Drug-free induction of a chloramphenicol acetyltransferase gene in Bacillus subtilis by stalling ribosomes in a regulatory leader

Abstract

The plasmid gene cat-86 is induced by chloramphenicol in Bacillus subtilis, resulting in the synthesis of the gene product chloramphenicol acetyltransferase. Induction is due to a posttranscriptional regulatory mechanism in which the inducer, chloramphenicol, activates translation of cat-86 mRNA. We have suggested that chloramphenicol allows ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the coding sequence. In the present report we show that cat-86 expression can be activated by stalling ribosomes in the act of translating a regulatory leader peptide. Stalling was brought about by starving host cells for specific leader amino acids. Ribosomal stalling, which led to cat-86 expression, occurred upon starvation for the amino acid specified by the leader codon located immediately 5' to the RNA stem-loop structure and was independent of whether that codon specified lysine or tyrosine. These observations support a model for chloramphenicol induction of cat-86 in which the antibiotic stalls ribosome transit in the regulatory leader. Stalling of ribosomes in the leader can therefore lead to destabilization of the RNA stem-loop structure.

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

“…pPL703-Thr is identical to pPL703, except that the first lysine codon (AAA) in the cat-86 leader was changed, by site-directed mutagenesis, to a threonine codon (ACA) (Fig. 1) (5). Throughout this report we do not refer to the plasmids, but rather we refer to the version of cat-86 under study.…”

“…Successful induction of cat-86 by amino acid starvation (i.e., lysine deprivation) requires that lysine residues become available during the starvation period to allow translation of the cat-86 coding sequence; there are 16 lysine codons in cat-86 (8). We previously proposed that the free lysines result from intracellular protein turnover that is triggered by the amino acid starvation regimen (5). In E. coli, starvation for a single amino acid has been shown to activate protein turnover, and this turnover did not occur in a relA mutant (20).…”

“…Induction by amino acid starvation was accomplished by growing cat-86 (or cat-86 Thr)-containing cells to the mid-log phase in a minimal medium supplemented with all amino acids (each at 50 ,ug/ml) (5). Cells were washed with cold, unsupplemented minimal medium and suspended in the same medium containing all amino acids except lysine.…”

“…cat-86 mRNA (5). It is evident, therefore, that the focus of the cat-86 induction mechanism is the ribosome itself.…”

Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli

“…The secondary structure in the regulatory domain of the wild-type cat-86 gene ensures the basal-level expression as well as induction. This structure allows the ribosome to stall on leader RNA and induce cat-86 gene expression if leader codon 6 (the codon which is immediately 5' to the RNA stem-loop) occupies the aminoacyl site of the ribosome even in the absence of chloramphenicol (8). The stalled ribosome destabilizes the RNA stem-loop structure, thereby liberating the cat-86 gene ribosomal binding site (RBS-3) (12).…”