Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

MamA essentiality in Mycobacterium smegmatis is explained by the presence of an apparent cognate restriction endonuclease.

OBJECTIVE: Restriction-Modification (R-M) systems are ubiquitous in bacteria and were considered for years as rudimentary immune systems that protect bacterial cells from foreign DNA. Currently, these R-M systems are recognized as important players in global gene expression and other cellular processes such us virulence and evolution of genomes. Here, we report the role of the unique DNA methyltransferase in Mycobacterium smegmatis, which shows a moderate degree of sequence similarity to MamA, a previously characterized methyltransferase that affects gene expression in Mycobacterium tuberculosis and is important for survival under hypoxic conditions. RESULTS: We found that depletion of mamA levels impairs growth and produces elongated cell bodies. Microscopy revealed irregular septation and unevenly distributed DNA, with large areas devoid of DNA and small DNA-free cells. Deletion of MSMEG_3214, a predicted endonuclease-encoding gene co-transcribed with mamA, restored the WT growth phenotype in a mamA-depleted background. Our results suggest that the mamA-depletion phenotype can be explained by DNA cleavage by the apparent cognate restriction endonuclease MSMEG_3214. In addition, in silico analysis predicts that both MamA methyltransferase and MSMEG_3214 endonuclease recognize the same palindromic DNA sequence. We propose that MamA and MSMEG_3214 constitute a previously undescribed R-M system in M. smegmatis.