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The DNA sequence of the ermC gene of plasmid pE194 is presented. This determinant is responsible for erythromycin-induced resistance to the macrolide-lincosamide-streptogramin B group of antibiotics and specifies a 29,000 dalton inducible protein. The locations of the ermC promoter, as well as that of a probable transcriptional terminator, are established both from the sequence and by transcription mapping. The sequence contains an open reading frame sufficient to encode the previously identified 29,000 dalton ermC protein. Between the promoter and the putative ATG start codon is a 141 base pair leader sequence, within which several regulatory (constitutive) mutations have been mapped and sequenced. The leader has a second open reading frame, sufficient to encode a 19 amino acid peptide. It is suggested that induction by erythromycin involves a shift between alternative ribosome-bound mRNA conformations, so that the ribosome binding sequence and the start codon for synthesis of the 29K protein are unmasked in the presence of inducer. Possible active and inactive folded configuration of the leader sequence are presented, as well as the effects on these configurations of regulatory mutations.

Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUB110, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 10(4) to 10(5) transformants per microgram of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iordănescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.

Antibiotic resistance chimeric plasmids have been constructed by in vitro enzymatic manipulation and introduced into Bacillus subtilis by transformation. The parental plasmids used had been introduced into B. subtiis from Staphylococcus aureus by transformation. Of the seven recombinant plasmids constructed using restriction endonucleases, one was made using EcoRI, another using Hpa II, and five with Xba I (from Xanthomonas badrii), demonstrating the utility of the latter enzyme for molecular cloning experiments. Although all of the recombinant plasmids we have made replicate and express their antibiotic resistance characters, three of them have suffered a loss of DNA, either in vitro or, more likely, in vivo. The deletion event in all cases involved-one of the two termini used to join the parental plasmids. The plasmid chimeras reported in this paper should prove useful for the study of plasmid replication, incompatibility, and recombination. In addition, the utility of the B. subtifis system for molecular cloning has been clearly illustrated.The ability to carry out molecular cloning in Bacillus subtilis would be useful for a variety of studies on sporulation, transformation, and gene expression. In addition, such a capability might be industrially significant, because Bacillus species are of considerable commercial importance. Ehrlich (1) has shown that several chloramphenicol and tetracycline resistance plasmids isolated from Staphylococcus aureus can be introduced by transformation into B. subtilis. This raised the possibility that S. aureus plasmids might be useful as vectors for molecular cloning in R. subtilis. We have transferred additional S. aureus plasmids to competent B. subtilis strains and have initiated a study of the molecular biology of these plasmids. This paper reports the construction of several plasmid chimeras by molecular cloning, thus demonstrating the utility of the B. subtilis system for recombinant DNA experiments and providing a collection of new plasmids for studies on replication, incompatibility, and transformation as well as for the engineering of better cloning vectors. MATERIALS AND METHODSThe B. subtilis host strain used in this study was BD170 (trpC2 thr-5). The plasmids used were originally isolated from S. aureus and were introduced into B. subtilis by transformation. All plasmid DNA preparations used in this study were isolated from B. subtilis. The plasmids are listed in Table 1 together with some relevant restriction endonuclease sites and molecular weights. The documentation of this data will be published elsewhere.'The isolation of covalently closed circular plasmid DNA was carried out by the sodium dodecyl sulfate/NaCl method of Guerry et al. (5) followed by dye-buoyant density centrifugation (6). The preparation of bacterial DNA and competent cells and the transformation procedure was as described previously (7), except that 1 mM ethylene glycol bis(#-aminoethyl ether)-N,N'-tetraacetic acid was added to the plasmid transformation mixtures, because this raise...

The DNA sequence of ermD , a macrolide-lincosamide-streptogramin B (MLS) resistance determinant cloned from the chromosome of Bacillus licheniformis, has been determined. ermD encodes an erythromycin inducible protein of molecular weight 32,796. S1 nuclease mapping of the ermD promoter has revealed the presence of an approximately 354 base leader sequence on the ermD transcript. This leader contains a short open reading frame sufficient to encode a 14 amino acid peptide, which is preceded by a potential ribosomal binding site. The leader sequence has the potential to fold into several base paired structures, in some of which the ribosomal binding site for the ermD product would be sequestered. Deletion analysis demonstrated that the leader contains regulatory sequences. Removal of the ermD promoter and fusion to an upstream promoter did not interfere with induction, strongly suggestion that ermD regulation is posttranscriptional. Based on these features it appears likely that ermD is regulated by a translational attenuation mechanism, analogous to that suggested for ermC , a resistance element from Staphylococcus aureus ( Gryczan et al. 1980; Horinouchi and Weisblum 1980). Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.