Expression cloning of three Rhizobium leguminosarum lipopolysaccharide core galacturonosyltransferases

The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo2-[4'-(32)P]lipid IV(A) and its 1-dephosphorylated derivative on the distal Kdo residue, as indicated by mild acid hydrolysis. The third enzyme modifies the mannose unit of the substrate mannosyl-Kdo2-1-dephospho-[4'-(32)P]lipid IV(A). Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.     

A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum. Purification,substrate specificity,and expression in Salmonella waaC mutants

The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IV(A). LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-(32)P]lipid IV(A) as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo(2)-[4'-(32)P]lipid IV(A). A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV(A) or Kdo-lipid IV(A). The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A). Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner l-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.     

Biosynthesis of a structurally novel lipid A in Rhizobium leguminosarum: identification and characterization of six metabolic steps leading from UDP-GlcNAc to 3-deoxy-D-manno-2-octulosonic acid2-lipid IVA.

Lipopolysaccharides (LPSs) are prominent structural components of the outer membranes of gram-negative bacteria. In Rhizobium spp. LPS functions as a determinant of the nitrogen-fixing symbiosis with legumes. LPS is anchored to the outer surface of the outer membrane by the lipid A moiety, the principal lipid component of the outer bacterial surface. Several notable structural differences exist between the lipid A of Escherichia coli and that of Rhizobium leguminosarum, suggesting that diverse biosynthetic pathways may also exist. These differences include the lack of phosphate groups and the presence of a 4'-linked GalA residue in the latter. However, we now show that UDP-GlcNAc plays a key role in the biosynthesis of lipid A in R. leguminosarum, as it does in E. coli. 32P-labeled monosaccharide and disaccharide lipid A intermediates from E. coli were isolated and tested as substrates in cell extracts of R. leguminosarum biovars phaseoli and viciae. Six enzymes that catalyze the early steps of E. coli lipid A biosynthesis were also present in extracts of R. leguminosarum. Our results show that all the enzymes of the pathway leading to the formation of the intermediate 3-deoxy-D-manno-2-octulosonic acid (Kdo2)-lipid IVA are functional in both R. leguminosarum biovars. These enzymes include (i) UDP-GlcNAc 3-O-acyltransferase; (ii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase; (iii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcN N-acyltransferase; (iv) disaccharide synthase; (v) 4'-kinase; and (vi) Kdo transferase. Our data suggest that the early steps in lipid A biosynthesis are conserved and that the divergence leading to rhizobial lipid A may occur at a later stage in the pathway, presumably after the attachment of the Kdo residues.     

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC. Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in lpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo(2)-lipid IV(A). We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A) at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo(2)-[4'-(32)P]lipid IV(A) in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC, is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.     

Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase

Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4'-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4' positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila.     

Expression cloning and characterization of the C28 acyltransferase of lipid A biosynthesis in Rhizobium leguminosarum

An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety. An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, and Sinorhizobium meliloti but not Escherichia coli. The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2 ((3-deoxy-d-manno-octulosonic acid)2)-lipid IV(A). We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening approximately 4000 lysates of individual colonies of an R. leguminosarum 3841 genomic DNA library in the host strain S. meliloti 1021. In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced. Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous. Nine other open reading frames around lpxXL were also sequenced. Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes. AcpXL purified from S. meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate. Expression of lpxXL in E. coli behind a T7 promoter results in overproduction in vitro of the expected R. leguminosarum acyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-d-manno-octulosonic acid)2-lipid IV(A) as the acceptor. These findings confirm that lpxXL is the structural gene for the C28 acyltransferase. LpxXL is distantly related to the lauroyltransferase (LpxL) of E. coli lipid A biosynthesis, but highly significant LpxXL orthologues are present in Agrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms.     

A phosphotransferase that generates phosphatidylinositol 4-phosphate (PtdIns-4-P) from phosphatidylinositol and lipid A in Rhizobium leguminosarum. A membrane-bound enzyme linking lipid a and ptdins-4-p biosynthesis

Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unusual phosphate-deficient lipid A found in this organism. The enzyme has been solubilized with Triton X-100 and purified 80-fold. As shown by co-purification and thermal inactivation studies, the 4'-phosphatase catalyzes not only the hydrolysis of (Kdo)2-[4'-32P]lipid IVA but also the transfer the 4'-phosphate of Kdo2-[4'-32P]lipid IVA to the inositol headgroup of phosphatidylinositol (PtdIns) to generate PtdIns-4-P. Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizobium meliloti, or the nodulation-defective mutant 24AR of R. leguminosarum. The specific activity for the phosphotransferase reaction is about 2 times higher than that of the 4'-phosphatase. The phosphotransferase assay conditions are similar to those used for PtdIns kinases, except that ATP and Mg2+ are omitted. The apparent Km for PtdIns is approximately 500 microM versus 20-100 microM for most PtdIns kinases, but the phosphotransferase specific activity in crude cell extracts is higher than that of most PtdIns kinases. The phosphotransferase is absolutely specific for the 4-position of PtdIns and is highly selective for PtdIns as the acceptor. The 4'-phosphatase/phosphotransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns. A phosphoenzyme intermediate may account for the dual function of this enzyme, since a single 32P-labeled protein species (Mr approximately 68,000) can be trapped and visualized by SDS gel electrophoresis of enzyme preparations incubated with Kdo2-[4'-32P]lipid IVA. Although PtdIns is not detected in cultures of R. leguminosarum/etli (CE3), PtdIns may be synthesized during nodulation or supplied by plant membranes, given that soybean PtdIns is an excellent phosphate acceptor. A bacterial enzyme for generating PtdIns-4-P and a direct link between lipid A and PtdIns-4-P biosynthesis have not been reported previously.     

Origin of the 2-amino-2-deoxy-gluconate unit in Rhizobium leguminosarum lipid A. Expression cloning of the outer membrane oxidase LpxQ

An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosamine-containing precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the approximately 20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O(2) for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.     

Characterization of galacturonosyl transferase genes rgtA, rgtB, rgtC, rgtD, and rgtE responsible for lipopolysaccharide synthesis in nitrogen-fixing endosymbiont Rhizobium leguminosarum: lipopolysaccharide core and lipid galacturonosyl residues confer membrane stability

Rhizobium lipopolysaccharide (LPS) contains four terminally linked galacturonic acid (GalA) residues; one attached to the lipid A and three attached to the core oligosaccharide moiety. Attachment of the GalA residues requires the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA), which is synthesized by the GalA transferase RgtE reported here. The galacturonosyl transferases RgtA, -B, and -C utilize Dod-P-GalA to attach GalAs on the LPS core region, and RgtD attaches GalA to the lipid A 4' position. As reported here, the functions of the rgtD and rgtE genes were determined via insertion mutagenesis and structural characterization of the mutant lipid A. The rgtE(-) mutant lacked Dod-P-GalA as determined by mass spectrometry of total lipid extracts and the inability of rgtE(-) mutant membranes to provide the substrate for heterologously expressed RgtA activity. In addition, we created single mutations in each of the rgtA, -B, -C, -D, and -E genes to study the biological function of the GalA residues. The structures of the core oligosaccharide region from each of the rgt mutants were elucidated by glycosyl linkage analysis. Each mutant was assayed for its sensitivity to sodium deoxycholate and to the antimicrobial cationic peptide, polymyxin B (PmxB). The rgt mutants were more sensitive than the parent strain to deoxycholate by varying degrees. However, the rgtA, -B, and -C mutants were more resistant to PmxB, whereas the rgtD and E mutants were less resistant in comparison to the parent strain.     

A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors

Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4'. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.     

An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.     

Purification and mutagenesis of LpxL, the lauroyltransferase of Escherichia coli lipid A biosynthesis

Escherichia coli lipid A is a hexaacylated disaccharide of glucosamine with secondary laurate and myristate chains on the distal unit. Hexaacylated lipid A is a potent agonist of human Toll-like receptor 4, whereas its tetra- and pentaacylated precursors are antagonists. The inner membrane enzyme LpxL transfers laurate from lauroyl-acyl carrier protein to the 2'- R-3-hydroxymyristate moiety of the tetraacylated lipid A precursor Kdo 2-lipid IV A. LpxL has now been overexpressed, solubilized with n-dodecyl beta- d-maltopyranoside (DDM), and purified to homogeneity. LpxL migration on a gel filtration column is consistent with a molecular mass of 80 kDa, suggestive of an LpxL monomer (36 kDa) embedded in a DDM micelle. Mass spectrometry showed that deformylated LpxL was the predominant species, noncovalently bound to as many as 12 DDM molecules. Purified LpxL catalyzed not only the formation in vitro of Kdo 2-(lauroyl)-lipid IV A but also a slow second acylation, generating Kdo 2-(dilauroyl)-lipid IV A. Consistent with the Kdo dependence of crude LpxL in membranes, Kdo 2-lipid IV A is preferred 6000-fold over lipid IV A by the pure enzyme. Sequence comparisons suggest that LpxL shares distant homology with the glycerol-3-phosphate acyltransferase (GPAT) family, including a putative catalytic dyad located in a conserved H(X) 4D/E motif. Mutation of H132 or E137 to alanine reduces specific activity by over 3 orders of magnitude. Like many GPATs, LpxL can also utilize acyl-CoA as an alternative acyl donor, albeit at a slower rate. Our results show that the acyltransferases that generate the secondary acyl chains of lipid A are members of the GPAT family and set the stage for structural studies.     

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.     

Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor

Sinorhizobium meliloti strain 1021 possesses the particularity to synthesize biologically inefficient capsular polysaccharides (KPS). It has been assumed that this class of compounds is not produced in high-molecular-mass (HMM) forms, even if many genetic analyses show the existence of expression of genes involved in the biosynthesis of capsular polysaccharides. The expression of these genes that are involved in the export of a KPS throughout the membrane and in the attachment of a lipid moiety has never been related to a structurally characterized surface polysaccharide. It is now reported that S. meliloti strain 1021 produces low-molecular-mass polysaccharides (4-4.5 kDa) that are exclusively composed of beta-(2-->7)-linked 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo) residues. These compounds are considered precursor molecules of HMM KPS, whose biosynthesis is arrested in the case of S. meliloti strain 1021. For the first time, the phospholipid anchor of a rhizobial KPS has been found, and its structure could be partially identified-namely, a phosphoglycerol moiety bearing a hydroxy-octacosanoic acid. When compared to other rhizobial KPS (composed of dimeric hexose-Kdo-like sugar repeating units), the Kdo homopolymer described here may explain why a complementation of S. meliloti strain 1021 Exo B mutant with an effective rkpZ gene restoring an active higher KPS size does not completely lead to the fully effective nitrogen fixing phenotype.     

An outer membrane enzyme that generates the 2-amino-2-deoxy-gluconate moiety of Rhizobium leguminosarum lipid A

The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria. Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4', R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species (designated A-E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2' acyloxyacyl moiety, is present in all five components. The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself. A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E. We now demonstrate that membranes of R. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1. The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IV(A). With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IV(A). The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R. leguminosarum as judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously.     

A novel 3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase that removes the outer Kdo sugar of Helicobacter pylori lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.     

ATPase activity of the MsbA lipid flippase of Escherichia coli

Escherichia coli MsbA, the proposed inner membrane lipid flippase, is an essential ATP-binding cassette transporter protein with homology to mammalian multidrug resistance proteins. Depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of E. coli. MsbA modified with an N-terminal hexahistidine tag was overexpressed, solubilized with a nonionic detergent, and purified by nickel affinity chromatography to approximately 95% purity. The ATPase activity of the purified protein was stimulated by phospholipids. When reconstituted into liposomes prepared from E. coli phospholipids, MsbA displayed an apparent K(m) of 878 microm and a V(max) of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mm Mg(2+). Preincubation of MsbA-containing liposomes with 3-deoxy-d-mannooctulosonic acid (Kdo)(2)-lipid A increased the ATPase activity 4-5-fold, with half-maximal stimulation seen at 21 microm Kdo(2)-lipid A. Addition of Kdo(2)-lipid A increased the V(max) to 154 nmol/min/mg and decreased the K(m) to 379 microm. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IV(A). MsbA containing the A270T substitution, which renders cells temperature-sensitive for growth and lipid export, displayed ATPase activity similar to that of the wild type protein at 30 degrees C but was significantly reduced at 42 degrees C. These results provide the first in vitro evidence that MsbA is a lipid-activated ATPase and that hexaacylated lipid A is an especially potent activator.     

Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti

Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for Sinorhizobium meliloti because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells. Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose. Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of Rhizobium leguminosarum biovars phaseoli and trifolii. Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism. All of the disaccharides that acted as powerful osmoprotectants in S. meliloti and R. leguminosarum also acted as very effective competitors of [14C]sucrose uptake in salt-stressed cultures of these bacteria. Conversely, disaccharides that were not osmoprotective for S. meliloti and R. leguminosarum did not inhibit sucrose uptake in these bacteria. Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria. Natural-abundance 13C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed S. meliloti cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide N-acetylglutaminylglutamine amide. The ecological implication of the use of these disaccharides as osmoprotectants is discussed.     

THE CELL WALL (THECA) OF TETRASELMIS STRIATA (CHLOROPHYTA): MACROMOLECULAR COMPOSITION AND STRUCTURAL ELEMENTS OF THE COMPLEX POLYSACCHARIDES

Isolated cell walls (thecae) from the scaly flagellate green alga Tetraselmis striata Butcher contain the unusual 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (Kdo), 3-deoxy-5-O-methyl-manno-2-octulosonic acid (5OMeKdo), and 3-deoxy-lyxo-2-heptulosaric acid (Dha). In addition, galacturonic acid, galactose, gulose, and arabinose are present. EDTA-extraction yielded an insoluble fraction that retains the shape of the cell walls and contains no 2-keto-sugar acids. Methylation analysis demonstrated the presence of terminal hexose, GalA, Dha, and Kdo as well as 2-substituted hexose, 4-or 8-substituted Kdo, and 4,8-disubstituted Kdo. However, most of the carbohydrate material (about 60%) was not methylated. Periodate oxidation of the cell wall preparation showed the presence of 2-substituted Gul, 4- or/and 5-substituted and 7- or/and 8-substituted Kdo, which is in agreement with the methylation analysis. Again, a significant amount of carbohydrate material was not degraded, indicating complex substitution patterns. Oligosaccharides were generated by partial hydrolysis and fractionated using gel permeation chromatography and high-pH anion-exchange chromatography. Oligosaccharides contained either GalA and Kdo, or Gal, Kdo, Dha, and Gul, respectively. The structure of a GalA and Kdo containing disaccharide was established using ¹ H NMR spectroscopy.