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.

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Induction of the synthesis of a plasmid-encoded polypeptide (E3) by erythromycin is known to be required for the inducible expression of resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in Bacillus subtilis strains carrying the plasmid pE194. This resistance is mediated by a specific N6-dimethylation of adenine in the 23S rRNA of the large ribosomal subunit. We show in this report that E3 induction is regulated posttranscriptionally in the sense that it can occur when RNA synthesis is b ocked and that induction is accompanied by an increase in the functional half-life of E3 mRNA but not of the mRNA species that code for the remaining four known pE194 polypeptides. The induction of E3 is subject to feedback regulation and involves the ribosome. Modification of the erythromycin binding site on the ribosome by methylation or by mutation interferes with induction. Plasmid-specified resistance to the macrolide-lincosamidestreptogramin B (MLS) group of antibiotics is due to a specific N6-dimethylation of an adenine residue in the 23S rRNA of the large ribosomal subunit, thereby reducing the affinity of these ribosomes for the MLS antibiotics (1-3). In certain strains this methylation is inducible by erythromycin (Em) at subinhibitory concentrations (4). Most MLS antibiotics, unlike Em, cannot induce this resistance, but when a culture is exposed to Em, resistance to all MLS antibiotics is induced (4-7). pE194 is a 3500-base pair plasmid isolated in Staphylococcus aureus (8) and introduced by transformation into Bacillus subtilis (9). This plasmid carries the ermC determinant, which confers inducible MLS resistance in both organisms via ribosome methylation (9). Isolation of clones that are resistant to tylosin, a noninducing macrolide, results in two classes of plasmid mutants (9). The first express resistance to the MLS antibiotics without induction. The second type (copy control, cop, mutants) are still inducible but possess elevated plasmid copy numbers (80-100 compared to about 10 for wild-type pE194). pE194 specifies five polypeptides detectable in B. subtilis minicells (10). One of these, a 29,000 molecular weight polypeptide (E3), is required for the expression of Em resistance (10). It is induced in minicells by exposure to Em but not by tylosin. It is produced constitutively by mutant plasmids that express resistance constitutively and inducibly by cop plasmids. Em-sensitive (Ems) deletion mutant plasmids produce altered E3 proteins that are still inducible (unpublished, and this report).As part of a continuing study of the molecular biology of pE194, we have addressed the mechanism of ES induction. This paper demonstrates that this regulation is mediated posttranscriptionally in the sense that induction occurs when transcription is blocked. Alteration of the Em binding site on the ribosome interferes with induction, suggesting that this site may be an essential component in the induction mechanism. MATERIALS AND METHODS The bacterial strains used in this study are BD170 (t...

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.

Reconstruction of ruptured anterior cruciate ligaments (ACLs) is limited by the availability and donor site morbidity of autografts. Hence, a tissue engineered graft could present an alternative in the future. This study was undertaken to determine the performance of lapine (L) ACL-derived fibroblasts on embroidered poly(l-lactide-co-ε-caprolactone) (P(LA-CL)) and polylactic acid (PLA) scaffolds in regard to a tissue engineering approach for ACL reconstruction. Surface modifications of P(LA-CL)/PLA by gas-phase fluorination and cross-linking of a collagen foam using either ethylcarbodiimide (EDC) or hexamethylene diisocyanate (HMDI) were tested regarding their influence on cell adhesion, growth and gene expression. The experiments were performed using embroidered P(LA-CL)/PLA scaffolds that were seeded dynamically or statically with LACL-derived fibroblasts. Scaffold cytocompatibility, cell survival, numbers, metabolic activity, ultrastructure and sulfated glycosaminoglycan (sGAG) synthesis were evaluated. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of collagen type I (COL1A1), decorin (DCN), tenascin C (TNC), Mohawk (MKX) and tenomodulin (TNMD). All tested scaffolds were highly cytocompatible. A significantly higher cellularity and larger scaffold surface areas colonized by cells were detected in HMDI cross-linked and fluorinated scaffolds compared to those cross-linked with EDC or without any functionalization. By contrast, sGAG synthesis was higher in controls. Despite the fact that the significance level was not reached, gene expressions of ligament extracellular matrix components and differentiation markers were generally higher in fluorinated scaffolds with cross-linked collagen foams. LACL-derived fibroblasts maintained their differentiated phenotype on fluorinated scaffolds supplemented with a HMDI cross-linked collagen foam, making them a promising tool for ACL tissue engineering.