Subsystem: Lipid A modifications

This subsystem's description is:

The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens.

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Literature ReferencesA phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant. Reynolds CM The Journal of biological chemistry 2005 Jun 315795227
Lipid A modification systems in gram-negative bacteria. Raetz CR Annual review of biochemistry 200717362200
DiagramFunctional RolesSubsystem SpreadsheetDescriptionAdditional Notes 

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PagPPagLLpxOPhoQPhoPPmrAPmrBEptAEptBYijP
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The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens.
Although the overall lipid A structure is relatively conserved among various bacteria, its structure is modified in response to the local environment, which, in turn, results in changes in the outer surface of the bacterium. In general, modifications of the lipid A structure include the removal or decoration of the lipid A phosphates and fatty acyl chains (ref.1,8).
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E. coli K-12 and S. typhimurium contain enzymes for modifying lipid A with phosphoethanolamine , L-Ara4N (see separate subsystem) and/or palmitate. Two selective deacylases and a dioxygenase are also present in S. typhimurium. Many of these enzymes are regulated in response to changes in growth conditions. For instance, the addition of palmitate by PagP and the removal of an acyl chain by PagL can be activated by cationic antimicrobial peptides acting through the PhoP transcription factor. Changes to the acylation pattern of lipid A can provide resistance to some cationic antimicrobial peptides and/or attenuate the endotoxic properties of lipid A. The attachment of phosphoethanolamine by the enzyme EptA and L-Ara4N by the enzyme ArnT is induced by activation of the PmrA transcription factor.

--------Palmitate Modification--------------------

Modification of the lipid A moiety of LPS with palmitate by PagP (CrcA) is under control of the PhoP/PhoQ system, which is activated by low Mg2+ concentrations or cationic antimicrobial peptides.
Two outer membrane enzymes, PagP and PagL, remodel lipid A. PagP catalyzes the addition of a phospholipid-derived palmitate chain to the hydroxyl of the R-3-hydroxymyristate chain at the 2 position of lipid A. PagL catalyzes the removal of the R-3-hydroxymyristate chain at position 3 of lipid A.

S.typhimurium contains several additional lipid A modification enzymes that are not present in wild-type E. coli K-12. PagL is an outer membrane lipase that is regulated by PhoP/PhoQ and removes the R-3-hydroxymyristoyl chain at position 3 of the lipid A moiety.
LpxR, a distinct outer membrane lipase, cleaves the intact 3'-acyloxyacyl moiety of Kdo2-lipid A.
In vitro studies have shown that LpxR activity is Ca2+ dependent and Kdo activated.LpxO is an inner membrane enzyme that hydroxylates the 3' secondary acyl chain of Kdo2-lipid A in the presence of O, using Fe2+ and α-ketoglutarate as cofactors. LpxO is not under the control of PhoP/PhoQ, and its active site faces the cytoplasm.

------------Phosphoethanolamine transfer------------------------------
Phosphoethanolamine transfer to core-lipid A by the enzyme EptA, predominantly to the 1 phosphate group, likewise occurs on the outer surface of the inner membrane. Under certain growth conditions or in the absence of L-Ara4N, EptA can also modify the lipid A 4'-position with a second phosphoethanolamine moiety. Phosphatidylethanolamine serves as the phosphoethanolamine donor substrate.
E. coli and S. typhimurium both contain a related enzyme, designated EptB, which is homologous to EptA but is not regulated by PmrA. Instead, EptB is induced by the addition of 5 mM Ca2+ to the growth medium and is under the control of the σE transcription factor. EptB transfers a phosphoethanolamine moiety from phosphatidylethanolamine to the outer Kdo residue.
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Addition, removal, and modification of lipid A fatty acyl chains PagP, PagL, LpxO, PhoP/PhoQ system:
Much of the diversity seen in the lipid A structures from various Gram-negative bacteria is due to differences in their acylation patterns.
The lipid A of some pathogenic bacteria can be further modified by the addition of a palmitoyl group (C16), which results in a hepta-acylated lipid A structure ( ref.2).
PagP, an outer membrane serine hydrolase , transfers the palmitate residue from the sn-1 position of phospholipids to the lipid A anchor of LPS. It has been shown in S. typhimurium that PagP is under the control of the PhoP/PhoQ two-component regulatory system that is activated during Mg2+ limitation ( ref.3).
In S. typhimurium, PhoP controls two additional enzymes, not found in E. coli, that modify its lipid A PagL and LpxO.
The outer membrane-localized lipid A 3-O-deacylase (PhoP activated lipase), encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified ( ref.4 ). Recently the presence of pagL homologs in a variety of Gram-negative bacteria was described (ref.5). Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. The biological function of PagL remains unclear.
Also absent in E. coli is the PhoP-activated lpxO gene. The lipid A of certain Gram- LpxO is thought to function as an aspartyl/asparaginyl beta-hydroxylase that is required for the direct hydroxylation of the 3 secondary myristoyl chain of S. typhimurium lipid A (ref.6). Homologues of LpxO can be found in several pathogenic bacteria, including P. aeruginosa, B. pertussis, L. pneumophila, and Klebsiella pneumonia.
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Modification and (or) removal of the lipid A phosphate groups - PmrA, PmrB, PhoP/PhoQ system:

As is the case with the fatty acyl chains of lipid A, the 1-and 4-phosphates attached to the dissacharide backbone can be modified and (or) removed. A number of Gram-negative bacteria contain latent enzymes capable of modifying the lipid A phosphates with 4-amino-4-deoxy-L-arabinose (LAra4N) and (or) phosphoethanolamine (pEtN). In Salmonella, the modification of the lipid A phosphates occurs upon activation of the transcriptional regulatory protein PmrA. PmrA activation is induced by extracytoplasmic iron via the iron-sensing protein PmrB. Additionally, PmrA is activated by the PhoP/PhoQ system during Mg2+ starvation.
The synthesis and attachment of the L-Ara4N moiety requires PmrA activation of at least seven genes at the pmrE(ugd) and pmrHFIJKLM loci (pmrM is not required). Based on bioinformatic analyses of the pmr loci, a pathway for the biosynthesis of UDP-LAra4N and transfer of the L-Ara4N unit to lipid A was proposed that is now supported by a significant amount of biochemical evidence (ref.7).
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References:

1. Trent MS. Biosynthesis, transport, and modification of lipid A.
Biochem Cell Biol. 2004 Feb;82(1):71-86. Review.

1a. Raetz CR, Reynolds CM, Trent MS, Bishop RE. Lipid A modification systems in gram-negative bacteria.Annu Rev Biochem. 2007;76:295-329. Review. PMID: 17362200.

2. Bishop, R.E., Gibbons, H.S., Guina, T., Trent, M.S., Miller, S.I.,and Raetz, C.R. 2000. Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria. EMBOJ. 19: 50715080.

3. Hwang PM, Choy WY, Lo EI, Chen L, Forman-Kay JD, Raetz CR, Prive GG, Bishop RE, Kay LE. Solution structure and dynamics of the outer membrane enzyme PagP by NMR. Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13560-5.

4. Trent, M.S., Pabich, W., Raetz, C.R., and Miller, S.I. 2001a. A PhoP/PhoQ-induced lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium. J. Biol. Chem. 276: 90839092.

5. Geurtsen J, Steeghs L, Hove JT, van der Ley P, Tommassen J. Dissemination of lipid A deacylases (pagL) among gram-negative bacteria: identification of active-site histidine and serine residues. J Biol Chem. 2005 Mar 4;280(9):8248-59.

6. Gibbons HS; Lin S; Cotter RJ; Raetz CR. Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A. Function of LpxO, A new Fe2+/alpha-ketoglutarate-dependent dioxygenase homologue. 2000, J. Biol. Chem., 275, 32940-32949.

7. Zhou, Z., Lin, S., Cotter, R.J., and Raetz, C.R. 1999. Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate. J. Biol.Chem. 274: 18 503 18 514.

8. Raetz CR. Regulated covalent modifications of lipid A. J Endotoxin Res. 2001;7(1):73-8.

9. Reynolds CM, Kalb SR, Cotter RJ, Raetz CR. A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant.
J Biol Chem. 2005 Jun 3;280(22):21202-11.PMID: 15795227.