Induction of ermC requires translation of the leader peptide.

ermC confers resistance to macrolide-lincosamide streptogramin B antibiotics by specifying a ribosomal RNA methylase, which results in decreased ribosomal affinity for these antibiotics. ermC expression is induced by exposure to erythromycin. We have previously proposed a translational regulation model in which erythromycin causes stalling of a ribosome, which is translating a leader peptide. Stalling causes a conformation shift in the ermC mRNA which in turn unmasks the methylase ribosomal binding site. A prediction of this translational attenuation model for ermC induction was tested by replacing the second codon of the putative ermC leader peptide coding region by TAA. As expected, the introduction of this mutation resulted in an uninducible phenotype which was suppressible by two ochre suppressor mutations in Bacillus subtilis. It is concluded that translation through the leader peptide coding region, in frame with the predicted leader peptide, is required for ermC induction.     

Identification of cis-acting sequences required for translational autoregulation of the ermC methylase.

ermC methylase gene expression has been shown to be limited by translational autorepression, presumably due to methylase binding to ermC mRNA. It was found that this repression occurs in trans, yielding a 50% reduction in translation of an ermC-lacZ fusion mRNA. We investigated the ermC mRNA sequences required for translational repression in vivo. A series of deletions identified sequences in the 5' regulatory region that were required for translational repression. These included sequences of the 5' stem-loop structure that were not required for induction, as well as some that were required. The implications of these results for regulation are discussed.     

Effect of ermC leader region mutations on induced mRNA stability.

Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression.     

Erythromycin-induced ribosome stalling and RNase J1-mediated mRNA processing in Bacillus subtilis

Addition of erythromycin (Em) to a Bacillus subtilis strain carrying the ermC gene results in ribosome stalling in the ermC leader peptide coding sequence. Using Δ ermC , a deletion derivative of ermC that specifies the 254 nucleotide Δ ermC mRNA, we showed previously that ribosome stalling is concomitant with processing of Δ ermC mRNA, generating a 209 nucleotide RNA whose 5' end maps to codon 5 of the Δ ermC coding sequence. Here we probed for peptidyl-tRNA to show that ribosome stalling occurs after incorporation of the amino acid specified by codon 9. Thus, cleavage upstream of codon 5 is not an example of 'A-site cleavage' that has been reported for Escherichia coli . Analysis of Δ ermC mRNA processing in endoribonuclease mutant strains showed that this processing is RNase J1-dependent. Δ ermC mRNA processing was inhibited by the presence of stable secondary structure at the 5' end, demonstrating 5'-end dependence, and was shown to be a result of RNase J1 endonuclease activity, rather than 5'-to-3' exonuclease activity. Examination of processing in derivatives of Δ ermC that had codons inserted upstream of the ribosome stalling site revealed that Em-induced ribosome stalling can occur considerably further from the start codon than would be expected based on previous studies.     

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

The ermC gene of plasmid pE194 specifies resistance to the macrolidelincosamide-streptogramin B antibiotics. This resistance, as well as synthesis of the 29,000 dalton protein product of ermC, has been shown to be induced by erythromycin. Weisblum and his colleagues have established that macrolide resistance is associated with a specific dimethylation of adenine in 23 S rRNA. We show that pE194 specifies an RNA methylase that can utilize either 50 S ribosomes or 23 S rRNA as substrates. Synthesis of this methylase is induced by low concentrations of erythromycin, and the enzyme is produced in elevated amounts by strains carrying a high copy number mutant of pE194. The methylase comigrates with the 29K ermC product on polyacrylamide gels. The purification and some properties of this methylase are described.     

A Staphylococcus aureus plasmid that specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel deletion in the ermC attenuator

A 2.5 kb plasmid, pA22, isolated from a naturally occurring S. aureus strain confers constitutive MLS-resistance. By restriction enzyme analysis, pA22 is indistinguishable from the S. aureus inducible MLS-resistance conferring plasmid, pT48, apart froma small deletion. DNA sequencing showed that the deletion, is in the leader/attenuator region of the ermC (MLS-resistance) gene and removes some of the complementary repeat regions required by the translational attenuation model in pT48 for inducible ermC expression. The deletion in plasmid pA22 is different from that found in similar 2.5kb constitutive MLS-resistance plasmids in other Gram-positive bacteria. It is suggested that plasmids conferring the constitutive phenotype have evolved from an inducible ancestor on several independent occasions.     

A rapid method to quantitate non-labeled RNA species in bacterial cells

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.     

Regulation of a macrolide resistance-beta-galactosidase (ermC-lacZ) gene fusion in Escherichia coli.

A fusion constructed between the putative attenuator plus the first 219 nucleotides of the ermC (erythromycin resistance) structural gene and a 5' terminally deleted lacZ gene produced a moderate, basal level of beta-galactosidase which was increased by erythromycin addition. Another construction containing an intact ermC gene in addition to the fusion produced lower levels of beta-galactosidase, suggesting that the ermC gene product exerts negative feedback control on expression.     

Mechanism of erythromycin-induced ermC mRNA stability in Bacillus subtilis.

In Bacillus subtilis, the ermC gene encodes an mRNA that is unusually stable (40-min half-life) in the presence of erythromycin, an inducer of ermC gene expression. A requirement for this induced mRNA stability is a ribosome stalled in the ermC leader region. This property of ermC mRNA was used to study the decay of mRNA in B. subtilis. Using constructs in which the ribosome stall site was internal rather than at the 5' end of the message, we show that ribosome stalling provides stability to sequences downstream but not upstream of the ribosome stall site. Our results indicate that ermC mRNA is degraded by a ribonucleolytic activity that begins at the 5' end and degrades the message in a 5'-to-3' direction.     

Mono- and dimethylating activities and kinetic studies of the ermC 23 S rRNA methyltransferase

The ermC 23 S rRNA methyltransferase converts a single adenine residue to N6,N6-dimethyladenine, both in vivo and in vitro. The ermC methyltransferase was demonstrated to produce both N6-mono and N6,N6-dimethylated adenine residues in Bacillus subtilis 23 S rRNA during the course of the reaction in vitro. An almost total conversion of monomethylated intermediates into dimethylated products was observed upon completion of the reaction. Data presented here demonstrate that the addition of the two methyl groups to each 23 S rRNA molecule takes place through a monomethylated intermediate and suggest that the enzyme dissociates from its RNA substrate between the two consecutive methylation reactions. The enzyme is able to utilize monomethylated RNA as substrate for the addition of a second methyl group with an efficiency approximately comparable to that obtained when unmethylated RNA was the initial substrate. Initial-rate data and inhibition studies suggest that the ermC methylase reaction involves a sequential mechanism occurring by two consecutive Random Bi Bi reactions.     

Ribosomal RNA methylation in Staphylococcus aureus and Escherichia coli: effect of the "MLS" (erythromycin resistance) methylase

Classical acquired resistance to erythromycin in Staphylococcus aureus ("MLS," or macrolide-lincosamide-streptogramin, resistance) was shown by Weisblum and colleagues to be a direct consequence of the conversion of one or more adenosine residues of 23S rRNA, within the subsequence(s) GA3G, to N6-dimethyladenosine (m62A). The methylation reaction is effected by a class of methylase, whose genes are typically plasmid- or transposon-associated, and whose synthesis is inducible by erythromycin. Using a recently obtained clinical MLS isolate of S. aureus, we have further defined the methylation locus as YGG X m62A X AAGAC; and have shown that this subsequence occurs once in the 23S RNA and that it is essentially completely methylated in all copies of 23S RNA that accumulate in induced cultures. Similar findings were obtained with laboratory S. aureus strains containing two well-characterized evolutionary variants (ermB, ermC) of MLS methylase genes. Analyses of a strain of E. coli containing the ermC gene indicated that the specificity of the methylase gene was unchanged, but that its expression was muted. Even after prolonged periods of induction, the strain manifested only partial resistance to erythromycin, and only about one-third of the copies of the MLS subsequence were methylated in such "induced" cultures. Since the E. coli 23S RNA sequence is known in its entirety, localization of the MLS subsequence is in this case unambiguous; as inferred by homology arguments applied earlier to the S. aureus data, the subsequence is in a highly conserved region of 23S RNA considered to contribute to the peptidyl transferase center of the ribosome.     

Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation.

The sequence of the Tn1696 determinant for inducible nonenzymatic chloramphenicol resistance has been determined. The cml region, the fourth insert of the Tn1696 integron, is 1547 bases and includes a 59-base element at the 3' end, as is typical of integron inserts. One gene, designated cmlA and predicting a polypeptide of 44.2 kDa, is encoded in the insert. However, the cmlA region shows one feature not previously found in an integron insert. A promoter is located within the cmlA insert, and translational attenuation signals related to those of the inducible cat and ermC genes found in gram-positive organisms are also present. The regulatory region includes a leader peptide of nine amino acids, a ribosome stall sequence related to those preceding cat genes, and two alternative pairs of stem-loop structures which either sequester or disclose the ribosome binding site and start codon preceding the cmlA gene.