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CMY-13, a Novel Inducible Cephalosporinase Encoded by an Escherichia coli Plasmid. V. Miriagou, 2004.An IncN plasmid (p541) from Escherichia coli carried a Citrobacter freundii-derived sequence of 4,252 bp which included an ampC-ampR region and was bound by two directly repeated IS26 elements . ampC encoded a novel cephalosporinase (CMY-13) with activity similar to that of CMY-2 . AmpR was likely functional as indicated in induction experiments . New Bacterial Pathway for 4- and 5-Chlorosalicylate Degradation via 4-Chlorocatechol and Maleylacetate in Pseudomonas sp . Strain MT1. Patricia Nikodem, 2003.Pseudomonas sp . strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway . 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases . However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone . Protoanemonin is obviously a dead-end product of the pathway . A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates . Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in . As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation . The maleylacetate formed in this way is reduced by maleylacetate reductase . Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH . Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila. Puneet Khandelwal, 2004.Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host . Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X . nematophila . Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X . nematophila isolated from that protein complex . The gene was amplified by PCR, cloned, and expressed in Escherichia coli . The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient . The protein oligomerized during in vitro refolding, forming multimers . Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation . The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase . The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives . The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein . The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H . armigera, causing extensive damage to the midgut epithelial membrane . To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium .
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