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Structural Features of Glycosyltransferases Synthesizing Major Bilayer and Nonbilayer-prone Membrane Lipids inAcholeplasma laidlawii and Streptococcus pneumoniae

2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês

10.1074/jbc.m211492200

1083-351X

Maria C. Edman, Stefan Berg, Patrik Storm, Malin Wikström, Susanne Vikström, Anders Öhman, Åke Wieslander,

Carbohydrate Chemistry and Synthesis

In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) α-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC2.4.1.157) and the (ii) α-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of theE. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes. In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) α-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC2.4.1.157) and the (ii) α-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of theE. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes. 1,2-diacyl-3-O-[α-d-glucopyranosyl-(1→2)-O-α-d-glucopyranosyl]-sn-glycerol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate 1,2-diacyl-3-O-(α-d-glucopyranosyl)-sn-glycerol 1,2-diacyl-3-O-[α-d-glucopyranosyl-(1→2)-O-α-d-galactopyranosyl]-sn-glycerol 3-DOG, 1,3-dioleoylglycerol 1,2-diacyl-3-O-(α-d-galactopyranosyl)-sn-glycerol 1,2-diacyl-3-O-[6-O-acyl(α-d-glucopyranosyl)]-sn-glycerol 1,2-diacyl-3-O-[α-d-glucopyranosyl-(1→2)-O-(6-O-acyl-α-d-glucopyranosyl)]-sn-glycerol disugar-glycolipid synthase monosugar-glycolipid synthase phosphatidylglycerol dioleoylphosphatidylglycerol dioleoylphosphatidic acid cardiolipin glycosyltransferase green fluorescent protein amino acids open reading frame Lipid bilayer properties, important for membrane barrier and protein function, are regulated at several levels in cells. Acholeplasma laidlawii andEscherichia coli membrane lipids are metabolically designed to yield a bilayer “window” with certain features between the gel and nonbilayer phases (1Lindblom G. Brentel I. Sjölund M. Wikander G. Wieslander Å. Biochemistry. 1986; 25: 16198-16207Google Scholar, 2Morein S. Andersson A.-S. Rilfors L. Lindblom G. J. Biol. Chem. 1996; 271: 6801-6809Google Scholar). In A. laidlawii this aims at maintaining (i) a certain lipid bilayer surface charge density and (ii) a constant radius of spontaneous curvature (elastic packing stress), including similar bilayer/nonbilayer transition temperatures (1Lindblom G. Brentel I. Sjölund M. Wikander G. Wieslander Å. Biochemistry. 1986; 25: 16198-16207Google Scholar, 3Österberg F. Rilfors L. Wieslander Å. Lindblom G. Gruner S.M. Biochim. Biophys. Acta. 1995; 1257: 18-24Google Scholar). Regulation of (i) and (ii) resides mainly at the polar headgroup level (4Wieslander Å. Karlsson O.P. Curr. Top. Membr. 1997; 44: 517-540Google Scholar), whereas in E. coli curvature is regulated at the acyl chain level (2Morein S. Andersson A.-S. Rilfors L. Lindblom G. J. Biol. Chem. 1996; 271: 6801-6809Google Scholar), with more or less constant headgroup composition. The bilayer-forming glucolipid α-diglucosyldiacylglycerol (DGlcDAG)1 is one of the major lipids in the small cell wall-less A. laidlawii, the other is the nonbilayer-prone α-monoglucosyldiacylglycerol (MGlcDAG),cf. pathway below. Another pathway leads from phosphatidic acid to phosphatidylglycerol (PG). Under certain circumstances more acylated, and more nonbilayer-prone, variants of MGlcDAG and DGlcDAG, i.e. MAMGlcDAG and MADGlcDAG, respectively, are synthesized (5Andersson A.-S. Rilfors L. Lewis R.N. McElhaney R.N. Lindblom F. Biochim. Biophys. Acta. 1998; 1389: 43-49Google Scholar). A. laidlawiican only synthesize saturated acyl chains, and to still be able to adapt to membrane stress and new environments, the amounts of the two major glucolipids are strongly adjusted to maintain a functional membrane bilayer. The MGlcDAG-synthesizing enzyme alMGS (EC 2.4.1.157) ((i) above) was recently cloned (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar) and found to be a surface-associating protein with no transmembrane segments, where bilayer binding was dependent on phosphatidylglycerol and stimulated by small amounts of nonbilayer lipids. 2L. Li, submitted for publication. This seemed logical given the strong involvement of this enzyme in the lipid surface charge regulation, i.e. (i) above (8Karlsson O.P. Dahlqvist A. Vikström S. Wieslander Å. J. Biol. Chem. 1997; 272: 929-936Google Scholar). The 1,2-diacylglycerol-3-α-glucose (1 → 2)-α-glucosyl transferase (DGS) (EC 2.4.1.208) catalyzes the consecutive transfer of glucose from UDP-Glc to MGlcDAG to yield DGlcDAG (cf. (ii) above). This reaction is activated in an essential manner by certain anionic lipids and stimulated by other additives promoting nonbilayer tendencies both in vivo (1Lindblom G. Brentel I. Sjölund M. Wikander G. Wieslander Å. Biochemistry. 1986; 25: 16198-16207Google Scholar, 9Wieslander Å. Rilfors L. Lindblom G. Biochemistry. 1986; 25: 7511-7517Google Scholar) and with crude or pure enzymes in vitro (10Vikström S. Li L. Karlsson O.P. Wieslander Å. Biochemistry. 1999; 38: 5511-5520Google Scholar, 11Dahlqvist A. Nordström S. Karlsson O.P. Mannock D.A. McElhaney R.N. Wieslander Å. Biochemistry. 1995; 34: 13381-13389Google Scholar), keeping or restoring bilayer packing conditions. Furthermore, an additional modulation of the activity is achieved by certain phosphorylated metabolites, double-stranded DNA (12Vikström S. Li L. Wieslander Å. J. Biol. Chem. 2000; 275: 9296-9302Google Scholar), and by a low redox potential (7Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Google Scholar). Due to their small polar headgroups, nonbilayer-prone lipids of the reversed type, like MGlcDAG, make the two monolayers in a bilayer each want to curl concavely toward the water phase. This induces a curvature elastic stress (increased spontaneous curvature), with an increased chain order and a closer approach to a bilayer-nonbilayer phase transition. Similar packing features can be inferred for the major galactolipids in the membranes of chloroplasts and photosynthetic bacteria (13Dörmann P. Benning C. Trends Plant Sci. 2002; 7: 112-118Google Scholar). A number of peripheral and integral membrane proteins are functionally affected by this lateral stress, but for none the exact mechanisms are known (14Ho C. Slater S.J. Stagliano B. Stubbs C.D. Biochemistry. 2001; 40: 10334-10341Google Scholar), although correlation with the lipid properties is very evident (e.g. Refs. 15Attard G.S. Templer R.H. Smith W.S. Hunt A.N. Jackowski S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9032-9036Google Scholar, 16Davies S.M.A. Epand R.M. Kraayenhof R. Cornell R.B. Biochemistry. 2001; 40: 10522-10531Google Scholar, 17Botelho A.V. Gibson N.J. Thurmond R.L. Wang Y. Brown M.F. Biochemistry. 2002; 41: 6354-6368Google Scholar). Hence, a question of large importance is how the A. laidlawii DGS is able to sense the curvature stress and respond by increased or decreased synthesis of DGlcDAG. The cloned gene (this work) yielded an active enzyme from E. coli, and the deduced amino acid sequence and the predicted secondary and three-dimensional structure models strongly indicate this enzyme to be, unexpectedly, very similar in structure and surface association as the preceding MGlcDAG synthase (glucosyltransferase) and several other glycosyltransferases. Hence, the bilayer/nonbilayer lipid balance, and the connected bilayer physical forces, are sensed at the bilayer surface by the DGlcDAG synthase. Data base searches revealed a new group of related lipid glycosyltransferases in Gram-positive bacteria and archaea. Functional cloning of CpoA from Streptococcus pneumoniae suggests this group to be responsible for synthesis of α-diglycosyldiacylglycerols in a number of species. However, CpoA responds differently to the properties of the surrounding bilayer, and the latter is substantiated by certain differences in the enzyme structure models. A. laidlawii strain A-EF22 was grown in 28 °C in tryptose/bovine serum albumin medium (11Dahlqvist A. Nordström S. Karlsson O.P. Mannock D.A. McElhaney R.N. Wieslander Å. Biochemistry. 1995; 34: 13381-13389Google Scholar), supplemented with 0.12 mm oleic acid (18:1c). The cells were harvested, and the DNA was prepared with the GenomicPrepTM kit (Amersham Biosciences). TheS. pneumoniae strain 19FCCUG 3030 was grown in Todd-Hewitt medium, pH 7.8 at 37 °C overnight. The DNA was prepared by heating the resuspended cell pellet at 95 °C for 10 min. After the subsequent centrifugation, the DNA was present in the supernatant. Degenerated DNA primers were made based on the N-terminal amino acid sequence of the purified A. laidlawii DGlcDAG synthase (alDGS) determined through Edman degradation (10Vikström S. Li L. Karlsson O.P. Wieslander Å. Biochemistry. 1999; 38: 5511-5520Google Scholar). An internal sequence was also determined after proteolytic cleavage and fragment purification by reverse phase-high performance liquid chromatography. The primers 5′-GGT CGT GCT TTT TAT CA(C/T) CA(A/G) AAA-3′ and 5′-AAT AAC AGC ACC (A/G)TC IAC (A/G)TG-3′ were used in a PCR amplification procedure that resulted in a ∼600-bp DNA fragment. The PCR products were purified and ligated into the pCR-Script SK(+) cloning vector (Stratagene) linearized withSrfI and then sequenced. The purified PCR product was also radioactively labeled by α-35S-dATP and used as a probe in a Southern blot procedure with completelyHindIII/EcoRI digested DNA from A. laidlawii. The hybridization results were visualized by electronic autoradiography (Packard Instant ImagerTM). The DNA fragments with corresponding size to the hybridization band were purified and ligated into the pCR-Script SK(+) cloning vector and transformed into E. coli TOP 10F heat-shock competent cells. Positive clones were found by colony replicating to nitrocellulose filter and hybridization (18$$Google Scholar) with the radioactively labeled probe. The nucleotide sequence of the inserted DNA fragment was determined using both gene-specific and vector-specific primers with ABI PRISM® Big Dye terminator cycle sequencing ready reaction kit (PE Applied Biosystems). Using these results, a construct was made where the geneALdgs from A. laidlawii A-EF22 strain was cloned into a pET15b-vector and transformed into E. coli BL21(DE3). This recombinant was used for the enzymatic assays. The gene coding for the spDGS in S. pneumoniae(cpoA), identified with the alDGS aa sequence in a computer search, was isolated by PCR with the primers 5′-TAG TTA TGG AGA AAA AGA AAT TAC G-3′ and 5′-TAC CTC ACT TTT TAC TTT CTC CC-3′, which bind to the Shine-Dalgarno and stop codon sequence regions, respectively. The PCR product was purified, ligated into a SmaI-digested pCR-script vector, and transformed into TOP10F. Syntheticrac-1,3-dioleoylglycerol (1,3-DOG) was purchased from Larodan (Malmö, Sweden). The MGlcDAG and DGlcDAG were prepared as described (8Karlsson O.P. Dahlqvist A. Vikström S. Wieslander Å. J. Biol. Chem. 1997; 272: 929-936Google Scholar). Synthetic α-MGalDAG was obtained from Dr. D. Mannock (cf. Dahlqvist et al. (11Dahlqvist A. Nordström S. Karlsson O.P. Mannock D.A. McElhaney R.N. Wieslander Å. Biochemistry. 1995; 34: 13381-13389Google Scholar)). Synthetic DOPA and DOPG were purchased from Avanti Polar Lipids (Alabaster, AL) and CHAPS detergent from Roche Molecular Biochemicals. Protein expression in the recombinant strains was performed in 1× LB medium supplemented with 100 μg of carbenicillin/ml. The strains were grown at 37 °C, and 0.3 mmisopropyl-1-thio-β-d-galactopyranoside was added at OD600 = 0.75. Cells were harvested by centrifugation after another 16 h at 23 °C. Harvested recombinant E. coli cells were solubilized in assay buffer (110 mmTris-HCl, pH 8.0, 22 mm MgCl2, and 22 mm CHAPS) to a total protein concentration of ∼1.3 mg/ml, by 4 × 30 s in a sonication bath and three times extensive vortexing during incubation on ice for 3 h, before enzymatic assays. In the standard assay for glycolipid synthesis 25 μl of protein solution (cf. above) was added to 20 μl of lipid micellar solution and incubated on ice for 30 min. The enzymatic reaction was started by addition of 5 μl of UDP-[14C]galactose or UDP-[14C]glucose to a concentration of 1 mm (30 GBq mol−1) in a total volume of 50 μl. Standard lipid concentration was 10 mm (1 mm MGlcDAG substrate in addition to the activator DOPG (or CL) and the nonbilayer-prone 1,3-DOG, with DGlcDAG as balance). After 30 min of incubation at 28 °C, the reaction was stopped with 375 μl of methanol/chloroform 2:1 (v/v), and the lipids were extracted and separated by TLC (10Vikström S. Li L. Karlsson O.P. Wieslander Å. Biochemistry. 1999; 38: 5511-5520Google Scholar, 19Karlsson O.P. Dahlqvist A. Wieslander Å. J. Biol. Chem. 1994; 269: 23484-23490Google Scholar). The lipid products on the TLC plates were visualized and quantified by electronic autoradiography (Packard Instant ImagerTM). All assays were done in duplicate. A variety of glycolipids was used as TLC references (20Fisher W. Arneth-Seifert D. J. Bacteriol. 1998; 180: 2950-2957Google Scholar). Full-size (398 aa), N- (aa 1–226), and C-domain (aa 227–398) alMGS, respectively, were cloned upstream and in-frame with the gene for green fluorescent protein (GFP) using a PCR primer approach based on the alMGS sequence (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar) and the vector pGFPuv (Clontech), in E. coli JM109. Ligation, transformation, and selection followed standard procedures, and the obtained constructs were verified by DNA sequencing. Enzymatic activities of the hybrid proteins were analyzed after induction with isopropyl-1-thio-β-d-galactopyranoside by labeling of E. coli lipids in vivo with radioactive acetate (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar) and by assay for MGlcDAG synthesis after detergent solubilization in vitro (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar). Localization of the MGS-GFP variants in E. coli was analyzed by fluorescence microscopy at various growth and induction conditions, using a Zeiss Axioplan2 microscope, fluorescein isothiocyanate filters, a CCD camera (Sony), and Image Access 3.0 software (Imagic Bildverarbeitung AG). To find homologs to the alDGS and to predict a function for the genes in flanking regions, PSI-BLAST (21Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar) at NCBI in the nonredundant data base and the data base for finished and unfinished microbial genomes, was used. Preliminary genomic sequence data were obtained from The Institute for Genomic Research. Features of the primary structure of the alDGS sequence were further analyzed with tools available at the ExPASy Molecular Biology Server (www.expasy.ch) (Swiss Institute of Bioinformatics) and with the Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI). Potential secondary and three-dimensional structure models were predicted from aa sequences by several local structure (PsiPred, Target99, and Jpred2) and fold recognition methods (FFAS, 3D-PSSM, GenTHREADER, mGenTHREADER, INBGU, Sam-T99, and FUGUE) and jointly evaluated using the Pcons consensus method (22Lundström J. Rychlewski L. Bujnicki J. Elofsson A. Protein Sci. 2001; 10: 2354-2362Google Scholar) at the MetaServer (www.bioinfo.pl) (23Bujnicki J.M. Elofsson A. Fischer D. Rychlewski L. Bioinformatics (Oxf.). 2001; 17: 750-751Google Scholar). The scores stating the similarity between the obtained models are derived from Levitt and Gerstein (cf. Ref. 22Lundström J. Rychlewski L. Bujnicki J. Elofsson A. Protein Sci. 2001; 10: 2354-2362Google Scholar). The aa sequences from several known glycosyltransferase structures were analyzed (“benchmarked”) in a similar manner (with PDB Test, www.bioinfo.pl) and the suggested PDB structures compared with the DALI method (24Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Google Scholar). Structural models of the glycosyltransferases were built, based on the sequence alignments and recognized folds given by the MetaServer (above), using the Swiss-PdbViewer program for alignment (Version 3.7; www.expasy.ch/spdbv/) (25Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Google Scholar) and the optimize-mode of the SWISS-MODEL program server (Version 3.5; www.expasy.ch/swissmod/SWISS-MODEL.html) for automatic model building. For the MGS sequences the three protein structures of ecMurG (PDB-ID 1F0K), ecEpim (PDB-ID 1F6D), and aoGtfB (PDB-ID 1IIR) were used as templates, while the DGS sequences were modeled with the first two (ecMurG and ecEpim) as templates. All five proteins were successfully modeled. Although the models obtained from the SWISS-MODEL server are already energy minimized, they were subjected to an additional global energy minimization. Prior to the final minimization a few side chains were adjusted as they were trapped within the ring of aromatic side chains, a situation an energy minimization would not be able to correct. For the global energy minimization the Swiss-PdbViewer implementation of the GROMOS96 43B1 force field was used, together with the SWISS-MODEL server minimization protocol, namely 200 steps of steepest descent, followed by 300 steps of conjugate gradient minimization. The quality of the models was accessed using the WhatCheck program (26Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Google Scholar). Identification of secondary structure elements, calculation of the electrostatic surface potential, and visualization were made using the program MOLMOL (27Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Google Scholar). The electrostatic potentials were calculated assuming point charges on heavy atoms, dielectric constants of 2 and 80 for protein and solvent, respectively, an ionic strength of 0.3 m, a 2-Å salt radius, and a boundary condition of zero potential at 15 Å. A solvent radius of 1.4 Å was used when the accessible van der Waals surfaces of the heavy atoms of each model were identified. The relative solvent accessibility of residues was calculated from their coordinates and divided into three groups; buried (0–5%), intermediate (5–25%) and exposed (25–100%) (28Pascarella S. de Persio R. Bossa F. Argos P. Proteins. 1998; 32: 190-199Google Scholar). Nucleotide sequence data for A. laidlawii DGlcDAG synthase have been deposited at GenBankTM with accession number AY078412. The N-terminal and an internal amino acid sequence of the purified DGlcDAG synthase from A. laidlawii (alDGS) were determined by Edman degradation (10Vikström S. Li L. Karlsson O.P. Wieslander Å. Biochemistry. 1999; 38: 5511-5520Google Scholar). Designed degenerated PCR primers yielded a gene-specific probe, which was used in a Southern blot procedure identifying a 2.2-kbp DNA fragment that was cloned. The sequence of this contig (TableI) revealed an open reading frame (ORF) of 999 bp coding for the alDGS sequence named ALdgs (Fig.1) with a potential Shine-Dalgarno sequence starting at position −16. The G+C content was 33%, which is typical for A. laidlawii DNA (31.7–35.7% (29Herrmann R. Maniloff J. McElhaney R.N. Finch L.R. Baseman J.B. Mycoplasmas: Molecular Biology and Pathogenesis. American Society for Microbiology, Washington D. C.1992: 157-168Google Scholar)). The translated ORF found just downstream the ALdgs gene was related to proteins from the aldo/keto-reductase family. The sequence further downstream show similarity to trigger factor, a conserved chaperone found in all prokaryotes. No ORFs in the opposite direction were found. The A. laidlawii contigs containing the preceding glucosyltransferase gene ALmgs cloned earlier (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar) and the ALdgs determined here (Table I) have no nucleotide sequence in common and were not localized to the same DNA fragment according to hybridization. Hence, it was concluded that these two glycosyltransferase (GT) genes are not adjacent to each other on the chromosome and do not belong to the same operon.Table IAnalysis of the content of flanking regions to DGS in A. laidlawiiPositionDbsourceaData base accession number; new GenBank™ entry for DGS is shown.Function/name, speciesE-valuebp−74 to −1Unknown+1 to +999gb AY078412DGS+1002 to +1803emb CAA11712Putative reductase protein Bacillus subtilis7E-42+1877 togb AAC82391Trigger factor (TF) Streptococcus pyogenes2E-5a Data base accession number; new GenBank™ entry for DGS is shown. Open table in a new tab BLAST searches in the finished/unfinished and the nonredundant data bases at NCBI with the alDGS amino acid sequence gave several significant hits in eubacteria and archaea (Table II). All the top hits were Gram-positive sequences, and most were from pathogenic species. The best score showed a gene in Enterococcus faecium with 49% aa identity to the alDGS, and sequences from streptococcal or other closely related species (Lactococcus) had an identity above 45%. These species do all contain an α-diglycosyldiacylglycerol identical to the A. laidlawii one (30Shaw N. Bacteriol. Rev. 1970; 34: 365-377Google Scholar, 31$$Google Scholar), and the sugar moieties are solely glucose. In the corresponding glycolipid from S. pneumoniae(cf. Table II) the outer glucose is replaced by a galactose. For the alMGS glucosyltransferase aa identities of 31 and 29% were recorded to similar enzymes in S. pneumoniae andBorrelia burgdorferi, and these were shown to encode homologous functions to the A. laidlawii enzyme (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar). Most important, alDGS and all the sequences of Table II contain the conserved EX 7E motif characteristic for the retaining (α) GTs of CAZy family 4 of glycosyltransferases (32Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-939Google Scholar); the preceding enzyme alMGS (cf. Introduction) also belongs to this family (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar). They also contain a typical motif for glycosyltransferases belonging to Pfam family 00534 (conserved domain search). Proteins in this family transfer nucleoside diphosphate-linked sugars, like glucose, galactose, mannose, and X-glucose, to a variety of acceptor substrates such as glycogen and lipopolysaccharides.Table IIDGS homologs/analogsRankaThese genes are followed by three genes fromListeria species and two from Pyrococcus species. Rank 25 is a gene from Thermotoga maritima (25% identity).DbsourcebData base accession number.SpeciescSpecies labeled by * contain a chemically verified α-DGlcDAG of identical structure to the A. laidlawiilipid, but in S. pneumoniae the outer Glc moiety is replaced by Gal.aa sequence identity1gb ZP 00036761Enterococcus faecium*165/336 (49%)2gb AAK33514.1Streptococcus pyogenes*163/335 (48%)3gb AAN59232Streptococcus mutans*166/334 (49%)4Streptococcus equi159/337 (47%)5Enterococcus faecalis*161/336 (48%)6gbAE005176Lactococcus lactis*152/337 (45%)7Streptococcus gordonii100/203 (49%)8gb AAM99596Streptococcus agalactiae*158/337 (46%)9gb ZP_00063752Leuconostoc mesenteroides*127/276 (46%)10gb ZP_00070248Oenococcus oeni135/338 (39%)11gbAAG19110Halobacterium sp. NRC-1106/351 (30%)12gb AE007798Clostridium acetobutylicum74/244 (30%)13embCAA72249Streptococcus pneumoniae TIGR4* (CpoA)58/189 (30%)13gb AE008471Streptococcus pneumoniae R6* (CpoA)57/189 (30%)The best hits found by BLAST in finished and unfinished microbial genomes data base at NCBI. Homologs/analogs are found in Gram-positive bacteria and archaea. Homologs to the alMGS are also found by similarity in almost all organisms in the table. All are members of CAZy family 4 of GTs.a These genes are followed by three genes fromListeria species and two from Pyrococcus species. Rank 25 is a gene from Thermotoga maritima (25% identity).b Data base accession number.c Species labeled by * contain a chemically verified α-DGlcDAG of identical structure to the A. laidlawiilipid, but in S. pneumoniae the outer Glc moiety is replaced by Gal. Open table in a new tab The best hits found by BLAST in finished and unfinished microbial genomes data base at NCBI. Homologs/analogs are found in Gram-positive bacteria and archaea. Homologs to the alMGS are also found by similarity in almost all organisms in the table. All are members of CAZy family 4 of GTs. The BLAST searches revealed no proteins with an established function, since many of them are from recently published genome projects and so far lack annotation. However, the S. pneumoniae gene in Table II was first sequenced by Grebe et al. (33Grebe T. Paik J. Hakenbeck R. J. Bacteriol. 1997; 179: 3342-3349Google Scholar) and namedcpoA. This protein was proposed to be involved in resistance to β-lactams and was isolated from first-step piperacillin-resistant mutants. The cpoA and the consecutive ORF5 gene, both part of the same operon, were suggested (but not shown) to be involved in formation of the linkage unit in lipoteichoic acid or polymerization of teichoic acid precursors prior to translocation (33Grebe T. Paik J. Hakenbeck R. J. Bacteriol. 1997; 179: 3342-3349Google Scholar). We showed recently that this ORF5 encoded an enzyme homologous in function to alMGS (cf. step (i) in Introduction) and that was named spMGS. This enzyme was of similarly high sequence identity to alMGS as the CpoA protein is to alDGS (cf. Table II and Fig. 1). Most intriguing, the majority of organisms with a potential alDGS homolog (Table II) also encode a homolog to alMGS, according to BLAST sequence analyses (data not shown) (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar). The fact that the homologs of DGS and MGS genes in S. pneumoniae were adjacent to each other (above), potentially in an operon organization, was true also for most of the species in Table II. However, not for A. laidlawii and the related species Clostridium acetobutylicum and not for Pyrococcus furiosus (data not shown). Since the function of the S. pneumoniae MGS enzyme (i.e. ORF5, see above) has been verified (6Berg S. Edman M. Li L. Wikström M. Wieslander Å. J. Biol. Chem. 2001; 276: 22056-22063Google Scholar), the adjacent gene cpoA was PCR-cloned (see “Materials and Methods”) to establish its function (cf. below). The BLAST searches also revealed analogs in the three archaeal speciesHalobacterium sp. NRC-1, Pyrococcus horikoshiiand P. furiosus (Table II); all sequences are of unknown function. The membrane of Halobacterium spp. contains the major glycolipid 3-HSO3-β-Galp-(1–6)-α-Manp