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

The transcription of sigB under stan-dard physiological growth conditions and its induction upon exposure to surface stress is dependent on rE[43], while its induction during heat shock

The sigma factors of Mycobacterium tuberculosis:

regulation of the regulators

Preeti Sachdeva1, Richa Misra1, Anil K Tyagi2and Yogendra Singh1

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

(http://www.who.int/tb/en/) 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 adaptedpathogen rests upon its ability to establish a persistentinfection in the hostile environment of the host cellthrough mechanisms involving transcriptional repro-gramming, ensuring metabolic slowdown and theupregulation 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 throughcoordinated regulation of its gene expression inresponse to signals encountered in the host body Geneexpression in bacteria is regulated primarily at the level

of transcription initiation, which is mediated bythe 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: ysingh@igib.res.in

(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 Mycobacteriumtuberculosis is adaptation to adverse conditions encountered in the hostcells The ability of Mycobacterium to successfully adapt to stress condi-tions is brought about by the expression of specific regulons effected by arepertoire of r factors The induction and availability of r factors inresponse to specific stimuli is governed by a complex regulatory networkcomprising 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, theirregulation and contribution to bacterial pathogenesis

Abbreviations

ECF, extracytoplasmic function; imp, immunopathology; M bovis BCG, Mycobacterium bovis Bacille Calmette–Gue´rin; PPE, 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.

proline-holoenzyme comprises a five-subunit core RNAP

(sub-unit composition, a2bb¢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 (r70) family and the sigma

54 (r54) family, named after the 70 kDa primary r

factor and the 54 kDa nitrogen-regulation r factor

both from Escherichia coli, respectively r54-type r

fac-tors are structurally different from the r70 family

members and utilize a distinct mechanism of open

complex formation There are no known

representa-tives of the r54 family in any GC-rich, Gram-positive

bacteria and cyanobacteria; however, r70-related r

fac-tors are encoded in all bacterial genomes [4,6] The r70

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 r70-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 centeredabout 35 bp upstream of the TSP and recognized bythe 4.2 subregion [3,10,11]; and (d) an AT-rich UPelement, generally located between 61 and 41 bp up-stream of the TSP [12,13] The upstream (UP) elementbinds the C-terminal domain of the a subunit of theRNAP and enhances the promoter activity by two-fold

to as much as 90-fold [12,14] Besides, the presence ofGC-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 thattranscribes the genes required for cell viability, andmost bacteria harbor alternative r factors that tran-scribe regulons in response to specific stimuli Thenumber of alternative r factors correlates generallywith the variability of the environments encountered

by a given bacterial species The number may rangefrom 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 soilactinomycete, Streptomyces coelicolor, whose naturalhabitat is highly variable in terms of nutrients, stressesand competing microbial flora [16] Also, while thenumber of r factors is generally found to increase withthe genome size, microorganisms that have developeddifferentiation programmes (e.g sporulation) tend tohave higher alternative r factor⁄ genome size ratiosthan most obligate pathogens or commensal bacteria.Interestingly, M tuberculosis has the highest alterna-tive r factor⁄ genome size ratio amongst the obligatepathogens, suggesting a highly complex regulatorymechanism for its transcription [17] Notably, a recentreport suggested endospore formation by mycobacteriaand speculated sporulation as a possible mechanismfor dormancy [18]

M tuberculosis encodes a repertoire of 13 r factors[19,20], of which rA, rB and rF are representatives ofgroups 1, 2 and 3 of the r70family, respectively, whilethe remaining 10 r factors belong to group 4.Amongst the ECF r factors, rG, rIand rJpossess anadditional carboxy-terminal extension in their struc-ture, which is presumed to provide a surface for inter-action with other regulatory molecules [17] The geneloci for the principal r factor in M tuberculosis, sigA,and for the principal factor-like r factor, sigB, are wellconserved 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 orthologshave been found to be present in all pathogenic Myco-bacterium spp sequenced to date, including Myco-bacterium leprae, but absent in nonpathogenicMycobacterium spp sequenced so far By contrast,

sigE is the only ECF r factor conserved across the

Mycobacteriumgenus 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

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’.]

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 rA (R515H)

resulted in attenuation of virulence of the M bovis

strain ATCC 35721 in a guinea pig model of infection

[27] The mutated rA 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

H37Rv, 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 rA 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 H37Rv 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

rA 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 rA in the expression of virulence genes is,

however, a host-specific feature, because

complementa-tion with a funccomplementa-tional rA 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

rA-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 rA-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 survivalare constitutively expressed rather than regulated bymacrophages [36] Interestingly, the role of rAin viru-lence gene expression goes very well with this report

SigB (rB)

rB, the principal factor-like r factor of M tuberculosis,

is very similar to the C-terminal portion of rA[17,37].But, unlike rA, it has been found to be dispensable forgrowth in both M smegmatis [22] and M tuberculosis[38] Moreover, the rAand rBregulons do not overlapmuch except for a few genes such as those belonging tothe PE_PGRS family [35,39] A comparison of aminoacid residues from the 2.4 and 4.2 subregions of rAand

rBshows that these r factors differ at four of 23 tions in the 2.4 subregion More significantly, the 4.2subregion of rA and rB differs at 15 of 39 positionsand nine of these changes are nonconserved Whilethese differences would definitely reflect on the pro-moter sequences recognized by these two r factors,other aspects that contribute to the nonoverlappingrepertoire of genes transcribed by the two r factorsremain to be elucidated [40] Besides, unlike sigA,expression of the M tuberculosis sigB gene increasesupon exposure to various environmental stresses such

posi-as low aeration [24], treatment with hydrogen peroxideand heat shock, with a more pronounced effect seen instationary phase than in logarithmic phase [26]

A recent report demonstrated that inactivation ofthe sigB gene did not affect the survival of M tubercu-losis during infection in human macrophages or inmouse and guinea pig models [38] However, deletion

of sigB in M tuberculosis results in its higher ity to SDS-induced surface stress, heat shock, oxidativestress, exposure to vancomycin and hypoxic conditions[17,38] Further evidence for the role of rBin adapta-tion to stationary phase and nutritionally poor condi-tions came from a report by Mukherjee et al [41] Thisgroup reported that upon overexpression of M tuber-culosis sigB in M smegmatis, the cell-surface glycopep-tidolipids found in the outer layers of M smegmatisbecome hyperglycosylated, similarly to what isobserved during carbon starvation Certain metabolicenzymes, 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 rB in M smegmatis [42] Overexpression

sensitiv-of sigB in M tuberculosis resulted in a significant

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 propro-teins, keto-acyl synthase, KasA

and the regulatory proteins, WhiB2, IdeR and rBitself

[39] Also, sigB is transcribed from a predicted rB

-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

hsp20by rB[38]

Previous studies have suggested that there are two

more promoters upstream of rB: one is recognized by

RNAP containing any of the three r factors, rE [43],

rH[44] and rL; while the other is recognized by RNAP

containing rF[45] The transcription of sigB under

stan-dard physiological growth conditions and its induction

upon exposure to surface stress is dependent on rE[43],

while its induction during heat shock or oxidative stress

is rH-dependent [44] In contrast to the rF-dependent

transcription of sigB observed in an in vitro

transcrip-tion assay [45], the upregulatranscrip-tion of sigB upon

overex-pression of sigF was not observed [46] The possibility

of certain rF-dependent genes being missed in the study

by Williams et al cannot be ruled out in view of

induc-tion of the anti-rFprotein, UsfX, which is expected to

significantly reduce the effective concentration of active

rFin the cell The conditions for rF- and rL-dependent

transcription of sigB are yet to be identified

rBseems 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 rB 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 (rC)

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,

rium vanbaalenii, Mycobacterium sp MCS,

Mycobacte-riumsp KMS and Mycobacterium sp JLS Despite the

sigC transcript being the most abundant transcript ofall r factors, most of the core RNAP during exponen-tial phase has been found to be associated with either

rAor rB Therefore, it was speculated that M losis sigCis either translated at a very low efficiency orhas a low affinity for RNAP [24] M tuberculosis sigC

tubercu-is downregulated during stationary phase and inresponse to heat shock and SDS-induced surface stress[24] Interestingly, sigC was also found to be downregu-lated in the M tuberculosis CDC1551 sigF mutantstrain [49], adding another r factor in the hierarchicalregulatory network of mycobacterial r factors (Fig 2)

rCis not required for the survival of Mycobacterium

in murine bone marrow-derived macrophages or inactivated J774A.1 macrophages [50] In a mouse model

of infection, the sigC mutant in both the CDC1551and H37Rv backgrounds grows and persists in lungsbut shows attenuated disease progression and fails toelicit the same degree of lethal immunopathology asthe wild-type strain [50,51] This phenotype of bacterialpersistence at high colony counts with reduced hostmortality is designated as the immunopathology (imp)defect and is associated with a significant reduction inthe number of infiltrating neutrophils and with theproduction of pro-inflammatory cytokines such astumor necrosis factor-a-a, interleukin-1b, interleukin-6and interferon-c in the lungs of the infected animal[51] The attenuation of the sigC mutant may resultfrom dysregulation of expression of several key viru-lence-associated genes, such as hspX (encoding ana-crystallin homolog), senX3 (a sensor kinase), mtrA (aresponse regulator), polyketide synthases and fbpC(antigen 85C) [50] Most importantly, in a guinea pigmodel of infection that best mimics the granuloma for-mation and disease progression in humans, the

M tuberculosis sigC mutant displayed delayed growthwith fewer caseating lesions compared with the wild-type strain [51] The two promoter-recognitiondomains of rC(rC

2 and rC

4) have been crystallized [52]and interact in vitro involving occlusion of the Pribnowbox recognition region This interdomain interaction issuggestive of an alternate mechanism of regulation of

rCactivity via autoinhibition in the absence of a nate anti-r factor [53]

cog-SigD (rD)

rD is expressed at a moderately high and constitutivelevel during exponential and stationary growth phasesand declines significantly, following hypoxia, in a pat-tern very similar to that of rA in an in vitro culture[54,55] The stringent response is modulated by the relAgene product via the synthesis of a key signaling

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 rDin physiological adaptations such as stringent

response and starvation The intracellular rD 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 H37Rv 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 rD-regulated genes include

those encoding proteins involved in lipid metabolism,

cell wall-related processes, stress response and DNAbinding 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 chalconesynthase), 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 ary phase [54,60] Notably, several rD-regulated geneswere reported to be highly expressed in the MprABTCS mutant strain under SDS stress, indicating thatmany of the rD-regulated genes are under the repres-sive effect of MprA [61]

station-SigE (rE)

rE is one of the two ECF r factors encoded by the

M leprae genome [48] sigE is upregulated in bacteria grown within human macrophages comparedwith those grown in an in vitro culture [23,62] Itsexpression also increases following exposure to heat

myco-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.

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

H37Rv 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

rE 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, rH-dependent

promoter, P3, induced under the conditions of

oxida-tive stress and heat shock The rH-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 rE

pro-tein [73] Various different sigE start codons have also

been characterized, which may give rise to different rE

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]

rE-dependent genes encode proteins belonging to

dif-ferent classes, such as transcriptional regulators

(includ-ing rB, 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 rE

-dependent genes mprA and mprB regulates the in vivo

expression of sigE as well as another stress-responsive rfactor, sigB, in M tuberculosis This regulation is medi-ated by binding of the response regulator, MprA, toconserved motifs in the upstream regions of rEand rB[47] A number of stress-responsive genes have beenreported to be downregulated in the mprA mutantstrain, some of which may actually be targets of these rfactors [61] In addition, the induction of mprA follow-ing exposure to stress also suggests a direct role of thisregulatory system in the stress-response pathways in

M tuberculosis [47] Surprisingly, some of the rEdent genes, such as Rv1129c, Rv1130 and Rv1131 (cit-rate synthase; gltA), are also under an indirect repressiveeffect of MprA, suggesting the MprAB–rE regulationnetwork to be highly complex [43,61]

-depen-Under conditions of stress (such as ATP depletion),inorganic polyphosphate polyP (synthesized by theenzyme PPK1) serves as a preferred donor for theMprB-mediated phosphorylation of MprA This results

in MprA-regulated transcription of the mprAB operon,which thereby facilitates rE-mediated transcription of astringent response gene, rel [74] As sigD is suggested to

be part of the RelA regulon [57], its expression is alsolikely to be under the indirect control of rE The positiveregulation of rEby MprAB, which results in bimodal relgene expression and thereby a phenotypic heterogeneity

in the bacterial population, may play a role in the opment of persistence in Mycobacterium [75] The bind-ing affinity of MprA for its promoter increases uponphosphorylation and is required for the upregulation ofthe mprA gene in vivo [76] By contrast, the binding ofMprA 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] islikely to be an indirect consequence of diminished poly-phosphate levels The direct effect of phosphorylation ofMprA on its binding to sigB and sigE upstream regionsremains to be studied The co-dependent transcriptionalregulation of rEand MprA [43,61], as well as the autore-gulation of MprA [76], may result in the maintenance ofthe levels of rE and MprA in a cyclic manner duringstress rE, in addition to a complex transcriptional regu-lation, is also subjected to translational regulation aswell as to post-translational regulation by a zinc-associ-ated anti-r factor (ZAS) family protein [71]

devel-SigF (rF)

M tuberculosis rF, the only group 3 r factor represented

in the Mycobacterium genus, bears significant homology

to the stress response r factors in Bacillus subtilis, ylococcus aureus and Listeria monocytogenes and to the

Staph-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 sigFis 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 H37Rv 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 rF in bacterial growth during stationary

phase and under stress conditions, as well as

suscepti-bility to antibiotics, the conditional overexpression of

sigFduring 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 H37Rv 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 rF is not

required for bacillary survival under nutrient

starva-tion condistarva-tions 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 mc2155 sigFmutant strain demonstrated that rF is required forresistance to heat shock and acid stress, but not for thesurvival of bacillus in human neutrophils M smegma-tisrFalso mediates resistance to oxidative stress, prob-ably in a KatG-independent and AhpC-independentmanner [82] As reported for M tuberculosis [80],

M smegmatis rFis also implicated in the regulation ofgenes involved in cell wall permeability, as evident bythe decreased transformation efficiency in the presence

of a functional sigF gene Also, rF is required for thebiosynthesis of carotenoids, complex lipids that act asfree-radical scavengers and protect the cells againstphotodynamic injury in M smegmatis [83]

In order to identify rF-dependent genes, tional profiling of an M tuberculosis CDC1551 sigFmutant 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

transcrip-‘CDC551’ was changed to ‘CDC1551’.] Disruption ofthe sigF gene resulted in the downregulation of a signifi-cantly larger number of genes in the late-stationaryphase compared with the exponential phase rF regu-lates the expression of genes involved in the biosynthesisand 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, intermediarymetabolism and information pathways, rF regulatesgenes encoding several transcriptional regulators, forexample, MarR, GntR and TetR family regulators,PhoY1 and Rv2884, as well as an ECF r factor, rC[46,49] Conditional overexpression of sigF during theearly exponential phase resulted in the upregulation ofseveral 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 transcriptionallevel via autoregulation of its promoter Besides, rFactivity 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 ofthese factors [86–88]

SigG (rG)sigG is one of the most highly induced genes in

M tuberculosis during macrophage infection [23,89]

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

sigGis 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 rG 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 rGin 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 rH, such as

clpB, dnaK and trxB2, were also found to be

down-regulated in this study Moreover, two other

rD-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 (rH)

The role of rH 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 sigHwas 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 rH-regulated genes, microarray

experi-ments were carried out at different phases of growth

[92] and following diamide stress [44] The rHregulon

includes its own structural gene and genes encoding

rB, rE, Rv0142 (putative transcriptional regulator)

DnaK, ClpB (heat shock proteins), TrxB and TrxC(thioredoxin reductase⁄ thioredoxin) [44,92] rH alsoinduces enzymes involved in cysteine biosynthesis and

in the metabolism of ribose and glucose, indicating anincreased need for the synthesis of mycothiol precur-sors [44] (mycothiols are known to be involved incellular protection during oxidative stress in actinomy-cetes [93]) In a recent report on the long-term effects

of rH induction following diamide stress, it wasobserved that in response to oxidative damage, certainvirulence⁄ detoxification genes were induced, whilemany lipid metabolism genes were repressed, as a part

of the stress-defense mechanism in M tuberculosis Asthe effect of stress diminished with time, the expression

of lipid metabolism and of cell wall-associated genesresumed, demonstrating a remarkable plasticity in geneexpression brought about by a mycobacterial r factor[94] rHactivity is regulated at the transcriptional levelvia autoregulation of the sigH promoter and post-translationally via interaction with its cognate anti-rfactor, RshA [95] The latter branch of regulation isintersected by PknB, a serine–threonine protein kinase(STPK), which further fine-tunes the stress responseregulon controlled by rH[96]

SigI (rI) and SigJ (rJ)sigI, and to a larger extent, sigJ, genes are expressed athigh levels in late stationary phase dormant cultures of

M tuberculosis The transcription of these two r tors continues following rifampicin treatment of thesecultures in an in vitro drug-persistent model of

fac-M tuberculosis[55] However, no difference in viabilitywas observed between the sigJ mutant and the wild-type strain in a late stationary phase culture followingrifampicin treatment, or in an immune stasis murinemodel The sigJ mutant is more susceptible to killing

by H2O2 than its parental strain As katG mRNAlevels remain unchanged upon deletion of sigJ, rJpossibly mediates resistance to H2O2 via a KatG-independent pathway [97] rJ may also contribute tothe survival of M tuberculosis in the host organism, assuggested by an increase in sigJ expression in humanmacrophages [89]

Using the E coli two-plasmid system, it was foundthat the expression of M tuberculosis sigI is regulated

by rJ [98], suggesting a possible relationship betweenthe two r factors It is likely that rImay be involved inregulating stress responses similar to those regulated by

rJ Based on the fact that the sigI transcript levelincreases after mild cold shock (room temperature), the

rIregulon has been speculated to be involved in the vival of M tuberculosis in aerosol particles, where the

sur-ambient temperature is usually lower than 37C [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 (rK)

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] rK 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 rK, may

have led to a state of constitutive activity of rK and

therefore constantly high levels of expression of its

reg-ulon observed in the study [100] Evolutionary analysis

indicates that the core regulatory system, rK⁄ 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 rKregulon 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 rK

regulon is atypically small; however, the number and

identity of rK-regulated genes varies across different

mycobacterial species [101] The role and trigger of this

regulon in M tuberculosis pathogenicity merits

investi-gation

SigL (rL)

sigLis constitutively expressed at a very low level from

a weak rL-independent promoter and is also transcribed

from a rL-dependent promoter [102] In a murineinfection model, the sigL mutant exhibited markedattenuation compared with the parental strain, suggest-ing a role of rL in virulence; however, there were nosignificant differences in the growth rate or in the sizeand extent of lesions in the infected organs [45,102].Microarray analysis was carried out by two groupsindependently in order to characterize the rL regulon[45,102] In one of the approaches, sigL overexpres-sion from an acetamide-inducible promoter led to thestrong 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 anextracellular oxidant required for proper folding ofreduced unfolded secreted proteins [103] While pks10and pks7 are polyketide synthase genes, the other

rL-regulated gene pair, Rv1139c-Rv1138c, encodes amembrane protein containing an isoprenyl cysteinecarboxy methyltransferase motif and a putative oxido-reductase, respectively [102] In another study, amutant strain lacking both sigL and rslA was comple-mented by integrating a single wild-type copy of sigLinto its chromosome, resulting in its constitutiveexpression [45] Some of the genes (pks10, pks7,Rv1138c, mpt53 and Rv2877c) were identified usingboth approaches Other genes identified using the lat-ter method included the remaining genes from thepks10 operon and the ppsA gene, involved in thebiosynthesis of dimycocerosyl phthiocerol (a cell wall-associated lipid found only in pathogenic mycobacte-ria [104]), mmpL13a and mmpL13b, involved in fattyacid transport [105], another r factor-encoding gene,sigB and two genes thought to be involved in hostcell invasion [45]

rL, like rE and rH, is post-translationally regulated

by a ZAS family protein with its gene located stream of the sigL gene However, unlike sigE andsigH, sigL does not play a role in the oxidative ornitrosative stress responses [45]

down-SigM (rM)sigM expression was found to be induced at high tem-perature and in the stationary phase during in vitrogrowth 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 thelate stationary phase in M tuberculosis [107] Bycontrast, Raman et al did not observe a significantchange in sigM expression at any growth stage in

M tuberculosis H37Rv [108] The M smegmatis sigMmutant is more susceptible to oxidative stress than the

parental strain [106] However, M tuberculosis

CDC1551 rM is dispensable for survival in the

pres-ence of oxidative and detergent stresses, suggesting

dif-ferent roles of the rMorthologs 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 rM

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 rM 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 rM, 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 rM 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 rM, M tuberculosis

H37Rv and M bovis AF2122⁄ 97 are fully virulent It is

possible that these mutations do not affect the function

of rM, or that rM may not be required for virulence

[17]

Promoter recognition by mycobacterial

r factorsDuring the last few years of work on mycobacterial rfactors, a major focus has been the identification ofgenes whose expression lies under the control of differ-ent r factors The promoters recognized by different rfactors 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 ofupstream regions of differentially expressed genes, led

to the selection of putative promoter sequences thatwere validated using in vitro assays Despite the accu-mulation of extensive data obtained by employing thisstrategy, the success in defining unambiguous arche-typal promoter consensus sequences recognized bymost of the mycobacterial r factors has been quitelimited A comprehensive analysis of the literature sug-gests a number of factors that may account for thepoor sequence conservation in promoter consensus andalso for certain inconsistencies in different promotersequences reported for some of the mycobacterial rfactors

A substantial body of research work on the tively expressed mycobacterial promoters, under thetranscriptional control of rA, has demonstrated thatthe -10 position of these promoters have sequencessimilar to their counterparts in E coli [40,111] How-ever, a majority of these mycobacterial promoters donot work efficiently in E coli This could be attributed

constitu-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 beenspeculated that the presence of an AT-rich sequence atthe -15 region of E coli promoters, which drasticallyinfluences the promoter strength [15], and its absence

in the GC-rich mycobacterial promoters, may beresponsible, at least partially, for the reduced activity

of mycobacterial promoters in E coli [111]

Another class of promoters, termed ‘extended -10promoters’, is more crucially dependent on the -10region 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 rD, rE, rH, rLand

rM, are very similar [54,72,102,108,114], often withonly single base differences, whereas their -10 consen-sus sequences are significantly divergent In theabsence of reports of genes commonly regulated byany of these r factors, it was suggested that the core-35 region is required for efficient promoter bindingand⁄ or transcription initiation, while binding specificity