PagP Activation in the Outer Membrane Triggers R3 Core Oligosaccharide Truncation in the Cytoplasm of Escherichia coli O157:H7

Abigail E Smith,Sang-Hyun Kim,Feng Liu,Wenyi Jia,Evgeny Vinogradov,Carlton L Gyles,Russell E Bishop

doi:10.1074/jbc.m708163200

Abstract

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPDelta5-14 allele, which mostly eliminates a periplasmic amphipathic alpha-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.

Highlights

Like most enteric Gram-negative bacteria, Escherichia coli surrounds its cytoplasmic membrane with a reticulated peptidoglycan exoskeleton and an outer membrane (OM),5 which demarcates the so-called periplasmic space
We demonstrate that a lipid A myristoylation mutant of enterohemorrhagic E. coli O157: H7, but not a similar mutant from E. coli K-12, necessarily triggers PagP activity in the OM to exert control on cytoplasmic enzymes that determine its characteristic R3 core oligosaccharide structure
The results identify a tion is a requirement for L-Ara4N addition in E. coli K-12 [45], complete lipid A-core that includes the attached O-antigen but this does not appear to be true in E. coli O157:H7

Summary

EXPERIMENTAL PROCEDURES

Materials—32Pi was purchased from PerkinElmer Life Sciences. Antibiotics and Gal were obtained from Sigma. Glassbacked Silica Gel 60 TLC plates were from Merck. The QIAprep spin miniprep, Qiaquick PCR purification, and QIAEX II gel extraction kits were obtained from Qiagen. Restriction endonucleases, T4 DNA ligase, and dNTP were obtained from Fermentas. Antibiotics were added when necessary at final concentrations of 12 ␮g/ml for tetracycline, 20 ␮g/ml for chloramphenicol and gentamycin (Gm), 100 ␮g/ml for ampicillin (Ap), and streptomycin (Str), and 40 ␮g/ml for kanamycin. Single colonies were inoculated from plates into 5 ml of liquid medium and grown at 37 °C overnight to stationary phase. A 1% inoculum was subcultured into the same medium and allowed to resume growth at 37 °C. GlcNAc creation of a pagP::aacC1 mutation in NH the double msbB mutant (4304-DM)

HepI 2

RESULTS

25 Ͼ100 Ͼ100 Ͼ100 Ͼ100

DISCUSSION