Posttranscriptional regulation of an erythromycin resistance protein specified by plasmic pE194.

Abstract

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...

A Macrolide-Lincosamide-Streptogramin B Resistance Determinant from Bacillus Anthracis 590: Cloning and Expression of ermJ

“…hour, when it remained constant. Others have invoked feedback inhibition or an autoregulation mechanism to account for the constant level of ermC product in bacterial cells (Shivakumar et al, 1980;Gryczan et al, 1978). The optimal culture time for methylase activity, following Em induction of ermJ was concluded to be The molecular mass of the purified rRNA methyltransferase was determined by gel permeation chromatography using Sephadex G-100.…”

“…(Weisblum,19SS), translational feedback inhibition (Shivakumar et al, 1980) or autoregulation (Breidt & Dubnau, 1990). Translational attenuation is the most important mechanism and has been studied intensively.…”

Characterization of a plasmid-specified ribosome methylase associated with macrolide resistance

“…The ermC gene, together with sequences responsible for inducibility has been localized on the physical map of pE194, the direction of transcription has been determined, and the ermC gene and flanking DNA regions have been sequenced (12,13,14). Recently we have reported that the induction of the 29K ermC product is regulated posttranscriptionally and that interaction of Em with a binding site on a sensitive (undermethylated) ribosome is required for induction (15). Based on these findings, a model for ermC regulation has been proposed (13,14,16).…”

pRj5: a naturally occurring Staphylococcus aureus plasmid expressing constitutive macrolide--lincosamide--stireptogramin B resistance contains a tandem duplication in the leader region of the ermC gene

“…Three plasmid-encoded elements are used in RCR: a plus origin, a replication protein (Rep) and a minus origin Plasmid pE194 encodes the ermC gene, which confers inducible MLS resistance, and it is to date the only member of its family. pE194 is 3.7 kb in size, and has been extensively studied in both S. aureus (Weisblum et al, 1979;Iordanescu & Surdeanu, 1980;Byeon & Weisblum, 1990;Sozhamannan et al, 1990) and Bacillus subtilis (Gryczan et al, 1980;Shivakumar et al, 1980;Horinouchi & Weisblum, 1982).…”