Abstract
In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM 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 σM. 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 σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM 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.
Highlights
The ability of cells to adapt to changing conditions relies in large part on the expression of specific stress responses controlled by transcription regulators
Bacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis
In Bacillus subtilis, the alternative σ factor σM is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis
Summary
The ability of cells to adapt to changing conditions relies in large part on the expression of specific stress responses controlled by transcription regulators. We explored the basis for σM toxicity by selecting for suppression of yhdL lethality in a sigM merodiploid strain to reduce the frequency of suppressors that had inactivated σM These studies led to the recovery of mutations in rpoB and rpoC, encoding the ß and ß’ subunits of RNA polymerase, that led to a reduction of σM activity sufficient to restore viability [10]. In the course of these studies we demonstrated that the toxicity from high σM could be alleviated by mutation of the autoregulatory promoter for the sigM operon, or by overexpression of the housekeeping σ factor, σA [10] These results suggest that the lack of a functional anti-σM factor (yhdL null mutant) leads to runaway activation of the sigM operon and a high level of σM activity that is incompatible with growth. It is unclear whether σM toxicity results from a decrease in activity of the essential housekeeping σA, overexpression of one or more σM-regulated genes, or both
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