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

Abstract

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.

Keywords: VLCFA; galacturonic acid; lipid A; lipopolysaccharide; malic acid; structure elucidation.

Figure 1

Fatty acid composition of O. carboxidovorans OM5 lipid A. Signals around 20 min and 21.30 min represent three isomers of the dehydrated form of 26:0(25-OH) and 28:0(27-OH), respectively, derived during strong acid hydrolysis (4 M HCl aq., 100 °C, 4 h). For conditions of gas chromatography, see Materials and Methods section. Artefacts mean hydrolytically dehydrated hydroxy fatty acids. Std—internal standard.

Figure 2

Part of the HSQC (a) and ¹H-¹H ROESY (b) spectra of lipid A from O. carboxidovorans OM5. Only signals important for structural analysis are marked.

Figure 3

Negative mode MALDI-TOF mass spectrum of native lipid A from O. carboxidovorans OM5 (a); proposed formulas of lipid A ions presented in the lipid A mass spectrum (with calculated m/z values) (b). The m/z values marked in red at part (a) refer to the relevant chemical formulas given in part (b) of the figure.

Figure 4

MALDI-TOF MS/MS spectrum of the parent ion at m/z 2478.775 (heptaacylated lipid A of O. carboxidovorans). Peaks between m/z 1600 and 2400 are magnified five times. Ions marked in red are discussed in the text.

Figure 5

Proposed structural formula for the most complex O. carboxidovorans OM5 lipid A molecule. M_mi = 2455.76435 u.