Mutations of the Bacillus subtilis YidC1 (SpoIIIJ) insertase alleviate stress associated with σM-dependent membrane protein overproduction

In Bacillus subtilis, the extracytoplasmic function sigma factor sigma(M) regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-sigma factors YhdL and YhdK form a complex that restricts the basal activity of sigma(M), and the absence of YhdL leads to runaway expression of the sigma(M) regulon and cell death. 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. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high sigma(M). Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the sigma(M) regulon. Derepression of the sigma(M) regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1(Q140K) substitution. We further show that the fitness defect caused by high sigma(M) activity is exacerbated in the absence of the SecDF protein translocase or sigma(M)-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific sigma(M)-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the sigma(M) regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.