
in certain cases is brought about by STPK-mediated
signaling. An emerging aspect in the post-translational
regulation of mycobacterial r factors is the involve-
ment of STPK-mediated phosphorylation events in
the modulation of regulatory interactions [88,96].
Although recent studies have provided evidence of the
interplay of r factors and their interaction with regula-
tors, how exactly these factors coordinate with one
another in the face of an adverse event, and the order
of events, still needs to be explored.
A combination of independent gene mutations that
may reduce the virulence of M. tuberculosis sufficiently
to provide safety in immunocompromised individuals,
yet allow the elicitation of an optimal immune response,
would be a rational approach for vaccine development.
The development of a vaccine strain that can persist, but
cannot disseminate, would be of particular utility to
immunocompromised individuals. The sigC deletion
strain exhibits both an imp and dissemination mutant
phenotype in a guinea pig model of infection and is
therefore likely to be an attractive candidate in the

development of safer and effective anti-TB vaccines.
Challenges ahead include unraveling the long-sought
role of r factors in the survival and virulence mecha-
nisms of Mycobacterium and to employ this knowledge
in generating novel attenuated strains that can serve as
effective therapeutic vaccines against TB.
Acknowledgements
The authors would like to thank Prof. Vani Brahma-
chari for critical reading of the manuscript. Financial
support was provided by the Council of Scientific and
Industrial Research (NWP0038). R. Misra is supported
by a CSIR fellowship.
References
1 Vergne I, Chua J, Singh SB & Deretic V (2004) Cell
biology of mycobacterium tuberculosis phagosome.
Annu Rev Cell Dev Biol 20, 367–394.
2 Manganelli R, Provvedi R, Rodrigue S, Beaucher J,
Gaudreau L & Smith I (2004) Sigma factors and global
gene regulation in Mycobacterium tuberculosis.
J Bacteriol 186, 895–902.
3 Gross CA, Chan C, Dombroski A, Gruber T, Sharp
M, Tupy J & Young B (1998) The functional and regu-
latory roles of sigma factors in transcription. Cold
Spring Harb Symp Quant Biol 63, 141–155.
4 Gruber TM & Gross CA (2003) Multiple sigma subun-
its and the partitioning of bacterial transcription space.
Annu Rev Microbiol 57, 441–466.
5 Smith I (2003) Mycobacterium tuberculosis pathogenesis
and molecular determinants of virulence. Clin Micro-
biol Rev 16, 463–496.

6 Wosten MM (1998) Eubacterial sigma-factors. FEMS
Microbiol Rev 22, 127–150.
7 Lonetto M, Gribskov M & Gross CA (1992) The
sigma 70 family: sequence conservation and evolution-
ary relationships. J Bacteriol 174, 3843–3849.
8 Paget MS & Helmann JD (2003) The sigma70 family
of sigma factors. Genome Biol 4, 203.
9 Murakami KS & Darst SA (2003) Bacterial RNA
polymerases: the wholo story. Curr Opin Struct Biol
13, 31–39.
10 Campbell EA, Muzzin O, Chlenov M, Sun JL, Olson
CA, Weinman O, Trester-Zedlitz ML & Darst SA
(2002) Structure of the bacterial RNA polymerase pro-
moter specificity sigma subunit. Mol Cell 9, 527–539.
11 Mitchell JE, Zheng D, Busby SJ & Minchin SD (2003)
Identification and analysis of ‘extended -10’ promoters
in Escherichia coli. Nucleic Acids Res 31, 4689–4695.
12 Ross W, Gosink KK, Salomon J, Igarashi K, Zou C,
Ishihama A, Severinov K & Gourse RL (1993) A third
recognition element in bacterial promoters: DNA bind-
ing by the alpha subunit of RNA polymerase. Science
262, 1407–1413.
13 Gourse RL, Ross W & Gaal T (2000) UPs and downs
in bacterial transcription initiation: the role of the
alpha subunit of RNA polymerase in promoter recog-
nition. Mol Microbiol 37, 687–695.
14 Ross W, Aiyar SE, Salomon J & Gourse RL (1998)
Escherichia coli promoters with UP elements of differ-
ent strengths: modular structure of bacterial promoters.
J Bacteriol 180, 5375–5383.

15 Liu M, Tolstorukov M, Zhurkin V, Garges S & Adhya
S (2004) A mutant spacer sequence between -35 and -
10 elements makes the Plac promoter hyperactive and
cAMP receptor protein-independent. Proc Natl Acad
Sci USA 101, 6911–6916.
16 Husson RN (2006) Leaving on the lights: host-specific
derepression of Mycobacterium tuberculosis gene
expression by anti-sigma factor gene mutations. Mol
Microbiol 62, 1217–1219.
17 Rodrigue S, Provvedi R, Jacques PE, Gaudreau L
& Manganelli R (2006) The sigma factors of Myco-
bacterium tuberculosis.
FEMS Microbiol Rev 30, 926–
941.
18 Ghosh J, Larsson P, Singh B, Pettersson BM, Islam
NM, Sarkar SN, Dasgupta S & Kirsebom LA (2009)
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 621
Sporulation in mycobacteria. Proc Natl Acad Sci USA
106, 10781–10786.
19 Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C,
Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE
III et al. (1998) Deciphering the biology of Mycobacte-
rium tuberculosis from the complete genome sequence.
Nature 393, 537–544.
20 Tekaia F, Gordon SV, Garnier T, Brosch R, Barrell
BG & Cole ST (1999) Analysis of the proteome of
Mycobacterium tuberculosis in silico. Tuber Lung Dis
79, 329–342.
21 Waagmeester A, Thompson J & Reyrat JM (2005)

Identifying sigma factors in Mycobacterium smegmatis
by comparative genomic analysis. Trends Microbiol 13,
505–509.
22 Gomez M, Doukhan L, Nair G & Smith I (1998) sigA
is an essential gene in Mycobacterium smegmatis. Mol
Microbiol 29, 617–628.
23 Volpe E, Cappelli G, Grassi M, Martino A, Serafino
A, Colizzi V, Sanarico N & Mariani F (2006) Gene
expression profiling of human macrophages at late time
of infection with Mycobacterium tuberculosis. Immunol-
ogy 118, 449–460.
24 Manganelli R, Dubnau E, Tyagi S, Kramer FR &
Smith I (1999) Differential expression of 10 sigma fac-
tor genes in Mycobacterium tuberculosis. Mol Microbiol
31, 715–724.
25 Dubnau E, Fontan P, Manganelli R, Soares-Appel S &
Smith I (2002) Mycobacterium tuberculosis genes
induced during infection of human macrophages. Infect
Immun 70, 2787–2795.
26 Hu Y & Coates AR (1999) Transcription of two
sigma 70 homologue genes, sigA and sigB, in station-
ary-phase Mycobacterium tuberculosis. J Bacteriol
181, 469–476.
27 Collins DM, Kawakami RP, de Lisle GW, Pascopella
L, Bloom BR & Jacobs WR Jr (1995) Mutation of the
principal sigma factor causes loss of virulence in a
strain of the Mycobacterium tuberculosis complex. Proc
Natl Acad Sci USA 92, 8036–8040.
28 Steyn AJ, Collins DM, Hondalus MK, Jacobs WR
Jr, Kawakami RP & Bloom BR (2002) Mycobacte-

rium tuberculosis WhiB3 interacts with RpoV to
affect host survival but is dispensable for in vivo
growth. Proc Natl Acad Sci USA 99, 3147–
3152.
29 Wu S, Howard ST, Lakey DL, Kipnis A, Samten B,
Safi H, Gruppo V, Wizel B, Shams H, Basaraba RJ
et al. (2004) The principal sigma factor sigA mediates
enhanced growth of Mycobacterium tuberculosis in
vivo. Mol Microbiol 51, 1551–1562.
30 Wu S, Barnes PF, Samten B, Pang X, Rodrigue S,
Ghanny S, Soteropoulos P, Gaudreau L & Howard ST
(2009) Activation of the eis gene in a W-Beijing strain
of Mycobacterium tuberculosis correlates with increased
SigA levels and enhanced intracellular growth. Microbi-
ology 155
, 1272–1281.
31 Collins DM, Kawakami RP, Buddle BM, Wards BJ &
de Lisle GW (2003) Different susceptibility of two
animal species infected with isogenic mutants of
Mycobacterium bovis identifies phoT as having roles
in tuberculosis virulence and phosphate transport.
Microbiology 149, 3203–3212.
32 Bagchi G, Chauhan S, Sharma D & Tyagi JS (2005)
Transcription and autoregulation of the Rv3134c-
devR-devS operon of Mycobacterium tuberculosis.
Microbiology 151, 4045–4053.
33 Roberts DM, Liao RP, Wisedchaisri G, Hol WG &
Sherman DR (2004) Two sensor kinases contribute to
the hypoxic response of Mycobacterium tuberculosis .
J Biol Chem 279, 23082–23087.

34 Basu S, Pathak SK, Banerjee A, Pathak S, Bhattachar-
yya A, Yang Z, Talarico S, Kundu M & Basu J (2007)
Execution of macrophage apoptosis by PE_PGRS33 of
Mycobacterium tuberculosis is mediated by Toll-like
receptor 2-dependent release of tumor necrosis factor-
alpha. J Biol Chem 282, 1039–1050.
35 Vallecillo AJ & Espitia C (2009) Expression of Myco-
bacterium tuberculosis pe_pgrs33 is repressed during
stationary phase and stress conditions, and its tran-
scription is mediated by sigma factor A. Microb Pathog
46, 119–127.
36 Rengarajan J, Bloom BR & Rubin EJ (2005) Genome-
wide requirements for Mycobacterium tuberculosis
adaptation and survival in macrophages. Proc Natl
Acad Sci USA 102, 8327–8332.
37 Doukhan L, Predich M, Nair G, Dussurget O,
Mandic-Mulec I, Cole ST, Smith DR & Smith I (1995)
Genomic organization of the mycobacterial sigma gene
cluster. Gene 165, 67–70.
38 Fontan PA, Voskuil MI, Gomez M, Tan D, Pardini
M, Manganelli R, Fattorini L, Schoolnik GK &
Smith I (2009) The Mycobacterium tuberculosis sigma
factor sigmaB is required for full response to cell
envelope stress and hypoxia in vitro, but it is
dispensable for in vivo growth. J Bacteriol 191,
5628–5633.
39 Lee JH, Karakousis PC & Bishai WR (2008) Roles
of SigB and SigF in the Mycobacterium tuberculosis
sigma factor network. J Bacteriol 190, 699–
707.

40 Bashyam MD, Kaushal D, Dasgupta SK & Tyagi AK
(1996) A study of mycobacterial transcriptional appa-
ratus: identification of novel features in promoter
elements. J Bacteriol 178, 4847–4853.
41 Mukherjee R, Gomez M, Jayaraman N, Smith I &
Chatterji D (2005) Hyperglycosylation of glycopeptid-
olipid of Mycobacterium smegmatis under nutrient
starvation: structural studies. Microbiology 151, 2385–
2392.
The r-factors of M. tuberculosis P. Sachdeva et al.
622 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
42 Mukherjee R & Chatterji D (2005) Evaluation of the
role of sigma B in Mycobacterium smegmatis. Biochem
Biophys Res Commun 338, 964–972.
43 Manganelli R, Voskuil MI, Schoolnik GK & Smith I
(2001) The Mycobacterium tuberculosis ECF sigma fac-
tor sigmaE: role in global gene expression and survival
in macrophages. Mol Microbiol 41, 423–437.
44 Manganelli R, Voskuil MI, Schoolnik GK, Dubnau E,
Gomez M & Smith I (2002) Role of the extracytoplas-
mic-function sigma factor sigma(H) in Mycobacterium
tuberculosis global gene expression. Mol Microbiol 45,
365–374.
45 Dainese E, Rodrigue S, Delogu G, Provvedi R, Lafl-
amme L, Brzezinski R, Fadda G, Smith I, Gaudreau
L, Palu G et al. (2006) Posttranslational regulation of
Mycobacterium tuberculosis extracytoplasmic-function
sigma factor sigma L and roles in virulence and in glo-
bal regulation of gene expression. Infect Immun 74,
2457–2461.

46 Williams EP, Lee JH, Bishai WR, Colantuoni C &
Karakousis PC (2007) Mycobacterium tuberculosis SigF
regulates genes encoding cell wall-associated proteins
and directly regulates the transcriptional regulatory
gene phoY1. J Bacteriol 189, 4234–4242.
47 He H, Hovey R, Kane J, Singh V & Zahrt TC (2006)
MprAB is a stress-responsive two-component system
that directly regulates expression of sigma factors SigB
and SigE in Mycobacterium tuberculosis. J Bacteriol
188, 2134–2143.
48 Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson
NR, Wheeler PR, Honore N, Garnier T, Churcher C,
Harris D et al. (2001) Massive gene decay in the
leprosy bacillus. Nature 409, 1007–1011.
49 Geiman DE, Kaushal D, Ko C, Tyagi S, Manabe
YC, Schroeder BG, Fleischmann RD, Morrison NE,
Converse PJ, Chen P et al. (2004) Attenuation of
late-stage disease in mice infected by the Mycobacte-
rium tuberculosis mutant lacking the SigF alternate
sigma factor and identification of SigF-dependent
genes by microarray analysis. Infect Immun 72,
1733–1745.
50 Sun R, Converse PJ, Ko C, Tyagi S, Morrison NE &
Bishai WR (2004) Mycobacterium tuberculosis ECF
sigma factor sigC is required for lethality in mice and
for the conditional expression of a defined gene set.
Mol Microbiol 52, 25–38.
51 Abdul-Majid KB, Ly LH, Converse PJ, Geiman DE,
McMurray DN & Bishai WR (2008) Altered cellular
infiltration and cytokine levels during early Myco-

bacterium tuberculosis sigC mutant infection are
associated with late-stage disease attenuation and
milder immunopathology in mice. BMC Microbiol 8,
151.
52 Thakur KG & Gopal B (2005) Crystallization and
preliminary X-ray diffraction studies of two domains
of a bilobed extra-cytoplasmic function sigma factor
SigC from Mycobacterium tuberculosis. Acta Crystal-
logr Sect F Struct Biol Cryst Commun 61, 779–781.
53 Thakur KG, Joshi AM & Gopal B (2007) Structural
and biophysical studies on two promoter recognition
domains of the extra-cytoplasmic function sigma factor
sigma(C) from Mycobacterium tuberculosis. J Biol
Chem 282, 4711–4718.
54 Raman S, Hazra R, Dascher CC & Husson RN (2004)
Transcription regulation by the Mycobacterium tuber-
culosis alternative sigma factor SigD and its role in vir-
ulence. J Bacteriol 186, 6605–6616.
55 Hu Y & Coates AR (2001) Increased levels of sigJ
mRNA in late stationary phase cultures of Mycobacte-
rium tuberculosis detected by DNA array hybridisation.
FEMS Microbiol Lett 202, 59–65.
56 Magnusson LU, Farewell A & Nystrom T (2005)
ppGpp: a global regulator in Escherichia coli. Trends
Microbiol 13, 236–242.
57 Dahl JL, Kraus CN, Boshoff HI, Doan B, Foley K,
Avarbock D, Kaplan G, Mizrahi V, Rubin H & Barry
CE III (2003) The role of RelMtb-mediated adaptation
to stationary phase in long-term persistence of Myco-
bacterium tuberculosis in mice. Proc Natl Acad Sci

USA 100, 10026–10031.
58 Betts JC, Lukey PT, Robb LC, McAdam RA & Duncan
K (2002) Evaluation of a nutrient starvation model of
Mycobacterium tuberculosis persistence by gene and pro-
tein expression profiling. Mol Microbiol 43, 717–731.
59 Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan
JA, Monahan IM, Dolganov G, Efron B, Butcher PD,
Nathan C et al. (2003) Transcriptional adaptation of
Mycobacterium tuberculosis within macrophages:
insights into the Phagosomal environment. J Exp Med
198, 693–704.
60 Calamita H, Ko C, Tyagi S, Yoshimatsu T, Morrison
NE & Bishai WR (2005) The Mycobacterium tuberculo-
sis SigD sigma factor controls the expression of ribo-
some-associated gene products in stationary phase and
is required for full virulence. Cell Microbiol 7, 233–244.
61 Pang X, Vu P, Byrd TF, Ghanny S, Soteropoulos P,
Mukamolova GV, Wu S, Samten B & Howard ST
(2007) Evidence for complex interactions of stress-asso-
ciated regulons in an mprAB deletion mutant of Myco-
bacterium tuberculosis. Microbiology 153, 1229–1242.
62 Graham JE & Clark-Curtiss JE (1999) Identification of
Mycobacterium tuberculosis RNAs synthesized in
response to phagocytosis by human macrophages by
selective capture of transcribed sequences (SCOTS).
Proc Natl Acad Sci USA 96, 11554–11559.
63 Pendzich J, Maksymowicz-Mazur W, Mazurek U,
Dworniczak S, Oklek K, Kozielski J & Wilczok T
(2004) [Quantitative analysis of sigma genes expression
in Mycobacterium tuberculosis cultures exposed to

rifampicin and isoniazid]. Wiad Lek 57, 233–240.
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 623
64 Provvedi R, Boldrin F, Falciani F, Palu G &
Manganelli R (2009) Global transcriptional response
to vancomycin in Mycobacterium tuberculosis.
Microbiology 155, 1093–1102.
65 Wu QL, Kong D, Lam K & Husson RN (1997) A
mycobacterial extracytoplasmic function sigma factor
involved in survival following stress. J Bacteriol 179 ,
2922–2929.
66 Fernandes ND, Wu QL, Kong D, Puyang X, Garg
S & Husson RN (1999) A mycobacterial extracyto-
plasmic sigma factor involved in survival following
heat shock and oxidative stress. J Bacteriol 181,
4266–4274.
67 Ando M, Yoshimatsu T, Ko C, Converse PJ & Bishai
WR (2003) Deletion of Mycobacterium tuberculosis
sigma factor E results in delayed time to death with
bacterial persistence in the lungs of aerosol-infected
mice. Infect Immun 71, 7170–7172.
68 Manganelli R, Fattorini L, Tan D, Iona E, Orefici G,
Altavilla G, Cusatelli P & Smith I (2004) The extra
cytoplasmic function sigma factor sigma(E) is essential
for Mycobacterium tuberculosis virulence in mice. Infect
Immun 72, 3038–3041.
69 Giacomini E, Sotolongo A, Iona E, Severa M, Remoli
ME, Gafa V, Lande R, Fattorini L, Smith I, Manga-
nelli R et al. (2006) Infection of human dendritic cells
with a Mycobacterium tuberculosis sigE mutant stimu-

lates production of high levels of interleukin-10 but
low levels of CXCL10: impact on the T-cell response.
Infect Immun 74, 3296–3304.
70 Fontan PA, Aris V, Alvarez ME, Ghanny S, Cheng J,
Soteropoulos P, Trevani A, Pine R & Smith I (2008)
Mycobacterium tuberculosis sigma factor E regulon
modulates the host inflammatory response. J Infect Dis
198, 877–885.
71 Dona V, Rodrigue S, Dainese E, Palu G, Gaudreau L,
Manganelli R & Provvedi R (2008) Evidence of com-
plex transcriptional, translational, and posttranslational
regulation of the extracytoplasmic function sigma fac-
tor sigmaE in Mycobacterium tuberculosis. J Bacteriol
190, 5963–5971.
72 Raman S, Song T, Puyang X, Bardarov S, Jacobs WR
Jr & Husson RN (2001) The alternative sigma factor
SigH regulates major components of oxidative and
heat stress responses in Mycobacterium tuberculosis.
J Bacteriol 183, 6119–6125.
73 Williams DL, Pittman TL, Deshotel M, Oby-Robinson
S, Smith I & Husson R (2007) Molecular basis of the
defective heat stress response in Mycobacterium leprae.
J Bacteriol 189, 8818–8827.
74 Sureka K, Dey S, Datta P, Singh AK, Dasgupta A,
Rodrigue S, Basu J & Kundu M (2007) Polyphos-
phate kinase is involved in stress-induced mprAB-
sigE-rel signalling in mycobacteria. Mol Microbiol 65,
261–276.
75 Sureka K, Ghosh B, Dasgupta A, Basu J, Kundu M &
Bose I (2008) Positive feedback and noise activate the

stringent response regulator rel in mycobacteria. PLoS
ONE 3, e1771.
76 He H & Zahrt TC (2005) Identification and character-
ization of a regulatory sequence recognized by Myco-
bacterium tuberculosis persistence regulator MprA.
J Bacteriol 187, 202–212.
77 DeMaio J, Zhang Y, Ko C & Bishai WR (1997) Myco-
bacterium tuberculosis sigF is part of a gene cluster
with similarities to the Bacillus subtilis sigF and sigB
operons. Tuber Lung Dis 78, 3–12.
78 Singh AK & Singh BN (2008) Conservation of sigma
F in mycobacteria and its expression in Mycobacterium
smegmatis. Curr Microbiol 56, 574–580.
79 Michele TM, Ko C & Bishai WR (1999) Exposure to
antibiotics induces expression of the Mycobacterium
tuberculosis sigF gene: implications for chemotherapy
against mycobacterial persistors. Antimicrob Agents
Chemother 43, 218–225.
80 Chen P, Ruiz RE, Li Q, Silver RF & Bishai WR
(2000) Construction and characterization of a Myco-
bacterium tuberculosis mutant lacking the alternate
sigma factor gene, sigF. Infect Immun 68, 5575–5580.
81 Karls RK, Guarner J, McMurray DN, Birkness KA &
Quinn FD (2006) Examination of Mycobacterium
tuberculosis sigma factor mutants using low-dose aero-
sol infection of guinea pigs suggests a role for SigC in
pathogenesis. Microbiology 152, 1591–1600.
82 Gebhard S, Humpel A, McLellan AD & Cook GM
(2008) The alternative sigma factor SigF of Mycobacte-
rium smegmatis is required for survival of heat shock,

acidic pH and oxidative stress. Microbiology 154,
2786–2795.
83 Provvedi R, Kocincova D, Dona V, Euphrasie D,
Daffe M, Etienne G, Manganelli R & Reyrat JM
(2008) SigF controls carotenoid pigment production
and affects transformation efficiency and hydrogen
peroxide sensitivity in Mycobacterium smegmatis.
J Bacteriol 190, 7859–7863.
84 Ramakrishnan L, Federspiel NA & Falkow S (2000)
Granuloma-specific expression of Mycobacterium viru-
lence proteins from the glycine-rich PE-PGRS family.
Science 288, 1436–1439.
85 Lamichhane G, Tyagi S & Bishai WR (2005) Designer
arrays for defined mutant analysis to detect genes
essential for survival of Mycobacterium tuberculosis in
mouse lungs. Infect Immun 73, 2533–2540.
86 Beaucher J, Rodrigue S, Jacques PE, Smith I,
Brzezinski R & Gaudreau L (2002) Novel Mycobacte-
rium tuberculosis anti-sigma factor antagonists control
sigmaF activity by distinct mechanisms. Mol Microbiol
45, 1527–1540.
87 Parida BK, Douglas T, Nino C & Dhandayuthapani S
(2005) Interactions of anti-sigma factor antagonists of
The r-factors of M. tuberculosis P. Sachdeva et al.
624 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS
Mycobacterium tuberculosis in the yeast two-hybrid
system. Tuberculosis (Edinb) 85, 347–355.
88 Greenstein AE, MacGurn JA, Baer CE, Falick AM,
Cox JS & Alber T (2007) M. tuberculosis Ser ⁄ Thr
protein kinase D phosphorylates an anti-anti-sigma

factor homolog. PLoS Pathog 3, e49.
89 Cappelli G, Volpe E, Grassi M, Liseo B, Colizzi V &
Mariani F (2006) Profiling of Mycobacterium tuber-
culosis gene expression during human macrophage
infection: upregulation of the alternative sigma factor
G, a group of transcriptional regulators, and proteins
with unknown function. Res Microbiol 157, 445–455.
90 Lee JH, Geiman DE & Bishai WR (2008) Role of
stress response sigma factor SigG in Mycobacterium
tuberculosis. J Bacteriol 190, 1128–1133.
91 Wu CW, Schmoller SK, Shin SJ & Talaat AM (2007)
Defining the stressome of Mycobacterium avium subsp.
paratuberculosis in vitro and in naturally infected
cows. J Bacteriol 189, 7877–7886.
92 Kaushal D, Schroeder BG, Tyagi S, Yoshimatsu T,
Scott C, Ko C, Carpenter L, Mehrotra J, Manabe
YC, Fleischmann RD et al. (2002) Reduced immuno-
pathology and mortality despite tissue persistence in
a Mycobacterium tuberculosis mutant lacking alterna-
tive sigma factor, SigH. Proc Natl Acad Sci USA 99,
8330–8335.
93 Newton GL, Arnold K, Price MS, Sherrill C, Delcard-
ayre SB, Aharonowitz Y, Cohen G, Davies J, Fahey
RC & Davis C (1996) Distribution of thiols in microor-
ganisms: mycothiol is a major thiol in most
actinomycetes. J Bacteriol 178, 1990–1995.
94 Mehra S & Kaushal D (2009) Functional genomics
reveals extended roles of the Mycobacterium tuberculo-
sis stress response factor sigmaH. J Bacteriol 191,
3965–3980.

95 Song T, Dove SL, Lee KH & Husson RN (2003)
RshA, an anti-sigma factor that regulates the activity
of the mycobacterial stress response sigma factor SigH.
Mol Microbiol 50, 949–959.
96 Park ST, Kang CM & Husson RN (2008) Regulation
of the SigH stress response regulon by an essential pro-
tein kinase in Mycobacterium tuberculosis. Proc Natl
Acad Sci USA 105, 13105–13110.
97 Hu Y, Kendall S, Stoker NG & Coates AR (2004) The
Mycobacterium tuberculosis sigJ gene controls sensitiv-
ity of the bacterium to hydrogen peroxide. FEMS
Microbiol Lett 237, 415–423.
98 Homerova D, Halgasova L & Kormanec J (2008)
Cascade of extracytoplasmic function sigma factors in
Mycobacterium tuberculosis: identification of a sigmaJ-
dependent promoter upstream of sigI. FEMS Microbiol
Lett 280, 120–126.
99 Charlet D, Mostowy S, Alexander D, Sit L, Wiker HG
& Behr MA (2005) Reduced expression of antigenic
proteins MPB70 and MPB83 in Mycobacterium bovis
BCG strains due to a start codon mutation in sigK.
Mol Microbiol 56, 1302–1313.
100 Said-Salim B, Mostowy S, Kristof AS & Behr MA
(2006) Mutations in Mycobacterium tuberculosis
Rv0444c, the gene encoding anti-SigK, explain high
level expression of MPB70 and MPB83 in Mycobacte-
rium bovis. Mol Microbiol 62, 1251–1263.
101 Veyrier F, Said-Salim B & Behr MA (2008) Evolution
of the mycobacterial SigK regulon. J Bacteriol 190,
1891–1899.

102 Hahn MY, Raman S, Anaya M & Husson RN (2005)
The Mycobacterium tuberculosis extracytoplasmic-
function sigma factor SigL regulates polyketide synth-
ases and secreted or membrane proteins and is required
for virulence. J Bacteriol 187, 7062–7071.
103 Goulding CW, Apostol MI, Gleiter S, Parseghian A,
Bardwell J, Gennaro M & Eisenberg D (2004) Gram-
positive DsbE proteins function differently from
Gram-negative DsbE homologs. A structure to func-
tion analysis of DsbE from Mycobacterium tuberculo-
sis. J Biol Chem 279, 3516–3524.
104 Kolattukudy PE, Fernandes ND, Azad AK, Fitzmau-
rice AM & Sirakova TD (1997) Biochemistry and
molecular genetics of cell-wall lipid biosynthesis in
mycobacteria. Mol Microbiol 24, 263–270.
105 Sonden B, Kocincova D, Deshayes C, Euphrasie D,
Rhayat L, Laval F, Frehel C, Daffe M, Etienne G &
Reyrat JM (2005) Gap, a mycobacterial specific
integral membrane protein, is required for glycolipid
transport to the cell surface. Mol Microbiol 58,
426–440.
106 Arraiz N, Salazar L, Lopez G, Rodriguez R, Casart Y
& Takiff H (2001) [Characterization of the expression
and function of SigM an ECF sigma factor in myco-
bacteria]. Acta Cient Venez 52(Suppl 1), 40–41.
107 Agarwal N, Woolwine SC, Tyagi S & Bishai WR
(2007) Characterization of the Mycobacterium tubercu-
losis sigma factor SigM by assessment of virulence and
identification of SigM-dependent genes. Infect Immun
75, 452–461.

108 Raman S, Puyang X, Cheng TY, Young DC, Moody
DB & Husson RN (2006) Mycobacterium tuberculosis
SigM positively regulates Esx secreted protein and non-
ribosomal peptide synthetase genes and down regulates
virulence-associated surface lipid synthesis. J Bacteriol
188, 8460–8468.
109 Camacho LR, Ensergueix D, Perez E, Gicquel B &
Guilhot C (1999) Identification of a virulence gene
cluster of Mycobacterium tuberculosis by signature-
tagged transposon mutagenesis. Mol Microbiol 34,
257–267.
110 Cox JS, Chen B, McNeil M & Jacobs WR Jr (1999)
Complex lipid determines tissue-specific replication of
Mycobacterium tuberculosis in mice. Nature 402,
79–83.
P. Sachdeva et al. The r-factors of M. tuberculosis
FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 625
111 Agarwal N & Tyagi AK (2006) Mycobacterial tran-
scriptional signals: requirements for recognition by
RNA polymerase and optimal transcriptional activity.
Nucleic Acids Res 34, 4245–4257.
112 Mulder MA, Zappe H & Steyn LM (1997) Mycobacte-
rial promoters. Tuber Lung Dis 78, 211–223.
113 Bashyam MD & Tyagi AK (1998) Identification and
analysis of ‘‘extended -10’’ promoters from mycobacte-
ria. J Bacteriol 180, 2568–2573.
114 Song T, Song SE, Raman S, Anaya M & Husson RN
(2008) Critical role of a single position in the -35
element for promoter recognition by Mycobacterium
tuberculosis SigE and SigH. J Bacteriol 190, 2227–

2230.
115 Rodrigue S, Brodeur J, Jacques PE, Gervais AL,
Brzezinski R & Gaudreau L (2007) Identification of
mycobacterial sigma factor binding sites by chromatin
immunoprecipitation assays. J Bacteriol 189, 1505–
1513.
116 Li MS, Waddell SJ, Monahan IM, Mangan JA, Martin
SL, Everett MJ & Butcher PD (2004) Increased tran-
scription of a potential sigma factor regulatory gene
Rv1364c in Mycobacterium bovis BCG while residing
in macrophages indicates use of alternative promoters.
FEMS Microbiol Lett 233, 333–339.
117 Hughes KT & Mathee K (1998) The anti-sigma
factors. Annu Rev Microbiol 52, 231–286.
118 Dutta R & Inouye M (2000) GHKL, an emergent AT-
Pase ⁄ kinase superfamily. Trends Biochem Sci 25, 24–28.
119 Kang JG, Paget MS, Seok YJ, Hahn MY, Bae JB,
Hahn JS, Kleanthous C, Buttner MJ & Roe JH (1999)
RsrA, an anti-sigma factor regulated by redox change.
EMBO J 18, 4292–4298.
120 Paget MS, Bae JB, Hahn MY, Li W, Kleanthous C,
Roe JH & Buttner MJ (2001) Mutational analysis of
RsrA, a zinc-binding anti-sigma factor with a thiol-di-
sulphide redox switch. Mol Microbiol 39, 1036–1047.
121 Sachdeva P, Narayan A, Misra R, Brahmachari V &
Singh Y (2008) Loss of kinase activity in Mycobacte-
rium tuberculosis multidomain protein Rv1364c. FEBS
J 275, 6295–6308.
122 Greenstein AE, Hammel M, Cavazos A & Alber T
(2009) Interdomain communication in the Mycobacte-

rium tuberculosis environmental phosphatase Rv1364c.
J Biol Chem 284, 29828–29835.
123 Hingley-Wilson SM, Sambandamurthy VK & Jacobs
WR Jr (2003) Survival perspectives from the world’s
most successful pathogen, Mycobacterium tuberculosis.
Nat Immunol 4, 949–955.
124 Sadagopal S, Braunstein M, Hager CC, Wei J, Daniel
AK, Bochan MR, Crozier I, Smith NE, Gates HO,
Barnett L et al. (2009) Reducing the activity and secre-
tion of microbial antioxidants enhances the immunoge-
nicity of BCG. PLoS ONE 4, e5531.
The r-factors of M. tuberculosis P. Sachdeva et al.
626 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS