Characterization of a novel lipid A containing D-galacturonic acid that replaces phosphate residues. The structure of the lipid a of the lipopolysaccharide from the hyperthermophilic bacterium Aquifex pyrophilus.

According to the 16 S rRNA phylogenetic tree, the hyperthermophilic bacterium Aquifex pyrophilus represents the deepest and shortest branching species of the kingdom Bacteria. We show for the first time that an organism, which is phylogenetically ancient on the basis of its 16 S rRNA and that exists at extreme conditions, may contain lipopolysaccharide (LPS). The LPS was extracted from dried bacteria using a modified phenol/water method. SDS-polyacrylamide gel electrophoresis and silver stain displayed a ladder-like pattern, which is typical for smooth-form LPS (possessing an O-specific polysaccharide). The molecular masses of the LPS populations were determined by matrix-assisted laser-desorption ionization mass spectrometry. Lipid A was precipitated after mild acid hydrolysis of LPS. Its complete structure was determined by chemical analyses, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser-desorption ionization mass spectrometry, and one- and two-dimensional NMR spectroscopy. The lipid A consists of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-D-glucopyranose (DAG) disaccharide carrying two residues each of (R)-3-hydroxytetradecanoic acid and (R)-3-hydroxyhexadecanoic acid in amide linkage and one residue of octadecanoic acid in ester linkage. Each DAG moiety carries one residue of each 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acid. In the nonreducing DAG, the octadecanoic acid is attached to the 3-hydroxy group of 3-hydroxytetradecanoic acid. Each DAG is substituted by one D-galacturonic acid residue, which is linked to O-1 of the reducing and to O-4 of the nonreducing end. This structure represents a novel type of lipid A.

Within the kingdom Bacteria, Aquifex pyrophilus (1) exhibits with 95°C the highest growth temperature. It does not belong to any of the known phyla and represents the deepest branching species of the kingdom Bacteria in the 16 S rRNA-based universal phylogenetic tree (2,3). Hyperthermophiles are represented among the deepest and shortest lineages of this tree and are discussed to be still rather primitive (4). A. pyrophilus was isolated from hot marine sediments (depth: 106 m) at the Kolbeinsey Ridge, Iceland and is growing microaerophilically under oxygen reduction at temperatures in the range of 67 to 95°C. Cells are Gram-negative highly motile rods exhibiting a complex envelope consisting of murein, an outer membrane, and a surface protein layer (1). However, it was not investigated whether A. pyrophilus contains lipopolysaccharide (LPS) 1 or LPS analogous structures.
LPS (5) are characteristic components of the cell wall of Gram-negative bacteria where they are located in the outer leaflet of the outer membrane. They contribute to the highly effective permeation barrier function of the outer membrane and, furthermore, participate in various physiological membrane functions essential for growth and survival of Gramnegative bacteria. LPS play also an important role in the interaction of the bacteria with suitable hosts. They are the endotoxins of Gram-negative bacteria and are responsible for a broad spectrum of biological activities. Chemically, LPS are composed of three regions, namely (a) the O-specific polysaccharide (6,7), which is built up of a varying amount of repeating oligosaccharide units, (b) the core oligosaccharide (8), and (c) the lipid A (9, 10), which anchors the molecule in the membrane and was shown to represent the toxic principle of LPS (11). Endotoxic active lipid A, e.g. enterobacterial lipid A, is known to possess a rather conserved structure, which is characterized by a ␤-(136)-linked 2-amino-2-deoxy-D-glucopyranose (D-GlcpN) disaccharide backbone with phosphate groups attached to O-1 and O-4Ј that carries (R)-3-hydroxy fatty acids and (R)-3-acyloxyacyl residues at positions 2 and 3, and 2Ј and 3Ј, respectively. LPS isolated from other bacterial species showed a greater variability of their lipid A including 2,3diamino-2,3-dideoxy-D-glucopyranose (DAG) instead of GlcpN and variations in the fatty acid and phosphate substitution pattern (9). LPS are discussed to be valuable (chemo-)taxonomic and also phylogenetic markers because of the compositions of their lipid A and inner core regions. This might also be true for the taxonomy of A. pyrophilus and related species. In this work we describe the isolation of the LPS from A. pyrophilus and the characterization of the structure of its lipid A moiety which is different from all previously known lipid A.

EXPERIMENTAL PROCEDURES
Bacterial Cultures and LPS Isolation-Batch cultures of A. pyrophilus were grown in 300-liter fermentors in modified SME medium under microaerophilic conditions at 85°C as described (1). After centrifuga-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
De-O-acylation of Lipid A-Lipid A was de-O-acylated by mild hydrazinolysis as described (15).
General and Analytical Methods-Analysis for neutral sugars, total GlcN, reducing GlcN, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), and phosphate were performed as described (16). The method for the analysis of neutral sugars was equally employed for the detection of uronic acids and amino sugars with the following variations: for the determination of uronic acids, samples were methanolysed (0.5 M methanolic HCl, 85°C, 45 min) and then carboxyl-reduced (NaB 2 H 4 , in H 2 O/CH 3 OH (1:1, v:v)) before continuing the protocol for neutral sugar determination. For the determination of amino sugars, samples were hydrolyzed (4 M HCl, 100°C, 4 h), neutralized, then N-acetylated (17), and reduced (NaB 2 H 4 ), followed by acetylation as described for neutral sugar analysis. The absolute configuration of sugars was determined by gas-liquid chromatography (GLC) analysis of the acetylated (R)-and (S)-2-hydroxybutyl glycosides (18). For the colorimetric detection of uronic acids, the method of Blumenkrantz and Asboe-Hansen (19) was employed. Protein was quantified according to Bradford (20). Impurities of DNA, RNA, and proteins were determined by UV photometric measurements at 190 -400 nm of sample concentrations of 0.1 mg ml Ϫ1 using an Uvikon 931 spectrometer. Thin-layer chromatography (TLC) of lipid A was carried out on precoated silica gel 60 plates (Merck) with CHCl 3 :CH 3 OH:H 2 O ϩ acetic acid (10:10:3 (v:v:v) ϩ 0.01% (w:v)) as solvent system. Spots were visualized by charring with 10% (v:v) ethanolic H 2 SO 4 , and compounds corresponding to each detected band were scraped out of the plate and extracted from silica gel with CHCl 3 : CH 3 OH:H 2 O ϩ acetic acid (10:10:3 (v:v:v) ϩ 0.01% (w:v)). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed as described (21) and gels were either stained with silver nitrate for the detection of LPS (22) or with Coomassie Brilliant Blue for the detection of proteins.
Fatty Acid Analysis-Fatty acid analyses were performed according to Wollenweber and Rietschel (23) and for ester bound fatty acids according to Rietschel (24). The absolute configuration of the hydroxy fatty acids was determined by GLC of the phenylethylamide derivatives (24). To characterize amide-linked acyloxyacyl groups, it was necessary to acetylate (first acetic anhydride, 37°C, 16 h, then pyridine/acetic anhydride (2:1, v:v), 37°C, 6 h) the lipid A to obtain better solubility. After reaction with trimethyloxonium-tetrafluoroborate and 2,6-t-butyl-4-methyl-pyridin (5 mg ml Ϫ1 in dioxan) (2:1, v:v) at 20 -22°C for 16 h and evaporation, the sample was treated with 100 l of 8 M NaOH. The chloroform phase of a chloroform extraction was evaporated and hydrolyzed with 1 M HCl (20 -22°C, 1 h). The chloroform extract of this sample was methylated with diazomethane and then analysis was performed using combined GLC-mass spectrometry (MS).
GLC and GLC-MS-GLC and GLC-MS were carried out as described (17,26). The temperature programs in GLC and GLC-MS (in parentheses) for sugars were 150°C for 3 min and then 3°C min Ϫ1 (5 C min Ϫ1 ) to 300°C (320°C). The temperature programs in GLC and GLC-MS (in parentheses) for fatty acids were 120°C for 3 min and then 5°C min Ϫ1 to 300°C (320°C). NMR Spectroscopy-NMR spectra were recorded of a solution of 5 mg in 0.5 ml Me 2 SO-d 6 with a Bruker DRX 600 (operating frequencies 600 MHz for 1 H, 150.9 MHz for 13 C, and 243 MHz for 31 P) or DRX 360 (operating frequency 90.6 MHz for 13 C) spectrometers at 47°C using Bruker standard software. The 1 H resonances were measured relative to the methyl group signal of Me 2 SO (2.5 ppm). The assignment of the proton chemical shifts was achieved by correlation spectroscopy, total correlation spectroscopy, and double-quantum-filtered correlated spectroscopy experiments. The assignment of carbon chemical shifts was achieved by 1 H, 13 C heteronuclear multiple quantum coherence experiments and, for galactopyranuronic acid (GalpA), by comparison to published 13 C NMR data (28). 13 C resonances were determined relative to the methyl group signal of Me 2 SO (40.0 ppm). Nuclear Overhauser effect contacts were identified using nuclear Overhauser effect spectroscopy experiments.

RESULTS
Isolation and Characterization of LPS-Extraction of dried bacterial cells using the phenol/chloroform/light petroleum method gave LPS in low yield together with a coextracted phospholipid. Therefore, the modified hot phenol/water method was applied. The LPS was isolated from the water phase after ultracentrifugation (450 mg, 1.7% of dry bacterial mass) and was found to be free of other lipids, proteins, DNA, and RNA.
On SDS-polyacrylamide gel electrophoresis of the LPS preparation, a ladder-like pattern indicating the presence of S-form LPS with different sizes of the O-specific polysaccharide moieties was identified (Fig. 1). The negative ion MALDI-TOF mass spectrum of the native LPS revealed the molecular masses of the LPS populations (Fig. 2, M 1 , M 2 , . . . . ). Each repeating unit was found to possess an average mass of 708 Da, which indicated that it is composed of two heptose and two hexose residues. Furthermore, the spectrum comprises prominent peaks originating from laser-induced cleavage of the labile linkage between lipid A and Kdo I of the core oligosaccharide (29,30). Thus, the fragment ion peak at m/z 1916 could be identified as the main free lipid A component (see below).
Quantitative sugar analysis of the isolated LPS revealed that it consists of GlcN, DAG, Kdo, galacturonic acid (GalA), Man, Glc, and L-glycero-D-manno-heptose (Table I) presence of 3-hydroxytetradecanoic acid (14:0(3-OH)) and 3-hydroxyhexadecanoic acid (16:0(3-OH)) in amide linkage as well as octadecanoic acid (18:0) in ester linkage (Table II).      (Table III) are based on one-dimensional 1 H and 13 C NMR-spectra and twodimensional double quantum filter correlated spectroscopy (Fig. 5), total correlation spectroscopy (not shown), and heteronuclear multiple quantum coherence experiments (Fig. 6). The chemical shifts assigned for the GalpA residues are comparable to those published in Ref. 28. In the 1 H NMR spectrum, four signals between 7.4 and 7.9 ppm were attributed to the four NH protons. Four other signals were identified in the anomeric region, of which three were attributed to H-1 of ␣-linked hexoses (residues A, B, and D; for labeling see Fig. 8) and one to H-1 of a ␤-linked (C) hexoses, as characterized by the chemical shifts and J H-1,H-2 coupling constants (A, 4.91 ppm (2.6 Hz); B, 4.80 ppm (3.5 Hz); D, 5.07 ppm (2.2 Hz); C, 4.39 ppm (8.4 Hz)). At 4.34 ppm and 4.10 ppm, the chemical shifts of H-5 of the two uronic acids were identified. The signals of the remaining ring protons are in the region 3.29 -4.05 ppm. The chemical shifts of the fatty acids are between 0.6 and 2.3 ppm. The signals of their ␣-CH 2 are between 2 and 2.2 ppm, and those of the other CH 2 -groups between 1 and 1.5 ppm. The signals of the CH 3 protons are between 0.7 and 0.9 ppm. In agreement with the results of other experiments the substance was found to be composed of four sugars, substituted with fatty acids.

Structure of A. pyrophilus Lipid A De-O-acylation of Lipid A and Characterization of the Product-Lipid
The 13 C NMR spectrum was assigned by an heteronuclear multiple quantum coherence experiment (Fig. 6, Table III). Six carboxyl resonances (of the two uronic acids and the four amide-bound 3-hydroxy fatty acids) are present between 170 and 173 ppm (spectrum not shown). In the anomeric region, four signals were identified between 92 and 103 ppm. Of the ring sugar signals in the region 50 -78 ppm, those four between 50 and 55 ppm are assigned to the C-2 and C-3 atoms of the two DAG residues. The intensive signals of the fatty acids are in the region 13-33 ppm, with that of the CH 3 groups at 13 ppm. The results confirm the presence of two DAG and two uronic acid residues. The complete assignment of chemical shifts and the determination of vicinal 1 H, 1 H-coupling constants confirmed that both uronic acids are 4  The sequence of the monosaccharides was established by nuclear Overhauser effect spectroscopy experiments (Fig. 7). Interresidual Nuclear Overhauser effect contacts were identified between protons C1 and B6a, b. Together with the downfield shift of the carbon B6 (68.4 ppm) it proved the (136)linkage between DAG residues B and C. The (131)-linkage between GalA A and DAG B was identified by a Nuclear Overhauser effect contact between protons A1 and B1, and the (134)-linkage between GalA D and DAG C by a Nuclear Overhauser effect contact between proton D1 and C4, together with the downfield shift of carbon C4 (74.0 ppm). Thus, the monosaccharide sequence D 3 C 3 B 3 A (Fig. 8) is unambiguously proven. A 31 P NMR spectrum gave no phosphate signals, which is in agreement with all other analyses. Taken together, our data establish the structure of the lipid A moiety of LPS from A. pyrophilus as shown in Fig. 8.

DISCUSSION
For the first time, LPS was isolated from a hyperthermophilic bacterium, namely from A. pyrophilus. The LPS was shown to be of the S-form, as indicated by varying numbers of repeating units in the O-specific polysaccharide resulting in a ladder-like banding pattern in SDS-polyacrylamide gel electrophoresis. The molecular masses of LPS populations were identified by MALDI-MS. The average mass of one repeating unit (708 Da) suggested the presence of two hexose and two heptose residues. Other detected sugars in the LPS are Glc, Man, GalA, GlcN, and DAG, and the common constituents of the core region, L-glycero-D-manno-heptose and Kdo (8).
The detailed part of our investigation deals with the structure of the lipid A moiety. The isolated lipid A was found to represent a mixture of compounds. Thus, for the final structural elucidation of the carbohydrate backbone, methylation analysis, MALDI-MS, and NMR spectroscopy of the de-O-acylated lipid A were employed, because this derivative was found to be a homogeneous compound. The complete structure of the carbohydrate backbone was characterized as ␣-D-GalpA-(134)-␤-D-DAG-(136)-␣-D-DAG-(131)-␣-D-GalpA. This is the first lipid A backbone that comprises two GalA residues. The complete structure, as revealed by MALDI-MS, is composed of this carbohydrate backbone, which is substituted by four (R)-3-hydroxy fatty acids in amide linkage and possesses a 14:0(3-O(18:0)) acyloxyacyl group at the nonreducing end. Each DAG residue carries one 14:0(3-OH) and one 16:0(3-OH); however, it was not possible to determine the exact position (N-2 or N-3) of each fatty acid. Smaller lipid A moieties were also present in the preparation, which lack either 18:0 or one GalA acid residue or both. Because only one lipid A fragmentation peak at m/z 1916 was found in the native LPS (see Fig. 3), it must be concluded that the observed heterogeneity in the free lipid A preparation does not originate from intrinsic biological heterogeneity but from cleavages during the chemical isolation procedure (33).
The DAG disaccharide is not substituted by phosphate but carries two D-GalA residues instead. DAG has been identified in lipid A from different LPS (9,10). A mixed ␤-(136)-linked GlcpN-DAG disaccharide was found in lipid A from Campylobacter jejuni and Rhodospirillum salinarum, and the homogenous ␤-(136)-linked disaccharide DAG-DAG is present in the lipid A from LPS of Bordetella pertussis and Legionella pneu-   (34). This precursor is then processed differently than in E. coli (35)(36)(37)(38), forming the lipid A-core region. During that process, the phosphate residues at O-1 and O-4Ј are removed by 1-and 4Ј-phosphatases followed in later steps by the transfer of D-GalpA to O-4Ј and the oxidation of O-1. From these and other data, it is discussed that this precursor represents a conserved structure in LPS biosynthesis of different bacteria. This could also be true for A. pyrophilus, as the gene for the lipid A 4Ј-kinase that inserts phosphate at O-4Ј of the lipid A backbone is present, despite the complete lack of phosphate in the final molecule (39) (GenBank TM accession number AE000657). A screening of the total genome sequence of Aquifex aeolicus (39), a close relative of A. pyrophilus, shows that all the enzymes required for the synthesis of the precursor as well as those required for the synthesis and transfer of heptose are present. However, no significant similarities to the genes lpxL (encodes the lauroyl transferase), lpxM (encodes the myristoyl transferase), and lpxP (encodes the palmitoleoyl transferase), that are involved in biosynthesis of acyloxyacyl groups in LPS of E. coli (40), were identified in the genome of A. aeolicus. Because each of these transferases exhibits a strong substrate specificity, the presence of a specific stearic acid transferase (transfers the 18:0 to one 14:0(3-OH) residue, compare Fig. 8) may be expected in Aquifex. It is possible that the gene encoding this transferase possesses no or only some sequence similarity to lpxL and lpxM, because lpxM displays already distant sequence similarities to lpxL (40). This may be the reason why at present no "lpxS" gene has been identified in Aquifex. Despite this, we hypothesize that in LPS biosynthesis of A. pyrophilus the same precursor as in E. coli is first furnished and then processed by specific enzymes to a structure comprising the unique lipid A. Notably, this process includes the replacement of phosphate groups by D-GalpA residues at O-1 and O-4Ј of the lipid A backbone.
Lipid A is considered a valuable chemotaxonomic and phylogenetic marker. From comparative analyses of presently known lipid A structures, the lipid A of A. pyrophilus is unique and, thus, reflects the separated position of this species in the phylogenetic tree, which is based on 16 S rRNA homology studies (2). In agreement with this, amino acid sequence alignments of KdsA (Kdo transferase) and WaaC and WaaF (heptosyl transferases) result in trees possessing Aquifex as a separate branch. Interestingly, Thermotoga maritima (41), the second deepest branching Bacterium according to the 16 S rRNA tree, does not possess the genes for LPS biosynthesis (42) (GenBank TM accession number AE000512). This finding is confirmed by our own finding 2 that no LPS can be isolated from this species.