Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 ( spoIIIJ), which encodes the major YidC membrane protein insertase in B. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σ M, and the absence of YhdL leads to runaway expression of the σ M regulon and cell death. In Bacillus subtilis, the extracytoplasmic function σ factor σ M regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. Some YidC1 variants exhibited reduced growth ability, and the variant containing R72 K140 R144 failed to grow, although the emergence of suppressors was noted. The negative control (None) has no yidC1 at thrC locus, while the positive control has the 168 version of yidC1 that contains a single positive charge at R73. Diluted cultures were spotted on LB without xylose (-Xyl) or with 1% final concentration of xylose (+Xyl).
The depletion strain has its native yidC1 and yidC2 deleted, and a xylose inducible copy of P xylA- yidC2. B) Spot dilution of YidC depletion strains with yidC1 variants at thrC locus. The positive charge of each variant was labelled next to the streaking, with a negative control “None” meaning no yidC1 variant at thrC locus (weak growth due to the depletion conditions, and cannot be restreaked), and a positive control “YidC1 PY79” meaning the native YidC1 mutated into the PY79 Q140K version (HB23719). S4 Fig: A) yhdL depletion strains with YidC1 variants were streaked on LB plates with or without xylose inducer for yhdL.