YtrG is a functional part of the ytrGABCDEF operon and contributes to phenotypic heterogeneity in Bacillus subtilis

ABSTRACT The Bacillus subtilis ytrGABCDEF operon consists of the transcriptional repressor YtrA, the ATP-binding cassette transporter subunits YtrB, YtrC, YtrD, YtrE, and YtrF, likely forming two distinct transmembrane complexes (YtrB 2 CD and YtrE 2 F 2 ), and the small monotopic transmembrane protein YtrG, which has so far remained uncharacterized. Overexpression of the operon leads to pronounced phenotypic heterogeneity characterized by a subpopulation of cells with strongly reduced length and width (“small cells”). Operon mutants that constitutively express ytrG , together with incomplete transporter variants, show pronounced lysis of these small cells that is not observed when the whole operon is overexpressed. Based on this observation, together with bioinformatic analyses of the YtrG protein sequence that revealed some structural features associated with membrane-active toxins, we investigated whether YtrG could function as a toxin underlying small cell lysis. Synthetic YtrG peptide displayed moderate antibacterial activity against Bacillus subtilis and Escherichia coli , but did not induce the characteristic small-cell or lysis phenotypes, and had only minor effects on the cell membrane. Likewise, overexpression of ytrG in the wild-type background did not impair growth and caused neither small cells nor cell lysis. In contrast, deletion of ytrG resulted in pronounced small-cell and lysis phenotypes that were partially complemented by ectopic ytrG expression, indicating that YtrG counteracts rather than promotes these effects. Together, our findings exclude YtrG as the cause of the small cell and lysis phenotypes, but identify it as an important factor required for small cell integrity. IMPORTANCE The Bacillus subtilis ytrGABCDEF operon is activated by cold shock and cell wall-targeting antibiotics and has been established as a reliable marker for interference with lipid-linked cell wall precursor synthesis in antibiotic mode of action studies. Despite its highly specific induction pattern, the function of the operon remains debated. Recent studies point toward a role in balancing cell wall synthesis and autolysis, possibly related to cannibalism. Despite thorough genetic, functional, and structural characterization of the operon, the function of its smallest component, YtrG, has remained unaddressed. Our findings provide the first insights into the role of YtrG and show that it is indeed a functional component of the operon.