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, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia 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 (Tc(r)). Interestingly, in the absence of continued selection for Tc(r), 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 two 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 Tc(r) 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. Tc(r) 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 Tc(r) 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. In contrast, transformation of S. sanguis (Challis) with pAM118 (erythromycin resistant [Em(r)] Tc(r)), constructed in E. coli from the shuttle vector pVA838 and the EcoRI F' fragment of pAM211, generated Tc(r) transformants at frequencies of 10-4 per survivor. The majority of tetracycline-selected transformants were Em(s), 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 more than one copy per chromosome. Interestingly, the majority of erythromycin-selected transformants (10-4 per survivor) were Tc(s); plasmid DNA was shown to consist of the pVA838 vector and the EcoRI F fragment of pAD1 regenerated after Tn916 had excised from pAM118. Em(r) Tc(r) transformants could be obtained 100-fold less frequently (10-6 per survivor), and in all such transformants examined the plasmid DNA was identical to that found in the Em(r) Tc(s) transformants (i.e., pVA838 harboring the EcoRI F fragment of pAD1). The presence of Tn916 insertions in the bacterial chromosome of these Em(r) Tc(r) transformants was demonstrated in filter blot hybridization experiments. These observations suggest 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.