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.

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Plasmids were constructed containing the regulatory regions and N-terminal portions of ermC and of ermD , fused in phase with the coding sequence of the Escherichia coli lacZ gene. ermC and ermD are erythromycin (Em) inducible macrolide-lincosamide-streptogramin B resistance elements derived from Staphylococcus aureus and Bacillus licheniformis, respectively. The fusion plasmids were introduced into B. subtilis and used to study ermC and ermD regulation. In both cases, beta-galactosidase synthesis could be induced by low levels of Em. Induction was prevented by introduction of ole-2, a chromosomal mutation which decreases ribosomal affinity for Em. Induction also did not occur in the presence of intact copies of ermC , suggesting that prior or concomitant methylation of 23S rRNA, a treatment known to decrease ribosomal affinity for Em, was capable of interfering with ermC and ermD induction. These experiments are consistent with the translational attenuation model of ermC regulation, and together with other evidence, suggest that ermD is regulated by a similar mechanism.

A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for suppression of a nonsense codon was studied for its ability to propagate phages T2, T4D, T5, phi K, f2, MS2, R17, Q beta, lambda as well as filamentous phages fl, fd and M13. Of all phages tested, only the growth of Q beta, lambda, and filamentous phages is inhibited in the mutant at 42 degree C. This selective inhibition suggests that, like Q beta, lambda and filamentous phages also require a read-through proten(s) which results from suppression of a termination codon.

Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD , an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD . The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD . It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis, another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yield a variety of related Staphylococcal and Streptococcal MLS determinants.