Membrane glycerophospholipid biosynthesis in Neisseria meningitidis and Neisseria gonorrhoeae: identification, characterization, and mutagenesis of a lysophosphatidic acid acyltransferase

Abstract

Lysophosphatidic acid (LPA) acyltransferases of Neisseria meningitidis and Neisseria gonorrhoeae were identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. In Escherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1-acyl-sn-glycerol-3-phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916-generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916 insertion in the meningococcal mutant was localized within a 771 bp open reading frame, The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. In N. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designated nlaA (neisserial LPA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity, >40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcal nlaA gene complemented in trans a temperature-sensitive E. coli PlsCts- mutant. Tn916 and omega-cassette insertional inactivations of the neisserial nlaA genes altered the membrane glycerophospholipid compositions of both N. meningitidis and N. gonorrhoeae but were not lethal. Therefore, the pathogenic Neisseria spp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcal nlaA mutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcal nlaA mutant, was increased in the meningococcal nlaA mutant. We postulate that the pathogenic Neisseria spp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence of nlaA enzymatic activity. Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on capsule and piliation.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

“…N. gonorrhoeae makes unsaturated fatty acids, and although the NGO1024 gene is required for unsaturated fatty acid synthesis under anaerobic conditions (22), the biochemistry is unknown. N. gonorrhoeae encodes for plsX (NGO2171), plsY (NGO0858), and two plsC (NGO0611 and NGO2069) genes (62,63). We show that N. gonorrhoeae encodes an acyl-ACP synthetase (AasN, NGO0530) that functions in the incorporation of exogenous fatty acids.…”

Rhodobacter capsulatus OlsA Is a Bifunctional Enyzme Active in both Ornithine Lipid and Phosphatidic Acid Biosynthesis

“…Furthermore, this species might have at least an additional enzyme with AGPAT activity as an NlaA Ϫ NlaB Ϫ double mutant is viable and has AGPAT activity (42,45). Indeed, R. capsulatus OlsA and PlsC316 show noteworthy similarities to NlaA (OlsA, ϳ21% identity and ϳ32% similarity; PlsC316, ϳ16% identity and ϳ30% similarity) and NlaB (OlsA, ϳ16% identity and 27% similarity; PlsC316, ϳ26% identity and ϳ32% similarity), as depicted in Fig.…”

“…Similarly, some bacterial species including N. meningitidis, N. gonorrhoeae, and P. fluorescens have multiple AGPATs, whereas others, like E. coli, appear to have only one such enzyme. It has been suggested that the different isozymes might play different roles, such as fine-tuning the membrane lipid and fatty acid profiles in diverse environments (14,42,45). Indeed, while P. fluorescens OlsA Ϫ mutants exhibited no major changes in the membrane phospholipid and fatty acid profiles, inactivation of P. fluorescens AGPAT isozymes PatB and HdtS did alter the fatty acid profile of phospholipids and some membrane properties (14), as seen here with R. capsulatus OlsA Ϫ and PlsC Ϫ mutants.…”

“…Thus, E. coli plsC mutants are temperature-sensitive and can be complemented for growth by heterologous expression of plant and mammalian AGPAT homologues (7,22,49). Unlike E. coli, certain bacteria such as Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas fluorescens, and Pseudomonas aeruginosa have multiple functional plsC homologues that function in diverse environments (4,14,42,45). Multiple AGPAT isozymes expressed in different tissues have been identified in eukaryotes, such as Limnanthes douglasii, Arabidopsis thaliana, human, and mouse as well (1,8,20,26,31,32,44,49,50).…”

“…Several membrane-associated AGPATs have been cloned and expressed from many bacteria, yeast, various plant species, and several mammals (6,7,8,12,14,22,27,29,37,42,44,45,49). The well-studied bacterium E. coli possesses only one AGPAT, and a deficiency in this enzyme is lethal, resulting in the accumulation of the LPA intermediate (11,12).…”