Streptococcus faecalis strain DS16 harbors the conjugative hemolysin-bacteriocin plasmid pADl (35 megadaltons) and the nonconjugative R-plnsmid pAD2 determining resistance to streptomycin, kanamycin, and erythromycin; a tetracycline resistance (Tetr) determinant is located on the chromosome. When strain DS16 was mated (on membrane filters) with the plasmid-free strain JH2-2, Tetr transconjugants could be obtained at a frequency of about 10 per recipient. Analyses of transconjugants showed that some contained the Tet' determinant linked to pAD1. Subsequent studies showed that the Tete determinant was located on a 10-megadalton transposon, designated Tn916, which could insert into two other hemolysin plamids: pAMyl and pOB1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring Tet' to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of Tete. Both transposition and transfer were found to be rec independent.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidaseprocessing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.

A plasmid determining resistance to erythromycin, lincomycin, and vernamycin Ba was isolated from a strain of Streptococcus pyogenes. The plasmid has a molecular weight of approximately 17 x 108 and is present to the extent of one to two copies per chromosomal genome equivalent.Erythromycin and lincomycin are antibiotics used in the treatment of group A beta-hemolytic streptococcus (Streptococcus pyogenes) infections in patients for whom penicillin is contraindicated. S. pyogenes strains are usually susceptible to these agents, although resistant clinical isolates have been reported (7,8,10,12,13,15). Whether resistance in this species is related to extrachromosomal (plasmid) determinants, as is often the case among drug-resistant staphylococci and enteric bacilli, has, to our knowledge, not been reported. In this communication we report on the isolation and characterization of a plasmid from a clinical isolate of S. pyogenes and show that it determines resistance to erythromycin, lincomycin, and vernamycin Ba (a streptogramin B-type antibiotic).S. pyogenes strain 10535/72 (designated here as AC-1), serotype M22: T12, was originally isolated by J. Dixon in Alberta, Canada. This strain, which was isolated from the inflamed throat of a 17-year-old Cree Indian woman, is resistant to erythromycin and lincomycin (greater than 1 mg/ml in each case), as well as vernamycin B, (no zone of inhibition with susceptibility disks containing 20 ug of drug). The patient had not been treated with antibiotics during the previous 12 months but had been in recent contact with a member of her family who was on erythromycin therapy.Todd-Hewitt broth (THB; Difco) was used as a growth medium in both liquid and agar forms. Cell growth (at 37 C) was followed by measuring turbidity with a Klett-Summerson colorimeter.Materials and methods of plasmid analyses by dye-buoyant density and sucrose density centrifugation were as described in detail previously (3, 4), as were the methods of plasmid elimination with intercalating dyes ("curing") and electron microscopy analysis (5). Extraction of deoxyribonucleic acid (DNA) was performed as follows. A 200-ml log phase culture isotopically labeled with 1 mCi of [methyl-PH]-thymidine (22 Ci/mmol from Amersham/Searle) was washed with 15 ml of 0.03 M tris(hydroxymethyl)aminomethane-0.005 M Na2 ethylenediaminetetraacetic acid (EDTA)-0.05 M NaCl, pH 8.0, (TES) and resuspended in 3.0 ml of 25% glucose (in TES). A 0.5-ml quantity of 0.25 M EDTA (pH 8.0) was added, followed by the addition of 1.0 ml of lysozyme (5 mg/ml in TES). The suspension was allowed to incubate for 3 h at 37 C, at which point 0.5 ml of Pronase (5 mg/ml in TES, preincubated at 37 C for 30 min) was added, followed by another 30-min period of incubation. The cells were then lysed by the addition of 2.0 ml of sodium dodecyl sulfate (a 2% solution in TES). NaClO, (from a 5 M solution) was added to make a final concentration of 1 M. The lysate was then twice extracted with an equal volume of TES-saturated phenolchloroform (2: 1). Two volum...

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