Tuberculosis remains a worldwide threat despite the availabilityof the BCG vaccine and antibiotic treatment . It is estimatedthat its etiologic agent, Mycobacterium tuberculosis, infectsalmost a third of the human population and kills two millionpeople every year [27] . The recent human immunodeficiency virus pandemic, the selection of multidrug-resistant strains of M. tuberculosis, and the increased immigration from countries witha high tuberculosis incidence, coupled with increasing povertyand homelessness in these countries, have awakened the developednations from the widespread apathy toward tuberculosis [36]. Indeed, recent years have seen great progress in the molecular characterization of this efficient human pathogen [26, 61].However, much work is still needed to understand how M . tuberculosiscopes with the numerous environments it encounters in the courseof a successful infection . Adaptation to such conditions mustrequire a complex regulation of gene expression.

The main stresses faced during infection can be summarized as follows . The first stress is exposure to oxidizing agents, principally represented by the reactive oxygen intermediates and reactive nitrogen intermediates, produced by activated macrophages . Thesecond is exposure to low pH . Even if M . tuberculosis is ableto block phagosome acidification, this block is not completeas the mycobacterial phagosome undergoes a slight decrease inpH [21] . The third is damage of surface structures . Alveolarsurfactant is a mild detergent with antibacterial activity andcould damage the structure of its fatty acid-rich cell envelope.In addition, toxic peptides and proteins like granulysin, thoughtto act at the level of the bacterial surface, are released byactivated macrophages and NK cells . Specifically, granulysinhas been recently shown to be essential for M . tuberculosiskilling after apoptosis of infected macrophages induced by NKcells [22] . Finally, toxic free fatty acids, secreted from macrophagesboth inside the mycobacterial phagosome and in the externalenvironment, exhibit their toxicity when interacting with themycobacterial surface [2] . The fourth is hypoxia, especially inside granulomas but also inside the phagosome . This environmental condition is actually the best candidate for the induction of persistence [also called dormancy or latency], a phenomenonof great importance in M . tuberculosis pathogenesis but stillnot well understood at the molecular level [68] . Recent experiments have implicated the transcriptional regulator DosR [dormancy survival regulator] [10, 51, 71], also known as DevR [18], and the response regulator MprA [72] in mycobacterial persistence.Interestingly, Voskuil et al . [67] recently showed that theDosR regulon is induced following NO-dependent inhibition ofaerobic respiration . The fifth is nutrient and essential-elementstarvation . Inside phagosomes and granulomas, the availabilityof nutrients and essential elements may be reduced, as was recentlyshown for iron [31; J . Timm et al., unpublished data] and Mg²⁺ [12; S . Walters and I . Smith, unpublished data] . Also, duringtransmission [between expulsion from an infected patient andinhalation by a new host], M . tuberculosis must face other environmentalstresses such as nutrient starvation, exposure to UV light,dehydration, and low temperature.

The M . tuberculosis genome [14, 29] encodes about 190 transcriptionalregulators: 13 factors, 11 two-component systems, 5 unpairedresponse regulators, 11 protein kinases [3], and more than 140other putative transcriptional regulators [9] . Several of these regulators have been characterized; some of them respond to environmental stresses such as cold shock [60], heat shock [41, 63], hypoxia [18, 51, 59], iron starvation [56], surface stress [41], and oxidative stress [42, 55], while others respond tostill unknown environmental conditions [28, 52, 72] . The resultingpicture is still incomplete, but it suggests very complex regulatorysystems with overlapping functions and redundancies . For example,the heat shock response is determined by the activation of fiveoverlapping regulons under the transcriptional control of three factors [SigB, SigE, and SigH] [41, 42, 43] and two other transcriptionalregulators [HspR and HrcA] [63].

In this review, we will principally discuss factors . Prokaryoticcore RNA polymerase [RNAP] is composed of four distinct subunits:ß, ß', , and an dimer . A fifth subunit,the factor, reversibly associates with RNAP, forming the RNAPholoenzyme, and provides the promoter recognition function.The number of factors encoded in a genome is quite variableand ranges from a minimum of one in Mycoplasma sp . [30, 37]to a maximum of 65 in Streptomyces coelicolor A3[2] [6] . M.tuberculosis encodes 13 different putative factors [14, 32, 34] . It is generally observed that every factor has its ownspecificity, allowing the initiation of transcription of differentsubsets of genes . Genes belonging to a defined regulon oftenparticipate in related cellular functions . Therefore, temporalvariation in active factor populations may represent a powerfulway for M . tuberculosis to modulate its gene expression profilesin accordance with physiological requirements and thus achievea successful infection.

CLASSIFICATION OF FACTORS factors can be divided in two groups that are phylogenetically distinct: those related to ⁷⁰ and those related to ⁵⁴ [70].While all eubacteria encode at least one factor belonging tothe ⁷⁰ class, not all of them encode one belonging to the ⁵⁴ class . Since the latter family of factors is not representedin mycobacteria, it will not be discussed any further . The ⁷⁰ family can be further divided into three groups, depending on their structure and function: [i] primary factors, [ii] nonessentialprimary factor-like factors, and [iii] alternative factors[70] . All eubacterial genomes encode one primary factor . Itis usually essential and allows the transcription of housekeepinggenes . Escherichia coli ⁷⁰ and Bacillus subtilis ^A are partof this category, and in M . tuberculosis, this group is representedby ^A [23, 33].

The factors belonging to the second group [primary factor-like factors] are nonessential under standard physiologic growth conditions and are highly similar to primary factors . Theycan be involved in different functions . In enterobacteria, theyare usually involved in stationary-phase survival [RpoS]; incyanobacteria, they are involved in the circadian cycle andin carbon and nitrogen utilization [^B and ^C of Synechococcussp.]; and they are involved in antibiotic biosynthesis in streptomycetes[HrdD] [70] . In M . tuberculosis, they are represented by ^B [23].

The third group, that of alternative factors, is the most heterogeneousand can be divided into numerous subgroups . In M . tuberculosis,they are represented by ^F [20], belonging to the subgroup thatalso contains the stress response-sporulation factors in bacilliand streptomycetes, and by ^C, ^D, ^E, ^G, ^H, ^I, ^J, ^K, ^L, and ^M, belonging to the subgroup of the extracellular function [ECF] factors . ECF factors are environmentally responsive regulators,and bacteria usually contain several members of the ECF familythat control a variety of functions in response to specificextracellular environmental signals, such as the presence ofmisfolded proteins in the periplasm, the presence of light,changes in osmolarity or barometric pressure, and the presenceof toxic molecules in the external environment [45, 70] . Examples are E . coli ^E, which controls the response to extreme heat shock[1]; AlgU, which controls alginate biosynthesis in Pseudomonas aeruginosa; FecI, which controls iron uptake in E . coli; CarQ, which controls carotenoid biosynthesis in Myxococcus xanthus; and P . syringae HrpL, which controls the synthesis of a virulence factor that functions during plant infections [45, 70].

MYCOBACTERIAL FACTOR GENOMICS In addition to the annotated M . tuberculosis H37Rv and CDC1551genomes [14, 29], almost complete DNA sequence data are nowavailable for several mycobacterial species . Examination ofthese genomes shows that factor genes and their loci are wellconserved across the genus, even though there are some exceptions.In Table 1 are listed the orthologs of the 13 M . tuberculosis factors in other mycobacterial species.

 
The locus containing the genes encoding the principal and principal factor-like factors [sigA and sigB] is well conserved in the completely sequenced mycobacterial genomes available for analysis[23; R . Provvedi, unpublished data].

Interestingly, M . leprae sigF is a pseudogene and M . avium and M . paratuberculosis have two genes encoding a ^F-like proteinin different chromosomal loci . Of the two sigF-like genes, onlythe one encoding a ^F-like protein with greater similarity toM . tuberculosis ^F is preceded by the gene encoding its own putativenegative regulator [anti- factor], UsfX, as in M . tuberculosis[Provvedi, unpublished].

The genes encoding the ECF factors show more variability . Aspreviously reported, massive gene decay has occurred in M . leprae,in which only sigC and sigE have functional homologs . All ofthe other ECF genes are pseudogenes, with the exception ofsigL, whose locus is deleted [15] . It has recently been proposed that the loss of functional factors initiated pseudogene accumulationin this bacterium [4] . In contrast, all M . tuberculosis ECF factor genes have an ortholog in M . bovis . The annotated sigM locus is a pseudogene, but this could be due to a mistake in the available sequence, which is not yet completely assembled. However, M . bovis BCG Pasteur lacks sigI as this locus is deleted, while in its genome, the sigH and sigM loci are duplicated [11].

Other differences we noted in the M . avium and M . paratuberculosis genomes are the lack of clear orthologs of sigK and sigI and the presence of four ECF factor genes encoding putative proteinssimilar to SigI/SigJ, suggesting that these species have moreECF factors than M . tuberculosis [Provvedi, unpublished] . InM . avium, sigL has a frameshift, but as is the case for sigM in M . bovis, this could also be due to a mistake in the available sequence . In M . marinum, we could find clear orthologs for all of the 13 M . tuberculosis factors, with the exception of sigI.As in M . avium and M . paratuberculosis, we also found four additionalECF factors genes . One of these encodes a protein very similarto ^C [Provvedi, unpublished].

The genome that shows the ECF factor gene pattern that is themost different from that of M . tuberculosis is that of the fast-growingmycobacterium M . smegmatis . It was not possible to find orthologsof sigC, sigI, and sigK, but there are at least seven or eight additional open reading frame showing similarity to ECF factorgenes in this genome [Provvedi, unpublished] . Also in this case,the genome sequence data, reportedly complete, are not yet assembled,preventing a complete and accurate analysis . The data extractedfrom these genomes support a rough correlation between the numberof factors encoded in a genome and the diversity of possibleniches for a given bacterium.

EXPRESSION OF M . TUBERCULOSIS FACTORS All of the 13 M . tuberculosis factors are expressed duringexponential growth [38, 41] . Quantitative reverse transcription-PCRshowed that the amount of sigA-specific mRNA is constant during exponential growth and that it can be used as an internal invariant standard for mRNA quantitation when cells are growing eitherin broth or in macrophages [25] . The mRNA levels of some factorschange when the cells are subjected to stress; e.g., both sigBand sigE mRNA levels increase when the cells are exposed tosodium dodecyl sulfate [SDS]-induced surface stress [41, 43].Levels of the same two mRNAs and that of sigH also increaseafter heat shock and exposure to diamide [a thiol-specific oxidizingagent] [41, 42] . It is interesting that sigF, sigE, and sigHwere induced during infection of macrophages, suggesting theirinvolvement in virulence [35] . Other studies have recently shownthat sigB, sigF, sigE, and sigD were induced after prolongednutrient starvation [8] . Finally, sigJ was recently shown tobe induced in stationary-phase cultures, and the high levelof sigJ mRNA was maintained after a 5-day treatment with rifampin[38].

FACTOR POSTTRANSLATIONAL REGULATION IN M . TUBERCULOSIS Even though mRNA levels of factor genes are frequently inducedunder a given condition, the activity at the protein level mayalso be regulated by a family of proteins called anti- factors.These proteins can bind to a specific factor, keeping it inan inactive form . In the presence of a specific stimulus, theanti- factor releases the factor, which becomes active . Moreover,another class of proteins, the anti-anti- factors, can inhibitanti- factor activity [39] . M . tuberculosis alternative factors ^E, ^F, ^H, and ^L are each closely linked to a gene encoding aputative anti- factor . The M . tuberculosis genome contains anotherputative anti- factor-encoding gene [Rv0093c], not associatedwith any factor gene, and seven genes encoding putative anti-anti- factors . The function of some of these molecules will be discussed later.

^A, THE PRIMARY FACTOR ^A [also known as RpoV] is believed to be the principal factorof M . tuberculosis because inactivation of its genetic determinant,sigA, has not been possible in both M . smegmatis and M . tuberculosis[33; J . Timms and I . Smith, unpublished data] . Its consensuspromoter sequence is shown in Table 2.

 
It was the first mycobacterial factor to be associated withvirulence . An arginine-to-histidine substitution at amino acidresidue 515 [R515H] caused attenuation of M . bovis virulencein a guinea pig model of infection [16] . This mutation was localizedto the C terminus of the protein, in a conserved domain knownto interact with transcriptional activators in other bacteria[24] . Since the mutant strain grew normally in vitro, it wassuggested that the mutant protein was still able to drive theexpression of the housekeeping genes but was deficient for bindingto some virulence-specific transcriptional activators . It wasrecently shown that ^A interacts with the putative transcriptionalregulator WhiB3 and that this interaction is lost in the R515Hmutant [64] . Interestingly, a deletion of whiB3 in M . bovisresulted in attenuation of M . bovis virulence as in the original sigA R515H mutant, but an M . tuberculosis whiB3 mutant was only partially attenuated for virulence . Since ^A is the same in M.tuberculosis and M . bovis, this different phenotype is probablydue to their different genetic backgrounds [64] . The WhiB familyin M . tuberculosis includes seven members . Related proteinsin Streptomyces coelicolor are involved in sporulation, septation, and cell wall deposition [62].

^B, A PRIMARY FACTOR-LIKE FACTOR sigB, the gene encoding ^B, is almost identical to the last 600bp of sigA and is localized approximately 3 kb downstream ofsigA in all of the mycobacterial species thus far analyzed [23].In contrast to the latter, sigB is dispensable for growth bothin M . smegmatis and in M . tuberculosis [M . Gomez and I . Smith, unpublished data] . An M . tuberculosis sigB knockout mutant is more sensitive to various environmental stresses, such as SDS-induced surface stress, heat shock, and oxidative stress, but it is still able to grow normally in human macrophages and is not attenuated in mice [Gomez and Smith, unpublished] . sigB regulation is complex; it is induced following exposure to surface or oxidative stress and after heat shock [41] . Moreover, its transcriptionunder physiological conditions and its induction after surfacestress are dependent on ^E, while during heat shock or oxidativestress, its induction is dependent on ^H [42, 55] . In vitro transcription experiments recently showed that the sigB promoter can be transcribedby RNAP containing ^E, ^H, or ^L, suggesting the necessity forits induction under very different stress conditions [S . Rodrigueet al., unpublished data].

The subdomains of ^B that are responsible for promoter recognitionare almost identical to those of ^A [23, 53] . It is thus tempting to speculate that ^A and ^B recognize similar promoter sequencesand that their respective regulons partially overlap, as isthe case with RpoS and ⁷⁰ in E . coli [65]. ^B could functionas a "backup" to maintain the transcription of essential housekeepinggenes during exposure to stress, when ^A could be inactive orits levels could be lowered . The role of ^B will be clarifiedwhen more genes that require it for their transcription areidentified . In this regard, it was reported that overexpressionof ^B in M . smegmatis and M . bovis BCG caused an increase in katG expression, but it is not known whether this is a direct transcriptional effect [46] . To generate a better ^B consensussequence, we are currently using DNA microarrays to find moregenes in the ^B regulon and preliminary results indicate thatseveral heat shock genes require ^B for their expression [P.Fontan and I . Smith, unpublished data].

^F, A FACTOR REQUIRED FOR FULL VIRULENCE sigF, encoding ^F, is part of a gene cluster with an organizationsimilar to that of the B . subtilis sigF and sigB operons . Inthis locus, the anti-sigma factor-encoding gene usfX [originallyannotated rsbW like in the Tuberculist database; RsbW is theB . subtilis ortholog] is directly upstream of the factor gene[19] . In B . subtilis, ^F is involved in sporulation, while ^B is a general stress response factor whose expression is activatedby heat, alcohol, osmotic stress, and entry into stationaryphase [70] . The M . tuberculosis gene encoding ^F is induced inM . smegmatis and M . bovis BCG after exposure to several antibiotics,hypoxia, cold shock, oxidative stress, and entry into stationaryphase [20, 44] . However, its induction was not observed in M. tuberculosis after cold shock, hypoxia, oxidative stress, or entry into stationary phase [38, 41] . This suggests that sigFis regulated differently in M . bovis and M . tuberculosis, despitethe similarity of these organisms . These findings, togetherwith those regarding the difference between the effects of whiB3inactivation in M . tuberculosis and M . bovis, discussed above,suggest that caution should be used when extrapolating resultsobtained in one species when coping with phenomena as complexas global gene regulation and virulence.

usfX and sigF are transcribed from the ^F-dependent promoterusfXP1 [Table 2], located directly upstream of usfX . The activityof ^F is posttranslationally regulated by its cognate anti-sigmafactor, UsfX . The latter protein is in turn posttranslationallyregulated by two anti-anti-sigma factors, RsfA and RsfB . Bothare able to disrupt the UsfX-^F complex, releasing ^F to allowits association with RNAP . The function of RsfA is regulated by redox potential, while it is postulated that the activityof RsfB is controlled by phosphorylation [5].

An M . tuberculosis CDC1551 mutant lacking sigF was produced to investigate its role in virulence and stress response . Interestingly, the mutant strain reached stationary phase later than the wild-type [WT] parental strain and did not exhibit the typical lag phase after dilution of a dense culture into fresh medium . The mutanthad the same sensitivity as the WT parent strain to heat shock,cold shock, hypoxia, and long-term stationary-phase growth;however, it was more sensitive than the WT to rifampin . Also,when used to infect human monocytes, the mutant did not showany difference from the WT . However, it was attenuated for virulencein mice when death was used as a criterion [13].

The ^F regulon was studied by using DNA arrays in order to identifygenes that require ^F for their expression [W . R . Bishai, personalcommunication], and a consensus sequence was formulated thatclosely resembles the usfX promoter previously shown to be transcribedby ^F-RNAP [5].

^C, AN ECF FACTOR REQUIRED FOR MOUSE LETHALITY ^C was recently inactivated in M . tuberculosis [Bishai, personal communication] . The resulting strain was more susceptible tohydrogen peroxide and diamide stress but was not altered forsurvival in activated mouse macrophages . However, it was significantlyattenuated in time-to-death experiments in the mouse model.Functional genomic studies with DNA arrays showed that at least38 genes are repressed in the sigC mutant at different pointof the growth curve . Those genes encode proteins involved ina broad range of cellular processes like fatty acids biosynthesis,phospholipid and cell wall biosynthesis, energy metabolism,and general stress response . A ^C consensus sequence has beenproposed from microarray data [Table 2] [Bishai, personal communication].

^E, AN ECF FACTOR ESSENTIAL FOR VIRULENCE INVOLVED IN RESPONSE TO SURFACE STRESS The gene encoding ^E is induced after exposure to various environmentalstresses, such as heat shock and detergent-induced surface stress[41], as well as during M . tuberculosis growth in human macrophages [35] . Interestingly, Schnappinger et al . [58] recently showedby functional genomics that a set of -dependent genes are inducedin the phagosomal environment . A mutant of M . tuberculosis H37Rvlacking a functional sigE gene is more sensitive than the WTparent strain to detergent, high temperature, and oxidativestress . This mutant is attenuated for growth in THP-1-derivedmacrophages and is more sensitive than the WT strain to thekilling activity of activated murine macrophages [43] . Moreover,the sigE mutant has reduced virulence both in BALB/C and inSCID mice [R . Manganelli et al., submitted for publication]. DNA array experiments comparing the transcriptome of the sigE mutant with that of the WT strain showed that 38 genes require ^E for their full expression during exponential growth, while13 putative transcriptional units containing 23 genes required ^E for their induction after exposure to a subinhibitory concentration of SDS [43] . Nine of the 13 putative transcriptional units werepreceded by a conserved ECF factor-like promoter [Table 2],suggesting their direct transcriptional dependence on ^E . Thegenes whose expression during exponential growth require ^E includegenes encoding proteins involved in translation, transcriptionalcontrol, mycolic acid biosynthesis, electron transport, andoxidative stress response . Interestingly, one of these genesis sigB, whose transcription under unstressed conditions isalmost totally due to ^E . Since sigB is the only gene of thisgroup to be preceded by an ECF factor-like promoter, this suggeststhat at least some of the other 37 genes downregulated in thesigE mutant are in the ^B regulon . Most of them are housekeepinggenes, supporting the hypothesis that ^B and ^A have overlappingregulons . This question is currently being investigated.

Genes requiring ^E for SDS-mediated induction encode heat shockproteins, proteins involved in fatty acid degradation, transcriptionalregulators [including ^B], and surface-exposed proteins withunknown function . The presence in this group of fadE23 and fadE24is of particular interest . They were previously found to beinduced after exposure to isoniazid, and it was hypothesizedthat their protein products could be involved in the degradationof the fatty acids accumulating on the surface as a consequenceof the block of mycolic acid biosynthesis [69] . The ^E-dependent induction of these genes [together with others encoding fatty acid degradation enzymes] after exposure to a detergent supportsthe hypothesis of their role [and that of ^E] in cell wall physiologyand structure.

The gene encoding ^E is followed by an operon including threegenes . The first, Rv1222, encodes a ^E-specific anti- factor[RseA] [Rodrigue et al., unpublished] . The second, htrA, encodesa putative membrane serine protease; the third, tatB, encodesa putative protein belonging to the Tween arginine translocator[Tat] secretion system . The Tat secretion system translocatesproteins showing at the N terminus a typical signal sequencecontaining a couple of adjacent arginine residues [7] . TatBwas suggested to be responsible for the association of the proteinssecreted by the Tat system to the membrane [57].

In E . coli, the anti- factor regulating ^E is a transmembraneprotein and it is degraded by a membrane-located serine proteasein the presence of misfolded proteins in the periplasmic space[54] . In M . tuberculosis, RseA is predicted to be a solubleprotein . We recently found that it has a putative Tat consensussequence at its N terminus . The fact that rseA is in the sameoperon with tatB suggests that their protein products couldinteract and that RseA could be secreted or associated to themembrane through TatB . The presence in the same operon of thegene encoding a membrane-located serine protease [HtrA] suggeststhat HtrA, with its proteolytic activity, could represent themolecular switch acting [directly or indirectly] on RseA activity.The interactions among RseA, HtrA, and TatB are currently beinginvestigated to better understand the mechanism of posttranslationalregulation of ^E.

^H, AN ECF FACTOR INVOLVED IN RESPONSE TO HEAT SHOCK AND OXIDATIVE STRESS ^H is very similar to the ECF factor ^R of S . coelicolor . Thelatter responds to intracellular formation of disulfide bondsdue to oxidation of cysteine thiol groups [49]. ^R activity isregulated at the posttranslational level by a cysteine-containinganti- factor [RsrA] whose gene is adjacent to sigR . In a reducing environment, RsrA binds ^R, keeping it inactive; however, inoxidizing environments, disulfide bonds can form between RsrAcysteine residues and, as a consequence, ^R is released in itsactive form from the ^R-RsrA complex [48] . Among the genes recognizedby the ^R-RNAP are sigR and the trx operon, which encodes thioredoxin and thioredoxin reductase, two proteins involved in disulfidebond reduction . Usually, cells have a second pathway by whichto reduce intracellular disulfide bonds, based on glutathione.Actinomycetes are an exception, as they do not synthesize glutathionebut use a different compound, mycothiol, for similar functions[47] . A sigR mutant of S . coelicolor produces less mycothiol than the WT parental strain, even if it is not known if thisis due to the direct control of mycothiol biosynthetic genesby ^R-RNAP [50].

The M . tuberculosis sigH gene is induced after heat shock, after treatment with the thiol-specific oxidizing agent diamide [42, 55], and during macrophage infection [35] . Similar to sigR inS . coelicolor, the M . tuberculosis sigH gene is followed bya gene encoding an anti- factor whose activity is regulatedby redox potential [Rodrigue et al., unpublished] . The geneencoding ^H was inactivated in three different laboratories [40, 42, 55] . The mutants are more sensitive than the WT to hightemperature and to diamide exposure . However, they are not restrictedfor growth in THP-1-derived macrophages and were as sensitiveas the WT parental strain to the killing activity of activatedmurine macrophages [42] . Interestingly, the sigH mutant hasa very subtle phenotype in a mouse model of infection: it isable to reach the same bacterial load as the WT parent strainin mouse organs [40, 42], but there are differences in lung histopathology, including fewer granulomas and a generally decreased pulmonary inflammatory response in mice infected with the sigH mutant [40].

DNA array experiments comparing the transcriptome of the sigH mutant with that of the WT parent strain do not show any gene requiring ^H for its expression during exponential growth, while26 putative transcriptional units including 39 genes require ^H for their induction after exposure to a subinhibitory concentration of diamide [42] . Sixteen of the 26 putative transcriptional units were preceded by a conserved ECF factor-like promoter,suggesting their direct transcriptional dependence on ^H, while4 were preceded by a potential consensus sequence for an unknownregulatory protein . The genes under ^H control included someencoding transcriptional regulators [^B, ^E, and ^H]; enzymes involvedin thiol metabolism, such as thioredoxin, thioredoxin reductase,and a protein of unknown function with a glutaredoxin activesite; and enzymes involved in cysteine and molybdopterin biosynthesis[42] . Work from two other laboratories [40, 55] also derived a similar consensus sequence for genes requiring ^H for theirexpression [Table 2].

OTHER ECF FACTORS Little information is available about the other seven ECF factorsencoded by the M . tuberculosis genome . sigD is induced followingtotal nutrient starvation [8] and in the M . tuberculosis Relmutant [17] . The Rel protein has been well studied in E . coliand is known to be a key enzyme in the stringent response, atransition process believed to shut down active metabolism.sigJ is induced in stationary-phase cultures [38] . Of particular interest is sigL . Its gene product, ^L, is the closest M . tuberculosishomolog of S . coelicolor ^E This protein in S . coelicolor controlscell wall structure, and its activity is posttranslationallyregulated by a two-component system encoded by an operon immediatelydownstream of its structural gene . In M . tuberculosis, however,this gene is followed by a gene encoding a transmembrane anti- factor, which specifically binds to and reversibly inactivates ^L [Rodrigue, unpublished data], suggesting its involvement withsurface processes.

CONCLUDING REMARKS factors, with their plethora of anti- factors and anti-anti- factors, are among the major and more complex players in the regulation of gene expression in bacteria . In the last few years, after the publication of the M . tuberculosis genome, the 13 factors of M . tuberculosis have become an important subjectof investigation . Mutations in six of the factor genes wereeither made [sigB, -C, -E, -F, and -H] or identified [sigA],and a role in virulence for five of them [all except sigB] wasdemonstrated.

The regulons of four of these factors, ^C, ^E, ^F, and ^H, werecharacterized by DNA array technology, and this analysis showed that many genes were represented only in one regulon . However, there was some overlap, which is typical in ECF factor regulons.As an example of this overlap, some of the sig genes are inthe regulon of other factors: sigE induction is ^H dependentfollowing oxidative stress but not after surface stress or heatshock [42, 55]; sigB expression, however, is ^E dependent understandard [unstressed] growth conditions. ^E is also requiredfor sigB induction after surface stress [43], but sigB inductionafter oxidative stress and heat shock is dependent on ^H [42,55] [Fig . 1] . Our observations, which indicate that sigB expressionis controlled by RNAPs containing different factors, suggestan important role for ^B in M . tuberculosis physiology and perhapsvirulence . Otherwise, why would this bacterium go through somuch trouble to make sure that this protein is available tocontrol transcription in different environments? However, wehave not seen any diminution of pathogenicity in sigB mutants,as yet . It is possible that there is a subtle change in virulencethat has been missed so far, and these studies are currentlybeing pursued.

 
The whole question of posttranslational regulation by anti-and anti-anti- factors makes the matter even more complicated.The resulting picture is that of a very intricate regulatorynetwork that will become even more complex as other factorsand other transcriptional regulators are characterized with their regulons . We predict that the understanding of global gene regulation in M . tuberculosis will help us to understand its physiology and virulence mechanisms and will help to designnew strategies to fight tuberculosis.

 
Our work cited in this article was supported by grants fromthe Istituto Superiore di Sanità [Progetto NazionaleAIDS 50D.20], from the Università di Padova [Assegnidi ricerca CPDR027593], from MIUR [PRIN 2001 2001053855 andPRIN 2002 2002067349] [awarded to R.M.], from the NSERC [awardedto L.G.], and from the NIH [grants AI-44856 and HL-68513] [awardedto I.S.].

We thank W . R . Bishai for sharing unpublished data and Patricia Fontan, Ryzsard Brzezinski, and Pierre-Étienne Jacquesfor valuable discussions . The literature survey for this articlewas completed in September 2003.

 
* Corresponding author . Mailing address: TB Center, The Public Health Research Institute at the International Center for Public Health, 225 Warren St., Newark, NJ 07103-3535 . Phone: [973] 854-3260 . Fax: [973] 854-3261 . E-mail: smitty@phri.org .

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