Stimulation of inositide degradation in clumping Stigmatella aurantiaca.

Numerous external signals which activate inositol phospholipid hydrolysis in eukaryotes are known; probably all of these signals are transduced by G proteins. So far, neither signal-transducing G protein nor receptor-regulated phospholipase C has been found in prokaryotes. However, a group of bacteria, the myxobacteria, displays cellular and tissue-like differentiation; therefore, it appeared that a search for the various activities involved in a signal-activated phosphatidylinositol cycle might be rewarding. Here, we report that in Stigmatella aurantiaca, under conditions which promote clumping, inositol phospholipid synthesis and degradation were stimulated with the resulting formation of inositol phosphate and inositol bisphosphate. The turnover was Ca2+ dependent and was increased by fluoride ions. Membrane preparations from these cells showed a phospholipase C activity which increased with the stage of incubation and which was stimulated by GTP gamma S, suggesting G protein dependency. To what extent this system in a prokaryotic cell shares properties of the phosphatidylinositol cycle in eukaryotes remains unexamined.

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees.

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314 degrees) and sacXY (333 degrees) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.

Immobilization and treatment of Streptococcus faecalis for the continuous conversion of arginine into citrulline.

Citrulline is one of the steps of the arginine dihydrolase system of Streptococcus faecalis. We have shown that the bacteria, immobilized in polyacrylamide gel and treated with Cetyl trimethyl ammonium bromide (CTAB) or heat, were able to convert arginine to citrulline. Used continuously in a column reactor, the entrapped cells have a stable enzymatic activity for at least 30 days at 45 degrees C.

Effect of 3,4-dihydroxybutyl-1-phosphonate on cardiolipin synthesis in B. subtilis.

Endogenous phosphatidylglycerol is rapidly transformed into cardiolipin when B. subtilis 168 cells were incubated in a buffer without an energy source. Upon addition of 3,4-dihydroxybutyl-1-phosphonate (DHBP), a synthetic glycerol 3-phosphate analogue, this synthesis was completely blocked after a short lag; if the cells were grown in the presence of the analogue, there was no lag. When membrane fractions were incubated with exogenous [32P]phosphatidylglycerol, free DHBP and glycerol 3-phosphate had no effect on [32P]cardiolipin synthesis, but phosphatidyl-DHBP and phosphatidylglycerolphosphate were potent inhibitors. These results are consistent with our hypothesis that phosphatidylglycerolphosphate, the phosphatidylglycerol precursor, might also be a physical inhibitor of cardiolipin synthesis.

Evidence for a membrane-associated GTP-binding protein in Stigmatella aurantiaca, a prokaryotic cell.

Signal transducing G proteins are present in all eukaryotic cells, but they have not been found in prokaryotes so far. Myxobacteria, especially Stigmatella aurantiaca, are prokaryotic organisms able to exchange signals. Moreover, they exhibit an active phosphoinositide metabolism, whose intensity is dependent on the physiological state of the cell. Therefore G proteins potentially involved in the activation of phospholipid metabolism or any other event stimulated by external signals were looked for in S. aurantiaca membranes. Using a photoaffinity technique based on cross-linking of radioactive GTP to membrane-associated proteins under UV irradiation, only one major band in the range of 54 kDa was detected. This GTP-binding protein present specifically in membrane preparations binds also GDP, whereas it does not react with other nucleotides, such as ATP, UTP and CTP. The membrane-bound G protein of S. aurantiaca needs further characterization but could be homologous to G alpha subunits found in cytoplasmic membranes of eukaryotes.

Specific extraction of bacterial cardiolipin from sporulating Bacillus subtilis.

A method for rapid purification of bacterial cardiolipin is presented. The cardiolipin level was first increased by suspending Bacillus subtilis cells in a buffer containing an uncoupling agent. At least 90% of the phosphatidylglycerol molecules were rapidly converted into cardiolipin. In sporulating strains, the accumulated cardiolipin appeared to be unextractable by conventional phospholipid extraction procedures. Sporulating bacteria were therefore treated first by a classical technique in order to eliminate lipids other than cardiolipin; a second extraction in a highly acidic medium then allowed us to quantitatively extract the remaining cardiolipin. Besides simplicity and rapidity, this method has the advantage of yielding cardiolipin in a nearly pure form from a relatively low number of bacteria.

Specific inhibition of phosphatidate cytidylyltransferase from Bacillus subtilis membranes by cytidine monophosphate.

In Bacillus subtilis, the phosphatidate cytidylyltransferase was localized exclusively in the membrane fraction prepared by sucrose density gradient fractionation. A single enzyme could synthesize the two liponucleotides: CDPdiacylglycerol and dCDPdiacylglycerol. Kinetic experiments and isotopic exchange reactions suggested a ping-pong mechanism. Among the nucleosides monophosphate, CMP specifically reduced the synthesis of both liponucleotides. This inhibition was non-competitive and might be involved in regulation of phospholipid synthesis.

Occurrence of dialkyl ether phospholipids in Stigmatella aurantiaca DW4.

We investigated the lipid composition of vegetative cells of Stigmatella aurantiaca. Four phospholipids were isolated and identified: phosphatidylethanolamine as the main component, phosphatidylglycerol, lysophosphatidylethanolamine in an exceptionally large amount (17%), and phosphatidylinositol (18 to 25%), rare in procaryotic cells. This composition did not change significantly during growth. The fatty acids of total lipids were found to be rather similar to those of other strains of myxobacteria; the main fatty acids found were unsaturated and branched. We noted a different fatty acid pattern for each phospholipid. The presence of unusual alkyl ether linkages, established by chemical hydrolysis and infrared spectroscopy, was unexpected in these bacteria. Diacyl ester, dialkyl ether, and monoacyl-monoalkyl structures were shown in phosphatidylethanolamine and phosphatidylglycerol. Lysophosphatidylethanolamine was essentially a monoacyl form, whereas phosphatidylinositol was a unique dialkyl ether phospholipid.

Fluorescence polarization immunoassay of estradiol.

Fluorescence polarization immunoassay (FPIA) offers a good alternative to isotopic methods for the determination of drug and hormone levels in biological fluids. We applied it to estradiol, using a fluoresceinated derivative of estradiol and the IgG fraction of a highly specific rabbit antiserum. After determining the optimal operational conditions, FPIA was used for measuring urinary concentrations of free estradiol in normal and pregnant women, after a purification step on a reversed phase cartridge. The method turned out to be rapid (1 min to reach the equilibrium state) and accurate.

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.

Effects of ethanol and methanol on lipid metabolism in Bacillus subtilis.

In Bacillus subtilis, the fatty acid moiety of the phospholipids was affected differently during growth in the presence of 1.1 M-methanol or 0.7 M-ethanol, though at these concentrations methanol and ethanol had the same effects on growth rate and completely inhibited sporulation. Synthesis of phosphatidylglycerol was also strongly inhibited and the amount of total cell phospholipids was reduced by 50% by both alcohols. The composition of fatty acids, especially the relative concentration of 12-methyltetradecanoic acid, was modified only by ethanol; in bacteria grown in the presence of methanol, changes in fatty acid composition were negligible. In non-sporulating mutants, synthesis of phosphatidylglycerol was much less affected than in the wild-type and synthesis of phosphatidylethanolamine was increased. In these strains, fatty acid composition was also modified by ethanol but unaffected by methanol.

[Characterization of the membrane attached to the folded chromosome isolated from Escherichia coli]. / Caractérisation de la membrane liée au chromosome replié isolé de Escherichia coli.

Phospholipid analysis of the membranes associated with fast sedimenting folded chromosomes prepared by lysis of E. coli CR 34 shows that both inner and outer membranes are parts of the complex, in proportions not very different from that found in the whole bacteria. During the preparation of the folded chromosomes, the most recently synthesized molecules of phosphatidylglycerol and phosphatidylethanoamine are more sensitive to solubilisation, particularly those from the cytoplasmic membrane. Identification of a dominant fraction, the outer membrane, in some complexes, results from a preferential solubilization of the inner membrane. These results do not favor any specific association between the folded chromosome and the membranes.