Plasmid-determined tetracycline resistance in Streptococcus faecalis: Tandemly repeated resistance determinants in amplified forms of pAM[alpha]1 DNA

Abstract

We recently reported that the six-megadalton plasmid pAM[alpha]1 (one of two plasmids in Streptococcus faecalis strain DS-5C1) undergoes a gene amplification as a result of extended growth (many generations) of cells in the presence of a subinhibitory concentration of tetracycline. In this communication we present a detailed analysis of this phenomenon using the newly constructed S. faecalis strain DT-11, a strain devoid of plasmid DNA other than pAM[alpha]1. It was found that during growth of strain DT-11 in the presence of tetracycline a gradual increase in plasmid size was observed, and this could be correlated with an increase in the level of tetracycline resistance. On the basis of analyses using sucrose density-gradient sedimentation, electron microscopy and the restriction endonuclease EcoRI, it was clearly established that the enlarged DNA contained repeated units of a 2.65 megadalton segment containing the tetracycline resistance determinant. This value (2.65 megadaltons) also corresponds to the amount of DNA deleted from the plasmid in easily obtainable tetracycline sensitive variants. EcoRI digestion of the amplified DNA structures generated three fragments designated [alpha]1,[alpha]2 and [alpha]3 with molecular weights of 4.20 x 106, 1.75 x 106 and 0.90 x 106, respectively. Unamplified pAM[alpha]1 was cleaved in only two places by the enzyme, while the plasmid of tetracycline sensitive variants (i.e. DNA missing the 2.65 megadalton sequence on which the tetracycline resistance determinant resides) is totally insensitive. In the case of amplified molecules the relative amount of the [alpha]2 fragment with respect to the [alpha]1 fragment is much higher than for normal pAM[alpha]1. The data indicate that the [alpha]2 fragment contains the tetracycline resistance determinant; and in the amplified state the additional copies are arranged in tandem connected together by the [alpha]3 fragment(s). The data support models which we proposed previously with regard to the mechanism of amplification.It was also observed that in the amplified state the total amount of closed circular plasmid DNA relative to chromosomal DNA was found to be similar to that for the case of unamplified DNA, indicating that when the molecules are larger there are fewer of them.