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Characterization of CmaA, an Adenylation-Thiolation Didomain Enzyme Involved in the Biosynthesis of Coronatine. Robin Couch, 2004.Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond . The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected . These efforts allowed overproduction of P . syringae pv . glycinea PG4180 CmaA in P . syringae pv . syringae FF5 as a FLAG-tagged protein and overproduction of P . syringae pv . tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form . Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain) . ATP-32PPi exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine . Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate . This covalent species was detected by incubating CmaA with ATP and L-[G-3H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA . The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid . The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of Aspergillus niger through Intracellular Acidification. Andrew Plumridge, 2004.The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid) . Conidia inoculated at 105/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower . The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size . A . niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies . The mechanism of action of sorbic acid was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy . We show that a rapid decline in cytosolic pH (pHcyt) by more than 1 pH unit and a depression of vacuolar pH (pHvac) in A . niger occurs in the presence of sorbic acid . The pH gradient over the vacuole completely collapsed as a result of the decline in pHcyt . NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A . niger mycelia to decrease dramatically, and they did not recover . The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth . Identification of Three New Genes Involved in Morphogenesis and Antibiotic Production in Streptomyces coelicolor. Ondrej Sprusansky, 2003.We report the isolation and partial characterization of three new mutants of Streptomyces coelicolor that are defective in morphogenesis and antibiotic production . The genes identified by the mutations were located and cloned by using a combination of Tn5 in vitro mutagenesis, cotransformation, and genetic complementation . Mutant SE69 produces lower amounts of antibiotics than the wild type produces, produces spores only after prolonged incubation on rich media, and identifies a gene whose predicted protein product is similar to the GntR family of transcriptional regulators; also, production of aerial mycelia on both rich and poor media is significantly delayed in this mutant . Mutant SE293 is defective in morphogenesis, overproduces antibiotics on rich media, fails to grow on minimal media, and identifies a gene whose predicted protein product is similar to the TetR family of transcriptional regulators . Preliminary evidence suggests that the SE293 gene product may control a molybdopterin binding protein located immediately adjacent to it . Mutant SJ175 sporulates sooner and more abundantly than the wild type and overproduces antibiotics on rich media, and it identifies a gene whose predicted protein product contains regions of predominantly hydrophobic residues similar to those of integral membrane proteins . Type II Protein Secretion in Pseudomonas aeruginosa: the Pseudopilus Is a Multifibrillar and Adhesive Structure. Eacute;ric Durand, 2003.The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase . This pathway requires the function of a macromolecular complex called the Xcp secreton . The Xcp secreton shares many features with the machinery involved in type IV pilus assembly . More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins . We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus . Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure . Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits . Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P . aeruginosa . In addition, heterologous pseudopilins can be assembled by P . aeruginosa into a type II pseudopilus . Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities . These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections . Effects of Essential Oils on Ruminal Microorganisms and Their Protein Metabolism. F. M. McIntosh, 2003.A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms . EO inhibited (P < 0.05) the rate of deamination of amino acids . Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi .
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