The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains D-glycero-D-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe2+/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVA
in vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.
Ko formation; Kdo hydroxylase; LPS inner core assembly; Burkholderia LPS Fe2+/O2/α-KG dependent dioxygenase
Bacterial lipases play important roles in bacterial metabolism and environmental response. Our laboratory recently discovered that a novel lipoprotein lysophospholipase, VolA, localizes on the surface of the Gram-negative aquatic pathogen Vibrio cholerae. VolA functions to cleave exogenous lysophosphatidylcholine, freeing the fatty acid moiety for use by V. cholerae. This fatty acid is transported into the cell and can be used as a nutrient and, more importantly, as a way to alter the membrane architecture via incorporation into the phospholipid biosynthesis pathway. There are few examples of Gram-negative, surface-exposed lipoproteins, and VolA is unique, as it has a previously undercharacterized function in V. cholerae membrane remodeling. Herein, we report the biochemical characterization of VolA. We show that VolA is a canonical lipoprotein via mass spectrometry analysis and demonstrate the in vitro activity of VolA under a variety of conditions. Additionally, we show that VolA contains a conserved Gly-Xaa-Ser-Xaa-Gly motif typical of lipases. Interestingly, we report the observation of VolA homologs in other aquatic pathogens. An Aeromonas hydrophila VolA homolog complements a V. cholerae VolA mutant in growth on lysophosphatidylcholine as the sole carbon source and in enzymatic assays. These results support the idea that the lipase activity of surface-exposed VolA likely contributes to the success of V. cholerae, improving the overall adaptation and survival of the organism in different environments.
Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from
Haloarcula marismortui, Halobacterium salinarum, and
Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform.
Two phosphorus-containing glycolipids have previously been observed in Clostridium acetobutylicum. We had shown that the concentration of one of them increases in response to increased unsaturation of the membrane lipid hydrocarbon chains, suggesting a potential role in the regulation of lipid polymorphism in this organism. Mass spectrometry shows that these glycolipids are ethanolamine phosphate (Etn-P)-containing derivatives of a mono- and diglycosyldiradylglycerol. The content of both diglycosyldiradylglycerol and the Etn-P-monoglycosyldiradylglycerol, which increase upon increased unsaturation of the membrane, also increase upon addition of octanol to the medium. Thus, it appears that the Etn-P-monoglycosyldiradylglycerol along with the diglycosyldiradylglycerol may serve to stabilize the membrane bilayer during membrane stress caused by the presence of the solvents produced during fermentation.
glycolipid; plasmalogen; phosphoglycolipid; solvent
We have examined the polar lipids of Clostridium psychrophilum, a recently characterized psychrophilic Clostridium isolated from an Antarctic microbial mat. Lipids were extracted from cells grown near the optimal growth temperature (+5 °C) and at −5 °C, and analyzed by two-dimensional thin layer chromatography and liquid chromatography coupled with mass spectrometry. The major phospholipids of this species are: cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol. Phosphatidylserine and lyso-phosphatidylethanolamine were found as minor components. The most abundant glycolipids are a monoglycosyldiradylglycerol (MGDRG) and a diglycosyldiradylglycerol (DGDRG). The latter was only seen in cells grown at −5 °C. An ethanolamine-phosphate derivative of N-acetylglucosaminyldiradylglycerol was seen in cells grown at −5 °C and an ethanolamine-phosphate derivative of MGDRG was found in cells grown at +5 °C. All lipids were present in both the all acyl and plasmalogen (alk-1′-enyl acyl) forms with the exception of PS and MGDRG, which were predominantly in the diacyl form. The significance of lipid changes at the two growth temperatures is discussed.
Phospholipid; Glycolipid; Phosphoglycolipid; Plasmalogen; Mass spectrometry
The S-layer glycoprotein is the sole component of the protein shell surrounding Haloferax volcanii cells. The deduced amino acid sequence of the S-layer glycoprotein predicts the presence of a C-terminal membrane-spanning domain. However, several earlier observations, including the ability of EDTA to selectively solubilize the protein, are inconsistent with the presence of a trans-membrane sequence. In the present report, sequential solubilization of the S-layer glycoprotein by EDTA and then with detergent revealed the existence of two distinct populations of the S-layer glycoprotein. Whereas both S-layer glycoprotein populations underwent signal peptide cleavage and N-glycosylation, base hydrolysis followed by mass spectrometry revealed that a lipid, likely archaetidic acid, modified only the EDTA-solubilized version of the protein. These observations are consistent with the S-layer glycoprotein being initially synthesized as an integral membrane protein and subsequently undergoing a processing event in which the extracellular portion of the protein is separated from the membrane-spanning domain and transferred to a waiting lipid moiety.
Archaea; Haloferax volcanii; lipid modification; membrane protein; S-layer glycoprotein
MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg2+ within the active site, and provide a structural basis of catalysis for this class of enzyme.
Mast cells play a critical role in the pathogenesis of allergic diseases. How mast cell function is regulated is still not well understood. Both phosphatidic acid (PA) and diacylglycerol (DAG) are important second messengers involved in mast cell activation. Lipin1 is a phosphatidate phosphatase that hydrolyzes PA to produce DAG. The role of lipin1 in mast cell function has been unknown. In this report, we show that lipin1 is an important and selective inhibitor of mast cell degranulation. Lipin1 deficiency enhanced FcεRI-mediated β-hexosaminidase and prostaglandin D2 release from mast cells in vitro and exacerbated the passive systemic anaphylaxis reaction in vivo. However, Lipin1 deficiency did not exert obvious effects on IL-6 or TNF-α production following FcεRI engagement. FcεRI-induced PKC and SNAP-23 phosphorylation was augmented in the lipin1-deficient mast cells. Moreover, inhibition of PKC activity reduced SNAP-23 phosphorylation and mast cell degranulation in lipin1 deficient mast cells. Together, our findings suggest that lipin1 may negatively control mast cell degranulation and anaphylactic response through inhibiting the PKC-SNAP-23 pathway.
Mast cells; lipin1; Phosphatidic acid; PKC; SNAP-23
N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile Haloferax volcanii have offered insight into the archaeal version of this universal protein-processing event. In the present study, AglQ was identified as a novel component of the pathway responsible for the assembly and addition of a pentasaccharide to select Asn residues of Hfx. volcanii glycoproteins, such as the S-layer glycoprotein. In cells deleted of aglQ, both dolichol phosphate, the lipid carrier used in Hfx. volcanii N-glycosylation, and modified S-layer glycoprotein Asn residues only presented the first three pentasaccharide subunits, pointing to a role for AglQ in either preparing the third sugar for attachment of the fourth pentasaccharide subunit or processing the fourth sugar prior to its addition to the lipid-linked trisaccharide. To better define the precise role of AglQ, shown to be a soluble protein, bioinformatics tools were recruited to identify sequence or structural homologs of known function. Site-directed mutagenesis experiments guided by these predictions identified residues important for AglQ function. The results obtained point to AglQ acting as an isomerase in Hfx. volcanii N-glycosylation.
N-glycosylation in Archaea presents aspects of this posttranslational modification not seen in either Eukarya or Bacteria. In the haloarchaeon Haloferax volcanii, the surface (S)-layer glycoprotein can be simultaneously modified by two different N-glycans. Asn-13 and Asn-83 are modified by a pentasaccharide, whereas Asn-498 is modified by a tetrasaccharide of distinct composition, with N-glycosylation at this position being related to environmental conditions. Specifically, N-glycosylation of Asn-498 is detected when cells are grown in the presence of 1.75 but not 3.4 M NaCl. While deletion of genes encoding components of the pentasaccharide assembly pathway had no effect on the biosynthesis of the tetrasaccharide bound to Asn-498, deletion of genes within the cluster spanning HVO_2046 to HVO_2061 interfered with the assembly and attachment of the Asn-498-linked tetrasaccharide. Transfer of the “low-salt” tetrasaccharide from the dolichol phosphate carrier upon which it is assembled to S-layer glycoprotein Asn-498 did not require AglB, the oligosaccharyltransferase responsible for pentasaccharide attachment to Asn-13 and Asn-83. Finally, although biogenesis of the low-salt tetrasaccharide is barely discernible upon growth at the elevated salinity, this glycan was readily detected under such conditions in strains deleted of pentasaccharide biosynthesis pathway genes, indicative of cross talk between the two N-glycosylation pathways.
In the haloarchaeon Haloferax volcanii, originally from the Dead Sea, the pathway responsible for the assembly and attachment of a pentasaccharide to the S-layer glycoprotein, a well-studied glycoprotein in this species, has been described. More recently, it was shown that in response to growth in low salinity, the same glycoprotein is modified by a novel tetrasaccharide. In the present study, numerous components of the pathway used to synthesize this “low-salt” tetrasaccharide are described. As such, this represents the first report of two N-glycosylation pathways able to simultaneously modify a single protein as a function of environmental salinity. Moreover, and to the best of our knowledge, the ability to N-glycosylate the same protein with different and unrelated glycans has not been observed in either Eukarya or Bacteria or indeed beyond the halophilic archaea, for which similar dual modification of the Halobacterium salinarum S-layer glycoprotein was reported.
Recent studies of Haloferax volcanii have begun to elucidate the steps of N-glycosylation in Archaea, where this universal post-translational modification remains poorly described. In Hfx. volcanii, a series of Agl proteins catalyzes the assembly and attachment of a N-linked pentasaccharide to the S-layer glycoprotein. Although roles have been assigned to the majority of Agl proteins, others await description. In the following, the contribution of AglR to N-glycosylation was addressed.
A combination of bioinformatics, gene deletion, mass spectrometry and metabolic radiolabeling served to show a role for AglR in archaeal N-glycosylation at both the dolichol phosphate and reporter glycoprotein levels.
The modified behavior of the S-layer glycoprotein isolated from cells lacking AglR points to an involvement of this protein in N-glycosylation. In cells lacking AglR, glycan-charged dolichol phosphate, including mannose-charged dolichol phosphate, accumulates. At the same time, the S-layer glycoprotein does not incorporate mannose, the final subunit of the N-linked pentasaccharide decorating this protein. AglR is a homologue of Wzx proteins, annotated as flippases responsible for delivering lipid-linked O-antigen precursor oligosaccharides across the bacterial plasma membrane during lipopolysaccharide biogenesis.
The effects resulting from aglR deletion are consistent with AglR interacting with dolichol phosphate-mannose, possibly acting as a dolichol phosphate-mannose flippase or contributing to such activity.
Little is known of how lipid-linked oligosaccharides are translocated across membrane during N-glycosylation. The possibility of Hfx. volcanii AglR mediating or contributing to flippase activity could help address this situation.
Archaea; Dolichylphosphate-mannose; Haloferax volcanii; N-glycosylation; S-layer glycoprotein
Clostridium botulinum has been classified into four groupings (groups I to IV) based on physiological characteristics and 16S rRNA sequencing. We have examined the lipid compositions of 11 representative strains of C. botulinum and a strain of Clostridium sporogenes by 2D-TLC and by MS. All strains contained phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylethanolamine (PE) in both the all-acyl and the alk-1′-enyl (plasmalogen) forms. Five strains in proteolytic group I, which are related to C. sporogenes, contained varying amounts of an ethanolamine-phosphate derivative of N-acetylglucosaminyl-diradylglycerol, which is also present in C. sporogenes. Three strains in group II, which are related to Clostridium butyricum, Clostridium beijerinckii and Clostridium acetobutylicum, contained lipids characteristic of these saccharolytic species: a glycerol acetal and a PG acetal of the plasmalogen form of PE. Two group III strains, which are related to Clostridium novyi, contained amino-acyl derivatives of PG, which are also found in C. novyi. A strain in group IV had PE, PG and CL, but none of the distinguishing lipids. This work shows that the lipidome of C. botulinum is consistent with its classification by other methods.
Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. It has been shown that OLs can be hydroxylated within the amide-linked fatty acyl moiety, the secondary fatty acyl moiety or within the ornithine moiety. These modifications have been related to increased stress tolerance and symbiotic proficiency in different organisms such as Rhizobium tropici or Burkholderia cenocepacia. Analysing the membrane lipid composition of the plant pathogen Agrobacterium tumefaciens we noticed that it forms two different OLs. In the present work we studied if OLs play a role in stress tolerance and pathogenicity in A. tumefaciens. Mutants deficient in the OLs biosynthesis genes olsB or olsE were constructed and characterized. They either completely lack OLs (ΔolsB) or only form the unmodified OL (ΔolsE). Here we present a characterization of both OL mutants under stress conditions and in a plant transformation assay using potato tuber discs. Surprisingly, the lack of agrobacterial OLs promotes earlier tumour formation on the plant host.
In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of Hfx. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C55 and C60 dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C60 dolichol phosphate than with C55 dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.
Archaea; dolichol phosphate; geranylgeranyl reductase; Haloferax volcanii; isoprene; reductase
Previous work from our laboratory showed that the Gram-negative aquatic pathogen Vibrio cholerae can take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability of V. cholerae to exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling the V. cholerae membrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrio outer membrane lysophospholipase A), required for these activities. The enzyme is well conserved in Vibrio species, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling.
Our understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogen Vibrio cholerae to utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.
VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV-1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β-elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4-derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5-N-formyllegionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid-like sugar, in an archaeal-derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N-acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC-ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4-derived peptides bearing the N-glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N-glycosylation apparatus, as determined by LC-ESI/MS of heterologously expressed VP4. Here, however, the N-linked pentasaccharide was the same as shown to decorate the S-layer glycoprotein in this species. Hence, N-glycosylation of the haloarchaeal viral protein, VP4, is host-specific. These results thus present additional examples of archaeal N-glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.
Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls– bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H+-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.
Osmotic stress; Bacteria; Cardiolipin; ProP; MscL
Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli, phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of Escherichia coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H]- ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.
mass spectrometry; E. coli; lipids; chloroform; phosgene; artifacts
Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of the α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.
Archaea; dolichol; electrospray ionization mass spectrometry; polyprenol; polyprenol reductase; Sulfolobus acidocaldarius
The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains.
Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs.
Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM.
Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.
Across evolution, dolichols and polyprenols serve as sugar carriers in biosynthetic processes that include protein glycosylation and lipopolysaccharide biogenesis. Liquid chromatography coupled with electrospray ionization mass spectrometry offers a powerful tool for studying dolichols and polyprenols in their alcohol or glycan-modified forms in members of all three domains of life. In the following, recent examples of the how different versions of this analytical approach, namely reverse phase liquid chromatography-multiple reaction monitoring, normal phase liquid chromatography/tandem mass spectrometry and normal phase liquid chromatography-precursor ion scan detection have respectively served to address novel aspects of dolichol or polyprenol biology in Eukarya, Archaea and Bacteria. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.
Liquid chromatography/tandem mass; spectrometry; Multiple reaction monitoring; Precursor ion scan; Dolichol; Polyprenol
To cope with life in hypersaline environments, halophilic archaeal proteins are enriched in acidic amino acids. This strategy does not, however, offer a response to transient changes in salinity, as would post-translational modifications. To test this hypothesis, N-glycosylation of the Haloferax volcanii S-layer glycoprotein was compared in cells grown in high (3.4 M NaCl) and low (1.75 M NaCl) salt, as was the glycan bound to dolichol phosphate, the lipid upon which the N-linked glycan is assembled. In high salt, S-layer glycoprotein Asn-13 and Asn-83 are modified by a pentasaccharide, while dolichol phosphate is modified by a tetrasaccharide comprising the first four pentasaccharide residues. When the same targets were considered from cells grown in low salt, substantially less pentasaccharide was detected. At the same time, cells grown at low salinity contain dolichol phosphate modified by a distinct tetrasaccharide absent in cells grown at high salinity. The same tetrasaccharide modified S-layer glycoprotein Asn-498 in cells grown in low salt, whereas no glycan decorated this residue in cells grown in the high-salt medium. Thus, in response to changes in environmental salinity, Hfx. volcanii not only modulates the N-linked glycans decorating the S-layer glycoprotein but also the sites of such post-translational modification.
Archaeal glycoproteins present a variety of N-linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific functions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Haloferax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S-layer glycoprotein is modified by an N-linked pentasaccharide. In the following, S-layer glycoprotein N-glycosylation was considered in cells in which AglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD and introduction of the AglJ homologue from Halobacterium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer glycoprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering ‘workshop’.
Recent insight into the N-glycosylation pathway of the haloarchaeon, Haloferax volcanii, is helping to bridge the gap between our limited understanding of the archaeal version of this universal post-translational modification and the better-described eukaryal and bacterial processes. To delineate as yet undefined steps of the Hfx. volcanii N-glycosylation pathway, a comparative approach was taken with the initial characterization of N-glycosylation in Haloarcula marismortui, a second haloarchaeon also originating from the Dead Sea. While both species decorate the reporter glycoprotein, the S-layer glycoprotein, with the same N-linked pentasaccharide and employ dolichol phosphate as lipid glycan carrier, species-specific differences in the two N-glycosylation pathways exist. Specifically, Har. marismortui first assembles the complete pentasaccharide on dolichol phosphate and only then transfers the glycan to the target protein, as in the bacterial N-glycosylation pathway. In contrast, Hfx. volcanii initially transfers the first four pentasaccharide subunits from a common dolichol phosphate carrier to the target protein and only then delivers the final pentasaccharide subunit from a distinct dolichol phosphate to the N-linked tetrasaccharide, reminiscent of what occurs in eukaryal N-glycosylation. This study further indicates the extraordinary diversity of N-glycosylation pathways in Archaea, as compared with the relatively conserved parallel processes in Eukarya and Bacteria.
PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1-deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.