Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis

Abstract

Tuberculosis (TB) is a major global health problem, infecting millions of people each year. The causative agent of TB, Mycobacterium tuberculosis, is one of the world's most ancient and successful pathogens. However, until recently, no work on small regulatory RNAs had been performed in this organism. Regulatory RNAs are found in all three domains of life, and have already been shown to regulate virulence in well-known pathogens, such as Staphylococcus aureus and Vibrio cholera. Here we report the discovery of 34 novel small RNAs (sRNAs) in the TB-complex M. bovis BCG, using a combination of experimental and computational approaches. Putative homologues of many of these sRNAs were also identified in M. tuberculosis and/or M. smegmatis. Those sRNAs that are also expressed in the non-pathogenic M. smegmatis could be functioning to regulate conserved cellular functions. In contrast, those sRNAs identified specifically in M. tuberculosis could be functioning in mediation of virulence, thus rendering them potential targets for novel antimycobacterials. Various features and regulatory aspects of some of these sRNAs are discussed.

Figure 1.

Strategy for sRNA discovery. This schematic shows the combination of cloning and computational approaches used to identify sRNAs in BCG, Mtb and M. smegmatis.

Figure 2.

sRNAs identified in BCG by cloning. (A) Properties of Mcr1-Mcr19. The approximate sRNA size observed by northern blot (predominant band), orientation relative to its flanking genes, corresponding Mtb H37Rv genes, co-transcription information and the type of sRNA are given. All of the gene names and numbers in the arrows are BCG designations. The black arrows represent the sRNA. ↑ = upstream ORF; ↓ = downstream ORF; Ind = independent transcript; Co = co-transcribed; n/d = not determined; intergenic = intergenic/trans-acting sRNA; 5′UTR = 5′ untranslated region; 3′UTR = 3′ untranslated region; 5′UTR AS = antisense to potential 5′ UTR; 3′UTR AS = antisense to potential 3′UTR; cds = overlapping coding sequence; RS = riboswitch. *See ref. (6). (B) Representative northern blots of cloned sRNAs. In each case the marker lane is on the left, labeled in nucleotides, while the sRNA lane is on the right. For Mcr5, s is the sRNA, while t is a tRNA control. All northerns shown were performed with 7-day shaking BCG RNA run on a 10% denaturing PAGE, except for Mcr4 and Mcr8, which were run on a 6% denaturing PAGE. Probes are given in Supplementary Table S3. (C) Representative co-transcription. The seven co-transcribed RNAs are shown, with the chromosomal DNA positive PCR control (C), and RT-PCR reactions spanning the junction of the sRNA and adjacent ORF with (+) or without (−) reverse transcriptase. The RT-PCR results for sRNAs that did not display co-transcription are not shown.

Figure 3.

sRNAs identified in BCG through computational predictions. (A) Properties of Mpr1-Mpr21 are depicted as for the Mcr RNAs in Figure 2A. Mpr7, Mpr13, and Mpr14 coincide with the cloned sRNAs Mcr3, Mcr14 and Mcr9, respectively. *See ref. (6). (B) Representative computationally predicted sRNAs verified in BCG by northern blot, depicted as in Figure 2B. Mpr5-11 were identified with 7-day shaking BCG RNA, while Mpr13 and Mpr17 were identified with 8-day shaking BCG RNA and 6-day standing BCG RNA, respectively. All northerns shown were run on 10% denaturing PAGE. Probes are given in Supplementary Table S3. (C) Representative co-transcription. Six of the nine co-transcribed RNAs are shown, with the chromosomal DNA positive PCR control (C), and RT-PCR reactions spanning the junction of the sRNA and adjacent ORF with (+) or without (–) reverse transcriptase.

Figure 4.

The Mcr and Mpr sRNAs are conserved within mycobacteria. (A) Summary of the BCG sRNAs and their presence in other mycobacterial genomes. For Mcr1-19, homology was determined by megablast using the cloned sRNA sequence, while for Mpr1-21 homology was determined by blastn using the candidate sRNA sequence (only results with an E value <1 are shown). sRNAs that did not show homology to M. smegmatis (Mpr5, 6, 9, 12, 13, 17, 18 and 20) were then rechecked by blastn restricted to Mycobacterium (taxid1763); a few of these had E values >1 (Mpr9, E = 5.0; Mpr11, E = 4.5; and Mpr17, E = 2.4). sRNAs were tested for expression by Northern blot in the three genomes listed; shaded boxes were not tested. For clarity, duplicate sRNAs are listed only once. + = positive signal by northern; – = negative signal by northern. *In this study TB-complex includes M. tuberculosis H37Rv, M. bovis subsp. bovis AF2122/97 and M. bovis Pasteur BCG; **previously identified in Mtb (6). (B) Representative sRNAs that are expressed in M. smegmatis. The lanes are as follows: M. Marker, in nucleotides, 1. log-phase BCG RNA, 2. log-phase M. smegmatis RNA. (C) Representative sRNAs that are expressed in Mtb. M. Marker, in nucleotides, 1. 7-day shaking Mtb RNA. (D) Schematic of the general division of mycobacteria. Species correspond to those in panel A, with homology to the Mcr and Mpr sRNAs.

Figure 5.

Differential expression of Mcr11. Representative blots showing that Mcr11 expression is responsive to environmental growth conditions, in both BCG (A) and Mtb (B). Lanes are as follows: M. Marker, in nucleotides, 1: shaking, 2: shaking, acid-treated, 3: shaking, low O₂. Differences in sRNA expression were determined by the percentage of tRNA signal for each sample; the average of three experiments is given, with the standard deviations listed below.