The lipid A palmitoyltransferase PagP: molecular mechanisms and role in bacterial pathogenesis

Palmitoylated lipid A can both protect pathogenic bacteria from host immune defences and attenuate the activation of those same defences through the TLR4 signal transduction pathway. A palmitate chain from a phospholipid is incorporated into lipid A by an outer membrane enzyme PagP, which is an 8-stranded antiparallel beta-barrel preceded by an amino-terminal amphipathic alpha-helix. The PagP barrel axis is tilted by 25 degrees with respect to the membrane normal. An interior hydrophobic pocket in the outer leaflet-exposed half of the molecule functions as a hydrocarbon ruler that allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen-bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. The mechanisms by which bacteria regulate pagP gene expression strikingly reflect the corresponding pathogenic lifestyle of the bacterium. Variations on PagP structure and function can be illustrated with the known homologues from Gram-negative bacteria, which include pathogens of humans and other mammals in addition to pathogens of insects and plants. The PagP enzyme is potentially a target for the development of anti-infective agents, a probe of outer membrane lipid asymmetry, and a tool for the synthesis of lipid A-based vaccine adjuvants and endotoxin antagonists.     

Gauging a hydrocarbon ruler by an intrinsic exciton probe

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.     

A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis‐specific lipid A

Strains of Pseudomonas aeruginosa (PA) isolated from the airways of cystic fibrosis patients constitutively add palmitate to lipid A, the membrane anchor of lipopolysaccharide. The PhoPQ regulated enzyme PagP is responsible for the transfer of palmitate from outer membrane phospholipids to lipid A. This enzyme had previously been identified in many pathogenic Gram‐negative bacteria, but in PA had remained elusive, despite abundant evidence that its lipid A contains palmitate. Using a combined genetic and biochemical approach, we identified PA1343 as the PA gene encoding PagP. Although PA1343 lacks obvious primary structural similarity with known PagP enzymes, the β‐barrel tertiary structure with an interior hydrocarbon ruler appears to be conserved. PA PagP transfers palmitate to the 3′ position of lipid A, in contrast to the 2 position seen with the enterobacterial PagP. Palmitoylated PA lipid A alters host innate immune responses, including increased resistance to some antimicrobial peptides and an elevated pro‐inflammatory response, consistent with the synthesis of a hexa‐acylated structure preferentially recognized by the TLR4/MD2 complex. Palmitoylation commonly confers resistance to cationic antimicrobial peptides, however, increased cytokine production resulting in inflammation is not seen with other palmitoylated lipid A, indicating a unique role for this modification in PA pathogenesis.     

Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli

The biogenesis of biological membranes hinges on the coordinated trafficking of membrane lipids between distinct cellular compartments. The bacterial outer membrane enzyme PagP confers resistance to host immune defenses by transferring a palmitate chain from a phospholipid to the lipid A (endotoxin) component of lipopolysaccharide. PagP is an eight-stranded antiparallel beta-barrel, preceded by an N-terminal amphipathic alpha-helix. The active site is localized inside the beta-barrel and is aligned with the lipopolysaccharide-containing outer leaflet, but the phospholipid substrates are normally restricted to the inner leaflet of the asymmetric outer membrane. We examined the possibility that PagP activity in vivo depends on the aberrant migration of phospholipids into the outer leaflet. We find that brief addition to Escherichia coli cultures of millimolar EDTA, which is reported to replace a fraction of lipopolysaccharide with phospholipids, rapidly induces palmitoylation of lipid A. Although expression of the E. coli pagP gene is induced during Mg2+ limitation by the phoPQ two-component signal transduction pathway, EDTA-induced lipid A palmitoylation occurs more rapidly than pagP induction and is independent of de novo protein synthesis. EDTA-induced lipid A palmitoylation requires functional MsbA, an essential ATP-binding cassette transporter needed for lipid transport to the outer membrane. A potential role for the PagP alpha-helix in phospholipid translocation to the outer leaflet was excluded by showing that alpha-helix deletions are active in vivo. Neither EDTA nor Mg(2+)-EDTA stimulate PagP activity in vitro. These findings suggest that PagP remains dormant in outer membranes until Mg2+ limitation promotes the migration of phospholipids into the outer leaflet.     

Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His(6)-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of YERSINIA: and BORDETELLA: species. PagP may provide an adaptive response toward both Mg(2+) limitation and host innate immune defenses.     

One membrane protein,two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane β-barrel and an N-terminal α-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal α-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP β-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.     

The N-terminal helix is a post-assembly clamp in the bacterial outer membrane protein PagP

The Escherichia coli outer membrane beta-barrel enzyme PagP and its homologues are unique in that the eight-stranded barrel is tilted by about 25 degrees with respect to the membrane normal and is preceded by a 19-residue amphipathic alpha-helix. To investigate the role of this helix in the folding and stability of PagP, mutants were generated in which the helix was deleted (Delta(1-19)), or in which residues predicted to be involved in helix-barrel interactions were altered (W17A or R59L). The ability of the variants to insert into detergent micelles or liposomes was studied in vitro using circular dichroism, fluorescence, Fourier transform infrared spectroscopy, electrophoretic mobility and gain of enzyme activity. The data show that PagP, initially unfolded in 5% (w/v) perfluoro-octanoic acid or 6 M guanidinium chloride, inserts spontaneously and folds quantitatively to an active conformation into detergent micelles of cyclofos-7 or into large vesicles of diC(12:0)-phosphatidylcholine (diC(12:0)PC), respectively, the latter in the presence of 7 M urea. Successful refolding of all variants into both micelles and liposomes ruled out an essential role for the helix or helix-barrel interactions in folding and membrane insertion. Measurements of thermal stability indicated that the variants R59L, W17A/R59L and Delta(1-19) were destabilised substantially compared with wild-type PagP. However, in contrast to the other variants, destabilisation of the W17A variant relative to wild-type PagP was much greater in liposomes than in micelles. Analysis of the kinetics of folding and unfolding of all variants in diC(12:0)PC liposomes suggested that this destabilisation arises predominantly from an increased dissociation of the refolded variant proteins from the lipid-inserted state. The data support the view that the helix of PagP is not required for folding and assembly, but instead acts as a clamp, stabilising membrane-inserted PagP after folding and docking with the membrane are complete.     

Structural biology of membrane-intrinsic beta-barrel enzymes: sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel beta-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three beta-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipidColon, two colonslipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane beta-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two beta-barrel enzymes of unknown structure; namely, the Salmonella enterica 3'-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O(2) to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how beta-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger beta-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7

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.     

Bordetella parapertussis PagP Mediates the Addition of Two Palmitates to the Lipopolysaccharide Lipid A

Bordetella bronchiseptica PagP (PagP_BB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagP_BPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagP_BPa is required for the modification of lipid A with palmitate. While PagP_BB transfers a single palmitate to the lipid A C-3′ position, PagP_BPa transfers palmitates to the lipid A C-2 and C-3′ positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagP_BPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.     

Molecular dynamics simulations of the Apo-, Holo-, and acyl-forms of Escherichia coli acyl carrier protein

Acyl carrier protein (ACP) is an essential co-factor protein in fatty acid biosynthesis that shuttles covalently bound fatty acyl intermediates in its hydrophobic pocket to various enzyme partners. To characterize acyl chain-ACP interactions and their influence on enzyme interactions, we performed 19 molecular dynamics (MD) simulations of Escherichia coli apo-, holo-, and acyl-ACPs. The simulations were started with the acyl chain in either a solvent-exposed or a buried conformation. All four short-chain (< or = C10) and one long-chain (C16) unbiased acyl-ACP MD simulation show the transition of the solvent-exposed acyl chain into the hydrophobic pocket of ACP, revealing its pathway of acyl chain binding. Although the acyl chain resides inside the pocket, Thr-39 and Glu-60 at the entrance stabilize the phosphopantetheine linker through hydrogen bonding. Comparisons of the different ACP forms indicate that the loop region between helices II and III and the prosthetic linker may aid in substrate recognition by enzymes of fatty acid synthase systems. The MD simulations consistently show that the hydrophobic binding pocket of ACP is best suited to accommodate an octanoyl group and is capable of adjusting in size to accommodate chain lengths as long as decanoic acid. The simulations also reveal a second, novel binding mode of the acyl chains inside the hydrophobic binding pocket directed toward helix I. This study provides a detailed dynamic picture of acyl-ACPs that is in excellent agreement with available experimental data and, thereby, provides a new understanding of enzyme-ACP interactions.     

Species-Specific Activation of TLR4 by Hypoacylated Endotoxins Governed by Residues 82 and 122 of MD-2

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.     

Lipid chain selectivity by outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is a unique, integral membrane enzyme found in Gram-negative bacteria and is an important virulence factor for pathogens such as Helicobacter pylori. This broad-specificity lipase degrades a variety of lipid substrates, and it plays a direct role in adjusting the composition and permeability of bacterial membranes under conditions of stress. Interestingly, OMPLA shows little preference for the lipid headgroup and, instead, the length of the hydrophobic acyl chain is the strongest determinant for substrate selection by OMPLA, with the enzyme strongly preferring substrates with chains equal to or longer than 14 carbon atoms. The question remains as to how a hydrophobic protein like OMPLA can achieve this specificity, particularly when the shorter chains can be accommodated in the binding pocket. Using a series of sulfonyl fluoride inhibitors with various lengths of acyl chain, we show here that the thermodynamics of substrate-induced OMPLA dimerization are guided by the acyl chain length, demonstrating that OMPLA uses a unique biophysical mechanism to select its phospholipid substrate.     

Spontaneous formation of detergent micelles around the outer membrane protein OmpX

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of approximately 18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic beta-barrel of OmpX rather than in a micellar-like oblate; approximately 40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the beta-barrel region. The phospholipids bind also on the extracellular, protruding beta-sheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, approximately 90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding beta-sheet as a "fishing rod".     

Structural enzymological studies of 2-enoyl thioester reductase of the human mitochondrial FAS II pathway: new insights into its substrate recognition properties

Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 Å resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

Comparative studies of detergent effects on the calcium adenosine triphosphatase of sarcoplasmic reticulum: protein isolation, lipid exchange and enzyme transport function.

The detergent effects of lysolecithin, Triton X-100, and cholate were compared in the calcium transport ATPase system of sarcoplasmic reticulum. Lysolecithin was found to act as a detergent in releasing the ATPase for subsequent purification, but did not strongly promote exchange of membrane lipid classes. Both Triton and cholate promoted exchange of membrane phospholipid. Higher concentrations of Triton and cholate inhibited the ATPase activity, but the enzyme could be reactivated by addition of phospholipid or fatty acid directly to the mixture. Under these conditions, reactivation depended on the presence of lipid acyl chains, rather than specific head groups. It was also found that Triton could be readily removed from the mixture by passing the enzyme through a hydrophobic bead column. Calcium transport was reactivated in the resulting membranes.     

Dynamic protein palmitoylation in cellular signaling

Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostasis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions.     

CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.     

Topology of the porin MspA in the outer membrane of Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.