The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii

Abstract

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In Gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in Gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of Gram-negative bacteria are also present in other mollicutes and Gram-positive bacteria.Essential cellular activities such as transport, sensing, signal transduction, growth, adhesion, and pathogenesis require proteins localized on the cell surface. One of the strategies to anchor a protein at the lipid membrane is covalent modification of proteins by fatty acids or other lipids. All bacteria rely on acylation of N-terminal Cys residues of lipoproteins to allow tight anchorage on the membrane surface (1). This uniquely bacterial modification was first observed in 1969 in a major outer membrane protein of Escherichia coli called Braun's lipoprotein and later detected in every bacterial organism studied (2). Adhesion proteins and substrate-binding subunits of ATPbinding cassette (ABC) 2 transporters are known to be attached to the membrane in this manner. Thousands of proteins predicted to be lipoproteins "by similarity" according to the presence of consensus sequences of Sec Type II or Tat Type II signal peptides are also thought to be acylated (3).Bacteria of the Mollicutes class are widespread in nature and are characterized by the lack of a rigid cell wall and drastically reduced genome sizes. In fact, representatives of this class are the simplest self-replicating organisms able to independently subsist, generate energy, and adapt to changing environments (4 and references therein). Most mollicutes depend on cholesterol and are partially or totally incapable of fatty acid synthesis and therefore depend on the host or the culture medium for a constant supply of these substances (5). Acholeplasma laidlawii is a convenient model mollicute because of its relatively simple nutritional requirements and fast growth rate. A. laidlawii can synthesize limit...

Complete Genome and Proteome of Acholeplasma laidlawii

“…It has been suggested earlier the Mollicutes are characterized by diacylation (45). However, we determined that A. laidlawii has three acyl residues (50).…”

Phosphatidylglycerol::Prolipoprotein Diacylglyceryl Transferase (Lgt) of Escherichia coli Has Seven Transmembrane Segments, and Its Essential Residues Are Embedded in the Membrane

“…The enzymes Lgt and Lsp are conserved in all classes of bacteria, whereas Lnt is only present in Proteobacteria, Actinobacteria, and Spirochetes (56,60,63). Recent reports, however, demonstrated that triacylated lipoproteins exist at least in the Staphylococcus species of Gram-positive bacteria (32) and in Mollicutes (50). This suggests that they might also possess N-acyltransferase activity.…”

Identification of the Lyso-Form N -Acyl Intramolecular Transferase in Low-GC Firmicutes

“…To prepare lipopeptides for MS analysis, the strategy of Serebryakova et al was utilized, with minor modifications (57). After SDS-PAGE separation (described above for immunoblotting), purified Lpp(K58A)-Strep-tag and enriched lipoproteins from E. faecalis and B. cereus were transferred onto BioTract NT nitrocellulose transfer membrane (PALL Life Sciences) and stained with Ponceau S (0.2% in 5% acetic acid).…”