The lipoprotein DolP affects cell separation in , but not as an upstream regulator of NlpD.

Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that Δ phenocopied Δ in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with revealed that ΔΔ and ΔΔ mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function , implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.