Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells.
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The identification of Mycobacterium tuberculosis genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis. By combining the natural fluorescence of the Aequoria victoria green fluorescent protein (GFP) with the counterselectable property of the Bacillus subtilis SacB protein, M. tuberculosis promoters displaying enhanced in vivo activity have been isolated. Macrophages were infected with recombinant Mycobacterium bovis bacille Calmette-Guerin containing a library of M. tuberculosis promoters controlling gfp and sacB expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting. The expression of sacB was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing M. tuberculosis promoters with greater activity intracellularly. The gene products identified displayed similarity to proteins from other organisms whose functions include nutrient utilization, protection from oxidative stress and defence against xenobiotics. These proposed functions are consistent with conditions encountered within the host cell and thus suggest that the augmented activity of the isolated promoters/genes may represent strategies employed by M. tuberculosis to enhance intracellular survival and promote infection. (+info)
Combined effects of pH and sugar on growth rate of Zygosaccharomyces rouxii, a bakery product spoilage yeast.
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The effects of citric acid-modified pH (pH 2.5, 2.75, 3, 3.5, 4, 4.5, 5, and 5.5) and a 30% glucose-70% sucrose mixture (300, 400, 500, 600, 700, 800, 875, and 900 g/liter) on an osmophilic yeast, Zygosaccharomyces rouxii, were determined by using synthetic medium. One hundred experiments were carried out; 50-ml culture flasks were inoculated with 10(3) CFU ml(-1) by using a collection strain and a wild-type strain cocktail. The biomass was measured by counting cell colonies, and growth curves were fitted by using a Baranyi equation. The growth rate decreased linearly with sugar concentration, while the effect of pH was nonlinear. Indeed, the optimal pH range was found to be pH 3.5 to 5, and pH 2.5 resulted in a 30% reduction in the growth rate. Finally, we evaluated the performance of two nonlinear predictive models developed previously to describe bacterial contamination. Equations derived from the Rosso and Ratkowsky models gave similar results; however, the model that included dimensionless terms based on the Ratkowsky equation was preferred because it contained fewer estimated parameters and also because biological interpretation of the results was easier. (+info)
Donnan potential of rabbit skeletal muscle myofibrils I: electrofluorochromometric detection of potential.
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The fluorescence of the dye CC-6 [(3-hexyl-2-(3-hexyl-2-benzoxazolinylidene)-1-propenyl)-benzoxazolium iodide] has been shown to indicate Donnan potentials in rabbit skeletal muscle myofibrils. These results are in agreement with previously published work in which the potentials were measured with microelectrodes on glycerol-extraced muscle fibers. The magnitude of the Donnan potential of the myofibrils has been shown to be dependent on the state (rigor or relaxed) of the system. (+info)
Microbacterium kitamiense sp. nov., a new polysaccharide-producing bacterium isolated from the wastewater of a sugar-beet factory.
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Two strictly aerobic, heterotrophic and mesophilic new organisms, strains Kitami A1 and Kitami C2T, were isolated from the wastewater of a sugar-beet factory in Kitami City, Hokkaido, Japan. In batch cultures, these organisms produced both insoluble and soluble exopolysaccharides (EPSs) utilizing sucrose as the sole carbon source. The G + C contents of the strains Kitami C2T and Kitami A1 were 69.2 mol%. Both strains had anteiso-C15:0 acid, anteiso-C17:0 acid and iso-C16:0 as major components. The major isoprenoid quinones from these strains included menaquinone-11 and menaquinone-12. Physiological and biochemical characterization, phylogenetic analysis and DNA-DNA relatedness indicated that these two organisms are new species of the genus Microbacterium, for which the name Microbacterium kitamiense is proposed. The type strain of M. kitamiense is strain Kitami C2T (= JCM 10270T). (+info)
Location and orientation of DODCI in lipid bilayer membranes: effects of lipid chain length and unsaturation.
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The location and orientation of a linear dye molecule, DODCI, in lipid bilayer membrane were determined by the effect of viscosity and refractive index of the aqueous medium on the fluorescence properties of the dye bound to the membrane. The membrane-bound dye is solubilized in two sites, one near the surface (short fluorescence lifetime) and another in the interior of the membrane (long lifetime). The ratio of the dye in the two locations and the orientation of the dye (parallel or perpendicular to the membrane) are sensitive to the lipid chain length and unsaturation in the alkyl chain. The fraction of the dye in the interior region is higher for short alkyl chains (C12>C14>C16>>C18C20) and in unsaturated lipids (C14:1>C14:0, C16:1>C16:0). These experimental results are consistent with the general principle that the penetration of an amphiphilic organic molecule in the interior region of the membrane is more when the structure of th bilayer is more fluid-like. (+info)
Ribosomal gene disruption in the extreme thermophile Thermus thermophilus HB8. Generation of a mutant lacking ribosomal protein S17.
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S17 is a primary rRNA-binding protein which has been implicated in ribosome assembly and translational fidelity. We describe the generation and biochemical characterization of an S17 minus ribosomal mutant, a ribosomal protein-lacking mutant obtained in Thermus thermophilus HB8. The S17 mutant was obtained by insertional inactivation of the target gene with the kanamycin adenyl transferase (kat) gene, making use of a Thermus-Escherichia shuttle vector and the natural ability of Thermus to transform. In the final construct used to transform Thermus cells, the S17 coding region was replaced with the kat gene cloned in-frame with the first three amino acids of S17. Hence, in vivo transcription of the kat gene was under the control of the ribosomal operon promoter. As in Escherichia coli, the Thermus S17 mutant exhibited a temperature-sensitive phenotype. Two-dimensional PAGE, Western blot, and ELISA confirmed the absence of S17 from the mutant ribosomes. Sucrose-gradient profiles of mutant cells showed a clear separation and normal proportions of 50S and 30S subunits and a normal ratio between them. In addition, the S17 mutant showed the presence of a 20S peak representing assembly-defective particles. The successful re-incorporation of protein S17 into the mutant ribosomes was demonstrated when reconstitution with isolated S17 was performed at 60 degrees C. (+info)
Independent in vitro assembly of all three major morphological parts of the 30S ribosomal subunit of Thermus thermophilus.
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Fragments of the 16S rRNA of Thermus thermophilus representing the 3' domain (nucleotides 890-1515) and the 5' domain (nucleotides 1-539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3' RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3' domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5' RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit [Agalarov, S.C., Zheleznyakova, E.N., Selivanova, O.M., Zheleznaya, L.A., Matvienko, N.I., Vasiliev, V.D. & Spirin, A.S. (1998) Proc. Natl Acad. Sci. USA 95, 999-1003], these experiments establish that all three main structural lobes of the small ribosomal subunit can be reconstituted independently of each other and prepared in the individual state. (+info)
Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils.
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The response to moderate salt stress of a Scytonema species isolated from a soil crust in the arid region of central Australia was studied. An increase in intracellular trehalose and sucrose concentrations was detected by NMR and HPLC analysis following salt stress, maximal amounts being produced by exposure to 150 mM NaCl after 48 h. When the organism was subsequently returned to normal growth conditions, the cellular concentrations of these solutes decreased. The biosynthesis of trehalose and sucrose was studied and found, in both cases, to involve both sugar phosphate synthase and phosphatase enzymes. The combined synthase activities and the individual phosphatase activities in cell extracts were increased by salt stress. Trehalose phosphorylase was the only catabolic enzyme detected for trehalose; neither trehalase nor phosphotrehalase activities could be detected. This is the first report of trehalose phosphorylase activity in cyanobacteria. Both trehalose and sucrose phosphorylase activities increased in salt-stressed cells, whereas the activity of invertase did not change. (+info)