The Mycobacterium tuberculosis extracytoplasmic-function sigma factor SigL regulates polyketide synthases and secreted or membrane proteins and is required for virulence

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.

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) β-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) β-Galactosidase activity measured in lysates from E. coli harboring the indicated pair of fusion constructs used to perform bacterial two-hybrid assays.

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.

FIG. 3.

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

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.

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::P_ace (−) and attB::P_ace-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.

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).

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 (σ^H) or SigL (σ^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.

FIG. 8.

(A) Infection of BALB/c mice with M. tuberculosis H37Rv wild-type (□), ΔsigL (•), and ΔsigL-complemented (▵) 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.