Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Found 1 result for 23372159
Filters applied. Clear all
. 2013 Mar 22;288(12):8111-8127.
doi: 10.1074/jbc.M112.445981. Epub 2013 Jan 31.

Molecular and structural basis of inner core lipopolysaccharide alterations in Escherichia coli: incorporation of glucuronic acid and phosphoethanolamine in the heptose region

Gracjana Klein  1 , Sven Müller-Loennies  2 , Buko Lindner  2 , Natalia Kobylak  3 , Helmut Brade  2 , Satish Raina  4
Affiliations

Molecular and structural basis of inner core lipopolysaccharide alterations in Escherichia coli: incorporation of glucuronic acid and phosphoethanolamine in the heptose region

Gracjana Klein et al. J Biol Chem. .

Abstract

It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn(2+).

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Proposed LPS structures from E. coli K12 in phosphate-limiting growth conditions. Schematic drawing of LPS glycoforms I, V, VI, and VII composition with various nonstoichiometric substitutions in the LPS core region is presented. Glycoforms VI and VII have GlcUA addition on the HepIII. The cognate genes, whose products are involved at different steps, are indicated.
FIGURE 2.
FIGURE 2.
Chemical structure of the main oligosaccharide from deacylated E. coli B LPS.
FIGURE 3.
FIGURE 3.
WaaH is required for the incorporation of the glucuronic acid. Charge-deconvoluted ESI FT-MS spectrum in negative ion mode of LPS obtained from the wild-type (A) and isogenic ΔwaaQ (B), ΔwaaY (C), and ΔwaaH (D) strains. LPS was extracted from cultures grown at 37 °C in the phosphate-limiting medium. The mass numbers refer to monoisotopic peaks. The predicted composition with varying numbers of substitutions of P-EtN and with Ara4N substitution is indicated. Mass peaks corresponding to the glycoform containing the third Kdo are marked as rectangular boxes and glycoform I with complete core derivatives as circles. Glycoforms VI and VII containing GlcUA are marked as GlcUA-P except in the case of ΔwaaY (C). Note that the mass peaks representing glycoforms corresponding to the presence of the GlcUA (4298.9 and 4489.9 Da) are absent in the LPS of ΔwaaY (D).
FIGURE 4.
FIGURE 4.
Requirement of the PhoB/R two-component system for the incorporation of P-EtN on the first heptose. Mass spectra of LPS obtained from phosphate-limiting growth conditions of the wild type (A), its isogenic derivatives ΔphoB (B), Δ(eptB basR) (C), and Δ(eptB basR phoB) (D) are depicted. Charge-deconvoluted ESI FT-MS spectra in negative ion mode are presented. The mass numbers refer to monoisotopic peaks with proposed composition. Unlabeled mass peaks mostly correspond to Na+ and/or with phosphate adducts. Mass peaks corresponding to glycoform containing the third Kdo are marked as rectangular boxes and glycoform I with complete core derivatives as circles.
FIGURE 5.
FIGURE 5.
Addition of ammonium metavanadate inducing the waaH transcription reveals the incorporation of GlcUA in different E. coli core types. Mass spectra of LPS were obtained from the wild-type E. coli K12 strain W3110 (A), E. coli strain F756 representing R2 core type (B), and E. coli strain F2513, representing R4 core type (C). LPS was extracted from culture grown in LB medium supplemented by 25 mm ammonium metavanadate at 37 °C and incubated with shaking for 24 h. Charge-deconvoluted ESI FT-MS spectra in negative ion mode are presented. The mass numbers refer to monoisotopic peaks with proposed composition.
FIGURE 6.
FIGURE 6.
Growth phase-dependent activity of the waaH promoter in phosphate-limiting conditions (A), which requires induction of the PhoB/R two-component system (C). Cultures of E. coli wild-type strain GK1111 carrying single copy chromosomal waaH-lacZ promoter fusion or its isogenic derivative with ΔbasR or ΔphoB mutation were grown to early log phase in LB medium at 37 °C, washed, and adjusted to an A595 of 0.02 in either LB medium or phosphate-limiting 121 medium. Aliquots of samples were drawn every 30 min and analyzed for β-galactosidase activity. Data corresponding to phosphate-limiting growth conditions are marked 121 in each case. The experiments were performed on four independent transductants. Error bars represent S.E. of four such cultures. B and D correspond to A595 indicating growth corresponding to different time intervals in which the β-galactosidase activity assay was performed.
FIGURE 7.
FIGURE 7.
Growth phase-dependent transcriptional activity of the promoter of the ugd gene in phosphate-limiting conditions (A), which requires the functional presence of PhoB/R and BasS/R two-component systems (C). Cultures of E. coli wild-type strain carrying single copy chromosomal ugd-lacZ promoter fusion or its isogenic derivative with ΔbasR or ΔphoB mutation were grown to early log phase in LB medium at 37 °C, washed, and adjusted to an A595 of 0.02 in either LB medium or phosphate-limiting 121 medium. Aliquots of samples were drawn every 30 min and analyzed for β-galactosidase activity. Data corresponding to phosphate-limiting growth conditions are marked 121 in each case. The experiments were performed on four independent isolates. Error bars represent S.E. of four such cultures. B and D correspond to A595 indicating growth corresponding to different time intervals in which the β-galactosidase activity assay was performed.
FIGURE 8.
FIGURE 8.
Incorporation of P-EtN on HepI requires the functional presence of the eptC gene. Mass spectra of LPS obtained from the wild-type strain W3110 (A) and its isogenic derivatives lacking eptC (B), Δ(eptA eptC) (C), and Δ(eptA eptB eptC) (D) are depicted. Cultures were grown at 37 °C in phosphate-limiting medium, and LPS was extracted under identical conditions. Charge-deconvoluted ESI FT-MS spectra in the negative ion mode are presented. The mass numbers refer to monoisotopic peaks with proposed composition.
FIGURE 9.
FIGURE 9.
Induction of transcription of the eptC promoter requires induction of the PhoB/R two-component system and the presence of Zn2+. Cultures of E. coli wild-type strain GK1111 carrying single copy chromosomal eptC-lacZ promoter fusion or its isogenic derivative with ΔbasR or ΔphoB mutations were grown to early log phase in LB medium at 37 °C, washed, and adjusted to an A595 of 0.02 in either M9 or phosphate-limiting 121 medium. Aliquots of samples were drawn at 35-min intervals and analyzed for the β-galactosidase activity. Data corresponding to phosphate-limiting growth conditions are marked 121 and phosphate-rich as M9 (A). The experiments were performed on four independent isolates. B, data with and without supplementation by Zn2+ in the phosphate-limiting 121 medium are presented.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nikaido H. (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 - PMC - PubMed
    1. Raetz C. R., Whitfield C. (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700 - PMC - PubMed
    1. Gronow S., Xia G., Brade H. (2010) Glycosyltransferases involved in the biosynthesis of the inner core region of different lipopolysaccharides. Eur. J. Cell Biol. 89, 3–10 - PubMed
    1. Amor K., Heinrichs D. E., Frirdich E., Ziebell K., Johnson R. P., Whitfield C. (2000) Distribution of core oligosaccharide types in lipopolysaccharides from Escherichia coli. Infect. Immun. 68, 1116–1124 - PMC - PubMed
    1. Vinogradov E. V., Van Der Drift K., Thomas-Oates J. E., Meshkov S., Brade H., Holst O. (1999) The structures of the carbohydrate backbones of the lipopolysaccharides from Escherichia coli rough mutants F470 (R1 core type) and F576 (R2 core type). Eur. J. Biochem. 261, 629–639 - PubMed

MeSH terms

LinkOut - more resources

-