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Acta Leprol ; 7 Suppl 1: 203-7, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2503991


Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.

Mycobacterium/genética , Vacina BCG/isolamento & purificação , Vacinas Bacterianas/isolamento & purificação , Genes Bacterianos , Vetores Genéticos , Mycobacterium/imunologia , Mycobacterium/patogenicidade , Plasmídeos , Transformação Genética , Virulência
Acta Leprol ; 7 Suppl 1: 256-7, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2503999


Pasteur 1173P2 and Japanese 172 BCG substrains were transformed with plasmid pAL5000::plJ666 by electroporation and assessed by the growth of kanamycin-resistant colonies. From the transformants pYUP plasmids were isolated containing plJ666 inserted into pAL5000. The introduction, persistence and the identity of the plasmid were monitored by reisolation from consecutive subcultures and restriction analysis. The effect of variables--age, viability, glycine pretreatment of BCG cultures, electroporation parameters--on transformation frequencies were analyzed. Consecutive transformation of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 10(1)-10(2) obtained with the initial library to 10(3)-10(4) colonies/micrograms DNA with the pYUB plasmids. Maintenance of plasmid and expression of kanamycin resistance in continuous subcultures were stable for 100 generations from the first successful transformation to the present. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.

Mycobacterium bovis/genética , Plasmídeos , Vacina BCG/isolamento & purificação , DNA Bacteriano/genética , Vetores Genéticos , Resistência a Canamicina/genética , Transformação Genética , Vacinas Sintéticas/isolamento & purificação
Proc Natl Acad Sci U S A ; 85(18): 6987-91, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2842799


Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.

Regulação da Expressão Gênica , Lisogenia , Mycobacterium/genética , Transformação Bacteriana , Clonagem Molecular , Escherichia coli/genética , Canamicina Quinase , Hanseníase/microbiologia , Métodos , Mycobacterium/enzimologia , Fosfotransferases/genética , Plasmídeos , Tuberculose/microbiologia