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Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria. Annelie Pernthaler, 2004.We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH . Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations . Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results . The quality of probes as well as a stringent hybridization temperature were determined with expression clones . To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG) . The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP) . Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides . p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach . After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols . To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH . By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene . Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures . Isoprenoid Biosynthesis in Synechocystis sp . Strain PCC6803 Is Stimulated by Compounds of the Pentose Phosphate Cycle but Not by Pyruvate or Deoxyxylulose-5-Phosphate. Yuri V. Ershov, 2002.The photosynthetic cyanobacterium Synechocystis sp . strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-D-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E . coli . However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds . Fosmidomycin (at 1 µM and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [14C]IPP incorporation stimulated by DHAP plus GA3P . To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium . The combined results suggest that the MEP pathway, as described for E . coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp . strain PCC6803 . Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis . Heat and Osmotic Stress Responses of Probiotic Lactobacillus rhamnosus HN001 (DR20) in Relation to Viability after Drying. Jaya Prasad, 2003.The viability of lactic acid bacteria in frozen, freeze-dried, and air-dried forms is of significant commercial interest to both the dairy and food industries . In this study we observed that when prestressed with either heat (50°C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 (also known as DR20) showed significant (P < 0.05) improvement in viability compared with the nonstressed control culture after storage at 30°C in the dried form . To investigate the mechanisms underlying this stress-related viability improvement in L . rhamnosus HN001, we analyzed protein synthesis in cultures subjected to different growth stages and stress conditions, using two-dimensional gel electrophoresis and N-terminal sequencing . Several proteins were up- or down-regulated after either heat or osmotic shock treatments . Eleven proteins were positively identified, including the classical heat shock proteins GroEL and DnaK and the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase, as well as tagatose 1,6-diphosphate aldolase of the tagatose pathway . The phosphocarrier protein HPr (histidine-containing proteins) was up-regulated in cultures after the log phase irrespective of the stress treatments used . The relative synthesis of an ABC transport-related protein was also up-regulated after shock treatments . Carbohydrate analysis of cytoplasmic contents showed higher levels (20 ± 3 µg/mg of protein) in cell extracts (CFEs) derived from osmotically stressed cells than in the unstressed control (15 ± 3 µg/mg of protein) . Liquid chromatography of these crude carbohydrate extracts showed significantly different profiles . Electrospray mass spectrometry analysis of CFEs revealed, in addition to normal mono-, di-, tri-, and tetrasaccharides, the presence of saccharides modified with glycerol .
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