Maintenance of phospholipid (PL) and lipopoly- or lipooligo-saccharide (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° C). Loss of this protein had pleiotropic effects. A
Y. pestis
Δail
mutant and KIM6
+
wild- type were systematically compared for (i) growth requirements at 37° C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernates, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild- type phenotype. The
Δail
mutant grew normally at 28° C but lysed at 37° C when it entered stationary phase as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immuno-fluorescent microscopy showed that the
Δail
mutant did not assemble Caf1 capsule. Expression of heat shock promoters
rpoE
or
rpoH
fused to a
lux
operon reporter were not induced when the
Δail
mutant was shifted from the 28° C to 37° 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 Ca
2+
, or 2.5 mM Mg
2+
to the growth medium, or by a mutation in the phospholipase A gene (
pldA
::miniTn
5
,
ΔpldA,
or PldA
S164A
). A model, accounting for the temperature-sensitive lysis of the
Δail
mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented.
IMPORTANCE
The Gram-negative pathogen,
Yersinia pestis
, transitions between a flea vector (ambient temperature) and a mammalian host (37° C). In response to 37° 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° 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.