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Evolution of Staphylococcus aureus by Large Chromosomal Replacements. D. Ashley Robinson, 2004.Conjugative transfer and replacement of hundreds of kilobases of a bacterial chromosome can occur in vitro, but replacements in nature are either an order of magnitude smaller or involve the movement of mobile genetic elements . We discovered that two lineages of Staphylococcus aureus, including a pandemic methicillin-resistant lineage, were founded by single chromosomal replacements of at least  244
and
557
kb representing
10
and
20%
of the chromosome, respectively, without the obvious involvement of
mobile genetic elements . The replacements are unprecedented in
natural populations of bacteria because of their large size and
unique structure and may have a dramatic impact on bacterial
evolution .
Inactivation of Mg Chelatase during Transition from Anaerobic to Aerobic Growth in Rhodobacter capsulatus. Robert D. Willows, 2003.The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism . As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue . A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg2+ to form Mg-protoporphyrin IX . Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R . capsulatus . We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts . BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX . Recombinant BchH is rapidly inactivated by light in the presence of O2, and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX . When photosynthetically growing R . capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions . The light- and O2-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration . Characterization of the CipA Scaffolding Protein and In Vivo Production of a Minicellulosome in Clostridium acetobutylicum. Fabrice Sabathé, 2003.The cipA gene encoding the Clostridium acetobutylicum scaffolding protein CipA was cloned and expressed in Escherichia coli . CipA contains an N-terminal signal peptide, a family 3a cellulose-binding domain (CBD), five type I cohesin domains, and six hydrophilic domains . The uniqueness of CipA lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain . Affinity-purified CipA was used in equilibrium-binding experiments to characterize the interaction of CipA with crystalline cellulose . A Kd of 0.038 µM and a [C]max of 0.43 µmol of CipA bound per g of Avicel were determined . A mini-CipA polypeptide consisting of a CBD3a and two cohesin domains was overexpressed in C . acetobutylicum, yielding the in vivo formation of a minicellulosome . This is to our knowledge the first demonstration of the in vivo assembly of a recombinant minicellulosome . Development of a Listeria monocytogenes EGDe Partial Proteome Reference Map and Comparison with the Protein Profiles of Food Isolates. Manilduth Ramnath, 2003.A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism . The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes . In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed . The method used provided partial fractionation of membrane and cytosolic proteins . The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L . monocytogenes EGDe proteome . An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome . The variation was greater for the less intense spots, and on average 28% of these spots were not matched . Two of the proteins identified in L . monocytogenes EGDe were missing in one or more of the food isolates . These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups . The two corresponding genes were found by PCR amplification to be present in the four food isolates . Our results show that the L . monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen . Assessment of the Effects of Holding Time and Temperature on Escherichia coli Densities in Surface Water Samples. Misty L. Pope, 2003.Escherichia coli is a routinely used microbiological indicator of water quality . To determine whether holding time and storage conditions had an effect on E . coli densities in surface water, studies were conducted in three phases, encompassing 24 sites across the United States and four commonly used monitoring methods . During all three phases of the study, E . coli samples were analyzed at time 0 and at 8, 24, 30, and 48 h after sample collection . During phase 1, when 4°C samples were evaluated by Colilert or by placing a membrane onto mFC medium followed by transfer to nutrient agar containing 4-methylumbelliferyl-ß-D-glucuronide (mFC/NA-MUG), three of four sites showed no significant differences throughout the 48-h study . During phase 2, five of seven sites showed no significant difference between time 0 and 24 h by membrane filtration (mFC/NA-MUG) . When evaluated by the Colilert method, five of seven sites showed no significant difference in E . coli density between time 0 and 48 h . During phase 3, 8 of 13 sites showed no significant differences in E . coli densities between time 0 and the 48-h holding time, regardless of method . Based on the results of these studies, it appears that if samples are held below 10°C and are not allowed to freeze, most surface water E . coli samples analyzed by commonly used methods beyond 8 h after sample collection can generate E . coli data comparable to those generated within 8 h of sample collection . Notwithstanding this conclusion, E . coli samples collected from surface waters should always be analyzed as soon as possible .
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