Initial Characterization of the FlgE Hook High Molecular Weight Complex of

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility

Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

Prediction of Antibacterial Activity from Physicochemical Properties of Antimicrobial Peptides

Consensus is gathering that antimicrobial peptides that exert their antibacterial action at the membrane level must reach a local concentration threshold to become active. Studies of peptide interaction with model membranes do identify such disruptive thresholds but demonstrations of the possible correlation of these with the in vivo onset of activity have only recently been proposed. In addition, such thresholds observed in model membranes occur at local peptide concentrations close to full membrane coverage. In this work we fully develop an interaction model of antimicrobial peptides with biological membranes; by exploring the consequences of the underlying partition formalism we arrive at a relationship that provides antibacterial activity prediction from two biophysical parameters: the affinity of the peptide to the membrane and the critical bound peptide to lipid ratio. A straightforward and robust method to implement this relationship, with potential application to high-throughput screening approaches, is presented and tested. In addition, disruptive thresholds in model membranes and the onset of antibacterial peptide activity are shown to occur over the same range of locally bound peptide concentrations (10 to 100 mM), which conciliates the two types of observations

Early evolution of the biotin-dependent carboxylase family

<p>Abstract</p> <p>Background</p> <p>Biotin-dependent carboxylases are a diverse family of carboxylating enzymes widespread in the three domains of life, and thus thought to be very ancient. This family includes enzymes that carboxylate acetyl-CoA, propionyl-CoA, methylcrotonyl-CoA, geranyl-CoA, acyl-CoA, pyruvate and urea. They share a common catalytic mechanism involving a biotin carboxylase domain, which fixes a CO₂molecule on a biotin carboxyl carrier peptide, and a carboxyl transferase domain, which transfers the CO₂moiety to the specific substrate of each enzyme. Despite this overall similarity, biotin-dependent carboxylases from the three domains of life carrying their reaction on different substrates adopt very diverse protein domain arrangements. This has made difficult the resolution of their evolutionary history up to now.</p> <p>Results</p> <p>Taking advantage of the availability of a large amount of genomic data, we have carried out phylogenomic analyses to get new insights on the ancient evolution of the biotin-dependent carboxylases. This allowed us to infer the set of enzymes present in the last common ancestor of each domain of life and in the last common ancestor of all living organisms (the cenancestor). Our results suggest that the last common archaeal ancestor had two biotin-dependent carboxylases, whereas the last common bacterial ancestor had three. One of these biotin-dependent carboxylases ancestral to Bacteria most likely belonged to a large family, the CoA-bearing-substrate carboxylases, that we define here according to protein domain composition and phylogenetic analysis. Eukaryotes most likely acquired their biotin-dependent carboxylases through the mitochondrial and plastid endosymbioses as well as from other unknown bacterial donors. Finally, phylogenetic analyses support previous suggestions about the existence of an ancient bifunctional biotin-protein ligase bound to a regulatory transcription factor.</p> <p>Conclusions</p> <p>The most parsimonious scenario for the early evolution of the biotin-dependent carboxylases, supported by the study of protein domain composition and phylogenomic analyses, entails that the cenancestor possessed two different carboxylases able to carry out the specific carboxylation of pyruvate and the non-specific carboxylation of several CoA-bearing substrates, respectively. These enzymes may have been able to participate in very diverse metabolic pathways in the cenancestor, such as in ancestral versions of fatty acid biosynthesis, anaplerosis, gluconeogenesis and the autotrophic fixation of CO₂.</p