Structural Analysis of Oligosaccharides from Lipopolysaccharide (LPS) of Escherichia coli K12 Strain W3100 Reveals a Link between Inner and Outer Core LPS Biosynthesis*

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115–154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for ∼70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4′-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional β-d-GlcN in 1→7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal l-α-d-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an α-Kdo-(2→4)-[α-l-Rha-(1→5)]-α-Kdo-(2→4)-α-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide l-α-d-Hep-(1→6)-α-d-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.

Lipopolysaccharide (LPS) 1 is the main constituent of the outer leaflet of the outer membrane in Gram-negative bacteria (1), and many LPS are highly toxic (endotoxins) in mammals (2,3). For the bacteria the outer membrane represents the first line of defense against e.g. antibiotics or bile salts, and therefore the integrity of the outer membrane is of prime importance for the survival of bacteria and as such is a target for the development of new antibacterial drugs. LPS from wild-type enterobacteria is made up of an O-antigen, the core-region, and lipid A (4). The structural features of LPS necessary for a functioning outer membrane are not well understood. However, conserved structural features of LPS have been recognized such as the lipid A moiety, the presence of heptose and Kdo in the inner core region, and the presence of conserved phosphate groups that result in a high number of negative charges at the surface of the membrane. It can be speculated that the chemical structure of the conserved parts of the LPS molecule has evolved to support specific functions of the outer membrane. Thus, the number and position of negative charges may be modulated by masking groups such as 2-aminoethanol or 4-amino-arabinose leading to enhanced resistance against polycationic antibiotics, e.g. polymyxin B (1). Experimental evidence has been obtained recently showing that certain LPS structural motifs are recognized by outer membrane proteins (Omp) and that LPS plays a role for the correct folding and activity of proteins, such as PhoE (5), OmpT (6), and FhuA (7,8). The isolation and detailed structural characterization of all different glycoforms present in LPS from Escherichia coli J-5 aided the interpretation of results from in vitro folding experiments of outer membrane proteins (5,6) and furthermore gave an insight into the biosynthesis of LPS (9). Whereas the presence and distribution of negative charges is undoubtedly very important, the highly conserved nature of carbohydrates that build the inner core region implies that they are particularly suited to fulfill certain functions. In order to better understand the structural requirements of LPS for a functioning outer membrane and compensatory mechanisms upon mutations, it is necessary to obtain a detailed knowledge about the molecular composition of LPS.
Serological and structural features of the LPS from E. coli K12 have been investigated earlier and led to the classification as a separate core type (4,10). Characteristic features are the occurrence of heptose in the outer (11) and rhamnose in the inner core (12), the latter being associated with the immunodominant antigen (13).
LPS consists of several different molecular species, and early studies indicated the presence of structurally heterogeneous LPS in E. coli K12 (13). E. coli K12 strains are widely used as hosts in molecular biology and were therefore the first used to elucidate the genetic details of LPS biosynthesis (14 -22). Although many details of LPS biosynthesis in E. coli K12 are known, there still remain open questions. During the course of this study, Frirdich et al. (23) raised the question whether the structure of the inner core in LPS has an influence on the biosynthesis of the outer core. Overexpression of the gene product of the waaZ gene performed in their study led to increased amounts of an ␣-Kdo-(234)-␣-Kdo-(234)-␣-Kdo trisaccharide in the inner core that was accompanied by a truncation of the outer core. At this stage, it remained unanswered whether the gene waaZ encoded a Kdo transferase that specifically transferred the third Kdo residue onto the common ␣-(234)-Kdodisaccharide inner core structure, which is known to be generated by a single bifunctional enzyme (24). It also remained an open question whether the truncation of the outer core was secondary because of the overexpression of the WaaZ gene product. The gene responsible for the transfer of rhamnose to the inner core of E. coli K12 has yet to be identified.
We have developed methods to purify the different glycoforms present in LPS (9) and for their conjugation to protein carrier molecules allowing their use in serological studies (25,26). The advantage of the developed procedures is the fact that they allow the determination of the relative distribution of the various glycoforms and allow the study of their immunoreactivities.
The complete carbohydrate chains of E. coli R1 to R4 LPS have been investigated (27,28); however, such data for E. coli K12 LPS are lacking. We have therefore now extended our studies to E. coli K12 W3100 LPS and show that it contains two major and two minor glycoforms that are subject to further modification by phosphorylation. Whereas methylation analyses were able to identify the main carbohydrate structure of different parts of the LPS molecule previously and to identify structural modifications (11,12,29), the applied methods failed to reveal how far non-stoichiometrically found substitutions were connected to each other. However, as seen for the E. coli J-5 mutant, from a biosynthetic point of view this information can be of importance in order to design experiments and understand the regulation of genes and enzymes involved in the biosynthesis of LPS and the factors influencing the function of the outer membrane. We now show that E. coli K12 W3100 LPS apart from the known structures contains a so far unknown LPS. The analysis of this structure proves a connection between the biosynthesis of the inner and outer core of LPS and additionally provides a basis for the cloning of the rhamnosyltransferase involved in E. coli K12 LPS assembly.

EXPERIMENTAL PROCEDURES
Bacteria and Bacterial LPS-E. coli K12 strain W3100 was cultivated and used for the isolation of LPS by phenol/chloroform/petrol ether extraction as reported (30).
Preparation of Deacylated LPS of E. coli K12 W3100 -LPS (1.09 g) was de-O-acylated by mild hydrazinolysis (32) (yield 797 mg) and sub-jected to alkaline de-N-acylation as described (33). After neutralization by addition of 4 M HCl, desalting by gel filtration on Sephadex G-10 in 10 mM NH 4 HCO 3 , and lyophilization (yield 366.6 mg), 180 mg of the deacylated oligosaccharide fraction was subjected to high performance anion-exchange chromatography (HPAEC; 9 runs of 20 mg each) using a semi-preparative CarboPak PA100 column (9 ϫ 250 mm) and a DX300 chromatography system (Dionex, Germany). Conditions for semi-preparative and analytical HPAEC were essentially as described . The corresponding molecular ions of oligosaccharides obtained after de-N-acylation (C) were numbered the same as in A but designated with M*. The mass numbers given are those of the monoisotopic peaks. Unannotated clusters of signals originated from molecular ions containing varying amounts of Na ϩ and K ϩ adduct ions. For the assignment of the structures see Table II, Fig. 9, and "Results." previously (9). Briefly, for semipreparative chromatography a gradient of 1-50% B in 60 min with eluents (A) H 2 O and (B) 1 M NaOAc was used. Aliquots of fractions (3 l) were spotted on silica TLC plates and charred with ethanolic sulfuric acid for detection. Two main fractions (fraction 3, yield 30.2 mg, oligosaccharide (OS) 1; fraction 4, yield 13.92 mg, OS 4) and two minor fractions (fraction 1, yield 4.6 mg, OS 2; fraction 2, yield 6.8 mg, OS 3) were collected, neutralized, and desalted by gel filtration on Sephadex G-10 as described. Analytical HPAEC ( Fig. 1) was carried out on a column of CarboPak PA1 (4 ϫ 250 mm, Dionex) using the eluents (eluent A) H 2 O and (eluent B) 1 M NaOAc and a gradient from 1 to 99% of eluent B in 80 min. The run was monitored by pulsed amperometric detection after post-column addition of NaOH.
NMR Spectroscopy-NMR spectra were recorded of samples of OS 1 (15 mg), OS 2 (4.6 mg), OS 3 (6.8 mg), and OS 4 (13.9 mg) in 0.5-ml solutions in D 2 O using a Bruker DRX 600 Avance spectrometer equipped with a multinuclear probe head with z-gradient. Chemical shift values were determined in reference to acetone 2.225 ( 1 H) and 31.5 ppm ( 13 C). All spectra were recorded at a temperature of 300 K using standard Bruker pulse programs. Sodium deutero-oxide was added to the samples prior to one-and two-dimensional 31 P NMR spectroscopy to achieve uniform signals as described (34).
Electrospray Ionization-Fourier Transform Ion Cyclotron-Mass Spectrometry (ESI-FT-ICR MS)-ESI-FT-ICR MS was performed in the negative and positive ion mode using an APEX II Instrument (Bruker Daltonics, Billerica, MA) equipped with a 7 tesla actively shielded magnet and an Apollo ion source. Mass spectra were acquired using standard experimental sequences as provided by the manufacturer. Samples were dissolved at a concentration of ϳ10 ng l Ϫ1 in a 50:50: FIG. 3. 600-MHz 1 H NMR spectrum and chemical structure of deacylated OS 1. In the high field region of the spectrum 2 pairs of signals were observed originating from axial (3a) and equatorial (3e) 3-deoxy protons with chemical shifts characteristic of ␣-Kdo residues (residues C and D). In addition, 10 signals of anomeric protons were present (labeled as indicated in the structure). Kdo is 3-deoxy-␣-D-manno-oct-2-ulopyranosonic acid; L-␣-D-Hep is L-glycero-␣-D-manno-heptopyranose; Gal is galactopyranose; Glc is glucopyranose; and GlcN is 2-amino-2-deoxyglucopyranose. 0.001 (v/v/v) mixture of 2-propanol, water, and triethylamine and sprayed at a flow rate of 2 l min Ϫ1 . Capillary entrance voltage was set to 3.8 kV and dry gas temperature to 150°C. Capillary skimmer dissociation was induced by increasing the capillary exit voltage from Ϫ100 V to Ϫ350 V. Infrared multiphoton dissociation (IRMPD) of isolated parent ions was performed with a 35-watt, 10.6-m CO 2 laser (Synrad, Mukilteo, WA). The duration of laser irradiation was adapted to generate optimal fragmentation and varied between 10 and 80 ms. The unfocused laser beam was directed through the center of the ICR cell, and fragment ions were detected after a delay of 0.5 ms.

RESULTS
Compositional analysis of PCP-extracted LPS from E. coli K12 (Table I) revealed the presence of Rha, Glc, Gal, Hep, Kdo, GlcN, and phosphate in a molar ratio of 0.2:2.8:1.0:4.1:1.9:2.1: 5.1. It has been shown previously that all sugars are present in the D-configuration apart from L-Rha and that all sugars are present as pyranoses (4). Fatty acid analysis by gas liquid chromatography also identified the presence of 3-OH-C14:0, tetradecanoic acid, and dodecanoic acid fatty acids indicating that the acylation pattern was the same as reported for other strains of E. coli (35). We have prepared de-O-acylated LPS by mild hydrazinolysis a part of which was further de-N-acylated under strong alkaline conditions (Fig. 1). The mixture of deac-ylated LPS oligosaccharides was then subjected to HPAEC and four oligosaccharides (OS 1 to OS 4) were obtained. ESI-FT-MS ( Fig. 2A) of the de-O-acylated LPS revealed the presence of 11 different LPS structures that had a composition as indicated in Table II. The major molecule had a mass of 2969 Da (M 1 ) which was consistent with a composition of four hexoses, four Hep, two Kdo, two GlcN, four phosphates, and two amide-linked 3-OH-C14:0 fatty acids (glycoform I). After deacylation NMR analysis of this oligosaccharide (OS 1) identified as hexoses one Gal and three Glc residues (see below). An additional molecular ion was observed which contained an additional GlcNAc (glycoform II, 3172 Da; M 4 ), and one molecular species was devoid of one Hep residue (glycoform III, 2777 Da; M 8 ). Another major component was identified that had a mass of 2981 Da, indicating a composition of three hexoses, three Hep, three Kdo, two GlcN, one Rha, four phosphates, and two amide-linked 3-OH-C14 fatty acids (glycoform IV; M 2 ). NMR analysis of the oligosaccharide (OS 4) after deacylation revealed that this molecule contained one Gal and only two Glc as hexoses.
Molecular ions M 3 , M 9 , M 5 , and M 6 showed that glycoforms I and II were also present as penta-and trisphosphorylated LPS (Table II) . Thus, ESI-FT-MS indicated the presence of four different glycoforms I-IV, which differed in their substitution with GlcNAc, Hep, and Rha. In addition, heterogeneity was observed for the phosphate substitution. Although the majority of all glycoforms contained four phosphates, all of them were also present with three phosphates. Only small amounts of glycoforms I and II contained five phosphates, and a very small amount of a pentaphosphorylated glycoform I LPS contained 2-aminoethanol.
Complete deacylation of LPS and ESI-FT-MS of the mixture of oligosaccharides (Fig. 2C) detected molecular ions with masses of 2436, 2448, 2516, 2528, 2596, and 2677 Da. The separation of the mixture by analytical HPAEC (Fig. 1) revealed the presence of two major (OS 1 and OS 4) and two minor (OS 2 and OS 3) oligosaccharides that were separated by semipreparative HPAEC and further analyzed by ESI-FT-MS and NMR.
Structural Analysis of OS 1 and OS 2-ESI-FT-MS analysis of the mixture of deacylated oligosaccharides (Fig. 2C) and of the isolated OS 1 (not shown) revealed a molecular mass of 2516.67 Da that was in agreement with a chemical composition of four hexoses, four Hep, two GlcN, two Kdo, and four phosphates (M theoretical ϭ 2516.59 Da). The one-dimensional 1 H NMR spectrum (Fig. 3) contained 10 signals of anomeric protons of carbohydrates and two pairs of signals of methylene groups belonging to two Kdo residues and identified this oligosaccharide as a dodecasaccharide. Furthermore, 31 P NMR confirmed the presence of four phosphates (Fig. 4). The full assignment (Tables III and IV)  The spectra were recorded after addition of sodium deutero-oxide (final concentration, ϳ10 mM) as described (34). Whereas OS 1, 2, and 3 contained four phosphates, OS 2 contained three phosphate less. 1 H, 31 P-HMQC spectroscopy revealed that the phosphate substitution was as depicted in Fig. 3 for OS 1, 3, and 4. In OS 2 the 4Јphosphate of the lipid A backbone was missing. the other in ␤-configuration (residue B, 3 J H-1,H-2 ϭ 8 Hz). NOE spectroscopy revealed that these were ␤-(136) connected and according to 31 P, 1 H-HMQC phosphorylated at positions 1 and 4Ј. Furthermore, HMBC revealed that one Kdo (residue C) was substituting position 6 of the ␤-GlcN. Thus, the GlcN-disaccharide represented the lipid A backbone. An NOE between H-6 of the second Kdo and H-3ax (weak) and H-3eq (strong) of Kdo C revealed that these were ␣-(234) connected (36). Three Hep residues (E, F, and H) were identified and NOEs between the anomeric proton of H and protons H-6 and H-7a/b of F in addition to an NOE between the anomeric proton of F and H-3 of E proved that they formed the Hep-(137)-Hep-(133)-Hep trisaccharide (residues H, F, and E, respectively) commonly found in enterobacterial LPS (4). The structure was confirmed by HMBC correlation signals between the anomeric protons of H and C-7 of F, and of F and C-3 of E, respectively. This trisaccharide substituted the inner Kdo (residue C) in position 5 leading to a NOE between the anomeric proton of E and protons H-5 and -7 of Kdo C. According to NOE data and HMBC three ␣ϪGlc residues (K, I, and G) formed the trisaccharide Glc-(132)-Glc-(133)-Glc which was connected to position 3 of Hep F. These residues thus belonged to the outer core (4). Additionally, the 3 J H-1,H-2 coupling constant of 4 Hz and the small 3 J H-3,H-4 (4 Hz) and 3 J H-4,H-5 (Ͻ 3 Hz) coupling constants of residue M identified it as a ␣-Gal residue that was bound to position 6 of Glc G (HMBC and NOE, low field chemical shift of C-6, and protons H-6a, H-5, and high field shift of H-6b). The fourth Hep (L) was connected to position 6 of Glc K. The configurations at the anomeric centers of all residues apart from Kdo were confirmed by a non-decoupled 1 H, 13 C-HMQC spectrum (Ͼ170 Hz for ␣ and Ͻ168 Hz for ␤; Fig. 5). The two remaining phosphates were located at position 4 of both Hep residues E and F ( Fig. 4 and Table V) because the 31 P resonances gave correlation signals to the respective protons in HMQC, and the cross-correlation signals of these protons in DQF-COSY showed an additional coupling of ϳ10 Hz due to 3 J P,H -coupling (9). Thus, OS 1 possessed the chemical structure as depicted in Fig. 3 and represented glycoform I. The mass spectrometric analysis of OS 2 (not shown) revealed a difference of 80 Da lower mass in comparison to OS 1, indicating the loss of one phosphate (mass ϭ 2436 Da). This was corroborated by the presence of only three phosphorous resonances in 31 P NMR spectroscopy (Fig. 4). The changes of the chemical shifts of protons H-3, -4, and -5 (Table III) and of carbons C-4 and C-5 (Table IV) of the ␤-GlcN indicated that the 4Ј-position was not phosphorylated. This was finally proven by 1 H, 31 P-HMQC spectroscopy and IRMPD-ESI-FT-ICR MS. Apart from these chemical shift differences, the same carbohy-drate composition and linkages were identified by NMR as in OS 1 which thus were identical in their carbohydrate structure (compare Fig. 3, glycoform I).
Structural Analysis of OS 3-The ESI-FT-MS of deacylated LPS oligosaccharides (Fig. 2C) and the isolated OS 3 (not shown) revealed a molecular mass 161 Da higher than OS 1 indicating the presence of an additional hexosamine residue. The only amino sugar identified by compositional analysis was GlcN. Accordingly, the 1 H NMR spectrum (Fig. 6) contained additional resonances (Tables III and IV) in comparison to OS 1 belonging to a GlcN (residue L). The 3 J H-1,H-2 coupling constant of 8.5 Hz and NOE connectivities between H-1, -3, and -5 revealed that it possessed ␤-configuration, and the far downfield shift of C-7 and H-6 of L proved that it was connected to position 7 of Hep L (glycoform II). 31 P NMR identified four phosphates (Fig. 4) at identical substitution sites as in OS 1. The corresponding molecular ion of the N-acylated sample in ESI-FT-MS had a molecular mass of 3172.09 Da ( Fig. 2A) and thus showed that this residue was N-acetylated in LPS. There was no molecular ion present that had an unsubstituted amino group at this position. Structural Analysis of OS 4 -The one-dimensional 1 H NMR spectrum of OS 4 (Fig. 7) contained three pairs of signals of CH 2 groups belonging to three Kdo residues (C, D, and P). In addition, the strong resonance of a methyl group was present at 1.2 ppm, indicating the presence of a 6-deoxy sugar. In the low field region of the spectrum four signals of anomeric protons were identified which had a small 3 J H-1,H-2 -coupling constant of less than 1 Hz and thus had an equatorial proton in position 2. The assignment of the spin systems revealed that only three of these belonged to Hep residues (E, F, and H) and one belonged to an ␣-Rha ( 1 J C-1,H-1 ϭ 171 Hz; R). Further signals of anomeric protons belonged to two ␣-Glc (G, and I) and one residue each of ␣-Gal (M), ␣-GlcN 1P (A) and ␤-GlcN 4P (B). Those residues that were also present in OS 1 formed the same primary structure according to chemical shift analysis, NOESY and HMBC spectra. However, the terminal Glc (K) and Hep (L) that were present in OS 1, OS 2, and OS 3 were missing in this oligosaccharide. The chemical shifts of carbons 4 and 5 of Kdo D (Table   IV) showed that these positions were substituted, and because the anomeric proton of the Rha (R) gave a strong NOE to proton 5 of Kdo D they were connected in 135-linkage and the additional Kdo (P) was located at position 4 of D. Thus, the side chain Kdo residue D was a branching point in this oligosaccharide and carried, in addition to a third Kdo (P) in position 4, a Rha attached to position 5. 31 P NMR (Fig. 4) spectroscopy revealed the presence of four phosphate residues that according to 1 (Fig. 8B) showed fragmentational loss of the terminal residue P (Kdo, Ϫ220 Da, mass of 2308.54 Da) and also of residue R (Rha, Ϫ142 Da, mass of 2162.48 Da). This unequivocally proved the middle Kdo as attachment site for Rha and Kdo. Further fragmentations gave rise to ions with masses of 1942.42 Da (Ϫ Kdo D) and 1442.34 Da (Ϫ GlcN 2 P 2 , Ϫ500 Da, lipid A backbone). The latter fragmentation was also observed directly after the loss of residues P and R which gave rise to an ion of 1662.39 Da. OS 4 thus possessed the chemical structure depicted in Fig. 7 and represented glycoform IV. DISCUSSION The investigation of deacylated LPS from E. coli K12 W3100 by ESI-FT-MS (Fig. 2) indicated the presence of 11 molecular species in this LPS. The presence of four different glycoforms was established, and these differed in their oligosaccharide    Tables III and IV. structures (Fig. 9). All glycoforms were heterogeneous with respect to their phosphate and 2-aminoethanol substitution. The main oligosaccharide accounting for 70% of the LPS (as determined by HPAEC of deacylated oligosaccharides, Fig. 1) was a tetraphosphorylated dodecasaccharide of the structure depicted in Fig. 9 (glycoform I). A minor component had the same carbohydrate structure but did not contain the 4Ј-phosphate on the lipid A backbone. This position was only partially substituted with phosphate in all different glycoforms as shown by ESI-FT-MS on the de-O-acylated sample prior to alkali treatment and therefore was not the result of an elimination of 2-aminoethanol monophosphate under the strong alkaline conditions applied for the N-deacylation reaction. One molecular ion indicated the substitution with 2-aminoethanol monophosphate of the main oligosaccharide. This substitution has been identified in several enterobacterial LPS in lipid A and on the 4-position of the first Hep residue as 2-aminoethanol diphosphate or the 7-position of Kdo as 2-aminoethanol monophosphate (Kdo-PE (4)). A molecular ion of 3092.01 Da ( Fig. 2A, M 6 ) was consistent with such a substitution of the main oligosaccharide (M calculated ϭ 3091.99 Da); however, the same ion could arise from the presence of a molecule containing GlcNAc devoid of phosphate at the 4Ј-position (M calculated ϭ 3092.09 Da), and the lack of phosphate at this position was found for all other glycoforms. Investigation of the expanded not charge-deconvo-luted ESI-FT-MS spectrum in the region of triple charged ions (Fig. 2B) showed the presence of both LPS species. We have isolated previously Kdo substituted in position 7 with 2-aminoethanol monophosphate or phosphate (37). Thus, the pentaphosphorylated LPS molecules may originate from the substitution with phosphate (Table II, M 3 and M 9 ) and 2-aminoethanol monophosphate (M 11 ) at this position. We have tried to identify Kdo phosphate and Kdo-PE by IRMPD-ESI-FT-MS in the de-O-acylated LPS but were unsuccessful due to the complexity of the spectrum in this region.
Another minor component was a tridecasaccharide which in comparison to the main component was additionally substituted by GlcNAc in ␤-(137)-linkage on the terminal 136linked Hep residue in the outer core (glycoform II), and ESI-FT-MS of the de-O-acylated LPS indicated that this molecule was also present with a higher degree of phosphorylation (Table II).
HPAEC led to the isolation of four phosphorylated oligosaccharides, two of which were present in larger amounts. The structural analysis of three of these oligosaccharides (glycoforms I to III), which were now isolated for the first time as complete carbohydrate chains from this LPS, confirmed the results of previous studies based on methylation analysis (4). We have isolated another component of this LPS (glycoform IV), which accounted for the remaining 20 -25% and which was FIG. 7. 600-MHz 1 H NMR spectrum and chemical structure of deacylated OS 4. In the high field region of the spectrum 3 pairs of signals were observed originating from axial (3a) and equatorial (3e) 3-deoxy protons with chemical shifts characteristic of ␣-Kdo residues. In addition only 9 signals originating from anomeric protons were observed. not known to be present in this LPS. The inner core structure consisted of a trisaccharide of Kdo residues connected in ␣-(234)-linkages and was further modified by an additional Rha residue located at the 5-position of the middle Kdo residue. Previous studies have identified in some strains of E. coli K12 the Rha substitution in the inner core (13), and the ␣-(135)linkage was shown by methylation analysis of the isolated disaccharide after mild acid hydrolysis of E. coli K12 W3100 LPS (12). Due to the acidic conditions used in the aforementioned study, the Kdo-trisaccharide structure was destroyed. Therefore, it was assumed that in this LPS the inner core structure may contain either a third Kdo in position 4 or a Rha substitution in position 5 of the side chain Kdo. We now isolated the complete oligosaccharide and unequivocally proved its structure by NMR (Fig. 7), ESI-FT-MS, and MS-MS studies (Fig. 8). The inner core structure of this LPS thus contains a second branched Kdo residue in the side chain. At the same time the terminal Hep-(136)-␣-D-Glc disaccharide of the outer core is missing. There was no indication that the Rha can be found on oligosaccharides containing only two Kdo, and it appears that the rhamnosyltransferase involved in the biosynthesis of this structure has an absolute requirement for the Kdo trisaccharide as substrate. Furthermore, there was no other glycoform in this LPS that contained three Kdo residues. Why the biosynthesis of the outer core is affected at the same time and does not go to completion cannot be answered at this stage, but it could be speculated that the additional sugars in the inner core have an influence on the conformation of the outer core sugars precluding an elongation. Additional evidence for this explanation may come from the early observation (13) that the Rha residue is part of the immunodominant epitope in this LPS. Although in other E. coli LPS parts of the deep inner core are usually inaccessible for antibodies when they are substituted by further sugars of the inner and outer core, this does not seem to hinder the reactivity with the inner core in this LPS. It can also not be answered at this stage why some E. coli K12 strains modify the inner core carbohydrate structure by Rha or 2-aminoethanol phosphate.
A recent study (23) provided genetic evidence for the correlation of the inner core and the outer core biosynthesis. It was found that the protein WaaZ with yet unknown function from E. coli K12 W3100 led to an increased amount of an ␣-Kdo-(234)-␣-Kdo-(234)-␣-Kdo-trisaccharide in the inner core when overexpressed in E. coli F470 which possesses an R1 type core oligosaccharide. Concomitantly, molecules containing three Kdo residues were truncated in the outer core. It remained unclarified whether this effect was secondary due to the overexpression of this enzyme in the R1 background. Inactivation of WaaZ in E. coli K12 W3100 led to indistinguishable banding patterns in Tricine SDS-PAGE from the wild-type LPS. Due to expected difficulties in obtaining large enough amounts for structural analysis and difficulties in analyzing complex inner core structures of LPS by NMR, it was not attempted to isolate and analyze the corresponding oligosaccharides from the parent strain. However, the investigation of the E. coli K12 W3100 LPS oligosaccharides described in this paper showed that a full structural characterization including NMR analysis was possible. According to our data, the results obtained by Frirdich et al. (23) must be interpreted in the sense that the presence of three Kdo residues connected in ␣-234-linkages in the inner core of E. coli LPS are associated with a truncated outer core, and this effect is not due to the overexpression of WaaZ; furthermore, we could show that the substitution of the inner core with Rha occurs only on the Kdo-trisaccharide and exclusively at the middle Kdo. Thus, the inner core structure had an effect on the biosynthesis of the outer core. Cloning experiments aiming at the identification of the gene responsible for rhamnose transfer in this LPS is recommended to be done in a host able to synthesize the inner core Kdo-trisaccharide.
Because much of the structural heterogeneity observed within LPS structures has been identified using derivatized partial structures obtained after hydrolysis and gas-liquid chromatography-mass spectrometry analysis, but also for the sake of simplicity, it has become common practice to depict all structural modifications within the same picture of a basic structure. This may misleadingly imply that all of these Glycoforms I (70%), II (5%), and IV (20%) were present as tetraphosphorylated LPS in larger amounts in this LPS and could be isolated as deacylated oligosaccharides (OS 1, 3 and 4) and further analyzed by NMR. ESI-FT-MS and NMR revealed that all glycoforms were present in small amounts lacking the phosphate in the 4-position of the ␤-GlcNAcyl residue, of which only the glycoform I could be isolated in larger amounts after deacylation (ϳ5% of LPS, OS 2). Only glycoforms I and II were present in small amounts as pentaphosphorylated LPS. A small amount of 2-aminoethanol was found only in pentaphosphorylated glycoform I LPS. A very minor component (glycoform III) did not contain the outer core heptose. modifications occur randomly and in all permutations. However, as can be seen from the results of this and previous studies on the LPS of E. coli strain J-5 (9), one has to keep in mind that this picture may not reflect the real situation because certain combinations of structural modifications may preclude each other. This may lead to false interpretations of experimental data.
The fact that only certain subfractions of the LPS of E. coli J-5 were able to promote folding of PhoE (5) and influence the activity of OmpT (6), it appears that E. coli, and probably other bacteria as well, employs structural modifications of LPS deliberately to support specific functions of the outer membrane. Thus, the observed heterogeneity of LPS may not be seen solely as a statistical distribution of possible modifications according to the fidelities of enzymes involved in LPS biosynthesis but rather as means of controlling function by structure. Determining the specific functions of individual LPS structures will be difficult but rewarding in the sense that a better understanding of the outer membrane assembly will open new doors for therapeutic treatment of infectious diseases.