02453nas a2200349   4500000000100000008004100001260001300042653002300055653002100078653003100099653002100130653001200151653001300163653001200176653001800188653002400206653001300230653003000243653001700273100001600290700001300306700001300319700001500332700001300347700001400360700001500374245008500389300001200474490000700486520159600493022001402089       1988                            d  c1988 Sep10aCloning, Molecular10aEscherichia coli10aGene Expression Regulation10aKanamycin Kinase10aleprosy10aLysogeny10aMethods10aMycobacterium10aPhosphotransferases10aPlasmids10aTransformation, Bacterial10aTuberculosis1 aSnapper S B1 aLugosi L1 aJekkel A1 aMelton R E1 aKieser T1 aBloom B R1 aJacobs W R00aLysogeny and transformation in mycobacteria: stable expression of foreign genes.  a6987-910 v853 a<p>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.</p>  a0027-8424