The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation

Abstract

Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.

FIG. 1.

Induction of catB and organization of the regulatory region. (A) Induction assays of various catB-containing strains of A. tumefaciens. BG53 is a wild-type strain of A. tumefaciens and contains a resident catB gene in the 2.1-Mb linear chromosome. All other induction assays were performed with strain LBA4404 with or without plasmids p1302, p745, and p1302 with the mutations ATG→ATC (a mutation of the leader initiation codon), TAG-5, TAA-6, and TAA-8 (stop codon mutations at leader codons 5, 6, and 8, respectively). Cells were grown to mid-log phase and exposed to 0.2 μg of fluorothiamphenicol per ml for 2 h at 30°C. Cells were lysed, and CAT assays were performed at 25°C as previously described (1, 18). Protein was measured by the Bradford method (5), and specific activities are expressed as micromoles of chloramphenicol acetylated per minute per milligram of protein. Mutations were made in E. coli by using the Quick Change Site Directed Mutagenesis Kit (Stratagene). Light bars represent CAT levels of cells grown without inducer, and dark bars show CAT levels of cells grown with fluorothiamphenicol. In the experiment depicted, CAT assays were performed in duplicate and the duplicates differed by less than 4%. The entire experiment was repeated more than six times with results comparable to those shown. (B) Diagram of the cloned catB gene in pBin19 and the sequence of the region involved in regulation. The diagram depicts the 1302 clone at the BamHI site of pBin19 with the location of the vector lac promoter and the site of transcription initiation indicated. The sequence shows the catB cloned fragment that begins at the site designated 1302; the endpoint of the 745 deletion derivative is designated. The sequence between the BamHI site and the site designated 1302 is from the linker in pNoTA/T7, and the sequence upstream of the BamHI site corresponds to the pBin19 vector plasmid. The leader ORF coding region consists of 10 sense codons preceded by an RBS, GAG, that is likely very weak because the spacing between this site and the initiator ATG is only two nucleotides. The first five codons of the catB structural gene are designated and are preceded by an RBS, GAG, spaced nine nucleotides upstream from the initiator ATG. The site of transcription initiation was determined by primer extensions with a 30-mer primer complementary to the leader ORF region of the mRNA.

FIG. 2.

Folding of the mRNA corresponding to the regulatory region of catB. Computer folding utilized algorithms of Zuker et al. (26). The leader sequence is overlined, and the catB coding region and the 5′ end point of the 745 deletion are indicated. The calculated ΔG of the structure is −64 kcal/mol.

FIG. 3.

Dot blot analysis of catB mRNA from BG53 or from LBA4404 carrying either p1302 or p745. RNA (10 μg) from uninduced cells (designated −) or cells grown with 0.2 μg of fluorothiamphenicol per ml for 2 h (designated +) was bound to GeneScreen Nylon Membrane (NEN Life Science Products). The filters were hybridized with end-labeled primers in 50% formamide-5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% sodium dodecyl sulfate-5× Denhardt's solution-100 μg of calf thymus DNA per ml at 42°C and washed at the same temperature. Probe 1 was a 30-mer complementary to the leader sequence, and probe 2 was a 24-mer probe complementary to codons 6 to 13 of the catB structural gene. Analysis was performed on a Storm PhosphorImager (Molecular Dynamics).