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Identification by Heterologous Expression and Gene Disruption of VisA as L-Lysine 2-Aminotransferase Essential for Virginiamycin S Biosynthesis in Streptomyces virginiae. Wises Namwat, 2002.The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics . Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety . The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35°C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into  1-piperidine 2-carboxylic acid . A visA deletion mutant of S . virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant . No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation . However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M1 at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis . These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine .
Regulation and Adaptive Evolution of Lactose Operon Expression in Lactobacillus delbrueckii. Luciane Lapierre, 2002.Lactobacillus delbrueckii subsp . bulgaricus and L . delbrueckii subsp . lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products . After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L . delbrueckii subsp . bulgaricus loses its ability to regulate lac operon expression . A series of mutations led to the constitutive expression of the lac genes . A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors . A lac repressor gene was discovered downstream of the ß-galactosidase gene of L . delbrueckii subsp . lactis and was shown to be inactivated by several mutations in L . delbrueckii subsp . bulgaricus . Regulatory mechanisms of the lac gene expression of L . delbrueckii subsp . bulgaricus and L . delbrueckii subsp . lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (ß-glucuronidase) in the presence or absence of the lac repressor gene . Insertion of the complex of IS elements in the lac promoter of L . delbrueckii subsp . bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter . Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L . delbrueckii subsp . bulgaricus . The Periplasmic Protein MppA Requires an Additional Mutated Locus To Repress marA Expression in Escherichia coli. Xiaowen Bina, 2003.Escherichia coli strain TP985, which has an insertional mutation in the gene for the periplasmic murein tripeptide binding protein MppA, was previously reported to overproduce MarA and exhibit a multiple-antibiotic resistance (Mar) phenotype (H . Li and J . T . Park, J . Bacteriol . 181:4842-4847, 1999) . We found that TP985 contained a previously unrecognized marR mutation which was responsible for the Mar phenotype . Transduction of the mppA mutation from TP985 to another wild-type strain did not affect antibiotic susceptibility . Overproduction of MppA repressed marA transcription in TP985 but not in other mppA or marR mutants . Therefore, TP985 contains an additional unknown mutation(s) that facilitates the repression of marA expression by MppA . Chlorine Inactivation of Spores of Encephalitozoon spp.. C. H. Johnson, 2003.This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E . hellem, associated with human infection . The 50% tissue culture infective doses of these three species were also determined . On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment .
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