Lipid A from Oligotropha carboxidovorans Lipopolysaccharide That Contains Two Galacturonic Acid Residues in the Backbone and Malic Acid A Tertiary Acyl Substituent

The free-living Gram-negative bacterium Oligotropha carboxidovorans (formerly: Pseudomonas carboxydovorans), isolated from wastewater, is able to live in aerobic and, facultatively, in autotrophic conditions, utilizing carbon monoxide or hydrogen as a source of energy. The structure of O. carboxidovorans lipid A, a hydrophobic part of lipopolysaccharide, was studied using NMR spectroscopy and high-resolution mass spectrometry (MALDI-ToF MS) techniques. It was demonstrated that the lipid A backbone is composed of two d-GlcpN3N residues connected by a β-(1→6) glycosidic linkage, substituted by galacturonic acids (d-GalpA) at C-1 and C-4’ positions. Both diaminosugars are symmetrically substituted by 3-hydroxy fatty acids (12:0(3-OH) and 18:0(3-OH)). Ester-linked secondary acyl residues (i.e., 18:0, and 26:0(25-OH) and a small amount of 28:0(27-OH)) are located in the distal part of lipid A. These very long-chain hydroxylated fatty acids (VLCFAs) were found to be almost totally esterified at the (ω-1)-OH position with malic acid. Similarities between the lipid A of O. carboxidovorans and Mesorhizobium loti, Rhizobium leguminosarum, Caulobacter crescentus as well as Aquifex pyrophylus were observed and discussed from the perspective of the genomic context of these bacteria.


Introduction
Oligotropha carboxidovorans strain OM5 is a Gram-negative slightly curved rod bacteria, possessing one lateral flagellum [1]. This bacterium was isolated from the soil of wastewater sewage treatment settling ponds near Götingen in Germany and was previously described as Pseudomonas carboxidovorans strain OM5 [2]. O. carboxidovorans can live in heterotrophic and chemolitho-autotrophic conditions. In the latter case, these bacteria can utilize carbon monoxide as a sole source of carbon and energy. Aerobically, they are able to oxidize approximately six molecules of CO to CO 2 and simultaneously assimilate one of the produced CO 2 molecules in the pentose phosphate pathway [3]. Oligotropha are economically important microorganisms, as they can oxidize syngas (i.e., a mixture of CO, CO 2 , and H 2 ) generated by biogasification of organic wastes [4]. Globally, such types of bacteria remediate more than 100 million tons of carbon monoxide from the atmosphere [5]. Heterotrophically, in aerobic conditions,

Isolation and Chemical Analysis of O. carboxidovorans Lipid A
The LPS of O. carboxidovorans strain OM5 was extracted from delipidated and enzymatically digested cells with the hot 45% phenol/water method [17,18]. The LPS was found mainly in the water phase. Lipid A was obtained by mild acid (1% acetic acid) hydrolysis of LPS and was subjected to fatty acid and sugar analyses. The sugar components of the O. carboxidovorans lipid A backbone were represented by 2,3-diamino-2,3-dideoxy-d-glucose (d-GlcN3N), and d-galacturonic acid (d-GalA). Among fatty acids, three amide-linked 3-hydroxy fatty acids (12:0(3-OH) and 14:0(3-OH) and 18:0(3-OH)) were identified, whereas ester-linked fatty acids were represented by a non-polar fatty acid (18:0) and two long (ω-1)-hydroxylated fatty acids (26:0(25-OH) and 28:0(27-OH)) ( Figure 1, Table 1). All fatty acids were identified based on their chromatographic properties (retention times) and characteristic mass spectra. Additionally, a short-chain organic acid (malic acid) was found in the lipid A preparation. This component has been identified based on GC-MS analysis of TMSi-derivatives of butyl esters

NMR Spectroscopy of O. carboxidovorans Lipid A
The native lipid A of O. carboxidovorans was dissolved in chloroform-d1/methanol-d4 (6:1, v/v) and structurally characterized by 1D and 2D NMR spectroscopy. As confirmed by 31 P NMR, the lipid A had no phosphate residues.
The HSQC-DEPT spectrum (Figure 2a) contained signals from four anomeric carbons (δc 92.57-102.69), four signals of nitrogen-bearing carbons (δc 51.65-54.12), signals of remaining sugar carbons (δc 76.94-61.24), signals for CH-OH and CH-OR groups from hydroxylated fatty acids, and from the CH-OH group of malic acid (δc 67. 16-73.39). Based on the 1 H-1 H COSY, TOCSY, and 1 H-13 C HMBC experiments four spin systems were identified, all deriving from hexopyranoses. Spin systems A and D derived from α-D-GalpA residues, B represented α-D-GlcpN3N, and C was assigned to β-D-GlcpN3N. All 1 H and 13 C chemical shifts for the sugar backbone of O. carboxidovorans lipid A were assigned and listed in Table 2. The anomeric configuration of all monosaccharides was confirmed by measuring the 1 J(C1,H1) coupling constants. Relatively large values of coupling constants (above 170 Hz) for anomeric signals were found for residues A, B, and D, thus identifying their α-configuration. Residue C was characterized by a small (161.1 Hz) 1 J(C1,H1) coupling constant value, indicating its βconfiguration.

NMR Spectroscopy of O. carboxidovorans Lipid A
The native lipid A of O. carboxidovorans was dissolved in chloroform-d1/methanol-d4 (6:1, v/v) and structurally characterized by 1D and 2D NMR spectroscopy. As confirmed by 31 P NMR, the lipid A had no phosphate residues.
The HSQC-DEPT spectrum ( Figure 2a) contained signals from four anomeric carbons (δ c 92.57-102.69), four signals of nitrogen-bearing carbons (δ c 51.65-54.12), signals of remaining sugar carbons (δ c 76.94-61.24), signals for CH-OH and CH-OR groups from hydroxylated fatty acids, and from the CH-OH group of malic acid (δ c 67. 16-73.39). Based on the 1 H-1 H COSY, TOCSY, and 1 H-13 C HMBC experiments four spin systems were identified, all deriving from hexopyranoses. Spin systems A and D derived from α-d-GalpA residues, B represented α-d-GlcpN3N, and C was assigned to β-d-GlcpN3N. All 1 H and 13 C chemical shifts for the sugar backbone of O. carboxidovorans lipid A were assigned and listed in Table 2. The anomeric configuration of all monosaccharides was confirmed by measuring the 1 J (C1,H1) coupling constants. Relatively large values of coupling constants (above 170 Hz) for anomeric signals were found for residues A, B, and D, thus identifying their α-configuration. Residue C was characterized by a small (161.1 Hz) 1 J (C1,H1) coupling constant value, indicating its β-configuration.  The chemical shift values of the α, β, and γ carbons and protons of the 3-OH-fatty acids (both 3-O-acylated and those with a free OH group) and for signals derived from ω, ω-1, ω-2, ω-3, and ω-4 protons and carbons of substituted as well as unsubstituted (ω-1)-hydroxylated long chain fatty acids were established and shown in Table 3. These data were similar to those reported for lipid A derived from B. elkanii or B. japonicum [12,13]. The NMR signals from α and β carbons and protons as well as signals from carbons of carboxyl groups belonging to malic acid residues were identified as well. Two signals derived from β-CH proton and carbon at δ 4.505/67. 16    The ROESY spectrum ( Figure 2b) showed the following connectivities between anomeric and linkage protons: A1/B1 (δ 5.248/5.053), C1/B6 (δ 4.395/3.710), and D1/C4 (δ 5.189/3.825). These data were confirmed by analysis of the HMBC spectrum, in which the following 1 H-13 C connectivities were found: A1/B1 (δ 5.248/92.57), C1/B6 (δ 4.395/68.70), and D1/C4 (δ 5.189/75.22). Taken together, the sugar backbone of O. carboxidovorans lipid A has the following structure: The chemical shift values of the α, β, and γ carbons and protons of the 3-OH-fatty acids (both 3-O-acylated and those with a free OH group) and for signals derived from ω, ω-1, ω-2, ω-3, and ω-4 protons and carbons of substituted as well as unsubstituted (ω-1)-hydroxylated long chain fatty acids were established and shown in Table 3. These data were similar to those reported for lipid A derived from B. elkanii or B. japonicum [12,13]. The NMR signals from α and β carbons and protons as well as signals from carbons of carboxyl groups belonging to malic acid residues were identified as well. Two signals derived from β-CH proton and carbon at δ 4.505/67.16 and 4.446/67.41 were recognized. The signals derived from carbons of malic acid carboxyl groups (δ 170.84 and 173.30) showed an interresidue correlation with (ω-1)-hydrogens (δ 4.929 and 4.996, respectively) from the long chain fatty acids in the HMBC spectrum. The signals of the β-CH group of unsubstituted 3-hydroxy fatty acids were identified at δ 3.833/68.80 and 3.942/68.80. Two signals derived from the β-CH proton and carbon of 3-O-substituted fatty acids were found at δ 5.262/68.0 and 5.145/71. 29. The proton/carbon chemical shifts at δ 4.996/73.39 and 4.929/72.36 were derived from the (ω-1) methine groups of the long chain fatty acids, which are connected to the 3-OH group of the amide-linked fatty acids and bear malic acid as a tertiary residue.

Mass Spectrometry of O. carboxidovorans Lipid A
Mass spectrometry experiments were performed in the positive and negative ion modes of native lipid A preparations. Figure 3 shows the negative-ion MALDI-TOF mass spectrum of the native lipid A sample.

Mass Spectrometry of O. carboxidovorans Lipid A
Mass spectrometry experiments were performed in the positive and negative ion modes of native lipid A preparations. Figure 3 shows the negative-ion MALDI-TOF mass spectrum of the native lipid A sample.   Based on the chemical analyses of the sugar components and fatty acid residues, the most abundant signal at m/z 2454.722 could be assigned to lipid A molecules containing two GlcpN3N residues, two GalpA units, 12:0(3-OH), 14:0(3-OH), two 18:0(3-OH) fatty acid residues, and two ester-linked fatty acids: 18:0 and 26:0(25-OH) acids. The last one was additionally esterified by malic acid. Around this signal, one can distinguish a number of others with a lower intensity. The mass differences between the next two (e.g., m/z 2441.708 and 2455.739 or 2427.696 and 2455.739) correspond to the mass of a single or double methylene groups (14 or 28 u), as a result of a different acylation pattern. Moreover, this spectrum ( Figure 3) contains signals corresponding to lipid A molecules lacking an octadecanoate (m/z at 2188.477) or acyloxyacyl (26:0(25-OH) + malate, m/z at 1944.382) residue or both substituents simultaneously (m/z at 1678.123, traces). As a result, all of these signals should be thought to prove the existence of the whole set of particles being variants of lipid A differing in acyl substitution and acyl substituents. It should be noted, however, that most of the molecules in the preparation are lipid A molecules substituted with seven acyl residues (four 3-OH-fatty acids, one VLCFA, one non-polar acid, and malic acid). Based on the analysis of the intensity of the respective ions in the mass spectrum of native lipid A, it was estimated that ca. 85% of the molecules in the preparation were heptaacylated lipid A. Each of the negative ions described above corresponds to two ions ( including only the C1, C2, and C3 carbons and one oxygen. The fragmentations described above clearly indicate that two acyls (18:0 and 26:0(25-O-(malic acid)) are ester-linked, thus demonstrating that they occur as secondary fatty acids. Moreover, this observation unambiguously proved again that both secondary O-substitutions occurred on the non-reducing GlcpN3N unit. Therefore, O. carboxidovorans OM5 displayed a lipid A structure with an asymmetric distribution of the acyl moieties with respect to the di-GlcpN3N backbone (symmetry 4+2 or, more precisely, taking into account malic acid as a separate residue, the symmetry should be written as follows: 5+2).
Summing up all the experimental data, a probable structural formula of lipid A from O. carboxidovorans OM5 can be proposed ( Figure 5). reducing GlcpN3N unit. Therefore, O. carboxidovorans OM5 displayed a lipid A structure with an asymmetric distribution of the acyl moieties with respect to the di-GlcpN3N backbone (symmetry 4+2 or, more precisely, taking into account malic acid as a separate residue, the symmetry should be written as follows: 5+2).
Summing up all the experimental data, a probable structural formula of lipid A from O. carboxidovorans OM5 can be proposed ( Figure 5).   reducing GlcpN3N unit. Therefore, O. carboxidovorans OM5 displayed a lipid A structure with an asymmetric distribution of the acyl moieties with respect to the di-GlcpN3N backbone (symmetry 4+2 or, more precisely, taking into account malic acid as a separate residue, the symmetry should be written as follows: 5+2).
Summing up all the experimental data, a probable structural formula of lipid A from O. carboxidovorans OM5 can be proposed ( Figure 5).

Genomic Studies
The experimental data showing the structure of O. carboxidovorans OM5 lipid A obtained with MALDI-TOF mass spectrometry and NMR spectroscopy were further supplemented by in silico analyses of the O. carboxidovorans OM5 genomic sequence aiming to identify putative genes encoding proteins/enzymes engaged in the lipid A biosynthesis pathway.
The sequence similarity of the 16S rRNA gene indicates that O. carboxidovorans is closely related to the members of the Bradyrhizobiaceae family [6]. Our results thereby confirm that the structure of the O. carboxidovorans lipid A sugar backbone is identical to that of Aquifex pyrophilus [20] and Caulobacter crescentus [21]. In turn, at its reducing end, it resembles some mesorhizobial lipids A (i.e., M. huakuii IFO 15243T and M. loti MAFF303099) [10,11]. Moreover, at the non-reducing end, it resembles some rhizobial lipids A [9]. The lipid A structure as well as gene clusters engaged in its biosynthesis have been recognized for M. loti MAFF303099 and Rhizobium leguminosarum 3841; therefore, respective protein sequences of these model strains were used as queries in BLAST similarity searches comprising the O. carboxidovorans OM5 genome sequence (Table 4). Using this approach, we recognized a set of putative genes coding for common enzymes required for the lipid A biosynthesis (i.e., lpxA-D, lpxH, lpxK, and kdtA, data for these genes are not shown) and genes encoding specific enzymes involved in the structural modifications of lipid A (lpxE, lpxF, rgtD, rgtF, rgtE and acpXL-lpxXL cluster) found in some Gram-negative bacteria (Tables 4 and 5). Additionally, using M. loti, lipid A biosynthesis related proteins as queries in the genome of O. carboxidovorans sequences coding for putative enzymes involved in the conversion of GlcpN to GlcpN3N (presumable homologs of gnnA and gnnB) were identified (Table 5). Such enzymes, essential for the biosynthesis of the GlcpN3N type of the lipid A disaccharide backbone, are specific not only for Mesorhizobium but also for the Azorhizobium and Bradyrhizobium genera. Putative ORFs detected in the O. carboxydovorans OM5 genome ascribed to lipid A biosynthesis/modification shared sequence similarity with the respective proteins of M. loti MAFF 303099 or R. leguminosarum bv. Viaciae 3841 (reaching even 78% for individual proteins encoded in the acpXL-lpxXL cluster) (Tables 4 and 5), strongly suggesting their engagement in this biosynthetic pathway. Due to the identical structures of the sugar part of lipid A of O. carboxidovorans OM5 and Aquifex pyrophilus, the database searches aiming to identify putative genes necessary for GlcpN3N-(1→6)-GlcpN3N backbone biosynthesis were extended to representatives of Aquifex and Caulobacter. In the Aquifex genus, Aquifex aeolicus VF5 is the only accessible fully sequenced genome (lack of genomic data for A. pyrophilus). In the A. aeolicus VF5 genome, putative homologues of the rgtD, rgtF, and rgtE genes were identified (Table 4). Surprisingly, we were not able to detect sequences similar to lpxE and lpxF (encoding hypothetical lipid A phosphatases) in the A. aeolicus VF5 genome. These enzymes were shown to be involved in dephosphorylation of the lipid A precursor during the biosynthesis of LPS in R. leguminosarum bv. Viciae 3841 [11]. It may be assumed that the process of phosphate residue removal from both ends of the GlcpN3N-disaccharide in representatives of the Aquifex genus is catalyzed by other types of unknown phosphatases.
As demonstrated by previously published structural data for lipid A of Caulobacter crescentus CB15 [21], putative homologues of lpxE, rgtD, rgtF, and rgtE were found in its genome (Table 4). In the reference genomes, the rgtDFE genes encode hypothetical 4 -α-GalpA, α-(1→1)-GalpA, and bactoprenyl-phosphate-GalpA transferases, respectively [11]. The C. crescentus CB15 ORF marked as CC_3019 displayed some sequence similarity with putative lipid A 1-phosphatase encoded by lpxE in R. leguminosarum bv. Viciae 3841 [22]. However, using this approach, no putative homologues of lipid A 4'-phosphatase were found in the C. crescentus CB15 genome. In Aquifex, Azorhizobium (data not shown), Mesorhizobium, and Oligotropha homologues of rtgE and rtgF are clustered; however, it seems not to be a prerequisite for respective protein products to participate effectively in lipid A modification (see R. leguminosarum, Table 4). On the other hand, the available data strongly suggest that genes responsible for the synthesis and incorporation of VLCFAs into lipid A always form a tight cluster [9]. Although a set of genes presumably engaged in VLCFA production was found in C. crescentus CB15 (Table 5), they were dispersed over the bacterial genome, and the formation and incorporation of VLCFAs was not observed in these bacteria. Table 4. Sequence similarity of putative proteins involved in lipid A modification in Oligotropha carboxidovorans OM5, Aquifex aeolicus and Caulobacter crescentus CB15 with enzymes of known functions from the reference strains: Rhizobium leguminosarum bv. Viciae 3841 and Mesorhizobium loti MAFF303099).  -(1,1)

Discussion
In this work, we have described the structure of O. carboxidovorans OM5 lipid A, which contains a β-d-GlcpN3N-(1→6)-α-d-GlcpN3N disaccharide decorated on both sides (at positions C-1 and C-4') by α-d-GalpA residues. This extended sugar backbone is unique in bacteria. To date, there have been only two reports of the existence of similar lipids A. They are synthesized by bacteria belonging to the hypertermophilic Aquifex pyrophilus species [20], and the stalk-forming Caulobacter crescentus [21]. The substitution of only the reducing end of lipid A by α-(1→1)-d-GalpA is also not common among bacteria and has been identified in lipids A from a few representative genera. These include the associative diazotroph Azospirillum lipoferum [23], and some rhizobia: M. huakuii [10], M. loti [11], and P. trifolii [24]. On the other hand, the substitution of exclusively the non-reducing end of lipid A by α-d-GalpA-(1→ at position C-4' is not common either, and has been identified in lipids A from a few representatives of rhizobia (R. etli CE3, R. leguminosarum bvs. Trifolii and Viciae) [9].
The amino groups of both GlcpN3N of O. carboxidovorans OM5 lipid A are symmetrically substituted by 3-hydroxyoctadecanoic fatty acid at position C-2 and C-2'. In the dominant type of lipid A molecules, the amino group at position C-3 is acylated with 3-hydroxylauric acid, while a 3-hydroxymyristoyl group is located at position C-3'. Due to the multiplicity of signals assigned to heptaacylated lipid A, another distribution of primary fatty acids in individual subgroups of lipid A molecules should also be expected. Even if the extended backbones of lipids A synthesized by Oligotropha, Aquifex, and Caulobacter are the same, the substituting primary fatty acids are significantly different. Lipid A contains mainly 18:0(3-OH) (or 14:0(3-OH) and 12:0(3-OH)):residues in Oligotropha, 16:0(3-OH) (and 14:0(3-OH)) residues in Aquifex, and almost exclusively 12:0(3-OH) residues in Caulobacter. This fact can be explained by the different basic metabolisms of fatty acids of these bacteria [7,25,26].
The 28:0(27-OH) fatty acid is the most often isolated VLCFA-type fatty acid among rhizobia [27], but the 26:0(25-OH) fatty acid took its place in Oligotropha. In addition, 25-hydroxyhexacosanoic acid was almost totally acylated with a malic acid residue. It should be emphasized that this type of tertiary substituent has not been described in the literature so far. Since ester-linked fatty acids (18:0 and 26:0(25-OH) are secondary substituents of the distal GlcpN3N, the complete lipid A is asymmetrically acylated and resembles the E. coli lipid A pattern denoted by formula 4+2 (or more precisely 5 + 2) [28].
We have found structural similarities in the acylation of lipid A with VLCFA between O. carboxidovorans and some rhizobia. Moreover, we have shown that the sugar backbone of lipid A in O. carboxidovorans is the same as in A. pyrophilus and C. crescentus. Additionally, structural similarities among the sugar backbones of lipid A of O. carboxidovorans, M. loti, R. leguminosarum, and P. trifolii, were shown. These observations were confirmed at the genomic level. Putative ORFs predicted for the lipid A biosynthesis pathway in Oligotropha shared sequence similarity with corresponding proteins of reference microorganisms. Further genetic and biochemical studies are necessary to elucidate which putative enzymes and at which stage of biosynthesis are involved in the esterification of the hydroxyl group of VLCFA by malic acid. This issue (on tertiary acyls) remains unexplored for all rhizobial lipids A.

Bacterial Strain and Culture Condition
Oligotropha carboxidovorans strain OM5 (type strain, DSM 1227; previously known as Pseudomonas carboxydovorans OM5) was obtained from the DSMZ culture collection (Leibniz-Institute, Braunschweig, Germany). The bacteria were cultivated in aerobic conditions, in a CMO (carbon monoxide oxidizer) medium containing 0.3% sodium acetate as a carbon source, at 30 • C, with aeration by vigorous shaking.

LPS and Lipid A Isolation and Purification
LPS was isolated from previously delipidated and enzymatically digested cells, using the hot 45% phenol/water extraction method [17,18], as described by Komaniecka and co-workers [29]. The LPS was obtained after ultracentrifugation (104,000× g, 4 h, 4 • C). It was found mainly in the water phase (90%). The lipid A was liberated from the LPS by mild acid hydrolysis of 210 mg of LPS using 1% acetic acid (3 h, 100 • C). After cooling in an ice bath, the hydrolysate was converted to the two-phase Bligh-Dyer system containing chloroform/methanol/water (2:2:1.8, v/v/v), and the resulting water and organic phases were separated by centrifugation (4000× g, 15 min, 20 • C) [30,31]. The organic phase containing a lipid A portion was collected, washed twice with a water phase from the freshly prepared two-phase Bligh-Dyer system, and evaporated to dryness. Crude lipid A was stored at −20 • C as a solution in chloroform/methanol (3:1, v/v).

Sugar and Fatty Acid Analysis
The sugar composition of lipid A was established by hydrolysis with 2 M trifluoroacetic acid (100 • C, 4 h) and conversion of liberated monosaccharides into (amino)alditol acetates [32]. Fatty acids were analyzed after acid hydrolysis (4 M HCl aq ., 100 • C, 4 h), extraction with chloroform (1:1, v/v), and methanolysis (0.5 M HCl/methanol, 85 • C, 2 h). Liberated hydroxy fatty acids were converted to trimethylsilyl (TMSi) derivatives of their methyl esters. Fatty acids and volatile components of lipid A obtained after methanolysis (2 M HCl/methanol, 85 • C, 18 h) were converted to butyl esters by weak butanolysis (1 M HCl/butanol, 65 • C, 2 h) and TMSi derivatized. All preparations were analyzed by GC-MS using the Agilent Technologies GC System 7890A connected to a mass selective detector (inert XL EI/CI MSD 5975C) equipped with a HP-5MS column (30 m × 0.25 mm). Helium was a carrier gas with a flow rate of 1 mL min −1 . The temperature program for sugar analysis was as follows: 150 • C for 5 min, raised to 310 • C at 5 • C·min −1 , and the final temperature was kept for 10 min. For analysis of fatty acid derivatives, the ramp of the temperature gradient was established at 10 • C·min −1 . The temperature program for volatile components was as follows: 50 • C for 5 min, raised to 310 • C at 5 • C·min −1 , and the final temperature was kept for 5 min.

Mass Spectrometry
MALDI-TOF mass spectrometry was performed with a SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 KHz Nd:YAG laser system (355 nm wavelength). Acquisition of the data was performed using MassLynx software version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with a multi-point external calibration using red phosphorous (Sigma). The lipid A samples were dissolved in chloroform/methanol (3:1, v/v) at a concentration of 10 µg/µL, and 2 mM of EDTA was added. A sample (1 µL) was transferred into the target plate wells covered with a thin matrix film. The matrix solution was prepared from 2',4',6'-trihydroxyacetophenone (THAP) (200 mg/ml in methanol) and mixed with nitrocellulose (NC) (15 mg/ml, suspended in 2-propanol/acetone (1:1, v/v)) in a proportion of 4:1 (v/v), as previously described by Silipo and co-workers [33]. Spectra were recorded in positive and negative ion modes.

Bioinformatics Analysis
Standard BLAST setting (with the cut-off E-value of 10 −5 ) was used in searching for putative proteins engaged in the biosynthetic pathway of Oligotropha lipid A. Protein sequences of M. loti MAFF 303099 and R. leguminosarum 3841 were used as queries in the TBLASTN searches against a genomic sequence of the O. carboxidovorans OM5 strain registered in the NCBI online Database. Individual BLAST protein subjects were then compared across their entire span with reference sequences using