Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
Centre for Computational Biology, Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK.
Microbiology (Reading). 2022 May;168(5). doi: 10.1099/mic.0.001197.
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.
细菌酰胺酶对于分裂附属子细胞的共有包膜以允许细胞分离是必不可少的。需要精确控制其活性以防止细胞溶解。酰胺酶活性由三种调节蛋白 NlpD、EnvC 和 ActS 控制。然而，最近的研究将外膜脂蛋白 DolP（以前称为 YraP）作为 NlpD 的潜在上游调节剂。在这项研究中，我们更详细地探讨了这种联系。令我们惊讶的是，DolP 不会调节酰胺酶活性，也无法在下拉和 MST（微量热泳动）测定中与 NlpD 相互作用。接下来，我们排除了 DolP 在一系列包膜应激下模拟Δ的假设。然而，酰胺酶（AmiA、AmiB 和 AmiC）和酰胺酶调节剂的双缺失突变体的形态分析表明，与亲本菌株相比，ΔΔ和ΔΔ突变体显示出更长的链长，这表明 DolP 在细胞分裂中起作用。总的来说，我们提供的证据表明 DolP 不影响 NlpD 功能，这意味着 DolP 不是 NlpD 的上游调节剂。然而，DolP 可能通过直接与 AmiA 或 AmiC 相互作用，或者通过尚未发现的机制，影响子细胞分离。
