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Journal of Bacteriology, October 2005, p. 7062-7071, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7062-7071.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor SigL Regulates Polyketide Synthases and Secreted or Membrane Proteins and Is Required for Virulence

Mi-Young Hahn,{dagger} Sahadevan Raman, Mauricio Anaya, and Robert N. Husson*

Division of Infectious Diseases, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115

Received 6 May 2005/ Accepted 2 August 2005


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ABSTRACT
 
Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the –35 region and CGTgtc in the –10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.


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INTRODUCTION
 
The Mycobacterium tuberculosis genome encodes 13 sigma factors, of which 10 fall into the extracytoplasmic function (ECF) subfamily. Three of these sigma factor genes are located 5' of genes that encode proteins containing an HXXXCXXC motif, which is found in several anti-sigma factors, including Streptomyces coelicolor RsrA (2, 15, 17, 25), Rhodobacter sphaeroides ChrR (23), and M. tuberculosis RshA (35). For both S. coelicolor SigR-RsrA and M. tuberculosis SigH-RshA, it has been demonstrated that this motif in the anti-sigma protein is a key element of a redox switch that affects the interaction between sigma and anti-sigma and thus regulates sigma factor activity in response to intracellular oxidative stress (24, 35). In the case of RsrA and ChrR, this cysteine-rich motif has been shown to bind zinc, and it has been suggested that this is a general property of this motif, leading to the term zinc-associated anti-sigma factor (ZAS) (17, 23, 24).

In addition to sigH, the other M. tuberculosis sigma factor genes linked to these putative ZAS protein genes are sigE and sigL. SigE, like SigH, has been shown to be a key regulator of the mycobacterial response to oxidative and heat stresses (21, 37). In addition, M. tuberculosis SigB, though it is not linked to a similar putative anti-sigma factor gene, is regulated by SigE and SigH and has been shown to be induced in response to these and other stresses and upon entry into stationary phase (13, 20). These data suggest that a major role of the ECF sigma factors of M. tuberculosis is to alter patterns of gene expression to allow adaptation to stress during infection of the host.

Though mycobacterial SigE and SigH and Streptomyces SigR regulate stress response genes, other sigma factors linked to ZASs, such as S. coelicolor RsuA and R. sphaeroides ChrR, do not. In S. coelicolor, an actinomycete which undergoes morphological differentiation, the SigU-RsuA pair was identified in a screening for mutants that fail to produce aerial mycelia; the stimulus sensed by RsuA is not known (11). The phototrophic proteobacterium R. sphaeroides SigE-ChrR pair plays a role in photosystem regulation, with the anti-sigma factor ChrR apparently functioning as a light sensor (5, 31).

On the basis of the role of the M. tuberculosis ZAS-associated ECF sigma factors SigH and SigE in regulating stress response genes, we hypothesized that the third putative member of this group, SigL, and its adjacent gene, Rv0736, might function similarly. We therefore undertook to determine whether SigL interacts with the intracellular domain of Rv0736. We investigated the susceptibility of a sigL mutant to a variety of stresses and identified genes regulated by this sigma factor. In contrast to SigE and SigH results, our findings suggest that SigL does not play a major role in responding to oxidative stress. On the basis of the data presented in this work, this ECF sigma factor appears to regulate the expression of genes involved in polyketide-lipid synthesis and genes encoding membrane-associated proteins that are involved in posttranslational protein modification. We also demonstrated that a sigL mutant of M. tuberculosis is severely attenuated in a mouse model of infection.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. M. tuberculosis H37Rv was used as the parental strain for generating {Delta}sigL::hyg/cm, complemented ({Delta}sigL::hyg/cm, attB::sigL), and sigL-overexpressing (attB::Pace-sigL) strains and as the wild type for all experiments. M. tuberculosis was grown in Middlebrook 7H9 broth supplemented with albumin dextrose complex plus 0.05% Tween 80 or 0.2% glycerol. Escherichia coli DH5{alpha} (Life Technologies) and XL1-Blue (Stratagene) were used as host strains for cloning experiments. E. coli was grown on LB agar or in LB broth. Ampicillin (50 µg/ml), chloramphenicol (25 µg/ml), hygromycin (Hyg) (50 to 100 µg/ml), kanamycin (Kan) (20 to 50 µg/ml), or tetracycline (25 µg/ml) was added to culture medium when required.

Analysis of Rv0736 (RslA) topology and interaction of SigL with RslA. The topology of RslA was analyzed by constructing a series of translational fusions between rslA and phoA or lacZ by use of the mycobacterial phoA and lacZ fusion vectors as previously described (1, 6). Fusions included the amino-terminal region, the amino-terminal region plus the predicted transmembrane region, and the entire protein. Interaction between SigL and the intracellular domain of RslA was assessed using a bacterial two-hybrid system as previously described except that ß-galactosidase activity assays were performed in a microtiter plate format (10, 35).

Construction of sigL mutant, complemented, and overexpressing strains. The M. tuberculosis {Delta}sigL::hyg/cm mutant was constructed by targeted mutagenesis as described previously except that the targeting construct was introduced into the temperature-sensitive shuttle phasmid phAE87 by use of the {lambda} Red recombinase system instead of by in vitro packaging (3, 9). Briefly, two 900-bp DNA fragments flanking sigL were amplified by PCR from M. tuberculosis genomic DNA and cloned into pYUB572, and a hygromycin-chloramphenicol resistance cassette was inserted between the two PCR fragments. The resulting plasmid was sequenced and used as template DNA to generate the linear DNA substrate for recombination by PCR using primers 572F (5'-GAATTAATTAATCTGCTGAAGCCAGTTACCTTCG-3') and 572R (5'-GAATTAATTAAGATCCTTTAGTGAGGGTTAATTG-3'). After DpnI digestion to remove template DNA, the targeting sequence was electroporated into E. coli DH5{alpha} cells, which carried both phAE87::572 and a temperature-sensitive RED helper plasmid carrying the {lambda} Red recombinase genes ({gamma} ß exo) under control of the arabinose-inducible promoter ParaB.

Recombinants were selected on chloramphenicol plates, and phasmid DNA was isolated. This sigL-disrupting construct in phAE87 was electroporated into M. smegmatis mc2155, and cells were plated for plaques at 30°C. Transducing phage isolation and transfection of M. tuberculosis were performed as described previously (3). Three Hygr colonies were obtained, and disruption of the sigL allele was identified in each by PCR analysis. The mutant used for subsequent experiments was confirmed by Southern blotting. To complement the {Delta}sigL::hyg/cm mutant, a 964-bp DNA fragment including the coding sequence of the sigL gene and 360 bp of 5' sequence (including its native promoters) was amplified by PCR with the primers 5'-ATGCTCTAGACCGTTATGGACGCTCGTTCG-3' and 5'-TGGAAGATCTCGTCATGTCATCTCTCTCCTGTC-3', cloned into pMV306, and sequenced. The M. tuberculosis {Delta}sigL::hyg/cm mutant was transformed with this construct. Hygr Kanr colonies were selected, and the presence of the intact sigL allele was identified by PCR and confirmed by Southern blotting.

For the sigL-overexpressing strain, the 600-bp sigL coding sequence was amplified by PCR with primers 5'-ATATATCATATGGCTCGTGTGTCGGGCGC-3' and 5'-TGGAAGATCTCGTCATGTCATCTCTCTCCTGTC-3' and cloned into pCKACEI, which contains the acetamide-inducible promoter Pace in pCRII (19). The insert was sequenced, and the 3.6-kb fragment containing sigL under the control of Pace was cloned into pMV306. This sigL-overexpressing construct was electroporated into M. tuberculosis H37Rv. The presence of the extra copy of the sigL gene at the attB site was verified by PCR analysis. Pace without sigL in pMV306 was also electroporated into M. tuberculosis, and the resulting strain was used as a control.

A sigL deletion mutant and a complemented strain of M. smegmatis were constructed in a similar manner, except that the temperature-sensitive sacB counterselection method was used as previously described to create the sigL deletion in this species (27).

Oxidative stress and nitrosative stress phenotypes. Susceptibility of the {Delta}sigL::hyg/cm mutant to diamide (1 M), cumene hydroperoxide (20 mM), H2O2 (100 mM), plumbagin (10 mM), GSNO (S-nitrosoglutathione) (100 mM), SNP (sodium nitroprusside dihydrate) (100 mM), DETA/NO (dimethylenetriamine nitric oxide adduct) (10 mM), and sodium dodecyl sulfate (1%) was determined using a disk diffusion assay as previously described (29).

RNA isolation and primer extension analysis. Total RNA was isolated from M. tuberculosis strains grown to mid-log phase (optical density value at 600 nm of 0.5) as previously described (28). For primer extension analysis, 0.5 pmol of {Upsilon}-32P-labeled primer (for sigA, 5'-CGACTTGGTGGCGGTGCGTTTTACC-3'; for sigL, 5'-TGACACGAGTCGAGTAAGAGTTTGG-3'; for mpt53, 5'-GTATTGGCCAGACCGAACATCAG-3'; for pks10, 5'-ATGTCTTCGTAGCCCTCGAAATCC-3'; for Rv1139c, 5'-GTAGTACACGGCCCTACCACCTAAGAA-3') was mixed with 5 µg of RNA in a 6.5-µl volume and reverse transcription (RT) was performed with Superscript II (Invitrogen) at 42°C for 1 h. Reaction products were electrophoresed on 6% polyacrylamide sequencing gels. Sequencing reactions with the same primers used for primer extension were performed in adjacent lanes to determine the size of the transcripts.

Microarray experiments and analysis. M. tuberculosis oligonucleotide arrays were obtained from The Institute for Genomic Research (TIGR) through the National Institute of Allergy and Infectious Diseases-sponsored Pathogen Functional Genomics Resource Center. This microarray contains 4,750 70-mer oligonucleotides representing 4,127 open reading frames from Mycobacterium tuberculosis strain H37Rv and 623 unique open reading frames from strain CDC1551. RNA was prepared as described above, and cDNA was synthesized by reverse transcription with Superscript II (Invitrogen) with amino-allyl dUTP (Sigma) incorporation, followed by coupling to Cy-3 or Cy-5 fluorescent dyes (Amersham). TIGR protocols were followed for probe synthesis, hybridization, and washing (36). The hybridized slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA), and spot intensities were defined and quantified using GenePix Pro software (Axon Instruments, Union City, CA).

Ten hybridization intensity values from microarray experiments (five replicates from five independent RNA preparations) were used for statistical analysis, which was performed using GeneSpring (Silicon Genetics, Redwood City, CA). The raw data median value for Cy3 and Cy5 data was normalized, and the induction ratio was calculated as the average fold change for the 10 data points.

Real-time PCR and mRNA quantification. Two-step quantitative PCR was carried out to analyze specific gene expression. RNA from M. tuberculosis H37Rv cells was prepared as described above except for an additional DNase I treatment with Turbo DNA-free (Ambion). Primers were designed using Primer 1 software (ABI), and reverse transcription and PCR were performed using iScript and iTaq SYBR green Supermix with ROX (Bio-Rad) and an ABI7000 sequence detection instrument. Duplicate reactions with 100 ng of template were performed for sigL by use of primers sigL F (5'-CGAGCATAGGGCCGTGATC-3') and sigL R (5'-ATCGCGACTTCACCGTTCCT-3'). Expression of sigA was quantified in each sample to allow comparison to the relatively stable expression of this gene. An RNA sample that had not been reverse transcribed was included in all experiments to exclude significant DNA contamination. Standard curves were obtained by performing PCR with SYBR green detection on serial dilutions of spectrophotometrically quantified genomic DNA.

Computer database searching. Searches of the M. tuberculosis H37Rv genome sequence for consensus promoter elements were performed utilizing the "search pattern" program available on the TubercuList web site of the Pasteur Institute (http://genolist.pasteur.fr/TubercuList) and utilizing the "motif finding" function in BioProspector program (http://ai.stanford.edu/~xsliu/BioProspector/) (18).

Purification of recombinant His-tagged SigL and in vitro transcription assays. The sigL coding sequence was amplified by PCR using two primers (5'-ATATATCATATGGCTCGTGTGTCGGGCGC-3' and 5'-GGCTGGATCCTCATCGAGTAACTCCCAG-3') and cloned into pET28a. The resulting construct was sequenced and used to transform E. coli BL21(DE3) pLysS (Novagen). Expression of sigL was induced at 15°C overnight, cells were harvested and disrupted by sonication, and His-tagged SigL protein (6X-his-SigL) was purified on a Ni-nitrilotriacetic acid column (Novagen) according to the manufacturer's protocol.

In vitro runoff transcription assays were performed as previously described (29). PCR products containing the sigL, pks10, mpt53, or Rv1139c promoter (10 nM in each reaction) were used as DNA templates. Transcripts were analyzed by autoradiography after separation on a 6% polyacrylamide gel containing 7 M urea.

Mouse infection and virulence assays. Six-week-old BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and infected with strain H37Rv, the sigL mutant strain, or the complemented strain. Infection was performed by lateral tail vein injection of 106 declumped bacteria, as previously described (28). Mice were housed in microisolator cages in a BL3 animal facility under specific-pathogen-free conditions. Twelve mice in each group were infected for survival analysis. Weights and morbidity of the mice were monitored for the duration of the survival experiment. Mice that became moribund were euthanized, with survival counted to the day of euthanasia. For CFU analysis, six mice were sacrificed at days 2, 8, 22, and 59, and one lung and the spleen were removed for CFU determination. The other lung was fixed in formalin, sectioned, and stained with hematoxylin and eosin. All work with mice was performed under approved protocols in compliance with federal guidelines and institutional policies.

With the exception of the mouse infection experiments, which were performed once each, all experiments were performed at least twice; representative data are shown.


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RESULTS
 
Topology of Rv0736 (RslA) and interaction with SigL. Rv0736, the gene immediately 3' of sigL, is a membrane protein that is coexpressed with sigL (see below). The predicted protein contains the HXXXCXXC motif characteristic of ZASs in the region amino terminal to the single transmembrane domain (Fig. 1A). To determine the topology of this protein in vivo, a series of phoA and lacZ fusions were made. Expression of these fusions in M. smegmatis and measurement of enzyme activity indicated that the amino-terminal region of this protein is intracellular and the carboxy-terminal region is extracellular (Fig. 1B).



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  		FIG. 1. Topology of Rv0736 (RslA) and its interaction with SigL. (A) Predicted domain structure of RslA and locations of junctions of translational fusions to lacZ or phoA. (B) ß-Galactosidase or alkaline phosphatase activity in lysates of M. smegmatis transformed with the rslA-lacZ or rslA-phoA fusion constructs shown in panel A. (C) ß-Galactosidase activity measured in lysates from E. coli harboring the indicated pair of fusion constructs used to perform bacterial two-hybrid assays.

On the basis of these data, we analyzed the interaction of SigL and the intracellular domain of RslA in a bacterial two-hybrid system (10). In this system, potential binding partners are fused to {lambda}CI or the amino-terminal domain of the alpha subunit of RNA polymerase. A protein-protein interaction causes activation of lacZ expression from a promoter located near a lambda operator, resulting in increased ß-galactosidase activity (10). As shown in Fig. 1C, these assays indicate that SigL and RslA interact strongly and specifically, consistent with the hypothesis that Rv0736 functions as an anti-sigma factor (designated RslA) that physically interacts with SigL.

Construction and stress phenotypes of M. smegmatis and M. tuberculosis sigL mutant and complemented strains. To study the function of SigL, we constructed sigL mutant strains based on M. smegmatis mc2-155 (34) and M. tuberculosis H37Rv. In both strains, the coding sequence of the sigL gene was replaced by a hygromycin and chloramphenicol resistance cassette. The absence of the sigL allele in these mutants was identified by PCR, and the deletion in the M. tuberculosis strain used in subsequent experiments was confirmed by Southern blotting (Fig. 2). A complemented strain of the {Delta}sigL::hyg/cm mutant used in subsequent experiments was constructed by integrating the sigL gene under the control of its native promoters at the phage L5 attB site (16).



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  		FIG. 2. (A) The M. tuberculosis sigL locus and strategy for deletion mutant construction. (B) Southern blot analysis of M. tuberculosis H37Rv and one of three independent sigL deletion mutants that were identified by PCR. Genomic DNA of each strain was digested with XmnI and ApaLI, electrophoresed in a 1% agarose gel, transferred to a nylon membrane, and probed as shown. Expected sizes for the wild-type (wt) and sigL mutant strains are 1.4 kb and 2.8 kb, respectively. In the complemented strain, the sigL coding sequence is linked to vector sequences resulting in a restriction fragment of 10.3 kb.

The growth curves of the sigL mutant strains of M. smegmatis and M. tuberculosis in broth culture were similar to those of the wild-type strains (not shown), and no clear differences from wild type in colony morphology were observed when the mutant strains were grown on solid medium. To examine stress phenotypes of the sigL mutant strains, we compared the susceptibility of the {Delta}sigL::hyg/cm strains to wild-type results by use of a disk diffusion assay (29). When exposed to oxidants (including H2O2, cumene hydroperoxide, diamide, and plumbagin), to nitric oxide-generating agents (including SNP, GSNO, and DETA/NO), and to the detergent sodium dodecyl sulfate, no consistent differences were observed between the wild-type and mutant strains (data not shown). Several of the same compounds were tested in a time-kill assay in liquid medium, again without showing differences between the wild-type and mutant strains.

M. tuberculosis sigL promoter identification and expression of the sigL gene during in vitro growth. Many ECF sigma factor genes, including M. tuberculosis sigH, are expressed from auto-regulated promoters. To examine the sigL promoter sequence, primer extension analysis of the sigL gene was carried out (Fig. 3). The signal from the sigL primer was extremely weak relative to the signal obtained with a sigA primer used as a control, and the sigL signal was only detectable after prolonged exposure. This sigL transcription start site, designated P1, was mapped 121 bp 5' of the annotated sigL initiation codon (8) and is located within the open reading frame of the mapA gene, whose termination codon is 72 bp 5' of the sigL gene.



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  		FIG. 3. Transcription of the sigL gene in the M. tuberculosis H37Rv wild-type (WT) strain and the {Delta}sigL::hyg/cm mutant. Primer extension analysis was performed using RNA isolated from M. tuberculosis H37Rv wild-type and {Delta}sigL::hyg/cm mutant strains with sigA and sigL primers. The transcription start site of the sigL gene is indicated by an arrow.

We also performed primer extension analysis with RNA prepared from the M. tuberculosis sigL mutant strain by use of a primer corresponding to the upstream region of the sigL gene. A band corresponding to the sigL P1 transcription start site was still present, indicating transcription initiation from this site in the sigL mutant strain and thus that this promoter is not completely autoregulated. The signal from this transcript is decreased in the sigL mutant, however, when normalized to the sigA transcript. We examined the region 5' of sigL P1 but did not identify –10 or –35 promoter elements similar to consensus sequences recognized by sigL (see below) or by other M. tuberculosis sigma factors. These data suggest that decreased sigL expression from this promoter in the sigL mutant results from indirect effects rather than direct recognition of this promoter by SigL.

We performed quantitative RT-PCR to examine the quantity and pattern of sigL gene expression during growth in vitro. M. tuberculosis cells were harvested at serial time points, and RNA was prepared and used for RT-PCR. An estimate of sigL mRNA copy numbers was calculated from a standard curve obtained from quantitative RT-PCR performed with a dilution series of known amounts of genomic DNA as a template. We found that the quantity of sigL mRNA is approximately 100-fold lower than that of sigA mRNA, the primary sigma factor in M. tuberculosis, throughout the course in vitro growth (Fig. 4). These results indicate that there is constitutive expression of the sigL gene from the very weak SigL-independent promoter P1 during in vitro growth.



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  		FIG. 4. Expression pattern of the M. tuberculosis sigL gene during growth. RNA was isolated from M. tuberculosis H37Rv at serial time points, and quantitative RT-PCR was performed using 100 ng of RNA as described in Materials and Methods. A standard curve for copy number of each gene was obtained by performing quantitative PCR using serial dilutions of genomic DNA. OD600, optical density at 600 nm.

Construction of a sigL overexpression M. tuberculosis strain and microarray analysis of SigL-regulated gene expression. To attempt to identify genes regulated by SigL, we performed global gene expression analysis in oligonucleotide microarray experiments. Initial experiments compared the {Delta}sigL::hyg/cm strain to the parental M. tuberculosis H37Rv strain. With one exception, the sigL mutant did not show a gene expression pattern significantly different from that seen with M. tuberculosis H37Rv. Signals from spots on the array corresponding to sigL were very weak, indicating that the expression level of the sigL gene is extremely low, consistent with the primer extension and RT-PCR results described above. The one clear difference observed between the wild type and the sigL mutant in these experiments was that the expression of rslA (Rv0736) was significantly increased in the sigL mutant. This increased transcription of rslA is likely the result of read-through from the promoters of the hygromycin and chloramphenicol resistance cassette, which replaced the sigL gene immediately 5' of rslA. This elevated rslA expression was not associated with other significant changes in gene expression in the sigL mutant, suggesting that it does not have a major role as an anti-sigma factor for other M. tuberculosis sigma factors.

Because of the low basal levels of sigL expression and the absence of consistent differences in the experiments comparing the sigL mutant to the wild type, to identify genes transcribed by SigL we constructed a sigL-overexpressing strain. The coding sequence of the sigL gene was cloned under the control of the acetamide-inducible promoter (Pace) (26), in the integrating vector pMV306, to create a strain with a single extra copy of sigL (M. tuberculosis H37Rv attB::Pace-sigL). The acetamide promoter region without any gene under its control was also cloned into pMV306 and used to make a control strain (M. tuberculosis H37Rv attB::Pace). The presence of the sigL allele at the attB site was confirmed by PCR using primers corresponding to the attB and acetamide promoter region.

The sigL-overexpressing and control strains were grown to an optical density at 600 nm of 0.5, RNA was prepared after 1 h of induction, and analysis of gene expression was performed using oligonucleotide microarrays. We performed the array experiments five times with five independent RNA preparations, and the results were analyzed statistically. The genes with significantly increased (P < 0.05; ≥threefold induction) expression in the sigL-overexpressing strain are listed in Table 1. The induced expression of several genes in the overexpression strain was confirmed by quantitative RT-PCR analysis (data not shown). We also tested a different set of induction conditions (24 h of induction with the same concentration of acetamide) and obtained a similar list of regulated genes. When subjected to the same stringent analysis, we did not identify genes that were significantly down-regulated in the sigL-overexpressing strain.


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  		TABLE 1. Genes highly expressed in the sigL over-expressing strain relative to H37Rv

The SigL regulon identified in these experiments includes four two-gene operons and a relatively small number of other genes. First, sigL itself and rslA (Rv0736) were both significantly up-regulated in the sigL-overexpression strain. The increased expression of rslA, which is not included in the overexpression construct, suggested that SigL regulates the sigL-rslA operon. A second putative SigL-dependent operon includes mpt53 and a gene of unknown function, Rv2877c. Mpt53 was originally identified as a major secreted protein of M. tuberculosis that is weakly immunogenic in natural M. tuberculosis infection (14, 22). Rv1139c and Rv1138c, both genes of unknown function, also form a SigL-regulated operon, with the start codon of Rv1138c overlapping the stop codon of Rv1139c. Rv1139c is predicted to have two to three membrane-spanning regions and to encode an isoprenyl cysteine carboxyl methyltransferase motif (7).

The pks10 and pks7 genes are also physically linked, and RT-PCR analysis confirmed the presence of a single transcript that includes pks10 and pks7 (not shown). Though both were up-regulated in the sigL-overexpression strain, no SigL consensus sequence (see below) is present 5' of pks7. The extent of up-regulation of pks7, however, was less than that of pks10. This result, and the presence of an 83-bp noncoding sequence between these genes, suggested that, in addition to being expressed as part of an operon from the pks10 promoter, pks7 might also be transcribed from a second SigL-independent promoter 5' of the pks7 gene. Primer extension experiments did not identify a transcription start site 5' of pks7 (data not shown), however, suggesting that this gene is expressed only as part of a transcript with pks10. PKS10 is characterized as a type III polyketide synthase and is similar to members of the plant chalcone synthase superfamily (30). PKS7 appears to be a multifunctional enzyme containing both beta-ketoacyl synthase active site and phosphopantetheine attachment site motifs.

In addition to these operon results, a notable finding of the microarray experiments was the up-regulation of four PE-PGRS genes in the sigL-overexpression strain. These genes encode proteins that are members of a large family of polymorphic proteins of unknown function.

Promoter analysis of SigL-dependent genes. To map the transcriptional start site of the SigL-dependent genes identified in the microarray experiments, we performed primer extension analysis with RNA prepared from the sigL-overexpressing strain, starting with sigL itself (Fig. 5A). In addition to the sigL P1 promoter identified in wild-type H37Rv, in the sigL-overexpressing strain we detected an additional sigL transcription start site, designated sigL P2. We next analyzed the in vivo transcription start sites of pks10, mpt53, and Rv1139c. These experiments demonstrated the presence of transcription start sites for each of these genes in the sigL-overexpressing strain that were not present in H37Rv. The aligned upstream sequences of the transcription start sites of these SigL-dependent genes showed two highly conserved consensus sequence elements, TGAACC in the –35 region and CGT in the –10 region, with 16-bp spacing between the 3' end of the –35 element and the 5' end of the –10 element (Fig. 5B).



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  		FIG. 5. Transcription start sites of SigL-dependent genes and consensus promoter sequence. (A) Primer extension analysis was performed for SigL-dependent genes using RNA isolated from M. tuberculosis attB::Pace (–) and attB::Pace-sigL (+). Transcription start sites are indicated by arrows. (B) Alignment of promoter sequences from SigL-dependent genes. The transcription start sites and consensus promoter sequences in the –10 and –35 regions are indicated in boldface, and the annotated initiation codons are underlined. The SigH promoter consensus sequence is also shown.

Exact matches were not found 5' of any of the other genes that were upregulated in the sigL-overexpression strain, although partial matches were observed. Primer extension experiments with several of these genes with the closest match to the SigL promoter consensus did not identify SigL-dependent transcription start sites that corresponded to these candidate promoters. These data suggest that the up-regulation of these genes in the sigL-overexpression strain is indirect and is not the result of recognition of these promoters by RNA polymerase containing SigL.

In vitro transcription from SigL-dependent promoters. To determine whether the in vivo SigL-dependent promoters were directly recognized by SigL, we affinity purified six-His-tagged M. tuberculosis SigL protein and performed in vitro transcription assays. Nonspecific transcripts were detected in assays performed with core RNA polymerase with or without SigL protein. Specific transcripts of the expected size were seen in assays performed with holoenzyme reconstituted with SigL for templates containing the promoter sequences of sigL, pks10, and mpt53 (Fig. 6). Surprisingly, we did not observe a specific transcript from Rv1139c, even though it has a well-conserved SigL-dependent promoter sequence, as shown in Fig. 5B. This assay was performed with several different Rv1139c templates, which extended 5' and 3' relative to the identified promoter, but in none of these experiments was a specific transcript observed. Of note, the in vivo transcription level of Rv1139c was very low even in the sigL-overexpressing strain compared to the results seen with the other SigL-dependent genes, pks10 and mpt53 (Fig. 5A). These data suggest that an additional factor, such as a transcriptional activator, may be required for initiation of transcription from the Rv1139c promoter, despite its containing the exact consensus –35 and –10 elements.



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  		FIG. 6. In vitro transcription assay with purified SigL protein. His6-tagged SigL protein was overproduced in E. coli and purified through a Ni-nitrilotriacetic acid column. Template DNAs including putative promoter sequences were amplified by PCR. In vitro transcription assays were performed as described in Materials and Methods. Specific transcripts are indicated by arrows. Lane M, RNA size markers (bases).

The SigL-dependent consensus promoter sequence that we identified in these experiments is highly similar to the SigH-dependent consensus promoter sequence that was previously identified in our laboratory (29). To determine whether SigL might also recognize SigH-regulated genes, we performed in vitro transcription assays with templates incorporating the SigH-dependent promoters of Rv2466c and sigB (Fig. 7A). RNA polymerase containing SigL initiated transcription from the sigB promoter, which differs from the SigL-dependent consensus at two positions in the –35 element, though the signal was substantially less intense than that produced from this promoter by RNA polymerase reconstituted with SigH. In contrast, no specific SigL-dependent transcript was observed from the Rv2466c promoter, which differs at two positions in the –35 element and one position in the –10 element from the SigL recognition consensus.



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  		FIG. 7. Transcription analysis of the Rv2466c and sigB promoters. (A) In vitro transcription analysis of these promoters was performed using core RNA polymerase (–) or RNA polymerase holoenzyme reconstituted with purified SigH ({sigma}H) or SigL ({sigma}L). Specific transcripts are indicated by arrows. (B) Primer extension analysis of the sigB and sigA promoters was performed using RNA from the sigL-overexpression strain (attB::Pace-sigL) and wild-type (WT) strain H37Rv.

To determine whether sigB might be regulated by SigL in vivo, primer extension analysis was performed using RNA from the sigL-overexpression strain and the control strain (Fig. 7B). This experiment demonstrated approximately 1.5- to 1.9-fold-greater transcription of sigB in the sigL-overexpression strain versus H37Rv after normalization to transcripts from the control sigA promoter. This result indicates that one or a few key residues within the –35 or –10 elements, or elsewhere in the promoter region, allow discrimination between these promoters by SigL and SigH.

Virulence analysis of the M. tuberculosis sigL mutant. To begin to assess the role of SigL in M. tuberculosis virulence, a mouse model of infection was used. BALB/c mice infected intravenously with the sigL mutant strain showed a significantly prolonged survival time relative to those infected with the parental H37Rv wild-type strain, with a median survival time of >145 days versus 99 days for H37Rv (P < 0.0001). The complemented strain showed virulence similar to that of the wild type in this experiment (median survival time = 86 days) (P < 0.0001 versus {Delta}sigL; P = 0.6 versus H37Rv) (Fig. 8A).



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  		FIG. 8. (A) Infection of BALB/c mice with M. tuberculosis H37Rv wild-type ({square}), {Delta}sigL (•), and {Delta}sigL-complemented ({triangleup}) strains. Mice were infected by lateral tail vein injection of 106 CFU. (A) Survival analysis. Morbidity and weight were monitored, and mice that became moribund were sacrificed. Twelve mice were infected in each group. (B and C) Replication and persistence of M. tuberculosis strains in mouse organs. Six mice were sacrificed at days 2, 8, 22, and 59, with plating for bacterial burden in lung (B) and spleen (C) performed as described in Materials and Methods.

We also investigated the ability of these three M. tuberculosis strains to proliferate and persist in the tissues of BALB/c mice after intravenous infection. The bacterial burden in lung and spleen was assessed by plating the dilutions of the organ homogenates at days 2, 8, 22, and 59. The growth kinetics of the three strains in the lung and spleen were very similar, as determined by plating at these time points following infection (Fig. 8B and C). Hematoxylin- and eosin-stained lung tissues from infected BALB/c mice were compared for extent of tissue pathology. Though subtle differences cannot be excluded, major variations in the amount of inflammation in the lungs of mice infected with the wild-type strain versus the {Delta}sigL strain were not observed (data not shown).


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DISCUSSION
 
Several alternative sigma factors of M. tuberculosis regulate gene expression in response to stress, including SigH and SigE, which have been shown (SigH) or are hypothesized (SigE) to be regulated by ZASs. Though sigL is also linked to a likely ZAS gene (rslA), we did not identify oxidative or nitrosative stress conditions that either induced expression of this sigma factor or resulted in decreased survival of a sigL mutant relative to wild-type survival. These results suggest that SigL is not a stress response regulator and that other conditions encountered by the bacillus during infection in vivo may activate sigL expression.

By performing microarray analysis followed by primer extension and in vitro transcription experiments, we identified a limited set of genes that are directly regulated by SigL. Despite the small number of regulated promoters, we identified clear –35 and –10 element consensus sequences (TGAACC-N16-CGT) in the promoters recognized by SigL. Though this is similar to the SigH recognition consensus (GGAACA-N16-GTT) that we previously defined (29), the inability of E{sigma}L to initiate transcription from the Rv2466c promoter, which has the exact SigH consensus, indicates that these differences are sufficient to distinguish the promoters recognized by these two ECF sigma factors.

It has been proposed that RNA polymerase containing SigL might transcribe the sigB promoter (33). We previously demonstrated this promoter to be SigE and SigH dependent, with no evidence of transcription initiation at this site in vivo in an sigE/sigH double mutant (29). In this work a SigL-dependent transcript was initiated from the sigB promoter in vitro, and a small increase in sigB transcription in the sigL-overexpression strain was observed in primer extension experiments. Expression of sigB was not significantly increased in the sigL-overexpression strain in the microarray experiments, however. Our interpretation of these data is that RNA polymerase incorporating SigL does not initiate transcription at the sigB promoter under non-sigL-inducing conditions. Based on our previous data and the lack of sigL induction in response to oxidative and nitrosative stresses observed in this work, SigL does not appear to play a role in sigB expression in response to these stresses. It remains possible, however, that under conditions in which sigL expression is highly induced, SigL-dependent upregulation of sigB may occur.

The SigL regulon we identified includes sigL-rslA and three other apparent operons: pks10-pks7, mpt53-Rv2877c, and Rv1139c-Rv1138c. While pks10 and pks7 are annotated as polyketide synthase genes, the functions of the PKS10 and PKS7 enzymes are not known. Available data, however, shed some light on the possible function of PKS10. PKS11 and PKS18, both chalcone synthase type III polyketide synthases of M. tuberculosis that are closely related to PKS10, have been shown to synthesize tri- and tetraketide {alpha}-pyrones with a preference for long-chain aliphatic-coenzyme A starter units (30). The sequence and predicted structural similarity of PKS10 to these proteins suggests that it is likely to have a similar enzymatic activity.

In a previous study, a pks10 mutant was found to be defective in synthesis of phthiocerol dimycocerosate, a major surface lipid of pathogenic mycobacteria (32). This pks10 mutant was also significantly attenuated for growth in mice following intranasal inoculation. Our data following intravenous infection showed a marked attenuation of the sigL mutant in terms of mouse survival but little effect on bacterial replication in vivo. While pks10 and/or pks7 likely contributes to this striking virulence phenotype, the role of these and other SigL-regulated genes in M. tuberculosis pathogenesis remains to be determined.

The functions of the proteins encoded by Rv1139c and Rv1138c are also unknown. In contrast to M. tuberculosis results, in M. avium and M. smegmatis, as well as in S. coelicolor, the Rv1139c-1138c operon is physically linked to pks10, suggesting that they function in a pathway with pks10. Rv1138c encodes a putative oxidoreductase. Rv1139c is a membrane protein that contains an isoprenylcysteine carboxylmethyltransferase motif. Enzymes with this motif in eukaryotes act on cysteines near the carboxy terminus of proteins and perform sequential isoprenylation and proteolytic cleavage after cysteine, followed by methylation of the carboxyl group of the now carboxy-terminal cysteine. Though several proteins that contain this motif are present in bacterial genome sequences, their function has not been characterized.

The other SigL-regulated gene pair, mpt53-Rv2877c, appears to encode a secreted protein disulfide oxidoreductase and a putative thiol-disulfide transporter with a conserved CcdA domain, respectively. Though a role for Mpt53 and Rv2877c in cytochrome c biogenesis has been postulated, other proteins corresponding to the gram-positive system II for cytochrome c biogenesis are encoded elsewhere [Rv0526 and ccdA (Rv0527)] in the M. tuberculosis genome (4, 8). Mpt53, annotated as a DsbE-like protein, has been characterized biochemically and structurally (12). It contains a thioredoxin active site and is a strong oxidant, in contrast to the weak reducing activity of E. coli DsbE. These biochemical data suggest a possible role for Mpt53 as an extracellular oxidant that may be required for proper folding of reduced unfolded secreted proteins, a function more similar to that of E. coli DsbA (12). Rv2877c has seven transmembrane regions and conserves two cysteines which are essential for the redox activity of CcdA, suggesting that, while it may not function as a CcdA orthologue, it is likely to be a redox-active membrane protein.

These data suggest that SigL regulates cell envelope lipid synthesis and envelope or secreted protein modification. Though we found that sigL is expressed at low levels during vegetative growth in vitro, the presence of an autoregulated sigL promoter suggests that significantly increased expression may occur under inducing conditions. Our data showing that SigL and RslA interact and the similarity of RslA to other ZASs suggest that RslA may function as a negative regulator of SigL. Taken together, these findings suggest a model in which RslA may sense as-yet-undefined extracellular signals that decrease its binding of SigL. The resulting increased activity of the SigL-regulated genes would then alter the surface characteristics of the mycobacterium. The marked attenuation of the sigL mutant suggests that such changes could be important adaptations that allow M. tuberculosis to survive and cause disease in the host during infection.


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grant RO1AI37901 to R.N.H.

We thank the National Institute of Allergy and Infectious Diseases Pathogen Functional Genomics Resource at TIGR for providing the oligonucleotide microarrays used in the work. We thank Kevin Conlon for assistance with the Southern blotting, Simon Dove for critical review of the manuscript, Chris Sassetti and Eric Rubin for providing phAE87::572 and pKD119, and Adrie Steyn for the phoA (pMB111) and lacZ (pVlacZ2) fusion vectors.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 919-2883. Fax: (617) 730-0254. E-mail: robert.husson{at}childrens.harvard.edu. Back

{dagger} Present address: Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea. Back


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Journal of Bacteriology, October 2005, p. 7062-7071, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7062-7071.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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