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Transcription Factor FnrP from Paracoccus denitrificans Contains an Iron-Sulfur Cluster and Is Activated by Anoxia: Identification of Essential Cysteine Residues. Matthew I. Hutchings, 2002.The Paracoccus denitrificans transcription factor FnrP has been characterized using artificial FNR-dependent promoter-lacZ fusion plasmids in Escherichia coli . FnrP can activate both class I and class II FNR-dependent promoters in response to anoxia but shows a marked preference for the class II promoter, where the FNR binding site is centered at -41.5 with respect to the transcription start site . FnrP was found to be inactive in an iscS mutant in vivo, demonstrating a requirement for cysteine desulfurase activity to assemble an iron-sulfur cluster in FnrP . Accordingly, an iron-sulfur cluster could be reconstituted into the purified protein in vitro using cysteine desulfurase, ferrous ions, and cysteine . Thus, FnrP is a true orthologue of FNR from E . coli and switches on target genes in response to anoxia . Inactivation of FnrP by oxygen very likely involves the oxidative disassembly of an iron-sulfur cluster . Possible ligands for the iron-sulfur cluster were identified by substituting each of the seven cysteine residues with serine and characterizing the altered proteins in vivo . Four substituted proteins showed activities less than 5% of the wild type, and so identify the four cysteines (Cys-14, Cys-17, Cys-25, and Cys-113) that are most likely to be involved in cluster ligation . The effects of N-oxides, NO-releasing compounds and a nitrosating agent on FNR and FnrP activity were investigated in vivo using the reporter system . Both proteins are very sensitive to the inclusion of sodium nitroprusside (a source of NO+) in defined growth media but are only moderately sensitive to those sources of NO that were tested . Filamentous Phage Active on the Gram-Positive Bacterium Propionibacterium freudenreichii. Marie-Christine Chopin, 2002.We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium . Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames . The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria . The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively . B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria . Microcystin Biosynthesis in Planktothrix: Genes, Evolution, and Manipulation. Guntram Christiansen, 2003.Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria . Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A . Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes . Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis . We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126 . The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ) . The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium . The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis . Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera . We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility . Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase . The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases . Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material. P. Langlois, 2003.Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions . S . coelicolor was grown in minimal medium with and without L . minor fronds . Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material . One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry . The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions . The repressed proteins were proteins synthesized under starvation stress conditions . These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants . Rapid Detection of Meat Spoilage by Measuring Volatile Organic Compounds by Using Proton Transfer Reaction Mass Spectrometry. D. Mayr, 2003.The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4°C was investigated over 11 days . We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system . Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers . The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time . We also observed a large difference in the emissions between vacuum- and air-packaged meat . We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination . The concentrations of these VOCs increased linearly with the bacterial numbers . This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days .
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