RESUMO
Terpenoids or isoprenoids constitute a vast family of organic compounds that includes sterols and carotenoids. The terpenoids in many organisms share early steps in their biosynthesis, including the synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) and its conversion to mevalonate. We have cloned and characterised the genes hmgS for HMG-CoA synthase and hmgR for HMG-CoA reductase from the Zygomycete Phycomyces blakesleeanus. Single copies of these genes are present in the Phycomyces genome. The predicted product of hmgS is largely hydrophilic and that of hmgR has eight putative transmembrane segments and a large hydrophilic domain. The hydrophilic domain suffices for catalytic activity, as shown by expressing it in Escherichia coli. Several features in the promoter of hmgS and in HMG-CoA reductase resemble motifs known to be involved in sterol-mediated regulation and sterol sensing. Carotene-overproducing mutants contain more hmgS mRNA than the wild type, possibly in response to an increased demand for HMG-CoA.
Assuntos
Genes Fúngicos , Ácido Mevalônico/metabolismo , Phycomyces/genética , Phycomyces/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Escherichia coli/genética , Genoma Fúngico , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Sintase/genética , Dados de Sequência Molecular , Mutação , Filogenia , Regiões Promotoras Genéticas , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de AminoácidosRESUMO
A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.