Deletion of Yersinia pestis ail Causes Temperature-Sensitive Pleiotropic Effects, Including Cell Lysis, That Are Suppressed by Carbon Source, Cations, or Loss of Phospholipase A Activity

Maintenance of phospholipid (PL) and lipopoly- or lipooligosaccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37 degrees C). Loss of this protein had pleiotropic effects. A Y. pestis Delta ail mutant and KIM61 wild type were systematically compared for (i) growth requirements at 37 degrees C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernatants, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild-type phenotype. The Dail mutant grew normally at 28 degrees C but lysed at 37 degrees C when it entered stationary phase, as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immunofluorescence microscopy showed that the Delta ail mutant did not assemble Caf1 capsule. Expression of heat shock promoter rpoE or rpoH fused to a lux operon reporter were not induced when the Delta ail mutant was shifted from 28 degrees C to 37 degrees C (P, 0.001 and P, 0.01, respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca2+, or 2.5 mM Mg2+ to the growth medium or by a mutation in the phospholipase A gene (pldA::miniTn5, Delta pldA, or PldAS164A). A model accounting for the temperature-sensitive lysis of the Delta ail mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37 degrees C is presented. IMPORTANCE The Gram-negative pathogen Yersinia pestis transitions between a flea vector (ambient temperature) and a mammalian host (37 degrees C). In response to 37 degrees C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of ail destabilizes the OM at 37 degrees C, causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.