Impaired envelope integrity in the absence of SanA is linked to increased Lipid II availability and an imbalance of FtsI and FtsW activities

In Gram-negative bacteria, the outer membrane (OM) acts in conjunction with the peptidoglycan (PG) cell wall as a barrier against physical, osmotic, and chemical environmental stressors including antibiotics. SanA, an inner membrane protein in Escherichia coli K-12, is required for vancomycin resistance at high temperatures (>42 {degrees}C) and impacts sodium dodecyl sulfate (SDS) resistance during stationary phase reached from carbon limitation. However, its function remains unknown. Here, we show that {Delta}sanA has a synthetic genetic interaction with {Delta}wecA, a mutation that increases the availability of the isoprenoid carrier for PG synthesis. Specifically, the {Delta}sanA {Delta}wecA strain demonstrated heightened SDS-EDTA sensitivity, activation of the Rcs stress response, and increased cell length. Further investigation tied the SDS-EDTA sensitivity to increased lipid II available for PG synthesis. Spontaneous suppressor mutants of this phenotype harbored point mutations in prc, which encodes tail specific protease, or ftsI, which encodes the cell division DD-transpeptidase, a target of Prc. We focused on the ftsI mutations and demonstrated that the ftsI mutations had increased cell length but nevertheless enhanced PG incorporation at the septum compared to the {Delta}sanA mutant, returning PG incorporation to wild-type levels. Moreover, other mutations affecting septal PG synthesis, but not divisome assembly, also suppressed the SDS-EDTA sensitivity. These findings suggest that, in the absence of SanA, increased lipid II availability perturbs the balance between septal PG synthesis, lateral PG elongation, and other envelope biogenesis pathways, which leads to increased OM permeability. IMPORTANCEThe Gram-negative cell envelope is a barrier that protects the cell from environmental stress. Therefore, the synthesis of each layer of this envelope needs to be closely coordinated throughout growth and division. Here, we investigated SanA, a protein in Escherichia coli K-12 that affects envelope permeability under cellular stress, including nutrient limitation and high temperature. We found that SanA plays a key role in maintaining the permeability barrier when precursor levels for peptidoglycan (PG) synthesis are elevated, linking envelope integrity to balanced septal PG production during cell division. Our results suggest that SanA modulates substrate availability to preserve envelope function, and that in its absence, imbalanced substrate flux to septal PG synthesis disrupts septum formation and compromises barrier integrity.