RESUMO
The fragment containing the carotenoid gene cluster from Mycobacterium aurum A+, a 3,3'-dihydroxy-isoneriatene and 3-monohydroxy-isoneriatene accumulator, has been sequenced and the exposed eight genes are organised in two operons. The function of three of these genes, a phytoene desaturase (crtI), a phytoene cyclase (crtY) and a beta-carotene desaturase (crtU), was demonstrated by complementation of M. aurum carotenoid mutants. The eight genes of the carotenoid cluster are highly homologous to other carotenoid gene clusters and thus this cluster is a candidate for its introduction into mycobacteria as a non-antibiotic reporter gene(s) as well as a source of new regulated promoters.
Assuntos
Carotenoides/biossíntese , Genes Bacterianos , Família Multigênica , Mycobacterium/genética , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Carotenoides/análise , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Geranil-Geranildifosfato Geranil-Geraniltransferase , Dados de Sequência Molecular , Mycobacterium/metabolismo , Oxirredutases/genética , Alinhamento de SequênciaRESUMO
This report describes the first successful transfer and complete expression of clustered mycobacterial genes controlling a biosynthetic pathway (carotenogenesis) in a homologous system. A genomic library of pigmented Mycobacterium aurum A+ (yellow-orange) DNA was constructed in shuttle vector pHLD-69. The colourless mutant A11 and the brick-red mutant NgR9 derived from M. aurum A+ were electroporated with the plasmid library. Among the transformants, colonies different in colour from the recipient mutants were detected, and were cloned. One of the clones from the transformed A11 mutant had a yellow-orange phenotype, and was designated A11T; one of the clones from the NgR9 (brick-red) mutant had a yellow-orange phenotype and was designated NgR9T. The carotenoid pigments from the A11T and NgR9T clones were analyzed and in both the end product of carotenogenesis in M. aurum (leprotene) was detected. A11T and NgR9T harboured the same recombinant plasmid (Cl) containing a 11-kb M. aurum fragment. pCl was used to transform the colourless Mycobacterium smegmatis MC2-155 strain. All the transformants were pigmented. A colony (MC2-T) was arbitrarily chosen and leprotene was detected. It was therefore concluded that M. aurum genes involved in carotenogenesis had been cloned, and were expressed not only in M. aurum mutants, but also in M. smegmatis.