Environmental context defines the physiological contribution of the Tol-Pal system to cell envelope integrity in Acinetobacter baumannii.

The gram-negative cell envelope requires coordinated remodeling of the peptidoglycan layer and outer membrane to preserve envelope integrity during growth and division. The conserved Tol-Pal system contributes to envelope maintenance, although the physiological consequences of its loss vary across environmental contexts and among gram-negative bacteria. Here, we examined the contribution of Tol-Pal across diverse growth environments in Acinetobacter baumannii. Loss of Tol-Pal did not cause a substantial population growth defect, and septal peptidoglycan incorporation remained largely preserved under standard laboratory conditions. In contrast, nutrient-rich media, conditions that alter osmotic balance, and a host-like environment exposed defects in cell division organization and cell morphology. Tol-Pal-deficient cells also exhibited modest but reproducible reductions in outer membrane barrier robustness, and disruption of the Mla phospholipid transport system further exacerbated these defects. Consistent with these phenotypes, loss of Tol-Pal reduced bacterial fitness under environmental stress and host-associated models. Together, these findings indicate that Tol-Pal contributes to outer membrane barrier robustness and that additional consequences of its loss become apparent under conditions that challenge the cell envelope.IMPORTANCEThe physiological consequences of disrupting conserved envelope systems can vary with the conditions in which bacteria grow. In Acinetobacter baumannii, loss of Tol-Pal has little effect on population growth under routine laboratory conditions but modestly compromises outer membrane barrier robustness, whereas specific environmental and host-associated conditions reveal additional defects in cell division organization and bacterial fitness. These findings highlight how environmental context shapes the physiological consequences of Tol-Pal deficiency and provide a framework for understanding why the contribution of this conserved envelope system differs among gram-negative bacteria.