Investigating the role of MlaA in SDS-EDTA resistance pathways in Escherichia coli

Abstract

The outer membrane (OM) in Gram-negative bacteria, like Escherichia coli, is asymmetric, with mainly lipopolysaccharide (LPS) molecules in the outer leaflet and phospholipids in the inner leaflet. Maintaining structural asymmetry of the OM is vital for cell survival in unfavourable conditions. The Mla pathway is a mechanism that restores membrane asymmetry upon exposure to external stressors, such as sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). Deletion of the OM porin OmpC, which forms a complex with MlaA, a component of the Mla pathway, results in hypersensitivity to SDS-EDTA treatment. However, these mutants are rescued when grown in phosphate deficient conditions. The phosphoporin PhoE, which is upregulated under phosphate limitation, likely complements OmpC for maintaining OM asymmetry. PhoE shares structural and functional similarities to OmpC, but its interaction with MlaA remains unclear. Thus, we sought to investigate the role of MlaA in the OmpC- and putative PhoE- dependent pathways of restoring membrane asymmetry. We hypothesized that since OmpC and PhoE appear to mediate independent pathways for maintaining membrane asymmetry that converge on MlaA,  deletion of MlaA would cause severely reduced cell growth compared to both ΔompC and ΔphoE mutants. To test this, we performed growth curve assays with wild type, ΔompC, ΔphoE, and ΔmlaA strains in minimal media containing SDS and increasing concentrations of EDTA. We found that growth of ΔmlaA mutants was not as severely inhibited as expected, and that ΔompC mutants were more sensitive to SDS-EDTA than ΔmlaA mutants, demonstrating the importance of MlaA-independent pathways of SDS-EDTA resistance.