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

Abstract

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.

Keywords: DNA methylation; MamA; Methyltransferase; Mycobacterium smegmatis; Restriction-Modification system.

Fig. 1

mamA depletion causes growth cessation in M. smegmatis. a Strains with depleted mamA (mamA KD), nonspecific CRISPRi control with a sgRNA sequence that does not target any specific part of M. smegmatis genome (Nonspecific sgRNA), or a rescue vector containing a synonymous mamA mutant that is not bound by dCas9 (mamA Rescue) were incubated in 7H9 in presence (+) or absence of ATc to activate or not the CRISPRi knockdown system, respectively. Three biological replicates of mamA KD, four biological replicates of mamA Rescue, and two technical replicates of each were used and values were averaged. b The strains were grown in 7H9 and CFUs were calculated at different timepoints

Fig. 2

Lowering mamA levels leads to cell filamentation and asymmetric DNA distribution. a DIC Microscopy showing WT (upper panels and mamA KD no ATc) and mamA depleted (mamA KD +ATc) phenotypes. b Cell length distribution determined by microscopy and analyzed with ImageJ software. c DIC microscopy and DNA staining with SYTO 24 in mamA depleted (+ ATc) and non-depleted (no ATc) cells. d Quantification of DNA occupation observed in SYTO 24-stained cells. Percent DNA occupation was calculated by dividing length of DNA fluorescence by cell length. ****p < 0.0001 Kruskal–Wallis followed by Dunn’s multiple comparisons test

Fig. 3

Morphologies of mamA depleted cells and rescue by deletion of MSMEG_3214. a Microscopy of the mamA KD strain treated with ATc for 12 h. Cells were stained with SYTO 24 (green) and FM 4-64FX (red) to visualize DNA and membranes, respectively. The images represent cells of three major phenotypic groups observed. The left column shows DNA stained only, the center column shows membrane stained only, and the right column shows the merged fluorescent images. b Strains with depleted mamA (mamA KD), nonspecific CRISPRi control with an sgRNA sequence that does not target any specific part of M. smegmatis genome (Nonspecific sgRNA), mamA KD in an MSMEG_3214-deleted background (mamA KD ΔMSMEG_3214), and the ΔMSMEG_3214 strain with a nonspecific sgRNA (Nonspecific sgRNA ΔMSMEG_3214) were incubated in 7H9 in presence (+) or in absence of ATc to activate or not the CRISPRi knockdown system, respectively