DNA sequence and regulation of ermD, a macrolide-lincosamide-streptogramin B resistance element from Bacillus licheniformis.

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.

Induction of macrolide-lincosamide-streptogramin B resistance requires ribosomes able to bind inducer.

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.

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.

Direct selection of recombinant plasmids in Bacillus subtilis.

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thyA thyB host. Thy- strains are resistant to trimethoprim (Tmp), and Thy+ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cmr Tmpr clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRI, BclI, PvuII, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed.

Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis.

pE194, a 3.5-kilobase multicopy plasmid, confers resistance to the macrolide-lincosamide-streptogramin B antibiotics in Bacillus subtilis. By molecular cloning and deletion analysis we have identified a replication segment on the physical map of this plasmid, which consists of about 900 to 1,000 base pairs. This segment contains the replication origin. It also specifies a trans-acting function (rep) required for the stable replication of pE194 and a negatively acting copy control function which is the product of the cop gene. The target sites for the rep and cop gene products are also within this region. Two incompatibility determinants have been mapped on the pE194 genome and their properties are described. One (incA) resides within the replication region and may be identical to cop. incB, not located in the replication region, expresses incompatibility toward a copy control mutant (cop-6) but not toward the wild-type replicon.

Translational attenuation of ermC: a deletion analysis.

ermC is a plasmid gene which specifies resistance to macrolide-lincosamide-streptogramin B antibiotics. The product of ermC was previously shown to be an inducible rRNA methylase, which is regulated translationally, and a mechanism for this regulation, termed the translational attenuation model, has been proposed. This model postulates that alternative inactive and active conformational states of the ermC mRNA are modulated by erythromycin-induced ribosome-stalling during translation of a leader peptide. In the present study the translational attenuation model was tested by constructing a series of deletants missing the ermC promoter and portions of the regulatory (leading) region. In these mutants, ermC transcription is dependent on fusion to an upstream promoter. Depending on the terminus of each deletion within the regulatory region, determined by DNA sequencing, ermC expression is observed to be either high level and inducible (like the wild-type), high level and noninducible, or low level and noninducible. The translational attenuation model predicts that as the deletions extend deeper into the leader region, successively masking and unmasking sequences required for translation of the methylase, an alternation of high and low level methylase expression will be observed. These predictions are confirmed. Based on this and other information, the model is refined and extended, and both direct translational activation and kinetic trapping of a metastable active intermediate during transcription are proposed to explain basal synthesis of methylase and to rationalize the effects of certain regulatory mutants.

Naturally occurring macrolide-lincosamide-streptogramin B resistance in Bacillus licheniformis.

Resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics is widespread and of clinical importance. B. Weisblum and his coworkers have demonstrated that this resistance is associated with methylation of the 23S ribosomal ribonucleic acid of the large ribosomal subunit which results in a diminished affinity of this organelle for these antibiotics (Lai et al, J. Mol. Biol. 74:67-72, 1973). We report that 10 of 15 natural isolates of Bacillus licheniformis, a common soil organism, are resistant to the MLS antibiotics. The properties of this resistance (high level of tolerance for erythromycin, broad cross-resistance spectrum, and inducibility) suggest that resistance is conferred as described above. The resistance determinant from one of these strains was cloned onto a B. subtilis plasmid vector, and the resulting hybrid plasmid (pBD90) was used to prepare radioactive probe deoxyribonucleic acid for hybridization studies. All of the resistance B. licheniformis strains studied exhibited homology with the pBD90 insert. Plasmid pBD90 showed no homology to the following staphylococcal and streptococcal MLS-resistance plasmids: pE194, pE5, pAM77, pI258. Plasmids pE194 and pE5, on the other hand, carry homologous MLS genes but showed no detectable homology to one another in their replication genes. pBD90 specified a 35,000-dalton erythromycin-inducible protein, detectable in minicells, which therefore appears different from the 29,000-dalton inducible resistance protein specified by pE194. We conclude that there are at least three distinct MLS resistance determinants to be found among gram-positive bacteria.

Conformational alteration of mRNA structure and the posttranscriptional regulation of erythromycin-induced drug resistance.

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.

Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis.

The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmid-free recipients (Canosi et al., 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus licheniformis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per microgram of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BglII and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.

Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.

Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.

Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.

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. Iordanescu, 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.

Construction and properties of chimeric plasmids in Bacillus subtilis.

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. subtilis 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. subtilis system for molecular cloning has been clearly illustrated.