Abstract
Insight into the mechanism of lipid transport to the outer membrane of gram-negative bacteria has been hampered by the lack of an effective genetic screen for defective mutants. This work demonstrates an enrichment of conditional mutants defective in lipopolysaccharide export by Ludox density gradient centrifugation and selection for detergent resistance. New temperature-sensitive mutants with lipid export defects were isolated with single missense mutations in msbA. The results demonstrate the power of this approach for the study of lipid export in Escherichia coli.
Highlights
The envelope of Escherichia coli and other gram-negative bacteria contains two distinct lipid bilayers
The results demonstrate the power of this approach for the study of lipid export in Escherichia coli
WD2 accumulates newly synthesized LPS and phospholipids at the cytoplasmic leaflet of the inner membrane after short growth at the nonpermissive temperature, resulting in the formation of inner membrane invaginations visible by electron microscopy [6, 9]. These results suggest that MsbA is a general lipid transporter and transports both LPS and phospholipids across the IM of E. coli
Summary
This work demonstrates the use of density gradient centrifugation with Ludox to enrich for temperature-sensitive E. coli mutants with defects in lipid export. This method may be useful in isolating novel conditional mutants with defects in membrane biogenesis. The conditional lpxA mutant SM101 [11] migrated to the parent strain on this gradient following growth at the nonpermissive temperature for 30 min (data not shown), indicating that loss of LPS transport, but not synthesis, results in altered mobility of cells on Ludox gradients.
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