NlpI‐mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in <i>Escherichia coli</i>

Abstract Outer membrane vesicles ( OMV s) are ubiquitously secreted from the outer membrane ( OM ) of Gram‐negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild‐type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan ( PG ) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr , consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin‐binding protein 4 ( PBP 4) during exponential growth. The data support a model in which NlpI negatively regulates PBP 4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane ( OM ‐ PG ) envelope stabilizing cross‐links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild‐type Gram‐negative bacteria can use to modulate vesiculation is by altering PG ‐ OM cross‐linking via localized modulation of PG degradation and synthesis.