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Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum. Françoise Blain, 2002.A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described . hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains . The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F . heparinum grown in heparin-only medium . The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F . heparinum grown in chondroitin sulfate A-only medium . The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain . The transcriptional start sites were determined for hepA in both the wild-type F . heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain . The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts . Two Separate Quorum-Sensing Systems Upregulate Transcription of the Same ABC Transporter in Streptococcus pneumoniae. Eivind Knutsen, 2004.Streptococcus pneumoniae secretes two different peptide pheromones used for intercellular communication . These peptides, which have completely unrelated primary structures, activate two separate signal transduction pathways, ComABCDE and BlpABCSRH, which regulate natural genetic transformation and bacteriocin production, respectively . Each signal transduction pathway contains a response regulator (ComE and BlpR, respectively) that activates transcription of target genes by binding to similar, but not identical, imperfect direct repeat motifs . In general the direct repeat binding sites are specific for one or the other of the two response regulators, ensuring that competence development and bacteriocin production are regulated separately . However, in the present study we show that the rate of transcription of an operon, encoding an ABC transporter of unknown function, can be stimulated by both peptide pheromones . We also show that this cross-induction is due to a hybrid direct repeat motif that can respond to both ComE and BlpR . To our knowledge this kind of convergent gene regulation by two separate two-component regulatory systems has not been described before in bacteria . Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection. Alexandra R. Keegan, 2003.Cryptosporidium parvum represents a challenge to the water industry and a threat to public health . In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C . parvum with disinfectants . The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine . The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst . Effective oocyst inactivation was achieved (>2 log10 units) with LP-UV (20 mJ/cm2) or 2 mg of ozone/liter (for 10 min) . MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log10 unit being inactivated . These results demonstrate the inability of MIOX to inactivate Cryptosporidium . The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water . Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis. Joanna C. Wilkins, 2003.Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque . We undertook to identify the most abundant surface-associated proteins of S . oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions . Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry . Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae . A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase . The most abundant protein, phosphocarrier protein HPr, was present as three isoforms . Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis . Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts . This study has extended the range of streptococcal proteins known to be expressed at the cell surface . Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions .
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