Evolution of the Kdo2-lipid A biosynthesis in bacteria

Abstract

Background: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps.

Results: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria.

Conclusions: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.

Figure 1

Structure of Kdo₂-lipid A from E. coli K12. Parts joined by the nine enzymes are indicated with shadow and dashed surrounding. The nine-enzyme pathway is illustrated at the bottom. Abbreviations for enzymes and substrates are as follows: LpxA: UDP-N-acetylglucosamine acyltransferase, LpxC: UDP-3-O-(3-hydroxymyristoyl) N-acetylglucosamine deacetylase, LpxD: UDP-3-O-(3-hydroxymyristoyl) glucosamine N-acyltransferase, LpxH: UDP-2,3-diacylglucosamine pyrophosphatase, LpxB: lipid-A-disaccharide synthase, LpxK: lipid A 4'-kinase, WaaA (or KdtA): 3-deoxy-D-manno-octulosonate-lipid A transferase (or Kdo-lipid A transferase), LpxL (or HtrB): Kdo₂-lipid IV_A lauroyl-ACP acyltransferase, LpxM (or MsbB): Kdo₂-lauroyl-lipid IV_A -myristoyl-ACP acyltransferase, ACP: acyl carrier protein, UDP: uridine diphosphate, UDP-GlcNAc: UDP-N-acetylglucosamine, and DS-1-P: tetraacyldisaccharide 1-phosphate. In the pathway, substrates are shown with numbers as follows: 1: UDP-3-O-(3-hydroxytetradecanoyl)-N-acetylglucosamine, 2: UDP-3-O-(3-hydroxytetradecanoyl)-glucosamine, 3: UDP-2,3-bis(3-hydroxytetradecanoyl)-glucosamine, 4: 2,3-bis(3-hydroxytetradecanoyl)-beta-D-glucosaminyl 1-phosphate, 5: tetraacyldisaccharide 1,4'-bis-phosphate (lipid IV_A), 6: Kdo₂-lipid IV_A, and 7: Kdo₂-(lauroyl)-lipid IV_A.

Figure 2

Distribution of Kdo₂-lipid A biosynthetic enzymes across bacteria genomes. Maximum likelihood phylogenies were reconstructed based on concatenated six protein sequences (A) and 16S rRNA nucleotide sequences (B). Enzymes identified are color-coded as follows: LpxA (A, red), LpxC (C, red), LpxD (D, red), LpxH (H, red), LpxH2 (H2, yellow), LpxK (K, blue), WaaA (W, blue), LpxL (L, blue), LpxM (M, purple), and LpxP (P, light blue). White boxes indicate the absence of the corresponding enzymes. Bacteria that have none of the enzymes and only LpxK are marked with white and blue stars, respectively. Red arrowheads indicate the points inferred to be the emergence of the Kdo₂-lipid A biosynthetic pathway with the last four enzymes (LpxK-LpxM). Circles at internal nodes indicate bootstrap-supporting values as follows: black circles ≥ 95%, gray circles ≥ 85%, and white circles ≥ 75%. Proteobacteria and Gram-positive bacteria are shown with green and orange letters/lines, respectively. Bacterial classifications (phylum, and for Proteobacteria, class) are shown in parentheses using the following abbreviations. Ac: Acidobacteria, Ba: Bacteroidetes, Ch: Chlamydiae, Cn: Cyanobacteria, Cr: Chlorobi, Di: Dictyoglomi, Fu: Fusobacteria, Ni: Nitrospire, Pl: Planctomycetes, Sp: Spirochaetes, Ve: Verrucomicrobia, α: Alphaproteobacteria, β: Betaproteobacteria, δ: Deltaproteobacteria, ε: Epsilonproteobacteria, and γ: Gammaproteobacteria. Group-II Gammaproteobacteria, those that have all nine enzymes, are shown with *.

Figure 3

The maximum-likelihood phylogenetic tree of LpxH, LpxH2, and related proteins. Circles at internal nodes indicate bootstrap-supporting values as follows: black circles ≥ 95%, gray circles ≥ 85%, and white circles ≥ 70%. Proteobacteria are shown with green letters and lines. Group-II Gammaproteobacteria are indicated by *. See Figure 2 legend for abbreviations used for bacterial classification. The red arrowhead shows where the LpxH/LpxH2 duplication happened. The inset shows the maximum likelihood phylogeny reconstructed from all of the similar proteins identified in this study. It shows that each protein family (indicated with triangles with InterPro accession numbers) forms a distinct cluster.

Figure 4

The maximum-likelihood phylogenetic tree of LpxM and LpxL proteins. Circles at internal nodes indicate bootstrap-supporting values as follows: black circles ≥ 95%, gray circles ≥ 85%, and white circles ≥ 70%. Proteobacteria are shown with green letters and lines. Group-II Gammaproteobacteria are indicated by *. See Figure 2 legend for abbreviations used for bacterial classification. Red arrowheads indicate where the duplication events happened. Light-red arrowheads are where the species-specific duplications are found.