Figure 7. M. smegmatis encodes a previously undescribed restriction-modification system.

(A) MSMEG_3213, a putative DNA methylase, is associated with genes involved in DNA replication and repair in UMAP space. (B) Knockdown of MSMEG_3213 leads to cellular filamentation. The morphological nearest neighbors are dnaE1, pyrG and dnaA. (C) Consensus heatmaps of ParB-mCherry localization demonstrate that oriC positioning is disrupted in the MSMEG_3213 knockdown mutant. (D) Nanopore-based RNA-Seq confirmed that knockdown of MSMEG_3213 was specific to the targeted sgRNA position. (E) MSMEG_3213 produces a transcriptional response comparable to treatment with the DNA damaging agent, mitomycin C (MMC). (F) MSMEG_3213 is located upstream of MSMEG_3214, a gene with weak homology, according to HHpred (Hildebrand et al., 2009), to an endonuclease subunit (REBASE: 3098 BbvCI). (G) Lethality of MSMEG_3213 knockdown is suppressed by simultaneous CRISPRi-mediated knockdown of MSMEG_3214.

Figure 7—figure supplement 1. Morphological profiling informs gene function.

(A) Genes with putative function were visualized in UMAP space. MSMEG_0317 (Cashmore et al., 2017) and MSMEG_6276 (murT) were found in the cell-wall cluster, supporting their suggested roles as components of cell-wall synthesis. The transcription factor, WhiA (Rustad et al., 2014), was found in the DNA-Cell Division-Translation Cluster, suggesting a role in cell-cycle regulation. (B) MSMEG_0317 produces a distinct phenotype on knockdown, with Dendra-tagged MSMEG_0317 localizing to the cell wall and cell septa. (C) Knockdown of whiA produces elongated, branching cells, with Dendra-tagged WhiA localized to the nucleoid. (D) MSMEG_6276, a gene predicted by HHpred (Hildebrand et al., 2009) to have strong homology to murT/gatD, causes cellular bulging and lysis on silencing.