Mutations of the YidC Insertase alleviate stress from σM-dependent membrane protein overproduction in Bacillus subtilis

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 spoIIIJ, which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 SpoIIIJ contains a single amino acid substitution in the substrate-binding channel (Q140K), and this allele suppresses the lethality of high SigM. Analysis of a library of YidC variants reveals that increased charge (+2 or +3) in the substrate-binding channel 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 YidCQ140K substitution. We further show that the fitness defect caused by high {sigma}M activity is exacerbated in the absence of 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.\n\nAuthor SummaryBacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In Bacillus subtilis, the alternative {sigma} factor {sigma}M is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the {sigma}M regulon, resulting from loss of the YhdL anti-{sigma}M protein, is lethal. We here identify charge variants of the SpoIIIJ(YidC) membrane protein insertase that suppress the lethal effects of high {sigma}M activity. Further analyses reveal that induction of the {sigma}M regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the {sigma}M regulon.