Autoregulation of AbsB (RNase III) expression in Streptomyces coelicolor by endoribonucleolytic cleavage of absB operon transcripts

Abstract

The Streptomyces coelicolor absB gene encodes an RNase III family endoribonuclease and is normally essential for antibiotic biosynthesis. Here we report that AbsB controls its own expression by sequentially and site specifically cleaving stem-loop segments of its polycistronic transcript. Our results demonstrate a ribonucleolytic regulatory role for AbsB in vivo.

FIG. 1.

absB mRNA abundance in the J1501 (wild-type) and C120 (absB mutant) strains of S. coelicolor. (A) Mycelia were collected after 48 h of incubation at 30°C of spore suspensions spread on cellophane membranes placed on R5 plates (11), and 20 μg of total RNA isolated with an RNeasy mini kit (Qiagen) was electrophoresed on denatured agarose gels. Following transfer to Zeta-Probe (Bio-Rad) membrane, RNA was analyzed by Northern blotting (NorthernMax kit; Ambion) with randomly radiolabeled absB ORF DNA as the probe. Phosphorimaging (Typhoon; GE) was used for RNA detection and quantification. A band corresponding in length to the full-length SCA7A1.14-rmpF-absB operon transcript is indicated by an arrow. hrdB transcripts which encode a constitutively expressed sigma factor (5) were used as internal loading controls. (B) J1501 and C120 spores germinated for 6 h in 2× YT medium (11) were inoculated into R5 liquid medium at an optical density at 450 nm of 0.05. After growth at 30°C for 16 h to an optical density at 450 nm of 1.6, 500 μg/ml rifampin (rifa or Rif) was added and Northern blotting analysis of total RNA which was isolated from aliquots of the cultures at the times indicated was performed as described above. 16S rRNA transcripts served as an internal loading control. (C) Strains J1501 and C120 expressing AbsB adventitiously from the inducible tipA promoter were constructed by introducing apramycin resistance plasmid pIJ6902 containing the intact absB ORF (11). RNA was isolated from strains harboring the empty pIJ6902 vector (J1501, lanes 1 and 2; C120, lanes 3 and 4) or pIJ6902-absB (J1501, lanes 5 and 6; C120, lanes 7 and 8) that were grown on R5-aparamycin plates containing (+) or lacking (−) thiostrepton for 48 h. hrdB transcripts which encode a constitutively expressed sigma factor (5) were the internal control. The values beside the gels are molecular lengths of RNA markers in thousands.

FIG. 2.

AbsB cleavage of absB-containing transcripts. (A) Total RNA was isolated from E. coli strains producing either wild-type or mutant absB transcripts after 4 h of treatment with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and analyzed by Northern blotting with random radiolabeled absB ORF DNA as the probe. (B) His-tagged wild-type or mutant AbsB protein was purified as previously described (2). DNA corresponding to the full-length SC7A1.14-rmpF-absB operon was amplified from S. coelicolor genomic DNA with 5′ primers that included the sequence of the bacteriophage T7 promoter. The primers are RNA A-forward (5′-TAATACGACTCACTATAGGGCCGGATTGGGCCGAGAGCGAATGG-3′), RNA A-reverse (5′-GCGGTCTGGGCCGGTGGATGAGCG-3′), RNA B-forward (5′-TAATACGACTCACTATAGGGGGCTCTTCGGTCGCGTGTATGCC-3′), and RNA B-reverse (same as RNA A-reverse). RNA substrates were synthesized with a MEGAscript T7 kit (Ambion) and purified. A 5-μg RNA sample was incubated with 100 ng purified protein in a 20-μl reaction mixture (30 mM Tris-Cl [pH 8.0], 160 mM NaCl, 0.01 μg/μl tRNA, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10 mM MgCl₂) as previously described (2). Purified cleavage products were electrophoresed in a denatured 1.5% agarose gel as described in the legend to Fig. 1A. Visualization of the reaction products was carried out by UV light. The arrows indicate the positions of uncleaved RNA and the cleavage products. (C) RNA A was treated with AbsB protein for the times indicated. (D) Approximate positions of cleavage sites in the absB operon transcript determined by this analysis. tsp, transcription start point; ter, terminus. The values beside the gels are molecular lengths of RNA markers in thousands.

FIG. 3.

Mapping of SC7A1.14-rmpF-absB operon cleavage sites by primer extension. Substrates were either 20 ng RNA synthesized as described above and treated with AbsB protein in vitro or 100 μg total S. coelicolor RNA. Primer I (5′-CGCCTCGTCGGGGTCCGCGT-3′), a synthetic deoxyribonucleotide corresponding to the sequence ∼150 nt 3′ to site I (as shown in Fig. 2D) and primer II (5′-GGTCGTCCGACAGGCGCAC-3′), located ∼150 nt 3′ to site II, were end labeled with [γ-³²P]ATP by using T4 polynucleotide kinase. A Primer Extension System (Promega) and a 7-deaza-dGTP sequencing kit (USB) were used for the extension reactions (left) and the generation of sequencing ladders (right). (A) Uncleaved RNA A (lane 1), RNA A treated with AbsB (lane 2), and RNA A treated with mutant AbsB protein (lane 3) were reverse transcribed with primer I. The products were separated on 10% denatured polyacrylamide gel and exposed to a PhosphorImager. (B) Primer extension analysis of cleavage site II. RNA A treated as described for panel A was reverse transcribed with primer II. (C) A 100-μg sample of RNA from the wild-type (lanes 1 and 3) or absB mutant (lanes 2 and 4) strain was reverse transcribed with the primers for sites I and II. No RNA was added to the gel lanes located between lanes 2 and 3. (D) Secondary structures of the RNA sequences of these two cleavage sites identified. The positions where AbsB cleaves are indicated. The molecular marker sizes beside the gels are in nucleotide bases.