Identification of small RNAs in Mycobacterium tuberculosis

Abstract

In spite of being one of our most prominent bacterial pathogens, the presence of small regulatory RNAs (sRNAs) has not previously been investigated in Mycobacterium tuberculosis. Post-transcriptional regulation of gene expression by sRNA molecules has been demonstrated in a wide range of pathogenic bacteria and has been shown to play a significant role in the control of virulence. By screening cDNA libraries prepared from low-molecular weight RNA from M. tuberculosis we have identified nine putative sRNA molecules, including cis-encoded antisense transcripts from within open reading frames and trans-encoded transcripts from intergenic regions. sRNAs displayed differential expression between exponential and stationary phase, and during a variety of stress conditions. Two of the cis-encoded sRNAs were associated with genes encoding enzymes involved in lipid metabolism, desA1 and pks12. These sRNAs showed complementarity to multiple M. tuberculosis genes, suggesting the potential to act as both cis-encoded and trans-encoded sRNAs. Overexpression of selected trans-encoded sRNAs had profound impact on growth of M. tuberculosis and M. smegmatis. This is the first experimental evidence of sRNAs in M. tuberculosis and it will be important to consider the potential influence of sRNA regulation when studying the transcriptome and the proteome of M. tuberculosis during infection.

Fig. 1

Genomic positions of sRNA candidates. Schematic showing genome locations of the four cis- and five trans-encoded sRNA candidates as determined by sequence analysis of the cDNA library clones. sRNAs are shown as black arrows with names of sRNA candidates. The pks12 gene contains two identical copies of the region encoding ASpks. Approximate distances between cloned sequences and ORF beginnings (cis candidates) or flanking ORFs (trans candidates) are indicated.

Fig. 2

Northern blots verify the presence of M. tuberculosis sRNAs. RNA from exponential (E) and stationary phase (S) cultures was analysed by Northern blotting using riboprobes complementary to cDNA clones identified as sRNA candidates; A shows trans-encoded sRNAs; B shows cis-encoded sRNAs. Transcript sizes are approximate and compared with Ambion's Decade marker (M).

Fig. 3

Mapping of 5′ ends with RACE. RLM-RACE analysis was used to map the 5′ ends of sRNA transcripts in TAP (+) and Mock (–) treated samples. Transcription start sites (indicated by arrows) were identified on the basis of a stronger signal in TAP-treated samples. Sequence analysis of TAP-dependent bands indicated by bracketed arrows in F6 and G2 identified them as concatamers with 5′ sequences identical to original cDNA clones.

Fig. 4

Alignment of putative sRNA promoters with M. tuberculosis promoter consensus sequences. Putative promoter motifs, found immediately upstream of the identified 5′ ends of the trans-encoded sRNAs, have been aligned to promoter motifs of the most likely consensus sequence.

Fig. 5

Northern blot of M. tuberculosis ASpks sRNA after stress. The blot illustrates of the appearance of a novel ASpks transcript upon induction with 10 mM H₂O₂. Lanes: exponential (–); 2: H₂O₂ treated; 3: Mitomycin C treated (MMC); 4: pH control; 5: acid stress (pH 5.0).

Fig. 6

Overexpression of M. tuberculosis sRNAs in M. smegmatis. A illustrates the growth on solid 7H11 of M. smegmatis expressing B11, F6, G2 compared with empty vector. B shows representative slides of stained M. smegmatis expressing B11 compared with cells with empty vector. Cells were grown overnight, resuspended in PBS + 5% Tween 80 and stained with carbolfuchsin.

Fig. 7

cis-encoded sRNA with trans-acting potential. Diagram illustrating the extent of complementarity between ASdes and ASpks and their respective alternative targets, desA2 and pks7, 8 and 15.