BackgroundDuring the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.ResultsSurprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.ConclusionsOur study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

BackgroundPhage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays.ResultsWe have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase.ConclusionsWe conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.

No abstract

To cite this article: Mutch NJ, Thomas L, Moore NR, Lisiak KM, Booth NA. TAFIa, PAI-1 and a 2 -antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots. J Thromb Haemost 2007; 5: 812-7.Summary. PAI-1 and a 2 -antiplasmin (a 2 AP) are the principal direct inhibitors of fibrinolytic proteases. Thrombin activatable fibrinolysis inhibitor (TAFI), a plasma procarboxypeptidase activated by thrombin-thrombomodulin to form TAFIa, also regulates fibrinolysis by modulating fibrin. In this study, the relative contributions of PAI-1, a 2 AP and TAFIa to inhibition of lysis were assessed. In platelet-poor plasma clots, a 2 AP, TAFIa and PAI-1 all inhibited lysis, as shown by the addition of neutralizing antibodies to a 2 AP and PAI-1 ± CPI, a potato carboxypeptidase inhibitor. a 2 AP played the largest role in regulating plasma clot lysis, but neutralization of inhibitors in combinations was more effective in shortening lysis times, with a maximal effect when all three inhibitors were neutralized. In platelet-rich clots, a larger contribution of PAI-1 was evident. Tissue plasminogen activator induced lysis of model thrombi, made from whole blood, was approximately doubled on incorporation of CPI, illustrating a substantial contribution of TAFIa to inhibition of thrombus lysis. Similar increases in thrombus lysis were observed on inclusion of neutralizing antibodies to PAI-1 and a 2 AP, with a 2 AP playing the dominant role. Maximal thrombus lysis occurred upon neutralization of all three inhibitors. These observations suggest that, despite the differences in concentrations and activities of inhibitors, and the different modes of action, the roles of the three are complementary in both plasma clot lysis and thrombus lysis.

Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6–9 residues representing 11 out of the 20 canonical amino acids.