Translational autoregulation of ermC 23S rRNA methyltransferase expression in Bacillus subtilis

Abstract

ermC specifies an rRNA methyltransferase that confers resistance to erythromycin. The expression of this determinant is induced by the addition of erythromycin. The induction mechanism has been shown to operate posttranscriptionally, and its mechanism has been elucidated. We now show that synthesis of the ermC gene product in Bacillus subtilis is also autoregulated by a mechanism operating on the level of translation. erythromycin-sensitive ermC mutations analyzed so far cause the overproduction of structurally altered methylase in vivo. However, as we have pointed out (11,29), this hyperinducibility is also consistent with another interpretation: feedback may be exerted by a direct interaction of the methylase protein with its own mRNA or with DNA. This possibility was also raised by Horinouchi and Weisblum (16), who pointed out that the ermC mRNA contains an AAAG sequence. The latter sequence is also found at the target for methylation of 23S rRNA by macrolideslincosamides-streptogramin B methylase. Autoregulation through direct protein-nucleic acid interaction has been described for a number of systems (3,8,19,33). In some cases, such as ribosomal proteins acting as translational repressors, the usual function of these proteins involves nucleic acid binding. In this sense the 29-kilodalton (kDa) product of ermC gene presents some particularly interesting similarities to the ribosomal proteins. The methylase is a basic protein with an intracellular function involving a direct interaction with rRNA (27,30) and may be associated with ribosomes intracellularly (27; C. Denoya and D. Dubnau, unpublished data). With these considerations in mind, we decided to explore further the regulatory mechanism involved in ermC gene expression. This paper presents in vivo data that support the existence of a direct autoregulatory mechanism operating at the posttranscriptional level and independently of the ermC methylase-mediated methylation of ribosomes. The structural similarities between the site of action of the methylase on the 23S rRNA and the ermC mRNA region around the methylase translation initiation site suggest that this autoregulation operates through a direct methylase-mRNA interaction. MATERIALS AND METHODSStrains and plasmids. All the B. subtilis strains used are derivatives of strain 168. They are described in Table 1. Escherichia coli JM109 was used as host for cloning in bacteriophage M13 (22). Plasmid pE194, which carries ermC, was originally isolated from Staphylococcus aureus by Iordanescu (18).

Modes and Modulations of Antibiotic Resistance Gene Expression

“…Finally, it has been shown in Bacillus subtilis that the Erm(C) methylase binds to its own mRNA at a site with structural similarity to the site of methylation in 23S rRNA. As a consequence, the methylase might block its own production when synthesized in excess (62).…”

Sequence analysis of conserved regA and variable orf43.1 genes in T4-like bacteriophages

“…Several translational repressor proteins and their cognate mRNA-binding sites have been characterized from Escherichia coli and its bacteriophages (2,7), although few such proteins are known from other organisms (4,22). All the repressor proteins appear to function through occlusion of a portion of the mRNA sequence recognized by the ribosome during translation initiation (2,7,30).…”

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

“…Uninduced control cultures were always kept at 30ЊC. Proteins were analyzed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (15). Cell extracts were prepared as described in the next section.…”