Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10–1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (−3 logs for synthetic wastewater; −6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.
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Amylomaltases or D-enzyme (4-α-glucanotransferases; E.C. 2.4.1.25) are carbohydrate-active enzymes that catalyze the transfer of glucan units from one α-glucan to another in a disproportionation reaction. These enzymes are involved in starch metabolism in plants or maltose/glycogen metabolism in many microorganisms. The amylomaltase of the hyperthermophilic bacterium Thermus thermophilus HB8 was overproduced in Escherichia coli, partially purified and used to modify potato starch. The action of amylomaltase caused the disappearance of amylose and the broadening of the side-chain length distribution in amylopectin, which resulted in a product with both shorter and longer side chains than in the parent starch. Amylomaltase-treated potato starch showed thermoreversible gelation at concentrations of 3% (w/v) or more, thus making it comparable to gelatin. Because of its animal origin, gelatin is not accepted by several consumer groups. Therefore, the amylomaltase-treated potato starch might be a good plant-derived substitute for gelatin. ? 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with beta-lactam nucleophile instead of water, mutants alphaR145G, alphaR145S and alphaR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic beta-lactam antibiotics.
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Implementation of reliable methodologies allowing Reduction, Refinement, and Replacement (3Rs) of animal testing is a process that takes several decades and is still not complete. Reliable methods are essential for regulatory hazard assessment of chemicals where differences in test protocol can influence the test outcomes and thus affect the confidence in the predictive value of the organisms used as an alternative for mammals. Although test guidelines are common for mammalian studies, they are scarce for non-vertebrate organisms that would allow for the 3Rs of animal testing. Here, we present a set of 30 reporting criteria as the basis for such a guideline for Developmental and Reproductive Toxicology (DART) testing in the nematode Caenorhabditis elegans. Small organisms like C. elegans are upcoming in new approach methodologies for hazard assessment; thus, reliable and robust test protocols are urgently needed. A literature assessment of the fulfilment of the reporting criteria demonstrates that although studies describe methodological details, essential information such as compound purity and lot/batch number or type of container is often not reported. The formulated set of reporting criteria for C. elegans testing can be used by (i) researchers to describe essential experimental details (ii) data scientists that aggregate information to assess data quality and include data in aggregated databases (iii) regulators to assess study data for inclusion in regulatory hazard assessment of chemicals.
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The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.
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The objective of this book ‘An introduction to Smart Dairy Farming’ is to provide insight in the development of the Smart Dairy Farming (SDF) concept and advise as to how to apply this knowledge in the field of activities of students from universities of applied science. The information in this book includes background information and comprehensive insight in the concept of SDF.
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