Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough-and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction. Lipopolysaccharides (LPSs) are the major components of the external membrane of almost all Gram-negative bacteria (1, 2); they are known as endotoxins and may cause several pathophysiological symptoms, such as fever, diarrhea, blood pressure decrease, septic shock, and death (3).The LPS molecular architecture consists of three different regions. The innermost, hydrophobic region, lipid A, is responsible for the major toxic and beneficial properties of bacterial endotoxins (4). Lipid A is the least variable part of the molecule among the different species of a genus, and its structure generally consists of a diglucosamine backbone substituted with varying numbers (usually four to seven) of ester-or amide-linked fatty acids. Phosphate and/or other substituents are linked to carbons at the C-1 and C-4 Ј positions of the glucosamine disaccharide (5). A 2-keto-3-deoxyoctonate (Kdo) unit links the lipid A to a core oligosaccharide (OS) composed of ‫ف‬ 10 sugar residues divided into two regions: a best conserved inner core part and a distal outer core. The core is linked to a third outermost region of a highly immunogenic and variable O-chain polysaccharide (PS) or O-antigen made up of repeating OS units. The latter region of the LPS molecule is responsible for bacterial serological strain specificity (6) and is present only in smooth-type bacteria. The so-called rough-type bacteria produce LPSs lacking O-antigens.Endotoxin can be isolated from Gram-negative bacteria by different methods, the most efficient and commonly used one being the hot phenol-water extraction procedure introduced by Westphal and Lüderitz (7). It was later modified by different authors (8, 9), and specific methods were developed for rough-type endotoxin extractions (10, 11). However, each method requires several days for the extraction and purification of endotoxins and further steps to isolate the lipid A moiety. New methods have been described to extract LPS from small quantities of cells, such as by mini phenol extraction (12) or using an RNAisolating reagent, but these methods still require 2 or 3 days and the use of phenol (13).In early experiments, mineral acid hydrolysis was used to liberate lipid A from endotoxins, splitting the acidolabile ketosidic bond of Kdo. It was followed in 1963 b...

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C 10 OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.