Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC (original) (raw)
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Journal of Bacteriology, 2005
In response to the in vivo environment, the Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) is modified. These modifications are controlled in part by the two-component regulatory system PmrA-PmrB, with the addition of 4-aminoarabinose (Ara4N) to the lipid A and phosphoethanolamine (pEtN) to the lipid A and core. Here we demonstrate that the PmrA-regulated STM4118 (cptA) gene is necessary for the addition of pEtN to the LPS core. pmrC, a PmrA-regulated gene necessary for the addition of pEtN to lipid A, did not affect core pEtN addition. Although imparting a similar surface charge modification as Ara4N, which greatly affects polymyxin B resistance and murine virulence, neither pmrC nor cptA plays a dramatic role in antimicrobial peptide resistance in vitro or virulence in the mouse model. Therefore, factors other than surface charge/electrostatic interaction contribute to resistance to antimicrobial peptides such as polymyxin B.
Escherichia coli Mutants that Synthesize Dephosphorylated Lipid A Molecules
Biochemistry, 2010
The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine that is phosphorylated at the 1 and 4 0 positions. Expression of the Francisella novicida lipid A 1-phosphatase FnLpxE in E. coli results in dephosphorylation of the lipid A proximal unit. Coexpression of FnLpxE and the Rhizobium leguminosarum lipid A oxidase RlLpxQ in E. coli converts much of the proximal glucosamine to 2-amino-2-deoxygluconate. Expression of the F. novicida lipid A 4 0 -phosphatase FnLpxF in wild-type E. coli has no effect because FnLpxF cannot dephosphorylate hexaacylated lipid A. However, expression of FnLpxF in E. coli lpxM mutants, which synthesize pentaacylated lipid A lacking the secondary 3 0 -myristate chain, causes extensive 4 0 -dephosphorylation. Coexpression of FnLpxE and FnLpxF in lpxM mutants results in massive accumulation of lipid A species lacking both phosphate groups, and introduction of RlLpxQ generates phosphate-free lipid A variants containing 2-amino-2-deoxygluconate. The proposed lipid A structures were confirmed by electrospray ionization mass spectrometry. Strains with 4 0 -dephosphorylated lipid A display increased polymyxin resistance. Heptose-deficient mutants of E. coli lacking both the 1-and 4 0 -phosphate moieties are viable on plates but sensitive to CaCl 2 . Our methods for reengineering lipid A structure may be useful for generating novel vaccines and adjuvants.
Journal of Bacteriology, 1981
The structural gene (pgsA) for the CDP-diacylglycerol:sn-glycero-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycerophosphate synthase) from Escherichia coli has been cloned, using pSC101 as the vector. The resulting hybrid plasmids not only correct the lack of in vitro synthase activity in pgsA strains but also cause an amplification (6- to 40-fold over wild-type levels) in enzymatic activity in direct proportion to the copy number of the plasmids found in vivo. The cloned gene also corrects the abnormally low level of polyglycerophosphatides found in pgsA strains and actually increases the level of phosphatidylglycerol to above that normally found in E. coli. The degree of alteration in phospholipid composition brought about by these hybrid plasmids is not of the order expected if fluctuations in enzyme levels in vivo were an important regulatory mechanism in phospholipid metabolism. The isolated hybrid plasmids have been mapped by restriction endonuclease analysi...
Journal of Biological Chemistry
Escherichia coli strain AD93 is unable to synthesize the nonbilayer lipid phosphatidylethanolamine and requires high concentrations of specific divalent cations for growth. Previous studies suggested that in this strain, cardiolipin in combination with divalent cations functionally replaces phosphatidylethanolamine, reflecting polymorphic regulation of membrane lipid composition. However, it is also possible that divalent cations are required for regulation of lipid packing or membrane surface potential. 2H NMR was employed to measure the effect of different divalent cations on lipid packing in aqueous dispersions of lipid extracts isolated from AD93 and the wild type parental strain W3899, which were grown with [ll,ll-2H2]oleic acid. The results indicate that a range of acyl chain order is compatible with growth and that Ba2+, which cannot support growth of AD93, can increase chain packing to the wild type level. By means of microelectrophoresis, it was shown that the growth-promoting cations and Ba2+ have a strong and comparable ability to screen the surface charge of large unilamellar vesicles prepared fromAD93 lipid extracts. Therefore, it is unlikely that the growthpromoting capacity of divalent cations is primarily due to their effect on lipid packing or their potency to decrease the surface potential. Furthermore, the addition of small amounts of Ba2+ to a AD93 lipid dispersion with excess Mg2' diminished H,, phase formation. This observation can explain the growth arrest in AD93 cultures upon the addition of Ba2+ and further supports the conclusion that the cation requirement of this strain arises mainly from polymorphic regulation of lipid composition.
Archives of biochemistry and biophysics, 2017
Phosphoethanolamine (pEtN) decoration of E. coli Lipopolysaccharide (LPS) provides resistance to the antimicrobial Polymyxin B (PolB). While EptA and EptB enzymes catalyze the addition of pEtN to the Lipid A and Kdo (pEtN-Kdo-Lipid A), EptC catalyzes the pEtN addition to the Heptose I (pEtN-HeptI). In this study, we investigated the contribution of pEtN-HeptI to PolB resistance using eptA/eptB and eptC deficient E. coli K12 and its wild-type parent strains. These mutations were shown to decrease the antimicrobial activity of PolB on cells grown under pEtN-addition inducing conditions. Furthermore, the 1-N-phenylnapthylamine uptake assay revealed that in vivo PolB has a reduced OM-permeabilizing activity on the ΔeptA/eptB strain compared with the ΔeptC strain. In vitro, the changes in size and zeta potential of LPS-vesicles indicate that pEtN-HeptI reduce the PolB binding, but in a minor extent than pEtN-Kdo-Lipid A. Molecular dynamics analysis revealed the structural basis of the Po...
Proceedings of the National Academy of Sciences, 1977
We have screened a bank of 2000 E. coli strains carrying hybrid ColE1 plasmids [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] for those that correct the temperature sensitivity of a mutant in CDP-1,2-diacyl sn-glycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase). Two hybrid plasmids of this kind (pLC34-44 and pLC34-46) were identified and characterized. Strains carrying these plasmids overproduce the synthase by 6- to 15-fold, as demonstrated by assays of extracts and purification to homogeneity of the overproduced enzyme. The overproduced synthase, like the wild-type enzyme, is found associated predominately with the ribosomal fraction of crude cell extracts. Because the membrane phospholipid composition of these overproducers is not greatly altered, we suggest that the synthase is normally present in excess.
Uptake and remodeling of exogenous phosphatidylethanolamine in E. coli
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2004
The fate of exogenous short-chain analogues of phosphatidylethanolamine and phosphatidylserine was studied in a deep-rough derivative of E. coli mutant strain AD93 that cannot synthesize phosphatidylethanolamine de novo. Using mass spectrometry, it was shown that dicaproyl(di 6:0)-phosphatidylethanolamine is extensively remodeled, eventually adopting the phosphatidylethanolamine species profile of the parental wild-type strain of AD93. Dicaproyl-phosphatidylserine was decarboxylated to form phosphatidylethanolamine, and yielded a species profile, which strongly resembled that of the introduced phosphatidylethanolamine. This demonstrates transport of phosphatidylserine to the cytosolic leaflet of the inner membrane. The changes of the species profile of phosphatidylethanolamine indicate that the short-chain phospholipids are most likely remodeled via two consecutive acyl chain substitutions, and at least part of this remodeling involves transport to the inner membrane. D