A conserved anti-repressor controls horizontal gene transfer by proteolysis

Abstract

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-PhrI sensory system activate ICEBs1 gene expression, excision and transfer by inactivating the ICEBs1 repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBs1-encoded anti-repressor ImmA (YdcM). ImmA was needed for the degradation of ImmR in B. subtilis. Coexpression of ImmA and ImmR in Escherichia coli or co-incubation of purified ImmA and ImmR resulted in site-specific cleavage of ImmR. Homologues of immR and immA are found in many mobile genetic elements. We found that the ImmA homologue encoded by B. subtilis phage phi105 is required for inactivation of the phi105 repressor (an ImmR homologue). ImmA-dependent proteolysis of ImmR repressors may be a conserved mechanism for regulating horizontal gene transfer.

Figure 1. Map of ICEBs1

Each of the 24 genes encoded by ICEBs1 is indicated by the thick black arrows oriented in the direction of transcription. Gene names are indicated under each arrow. Single letter designations are given for the genes of unknown function (ydcO-ydcT; yddA-yddK; yddM). Thin arrows indicate the positions of the characterized promoters. White boxes denote the ends of the element.

Figure 2. ImmA is required for de-repression of Pxis-lacZ

Expression of Pxis-lacZ was monitored in cells grown in defined minimal medium and treated at time 0 with either MMC to induce the SOS response (A & C), or with IPTG to induce expression of Pspank(hy)-rapI (B & D). β-galactosidase specific activity is plotted as a function of time after the indicated treatment. A. Effects of MMC on expression of Pxis-lacZ. ICEBs1⁺ cells (JMA201, □, ICE⁺); ICEBs1⁰ cells expressing only immR from its own promoter (JMA421, ●, ICE⁰/immR⁺); ICEBs1⁰ cells co-expressing immR and immA from the immR promoter (JMA436, ○, ICE⁰/immR⁺A⁺). Data for JMA201 were published previously (Auchtung et al., 2007) and are re-plotted for comparison. B. Effects of overproducing RapI on expression of Pxis-lacZ. ICEBs1⁺ (KLG126, □, ICE⁺); ICEBs1⁰ cells expressing immR (JMA444, ◆, ICE⁰/immR⁺); ICEBs1⁰ cells expressing both immR and immA (JMA446, ◊, ICE⁰/immR⁺A⁺). C-D. Effects of ImmA on expression of Pxis-lacZ induced with MMC (C) or RapI (D). All cells contained the Δint∷cat mutation and the indicated immA allele. C. immA⁺ (CAL16, □); ΔimmA (JMA726, σ); ΔimmA Pspank-immA (JMA840, ). IPTG was present throughout growth of JMA840. D. immA⁺ (JMA836, □); ΔimmA (JMA838, τ); ΔimmA Pspank-immA (JMA842, ∇).

Figure 3. The bacteriophage ø105 homolog of ImmA, (ø105)ImmA, is needed for de-repression of phage gene expression in response to DNA damage

A. Diagram of the region of ø105 that contains immA(ø105) {aka orf2}, cø105, and orf4. Genes are indicated by thick black arrows, with the name of each gene indicated above. Thin arrows indicate the positions of the orf4 and cø105 promoters (Van Kaer et al., 1987). Black rectangles indicate the positions of the six ø105 repressor bindings sites, three upstream from and adjacent to the leftward promoter Pcø105, two upstream from and adjacent to the rightward promoter Porf4, and one internal to orf4 (Van Kaer et al., 1989). The white box underneath the map indicates the region of the orf4 promoter cloned upstream of lacZ that was used to assay gene expression in B. B. Porf4-lacZ expression was monitored in a ø105 lysogen (BOSE447, ◆, ø105⁺), and in cells lacking ø105 that were otherwise wild-type (BOSE446, □, ø105⁰), expressed the ø105 repressor from its native promoter (BOSE451, ○, ø105⁰/R⁺), or expressed the ø105 repressor from its native promoter and also expressed ImmA(ø105) from the IPTG-inducible promoter Pspank (BOSE567, , ø105⁰/R⁺A⁺). Cells were grown in minimal medium containing IPTG and were treated with mitomycin C at OD600 ∼ 0.5. Samples were collected at the times indicated and β-galactosidase specific activity was determined.

Figure 4. ImmA promotes degradation of ImmR in vivo

A. ImmR stability was monitored through pulse-chase experiments. ICEBs1⁰ cells that co-expressed immR and immA from their native promoter and were otherwise wild-type (JMA436, Lanes 1-4) or expressed rapI from a xylose-inducible promoter (CAL746, Lanes 5-8) were grown in defined minimal medium lacking xylose. At OD600 ∼ 0.5, ³⁵S-met was added to the cultures. One minute later >1000-fold excess unlabeled methionine was added along with xylose to induce expression of rapI. Samples were collected just prior to addition of unlabeled methionine and xylose, and 5, 10, and 20 minutes after addition. Samples were immunoprecipitated with anti-ImmR antibodies and analyzed by polyacrylamide gel electrophoresis followed by phosphorimaging. B-E. ImmR levels were monitored with Western blots using anti-ImmR antibodies. Cultures were grown in minimal medium to an OD600 ∼ 0.5 and split in two. Cells were untreated (-) (B-E), or treated with MMC to induce the SOS response (+) (B, D), or treated with IPTG to overproduce RapI (+) (C, E). Samples were collected 60 minutes after the indicated treatment. B. ICEBs1⁺ (JMA201); ICEBs1⁰ PimmR-immR (JMA421); ICEBs1⁰ PimmR-immR immA (JMA436) C. All three strains contained Pspank(hy)-rapI. ICEBs1⁺ (KLG126); ICEBs1⁰ PimmR-immR (JMA444); ICEBs1⁰ PimmR-immR immA (JMA446) D. ICEBs1⁺ ΔrecA (IRN444) E. ICEBs1⁺ Pspank(hy)-rapI ΔrecA (CAL92).

Figure 5. ImmA-mediated cleavage of ImmR in E. coli and in vitro

A. ICEBs1 proteins were overexpressed in E. coli and lysates were analyzed by Western blot using anti-ImmR antibodies. Lysates were collected two hours after induction from cells that overexpressed His6-ImmR with ImmA (BOSE799, lane 1), with ImmA and RapI (BOSE817, lane 2), or with RapI (BOSE819, lane 3). B. His6-ImmR (17μM) was incubated in vitro overnight at 37°C: alone (lane 1); with 22μM ImmA (lane 2); with 18μM ImmA(H75A) (lane 3); or with 11μM ImmA (lane 4). Reactions were run on SDS-PAGE and stained with Coomassie brilliant blue. Bands representing ImmA, His6-ImmR, and the ImmR fragment are indicated. C. Products of ImmR cleavage assays in vitro and in E. coli were analyzed by MALDI-TOF. The form of the ImmA, ImmR, and RapI present in each reaction is indicated. Experimentally determined masses are shown alongside expected masses for the cleavage occurring between F95 and M96 of ImmR. The smaller ImmR fragment was detected from the in vitro reactions by not from E. coli. D. The cleavage site in ImmR. The helix-turn-helix domain, contained in the first 61 N-terminal residues, is the presumed DNA binding domain that is similar to that of other phage-like repressors. The amino acid sequence from residue 62 to the C-terminus is shown with the cleavage site between F95 and M96 marked with a slash.