Acyl chain specificity of the acyltransferases LpxA and LpxD and substrate availability contribute to lipid A fatty acid heterogeneity in Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis lipid A is heterogeneous with regard to the number, type, and placement of fatty acids. Analysis of lipid A by matrix-assisted laser desorption ionization-time of flight mass spectrometry reveals clusters of peaks differing by 14 mass units indicative of an altered distribution of the fatty acids generating different lipid A structures. To examine whether the transfer of hydroxy fatty acids with different chain lengths could account for the clustering of lipid A structures, P. gingivalis lpxA (lpxA(Pg)) and lpxD(Pg) were cloned and expressed in Escherichia coli strains in which the homologous gene was mutated. Lipid A from strains expressing either of the P. gingivalis transferases was found to contain 16-carbon hydroxy fatty acids in addition to the normal E. coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity. Both LpxA and LpxD, from either E. coli or P. gingivalis, were also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. This indicates that E. coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available. We conclude that the relaxed specificity of the P. gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in P. gingivalis lipopolysaccharide preparations.

FIG. 1.

Characterization of P. gingivalis lipid A by negative ion mass spectrometry. Lipid A was cleaved and separated from the LPS as described by Caroff et al. (8). MALDI-TOF mass spectra were determined as previously described (15). P. gingivalis displays lipid A heterogeneity, in that LPS isolated from a single species contains multiple lipid A structures. Kumada et al. (26) elucidated the major structures of several of the major lipid A mass ions. In this figure, one major lipid A “cluster” centered around m/z 1,690 is shown. All values given are average mass rounded to the nearest whole number for singly charged deprotonated molecules. Note that peaks varying by 14 amu may be due to changes in the fatty acid composition.

FIG. 2.

Negative ion MALDI-TOF mass spectra of lipid A from the E. coli lpxA temperature-sensitive mutant (SM101) with a plasmid carrying either E. coli lpxA (plpxA_Ec) (A) or P. gingivalis lpxA (plpxA_Pg) (B). The bacteria were cultured at the restrictive temperature 42°C in the presence of 100 mM IPTG.

FIG. 3.

Negative-ion MALDI-TOF mass spectra of lipid A from the E. coli lpxD temperature-sensitive mutant (RL25) with a plasmid carrying either E. coli lpxD (plpxD_Ec) (A) or P. gingivalis lpxD (plpxDPg) (B). The bacteria were cultured at the restrictive temperature 42°C in the presence of 100 mM IPTG.

FIG. 4.

Fatty acid composition of LPS from E. coli transformed with P. gingivalis lpxA or lpxD mutant. (A) Strain SM101 expressing either E. coli lpxA (▪) or P. gingivalis lpxA (□); (B) strain RL25 expressing either E. coli lpxD (▪) or P. gingivalis lpxD (□). Fatty acid methyl esters were determined by gas chromatography and are expressed in the figure as a percentage of the total fatty acids identified. Determinations were done on bacteria grown on three separate occasions, and typical results are presented.

FIG. 5.

Proposed lipid A structures for chimeric lipid A generated when E. coli is complemented with plpxA_Pg or plpxD_Pg. The sites for the addition of lipid A fatty acids by lpxA and lpxD are indicated. Note that since lpxA and lpxD transfer fatty acids at the monosaccharide stage in lipid A biosynthesis, an asymmetric distribution of fatty acids with different chain lengths can occur. We did not determine whether the C₁₆OH was added to the 2 or 2′ position or to the 3 or 3′ position.

FIG. 6.

Negative ion MALDI-TOF mass spectrum of lipid A from wild-type E. coli grown overnight in medium containing 1% propionate. Lipid A was cleaved and separated from the LPS as described by Caroff et al. (8). MALDI-TOF mass spectra were determined as previously described (15). Note that numerous peaks were observed that centered around m/z 1,798 and differed by 14 amu.

FIG. 7.

Negative ion MALDI-TOF mass spectra of lipid A from mutant strains expressing E. coli or P. gingivalis acyltransferases and cultured in medium containing 1% propionic acid. (A) SM101 plpxA_Ec; (B) SM101 plpxA_Pg. Lipid A was cleaved and separated from the LPS as described by Caroff et al. (8). MALDI-TOF mass spectra were determined as previously described (15). Note that numerous peaks were observed that differed by 14 amu.

FIG. 8.

Negative ion MALDI-TOF mass spectra of lipid A from mutant strains expressing E. coli or P. gingivalis acyltransferases and cultured in medium containing 1% propionic acid. (A) RL25 plpxD_Ec; (B) RL25 plpxD_Pg. Lipid A was cleaved and separated from the LPS as described by Caroff et al. (8). MALDI-TOF mass spectra were determined as previously described (15). Note that numerous peaks were observed that differed by 14 amu.

FIG. 9.

Fatty acid composition of LPS from mutant strains expressing E. coli or P. gingivalis acyltransferases and cultured in medium containing 1% propionic acid. Fatty acid methyl esters were determined by gas chromatography. (A) Wild-type strain JM83. The data are expressed as a percentage of the total fatty acids identified. (B) Strain SM101 expressing lpxA from either E. coli (▪) or P. gingivalis (□). The data are expressed as a percentage of the total hydroxy fatty acids; (C) Strain RL25 expressing lpxD from either E. coli (▪) or P. gingivalis (□), the data are expressed as a percentage of total hydroxy fatty acids. Determinations were done on bacteria grown on three separate occasions, and typical results are presented.