Abstract
ObjectiveRestriction-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.ResultsWe 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.
Highlights
The primary role of bacterial Restriction-Modification (R-M) systems has typically been viewed as protection from foreign DNA, such as plasmids and phages
No complete R-M systems have been defined in M. tuberculosis, the causative agent of tuberculosis, or in its non-pathogenic relative M. smegmatis, a model system widely used to study the basic biology of M. tuberculosis
We previously identified and characterized one of the three predicted DNA methyltransferases in M. tuberculosis, MamA [5], which is non-essential for M. tuberculosis survival in vitro and during infection [6,7,8]
Summary
Depletion of MamA impairs growth and reduces viability in M. smegmatis We generated an ATc-inducible mamA KD strain and a control with a non-specific CRISPRi construct containing an sgRNA that does not target the M. smegmatis genome [11]. As CRISPRi represses transcription of co-transcribed genes, we constructed a rescue vector expressing a second copy of mamA with two synonymous point mutations that disrupt the PAM adjacent to the sgRNA binding site and prevent dCas binding This vector was integrated into the genome of M. smegmatis that contained the mamA KD construct. We generated an MSMEG_3214 deletion strain containing the mamA CRISPRi construct and evaluated its growth in the presence and absence of ATc (Fig. 3b). In the absence of MSMEG_3214, mamA KD does not affect growth, supporting the hypothesis that MamA and MSMEG_3214 are part of an active R-M system in M. smegmatis
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