Length matters: the disordered N-terminus of Pal coordinates Lpp exclusion for outer membrane constriction in <i>E. coli</i>

The peptidoglycan-associated lipoprotein (Pal) is essential for outer membrane integrity in gram-negative bacteria, but how its N-terminal disordered region contributes to cell division remains unclear. Here, we truncated this region in <i>Escherichia coli</i> (<i>pal</i>^-8: 8-aa deletion, <i>pal</i>^-24: 24-aa deletion) and found that while neither truncation impaired Pal localization or growth, both mutants exhibited graded sensitivity to membrane stressors (1 mM EDTA/1% SDS), with <i>pal</i>^-24 showing intermediate susceptibility between <i>pal</i>^-8 and Δ<i>pal</i>. Atomic force microscopy revealed that the disordered region length fine-tunes Pal's ability to exclude Braun's lipoprotein (Lpp) from division sites: <i>pal</i>^-8 maintained wild-type Lpp-exclusion zones, whereas <i>pal</i>^-24 exhibited either absent or narrowed grooves. These results suggest that the disordered region acts as a length-dependent molecular spacer, ensuring efficient Pal-Lpp spatial exclusion during cell division. Our work establishes a mechanistic paradigm by which Pal's flexible N-terminus coordinates outer membrane constriction through dynamic competition with Lpp, offering new insights into the functional plasticity of disordered domains in bacterial morphogenesis.IMPORTANCEThe outer membrane of bacteria like <i>E. coli</i> must constrict during division, but the full mechanism is unclear. We studied peptidoglycan-associated lipoprotein (Pal), a protein that helps coordinate this process. Pal has a disordered linker region, and we found that the length of this linker acts as a molecular ruler. Shortening the linker disrupts Pal's ability to properly organize the division site and create a zone required for outer membrane remodeling. This reveals how a seemingly unstructured protein region can perform a precise, measurement-dependent function. Since disordered linkers are common in bacterial outer membrane lipoproteins, our work suggests a general design principle for fine-tuning cell envelope dynamics during growth and division.