Analysis of the regulatory sequences needed for induction of the chloramphenicol acetyltransferase gene cat-86 by chloramphenicol and amicetin

Abstract

Induction of the chloramphenicol acetyltransferase gene cat-86 In Bacillus subtilis results from the activation of translation of cat-86 mRNA. The inducers, chloramphenicol and amicetin, are thought to enable ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome binding site for the cat-86 coding sequence, designated RBS-3. The region of cat-86 mRNA which is 5' to the stem-loop contained two additional ribosome binding sites, RBS-1 and RBS-2, located 84 and 56 nucleotides, respectively, upstream from RBS-3. RBS-1 and RBS-2 were each followed by a potential translation initiation codon and a short open reading frame. Bal 31-generated deletions into the 5' end of the regulatory region that removed RBS-1 but did not enter RBS-2 caused a fourfold decrease in the uninduced and chloramphenicol-induced level of cat-86 expression and a -more than 10-fold reduction in the amicetin-induced level of expression. Deletions that removed both RBS-1 and RBS-2 but did not enter the stem-loop abolished both chloramphenicol-and amicetin-inducible expression. These data indicate that RBS-2 and sequences 3' to RBS-2 are minimally essential to chloramphenicol induction. However, the presence of RBS-1 in the mRNA elevated the maximum level of expression obtained during chloramphenicol induction. These studies also demonstrate that induction of cat-86 by amicetin is highly dependent on RBS-1. To determine whether a correlation existed between RBS-1 and amicetin inducibility, we examined the sequence of the regulatory regions for two natural variants of cat-86, cat-66 and cat-57, which are chloramphenicol inducible but are very poorly induced by amicetin. Both contained nucleotide sequence differences fromn cat-86 in the vicinity of RBS-1 that would prevent translation of the leader peptide associated with RBS-1 in cat-86. In contrast, the regulatory regions for the three genes were virtually identical in the vicinity of RBS-2. These data indicate that efficient induction by amicetin requires sequences that are not essential for induction by chloramphenicol.Chloramphenicol acetyltransferase (CAT) in grampositive bacteria is specified by genes designated cat, whose expression is induced by chloramphenicol (Cm) (23). One such gene, cat-86, was cloned from Bacillus pumilus DNA into Bacillus subtilis by using plasmid pUB110 as a vector (28). Subsequent replacement of the natural cat-86 promoter with a short multicloning site linker generated the promotercloning plasmid pPL703 (17) (Fig. 1). By use of pPL703 it has been shown that the sequences necessary for the Cminducible expression of cat-86 are independent of both the promoter that is selected to activate the gene and the cat-86 coding sequence (16). Accordingly, the inducible regulation of cat-86 results from sequences located within a 144-basepair (bp) regulatory region that intervenes between the coding sequence and the upstream site of promoter insertion.The nucleotide sequence of the 144-bp regulatory region demonstrates that the ribosofme bind...

A gratuitous inducer of cat-86, amicetin, inhibits bacterial peptidyl transferase

“…Amicetin and bamicetin are inducers of cat-86, and plicacetin is not (1). The structures of the three antibiotics offer no obvious basis for the difference in inducing activities (1). We observed that amicetin and bamicetin were highly inhibitory for PT of Bacillus subtilis ribosomes and plicacetin was only marginally active as an inhibitor (Fig.…”

“…Amicetin and bamicetin are inducers of cat-86, and plicacetin is not (1). The structures of the three antibiotics offer no obvious basis for the difference in inducing activities (1).…”

Translational attenuation as the regulator of inducible cat genes

“…The minimum region on the cloned cat-86-containing fragment from B. pumilus that is responsible for chloramphenicol inducibility consists of 84 base pairs (bp) and is located immediately 5' to the cat-86 coding sequence (3,26). The 84-bp regulatory region contains two overlapping but functionally distinct domains (Fig.…”

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

“…In addition, an open reading frame of nine codons (the regulatory leader, crb) that ends with a TGA stop codon is within the same regulatory domain. Structures similar to this one were found at the 5' end of the other chloramphenicol-inducible cat genes, but not upstream of the constitutively expressed Tn9 cat gene (2,4,6).…”

“…Ribosome stalling destabilizes an adjacent hairpin structure in cat-86 mRNA, making an adjacent RBS upstream from the cat-86 gene coding sequence available for interaction with a ribosome, i.e., translation of the cat-86 gene (4). cat-86 is, therefore, regulated through translational attenuation (18).…”