CONSTRUCTION AND EVALUATION OF NEW DRUG-RESISTANCE CASSETTES FOR GENEDISRUPTION MUTAGENESIS IN STREPTOCOCCUS-PNEUMONIAE, USING AN AMI TESTPLATFORM

Although drug-resistance markers have been used frequently for gene-di sruption mutagenesis in Streptococcus pneumoniae, none has yet been sh own to be free of dependence on local transcription for its expression . Indeed, the erythromycin-resistance marker (erm), originating in pAM beta 1, has been used as an indicator of local transcription on sever al occasions, A procedure is demonstrated for evaluation of the autono mous expression of such a marker by placing it in a consistent backgro und, at the pneumococcal anti (aminopterin resistance) locus, in combi nation with active or inactive alleles of the ami promoter (pA). Using this test platform, a chloramphenicol-resistance marker (cat) and a s pectinomycin-resistance marker used in streptococcal gene disruption s tudies and derived from pJS3 and pDL269, respectively, were shown to d epend on local transcriptional signals for expression when placed in t he pneumococcal chromosome as single-copy genes. To overcome this limi tation, new drug-resistance cassettes were designed and constructed, u sing pA as a model for synthetic promoters for the erm and cat genes. Both new cassettes were shown, by the same procedure, to be expressed after insertion in the pneumococcal chromosome, independent of local t ranscription. A new insertion-duplication vector, pEVP3, incorporating the new cat cassette and a lacZ reporter derived from pTV32, was also constructed. The ami test platform was used to demonstrate both the a utonomous expression of cat and the reporter function of lacZ in chrom osomal copies of pEVP3.