Purification and mass spectrometry of six lipid A species from the bacterial endosymbiont Rhizobium etli. Demonstration of a conserved distal unit and a variable proximal portion.

Lipid A of Rhizobium etli CE3 differs dramatically from that of other Gram-negative bacteria. Key features include the presence of an unusual C28 acyl chain, a galacturonic acid moiety at position 4', and an acylated aminogluconate unit in place of the proximal glucosamine. In addition, R. etli lipid A is reported to lack phosphate and acyloxyacyl residues. Most of these remarkable structural claims are consistent with our recent enzymatic studies. However, the proposed R. etli lipid A structure is inconsistent with the ability of the precursor (3-deoxy-D-manno-octulosonic acid)(2)-4'-(32)P-lipid IV(A) to accept a C28 chain in vitro (Brozek, K. A., Carlson, R. W., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32126-32136). To re-evaluate the structure, CE3 lipid A was isolated by new chromatographic procedures. CE3 lipid A is now resolved into six related components. Aminogluconate is present in D-1, D-2, and E, whereas B and C contain the typical glucosamine disaccharide seen in lipid A of most other bacteria. All the components possess a peculiar acyloxyacyl moiety at position 2', which includes the ester-linked C28 chain. As judged by mass spectrometry, the distal glucosamine units of A through E are the same, but the proximal units are variable. As described in the accompanying article (Que, N. L. S., Ribeiro, A. A., and Raetz, C. R. H. (2000) J. Biol. Chem. 275, 28017-28027), the discovery of component B suggests a plausible enzymatic pathway for the biosynthesis of the aminogluconate residue found in species D-1, D-2, and E of R. etli lipid A. We suggest that the unusual lipid A species of R. etli might be essential during symbiosis with leguminous host plants.


Introduction
Gram-negative bacteria possess outer membranes consisting of a lipid bilayer in which the outer leaflet is composed largely of lipopolysaccharide (LPS) (1)(2)(3)(4)(5)(6)(7). The structure of LPS can be divided into three regions: 1) the lipid A moiety that serves as the hydrophobic anchor of LPS in the outer membrane; 2) the core region, which consists of a non-repeating oligosaccharide; and 3) the immunogenic O-antigen, a distinctly different, but repeating oligosaccharide.
While the core region confers upon the outer membrane the capacity to act as an effective barrier to many antibiotics (8,9), the lipid A moiety is needed for cell viability (10)(11)(12)(13)(14). Furthermore, lipid A is the portion of LPS that elicits many of the diverse pathophysiological responses associated with severe Gram-negative infections of animals, such as cytokine production, inflammation and shock (1,4,6,(15)(16)(17).
The lipid A moiety found in typical Gram-negative bacteria, like 7 (44), permitting the identification of at least six distinct, but related, lipid A species. In contrast to previous studies (33,34), we obtained these R. etli lipid A fractions without resorting to strong acid or base hydrolysis, thereby greatly facilitating the analysis of the intact species by MALDI/TOF mass spectrometry, NMR spectroscopy, and GC/MS. With this information, we were able to deduce logical structural skeletons for these molecules. All six purified components possess the same distal ends, but are intriguingly heterogeneous in their proximal units. Some of the purified species contain the novel aminogluconate residue (33, 34), but others consist of a more conventional glucosamine disaccharide backbone. Evidence is presented for a single acyloxyacyl group in the distal unit of each of the six components. The secondary fatty acyl chain of this acyloxyacyl residue is the 27-hydroxyoctacosanoate moiety. Our revised structures are consistent with previous enzymatic studies of Kdo2-lipid IVA acylation in R. etli extracts (42). The accompanying manuscript presents an in depth NMR analysis of the purified substances. by guest on  http://www.jbc.org/ Downloaded from 9 chloroform and 800 ml methanol. After incubation for one hour at room temperature with occasional stirring, the mixture was centrifuged at 7520 x g, for 15 min. The insoluble material was recovered and washed once with 380 ml of a fresh, single phase Bligh-Dyer mixture, consisting of CHCl3/MeOH/H2O (1:2:0.8, v/v). The insoluble material was again recovered by centrifugation, and the supernatant was discarded. The washed pellet, which contains the LPS with its covalently bound lipid A moiety, was then suspended in a 120 ml portion of 12.5 mM sodium acetate, pH 4.5, containing 1 % SDS. Dispersion was facilitated by a brief sonic irradiation in a Virsonic cell disrupter. The volume was then adjusted to 360 ml with 12.5 mM sodium acetate, pH 4.5, containing 1 % SDS. At this point, the pH was re-adjusted to 4.5 by careful drop-wise addition of glacial acetic acid. The suspension was divided between two 500 ml glass bottles covered with loose caps. The glycosidic bond between the inner Kdo of the core and the lipid A moiety was cleaved by heating the suspension to 100 °C in a boiling water bath for 30 min (29,(45)(46)(47)(48)(49). After cooling, the suspension was divided into ten 36 ml aliquots, each of which was placed into a 150 ml Corex glass tube. The contents of each tube were converted to a two-phase Bligh-Dyer mixture (50) by addition of 40 ml of chloroform and 40 ml methanol. The phases were mixed thoroughly, and were separated by centrifugation at 7520 x g for 15 min at 25 °C. The lower phases, containing the released lipid A, were combined and passed through a funnel plugged with glass wool to remove insoluble cell debris. A second extraction of the remaining upper phases was done by adding to each tube 40 ml of a pre-equilibrated lower phase, which was obtained from a fresh two-phase Bligh-Dyer mixture, consisting of CHCl3/MeOH/H2O (2:2:1.8, v/v). These mixtures were again centrifuged to separate the phases (as above), and the lower phases from the second extraction were filtered and pooled with the lower phases from the first extraction.
After drying the pooled lower phases by rotary evaporation in a 500 ml round bottom flask at room temperature, another two phase partitioning was carried out to reduce the amount of SDS in the sample. The dried material was re-dissolved in 240 ml of chloroform/methanol (1:1, v/v), and was then equally divided between six 150 ml Corex glass bottles. The round bottom flask was rinsed with a second 240 ml solution of chloroform/methanol (1:1, v/v), and the contents equally distributed between the six Corex bottles. The portion in each Corex bottle was then converted to a two-phase Bligh-Dyer system by adding 36 ml water. The phases were mixed thoroughly, and separated by centrifugation at 7520 x g for 15 min at room temperature. The lower phases were recovered and filtered as above. The filtrate was collected in a 500 ml round bottom flask, dried by rotary evaporation, sealed, and stored at -20 °C. About 160 mg of crude lipid A (contaminated with SDS and residual glycerophospholipids) was obtained.

Resolution of multiple R. etli lipid A species by ion exchange
chromatography-The first step in the purification of the lipid A species of R. etli 11 previously for E. coli lipid A (25,29,48,51). A 60 ml DEAE cellulose (Whatman DE52) column (2.5 x 13 cm) in the acetate form (25,51) was equilibrated with the solvent CHCl3/MeOH/H2O (2:3:1, v/v). The entire crude lipid A sample, prepared as described above, was dissolved in 100 ml CHCl3/MeOH/H2O (2:3:1, v/v) and loaded onto the column by gravity flow. The same solvent mixture (10 ml) was used to rinse the flask, and the additional material was also loaded onto the column. The run-through was collected as a single fraction. Next, the column was washed with 80 ml of CHCl3/MeOH/H2O (2:3:1, v/v), also collected as a single fraction. The various lipid A components were then eluted by increasing the salt concentration of the aqueous portion stepwise in the

Purification and separation of D-1, D-2, and E-Components D-1, D-2
and E, which elute together during the anion exchange step, were resolved by preparative thin layer chromatography using the solvent chloroform/ pyridine/88% formic acid/MeOH/H2O (60:35:10:5:2, v/v). Chromatography and elution from the silica chips was carried out as described above for A and B.
Passage over a final 1-ml DEAE-cellulose column was also carried out, as described above for A and B, and all the purified lipid A samples were stored dry at -20 °C. it was held at 300 °C for 60 min for detecting the C28 fatty acid species.
Chemical ionization mass spectrometry was performed with ammonia as the reactant gas. Electron-impact mass spectra were recorded at 70 eV.  (10). In our procedure, the lipid A is then released from the LPS that remains associated with the extracted cell pellet by mild hydrolysis in sodium acetate buffer at pH 4.5 (29,45,48). Under these conditions, the labile glycosidic linkage between the 3deoxy-D-manno-octulosonic acid (Kdo) and lipid A is cleaved selectively without damage to ester substituents (29,(45)(46)(47)(48).

Micro-heterogeneity of R. etli lipid A released from cells by hydrolysis
When analyzed by thin layer chromatography on silica plates, followed by charring, the lipid A components released from cells by pH 4.5 hydrolysis were surprisingly complex. At least five bands were resolved (Fig. 3   and 1871.7 respectively seen for B (Fig. 5). Similarly, the molecular ions, is mainly a property of the proximal unit. However, partial substitution with βhydroxbutyrate (86 amu) appears to be a distinct feature of the distal unit, since a smaller B1 + ion peak is observed near m/z 1213.4 in most of the samples (Fig. 6).

Positive-ion MALDI/TOF mass spectrometry-
In this interpretation, the peak seen at m/z 1299.9 in component B is derived by glycosidic bond cleavage of either of the two molecular species that give rise to the sodium adduct ions at m/z 2008.5 and 1980.2 (Fig. 6). The peak at m/z 1213.4 is derived from the two species that generate the sodium adducts seen at m/z 1922.1 and 1894.4 (Fig. 6). with certain glycerol based lipids (55). amu (see Fig. 6), indicative of the loss of a β-hydroxymyristoyl group. These assignments are confirmed in the negative-ion mode spectra ( Fig. 7 and 8 respectively) are 226 amu smaller than the B1 + ions of intact C and B (Fig. 6).

MALDI/TOF mass spectrometry of mild base-hydrolyzed components C and B-When fractions containing
The sizes of the B1 + fragment ions therefore indicate that the C28 chain is still present in the distal units of deacylated C and B, and that it must comprise part of an acyloxyacyl moiety at N-2', since the C28 chain itself is ester-linked (33).  Fig. 9. Peak assignments were based on the patterns observed with standards that were prepared and run in parallel. In addition, the masses for each of the peaks derived from B and D-1, observed in the GC traces, were determined by on-line electron impact and chemical ionization mass spectrometry. These spectra were compared to those of the standards, further validating the assignments. The sugar and fatty acid constituents that comprise the individual purified R. etli lipid A components are unambiguously revealed by this procedure (Fig. 9).

GC/MS analysis of components B and D-1-
Interestingly, six fatty acids were detected: 3-OH C14:0, 3-OH C16:0, 3-OH C18:0, two isomers of 3-OH C15:0, and 27-OH C28:0. The latter emerges after 50 min and is not shown in Fig. 9. These results are consistent with the 24 heterogeneity in the fatty acyl chain composition of the proximal unit of B (Fig. 5 and Fig. 6). The fatty acid composition of D-1 is similar to that of B (Fig. 9).
What is important, however, is that the sugars present in B consist only of galacturonic acid and glucosamine, while D-1 clearly contains an additional sugar not present in B. The extra peak, eluting at 28'50" (Fig. 9), is attributed to  Fig. 9). In contrast to the previously published structure (Fig. 1) (33,34), we suggest that the 27-hydroxyoctacosanoate chain is attached to the distal unit as part of an acyloxyacyl moiety. The predicted size of the B1 + ion (1299.8 amu, Fig. 10), which is within experimental error of the 25 observed values (Fig. 6), together with the presence of the β-hydroxybutyryl group on the distal unit, strongly support our idea. A complete comparison of the predicted molecular weights and the observed peaks for the largest of the major species present in B, C, D-1/D-2, and E is shown in Table I. All of the observed peaks are within one mass unit of the values predicted by the structural formulas shown in Fig. 10. In contrast, the molecular weight of the published R.
etli lipid A structure (33,34) is predicted to be 2313.31 (Fig. 1). Ions corresponding to the latter value are not observed (Figs. 5 and 6). Lastly, the more conventional non-phosphorylated glucosamine disaccharide backbone that we have found in components B and C was not originally proposed for R. etli lipid A (33,34). Although the purified components migrate as single bands during TLC in two solvent systems (Fig. 4), the MALDI/TOF mass spectra display peaks corresponding to additional sub-species with or without the β-hydroxybutyryl            Table I are  38 those of the species with the longer acyl chains at position 2, and that are decorated with the β-hydroxybutyrate group.