RESUMO
The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Fases de Leitura Aberta , Plasmídeos , Replicon , Rhodococcus/genética , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo II , Hexosiltransferases/biossíntese , Hexosiltransferases/genética , Dados de Sequência Molecular , Plasmídeos/biossíntese , Plasmídeos/química , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sacarose/metabolismoRESUMO
The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.
