Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested. .Tn916 has been cloned in Escherichia coli on plasmid vectors, where it has been mapped and studied genetically by using TnS as an insertional mutagen (35,41). In the case of certain plasmid vectors, Tn916-containing chimeras are unstable under nonselective conditions; the transposon excises (RecA independent), giving rise to tetracycline-sensitive segregants. The sequences that flanked the transposon are spliced together during the excision process.Tn916 can be reintroduced into E. faecalis via transformation of protoplasts (19, 40) by appropriate chimeric plasmids (41). A high-frequency, zygotically induced transposition gives rise to insertions into the recipient chromosome; the plasmid from which the element transposed is generally lost in the process. Genetic studies have shown (35) that when Tn5 is inserted near the left end of Tn9J6, the tendency to excise under nonselective conditions is eliminated; these derivatives cannot be reintroduced into E. faecalis, presumably because they cannot undergo zygotic induction. Since the mutated excision function could be complemented in * Corresponding author. t Present address: Ecogen, Inc., Langhorne, PA 19047. trans, it is conceivable that the related determinant encodes a specific excisase that is integrally related to transposition.It is clear that the ends of Tn916 must play a key role in the mechanism of transposition. To gain insight into the nature of this role, we have conducted sequence analyses, the results of which are presented here. We also report analyses of target sequences both before insertion in E. faecalis and after excision in an E. coli background. The data suggest a possible mechanism for transposition.(A preliminary account of some of these data was presented at the Second International Conference on Streptococcal Genetics held in May 1986 [23].) MATERIALS AND METHODS Strains, plasmids, media, and reagents. Bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains used for plasmid preparation were grown in LB medium (13). When antibiotics were used, they were incorporated in the medium at the following concentrations unless o...

Tn916, which encodes resistance to tetracycline, is a 16.4-kilobase conjugative transposon originally identified on the chromosome of Streptococcus faecalis DS16. The transposon has been cloned in Escherichia coli on plasmid vectors, where it expresses tetracycline resistance; it can be reintroduced into S. faecalis via protoplast transformation. We have used a lambda::Tn5 bacteriophage delivery system to introduce Tn5 into numerous sites within Tn916. The Tn5 insertions had various effects on the behavior of Tn916. Some insertions eliminated conjugative transposition but not intracellular transposition, and others eliminated an excision step believed to be essential for both types of transposition. A few inserts had no effect on transposon behavior. Functions were mapped to specific regions on the transposon.

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The Streptococcus faecalis pheromone-dependent conjugative plasmid pAD1::Tn916 and the membrane ifiter-dependent conjugative plasmid pPD5::Tn916 were used to introduce Tn916 into Staphylococcus aureus by intergeneric protoplast fusions and intergeneric membrane-filter matings. In recombinants obtained by protoplast fusion where no plasmid DNA could be detected, tetracycline resistance resulted from transposition of Tn916 from pADi to the S. aureus chromosome. Transformation analyses showed that S. aureus Tn916 chromosomal insertions occurred near pig, ilv, uraA, tyrB, fus, ala, and the trp operon. DNA hybridization analyses of EcoRI-and HindIII-digested chromosomal DNAs confirmed the diversity of chromosomal sites involved and demonstrated that the inserts were Tn916 insertions rather than integrations of all or part of pAD1::Tn916. Both pAD1::Tn916 and pPD5::Tn916 were transferred to S. aureus by membrane-filter matings. These plasmids remained intact and expressed tetracycline resistance in S. aureus. S. aureus strains carrying pAD1::Tn916, but not a chromosomal insert of Tn916, and any one of several conjugal gentamicinresistance plasmids lost their ability to serve as conjugal donors of the gentamicin-resistance plasmids.

MICs of 14 antimicrobial agents for 29 strains of Edwardsiella tarda were determined by an agar dilution method. Of the agents tested, ciprofloxacin, enoxacin, and norfloxacin were the most active on a weight basis. All strains were also susceptible to clinically achievable concentrations of ampicillin, chloramphenicol, tetracycline, trimethoprim, sulfamethoxazole, trimethoprim plus sulfamethoxazole, cefotaxime, and gentamicin. Ninety percent of the strains demonstrated high-level resistance to polymyxin B and colistin.