Components Subcellular Localization: Identification of Lipoproteins Using Alkyne Fatty Acids and Click Chemistry.

Lipoproteins contain fatty acids at their amino termini through which they are anchored in lipid membranes. Here, we describe the use of alkyne fatty acids and click chemistry to label lipoproteins in bacterial cells. Exogenous fatty acids containing an alkyne group are incorporated in phospholipids and lipoproteins during bacterial growth.

Components Subcellular Localization: Identification of Lipoproteins Using Globomycin and Radioactive Palmitate.

Bacterial lipoproteins are characterized by fatty acids, derived from membrane phospholipids, which are covalently attached to their amino terminus via posttranslational modification in the cytoplasmic membrane. Here, I describe the detection of one of the intermediate forms of lipoprotein, diacylglyceryl-prolipoprotein, using H-palmitate labeling and inhibition of signal peptidase II (Lsp) by globomycin and detection by fluorography.

A Defect in Lipoprotein Modification by Lgt Leads to Abnormal Morphology and Cell Death in Escherichia coli That Is Independent of Major Lipoprotein Lpp.

Lgt is an essential enzyme in proteobacteria and therefore a potential target for novel antibiotics. The effect of Lgt depletion on growth, morphology, and viability was studied in Escherichia coli to assess whether absence of Lgt leads to cell death. Two Lgt depletion strains were used in which was under the control of an arabinose-inducible promoter that allowed regulation of Lgt protein levels.

Mode of action of lipoprotein modification enzymes-Novel antibacterial targets.

Lipoproteins are characterized by a fatty acid moiety at their amino-terminus through which they are anchored into membranes. They fulfill a variety of essential functions in bacterial cells, such as cell wall maintenance, virulence, efflux of toxic elements including antibiotics, and uptake of nutrients. The posttranslational modification process of lipoproteins involves the sequential action of integral membrane enzymes and phospholipids as acyl donors.

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid.

A sensitive fluorescence-based assay to monitor enzymatic activity of the essential integral membrane protein Apolipoprotein N-acyltransferase (Lnt).

Lipoprotein modification is an essential process in Gram-negative bacteria. The action of three integral membrane proteins that catalyze the transfer of fatty acids derived from membrane phospholipids or cleave the signal peptide of the lipoprotein substrate result in the formation of mature triacylated proteins. Inactivation of the enzymes leads to mis-localization of immature lipoproteins and consequently cell death.

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics.

Identification of Lipoproteins Using Globomycin and Radioactive Palmitate.

Bacterial lipoproteins are characterized by fatty acids that are covalently attached to their amino terminus via posttranslational modification in the cytoplasmic membrane. Three enzymatic steps are involved in the synthesis of mature triacylated lipoprotein: prolipoprotein converts into diacylglyceryl-prolipoprotein that in turn converts into apolipoprotein, which is finally converted into mature triacylated lipoprotein. Here we describe the detection of one of these intermediate forms of lipoprotein, diacylglyceryl-prolipoprotein, using H-palmitate labeling and inhibition by globomycin and detection by fluorography.

The molecular mechanism of bacterial lipoprotein modification--how, when and why?

Posttranslational modification of proteins by lipidation is a common process in biological systems. Lipids provide protein stability, interaction with other membrane components, and in some cases, due to reversibility of the process, a mechanism for regulating protein localization and function. Bacterial lipoproteins possess fatty acids at their amino-termini that are derived from phospholipids, and this lipid moiety anchors the proteins into the membrane.

Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction.

Apolipoprotein N-acyltransferase (Lnt) is an essential membrane-bound enzyme that catalyzes the third and last step in the post-translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non-functional variants of Lnt obtained by error-prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain.

Phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.

Lgt of Escherichia coli catalyzes the transfer of an sn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins. The enzyme is essential for growth, as demonstrated here by the analysis of an lgt depletion strain. Cell fractionation demonstrated that Lgt is an inner membrane protein.

Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase.

The enzyme apolipoprotein N-acyltransferase (Lnt) is an integral membrane protein that catalyzes the last step in the post-translational modification of bacterial lipoproteins. Lnt undergoes covalent modification in the presence of phospholipids resulting in a thioester acyl-enzyme intermediate. It then transfers the acyl chain to the α-amino group of the N-terminal diacylglyceryl-modified cysteine of apolipoprotein, leading to the formation of mature triacylated lipoprotein.

The essential Escherichia coli apolipoprotein N-acyltransferase (Lnt) exists as an extracytoplasmic thioester acyl-enzyme intermediate.

Escherichia coli apolipoprotein N-acyltransferase (Lnt) transfers an acyl group from sn-1-glycerophospholipid to the free alpha-amino group of the N-terminal cysteine of apolipoproteins, resulting in mature triacylated lipoprotein. Here we report that the Lnt reaction proceeds through an acyl-enzyme intermediate in which a palmitoyl group forms a thioester bond with the thiol of the active site residue C387 that was cleaved by neutral hydroxylamine. Lnt(C387S) also formed a fatty acyl intermediate that was resistant to neutral hydroxylamine treatment, consistent with formation of an oxygen-ester linkage.

Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane.

The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS.

Identification of essential residues in apolipoprotein N-acyl transferase, a member of the CN hydrolase family.

Apolipoprotein N-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of Escherichia coli were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family.

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane.

Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM.

The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci.

Traffic spotting: poles apart.

Finding out where specific functions are carried out within a bacterial cell has now become technically feasible. Here we consider recent experiments aimed at determining where bacteria translocate proteins across the cytoplasmic membrane using the Sec machinery.

A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region.

Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB.

Assembly of cell division proteins at the E. coli cell center.

Cell division in Escherichia coli requires the coordinated action of at least ten proteins. In recent years, substantial progress has been made in understanding the assembly of these proteins at the cell septum. These findings suggest a largely stepwise appearance of cell division proteins at the centre of the cell.

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae, localizes in codependent fashion with FtsL to the division site.

YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae. In E. coli the ygbQ gene was discovered as a result of a computer search of the E.