Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria

Abstract

The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, was cloned as an intact element on the pBR322-derived vector pGL101 in E. coli. The EcoRI F'' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera. pAM170, generated by ligating the EcoRI D'' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the 2 fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368. Even the presence in the polA1 background of a DNA segment known to acquire insertions of Tn916 in S. faecalis did not allow the detection of any Tcr transformants of E. coli C2368 after transformation with pAM170 DNA. Tn916 insertions into the E. coli chromosome were only observed after prolonged growth of E. coli CG120 [DH1(pAM120)] in the absence of drug. Transformation of S. sanguis (Challis) with pAM118 (erythromycin resistant [Emr] Tcr), constructed in E. coli from the shuttle vector pVA838 and the EcoRI F'' fragment of pAM211, generated Tcr transformants at frequencies of 10-4/survivor. The majority of tetracycline-selected transformants were Em8, however, and shown to lack plasmid DNA. Hybridization profiles obtained from Southern experiments showed that these transformants contained Tn916 insertions in the bacterial chromosome; Tn916 insertions could occur at various sites and in some cases in > 1 copy/chromosome. Interestingly, the majority of erythromycin-selected transformants (10-4/survivor) were Tcs; plasmid DNA was shown to consist of the pVA838 vector and the EcoRI F fragment of pAD1 regenerated after Tn916 had excised from pAM118. Emr Tcr transformants could be obtained 100-fold less frequently (10-6/survivor), and in all such transformants examined the plasmid DNA was identical to that found in the Emr Tcs transformants (i.e., pVA838 harboring the EcoRI F fragment of pAD1). The presence of Tn916 insertions in the bacterial chromosome of these Emr Tcr transformants was demonstrated in filter blot hybridization experiments. These observations suggests a strategy for the cloning of gram-positive genes which capitalizes on the unique aspects of Tn916 regarding its transposition mechanism and behavior when cloned in E. coli.