GTA ATT ACG GTT ATG TCC) and rhsB-Spe (59 -AAA CTA GTA ATT TTT CTT GAT TTA TAT TTT ACA AGC); rhs-orph1-Nco (59 - TCC CAT GGG GTT GGT GGG ATG TCC GC) and rhs-orph1-Spe (59 - AAA ACT AGT GCC ATC AAG GTA TAC AGA AGG); and rhs-orph2-Nco (59 - ACC CCA TGG GGC TGG CAG GGG GGC TG) and rhs-orph2- Spe (59 - TTT ACT AGT AAC AGC TTT GTA ATA ATC GTG). All PCR products were digested with NcoI and SpeI and ligated to pET21S. The D. dadantii rhsI3937 genes were amplified from pET21S constructs using primer pET-Pst (59 - CGG CTG CAG CAG CCA ACT CAG TGG) in conjunction with primers: rhsIB-Nco (59 - TAA CCA TGG ATA TTG AAA ATG C), rhsIo1-Nco (59 - TCA CCA TGG ATT CTA GTG ATA AG), and rhsIo2-Nco (59 - AAT CCA TGG ATG CTG AAC AAT TTG). The resulting PCR products were digested with NcoI and PstI and ligated to plasmid pCH450 [35]. A cassette encoding the ssrA(DAS) peptide tag (AANDENY- SENYADAS) was generated from oligonucleotides, DAS-top (59 - CTA GTG CTG CGA ACG ATG AAA ATT ACT CCG AAA ATT ATG CGG ATG CGT CTT AAT G) and DAS-bot (59 - GAT CCA TTA AGA CGC ATC CGC ATA ATT TTC GGA GTA ATT TTC ATC GTT CGC AGC A), and ligated to SpeI/ BamHI digested plasmid pKAN [36]. As part of an unrelated study, a fragment of the E. coli hisS gene was cloned into pKAN(DAS) using SacI and SpeI sites. The resulting hisS-(DAS) SacI/BamHI fragment was subcloned into plasmid pTrc99A (Amersham Pharmacia) to generate plasmid pTrc(DAS). All cdiA- CT/cdiI and rhs-CT/rhsI modules were subcloned into pTrc(DAS) using NcoI and SpeI restriction sites. The sspB and sspB(D47) genes were amplified using SspB-Nde (59 - GAG TTA ATC CAT ATG GAT TTG TCA CAG C) in combination with SspBD47-Bam (59 - TGC GGA TCC TTA ATT CAT GAT GCT GGT ATG TTC ATC GTA GGC) and SspB-Bam (59 - ATA TGA TTG CCA GGA TCC CGC TAT TTT ATT AAG TC), respectively. Both PCR products were digested with NdeI and BamHI and ligated to plasmid pCH410, allowing L-arabinose control of SspB and SspB(D47) expression [37]. The orphan cdiA-CTo1EC93/cdiIo1EC93 module from E. coli EC93 was fused to the full-length EC93 cdiAEC93 gene in multiple steps. A fragment of cdiAEC93 (upstream of the VENN encoding sequence) was amplified using primers, #1527 (59 - GAA CAT CCT GGC ATG AGC G) and #1758 (59 - CAA GCT CAG CGA GTT ATT CTC AAC CGA GTT CCT ACC TGC CTG). The EC93 orphan module was amplified using primers, #1759 (59 - CAG GCA GGT AGG AAC TCG GTT GAG AAT AAC TCG CTG AGC TTG) and #1663 (59 - GGT CTG GTG TCT AAC CTT TGG G). The two products were combined by overlapping-end PCR using primers #1527 and #1663, and the resulting product digested with SphI and AvrII and ligated to plasmid pDAL660D1-39 [4]. In Vitro Characterization of Orphan Modules All CdiA-CT/CdiI-His6 complexes were overproduced and purified by Ni2+-affinity chromatography as described [5]. Complexes were eluted from Ni2+-nitrilotriacetic acid resin with native elution buffer [20 mM sodium phosphate (pH 7.0) –10 mM b-mercaptoethanol – 250 mM imidazole], followed by dialysis in storage buffer [20 mM sodium phosphate (pH 7.0) – 150 mM NaCl – 10 mM b-mercaptoethanol]. CdiA-CT and CdiI-His6 proteins were separated from one another by Ni2+-affinity chromatography with denaturing buffer [20 mM sodium phos- phate (pH 7.0) – 10 mM b-mercaptoethanol – 6 M guanidine- HCl]. Denatured proteins were refolded by dialysis into storage buffer. All purified proteins were quantified by absorbance at Toxin Effectors of CDI and Rhs Systems 260 nm using the following molar extinction coefficients: CdiA- CTUPEC536, 12,950 M21 cm21; CdiIUPEC536, 8,480 M21 cm21; CdiA-CTo1EC93, 11,460 M21 cm21; and CdiIo1EC93, 11,460 M21 cm21. The specificity of CdiA-CT/CdiI-His6 bind- ing interactions was determined by Ni2+-affinity co-purification as described [5]. The tRNase activity of isolated and refolded CdiA- CTUPEC536 and CdiA-CTo1EC93 proteins was determined as described [5,37]. In Vivo Activity of Orphan Modules E. coli strain CH4180 (690 DsspB) was co-transformed with pTrc(DAS) orphan module constructs and either the SspB or SspB(D47) arabinose-inducible expression plasmids. The resulting strains were grown at 37uC with aeration in LB media supplemented with 150 mg/mL ampicillin and 10 mg/mL tetra- cycline (to maintain plasmids) to mid-log phase and re-diluted into fresh media to an optical density at 600 nm (OD600) of 0.05. After 40 min, SspB or SspB(D47) expression was induced by the addition of 0.4% L-arabinose. Cell growth was tracked by measuring the OD600 every 30 min after induction. Cells expressing CdiA-CTo1EC93/CdiIo1EC93-DAS were harvested into an equal volume of ice-cold methanol and RNA extracted as described [38]. Northern blot hybridizations were conducted as described [37], using radiolabeled oligonucleotides tRNAHis probe (59 - CAC GAC AAC TGG AAT CAC) and tRNA1BAla probe (59 - TCC TGC GTG AGC AG) as probes. E. coli sspB+ cells expressing RhsIB3937, RhsIo13937 and RhsIo23937 were transformed with plasmids encoding rhs-CT/ rhsI(DAS) modules under control of the PBAD promoter [39]. Competent cells were incubated with 0.5 mg of purified super- coiled plasmid for 20 min on ice, then heat-shocked at 42uC for 45 s. The treated cells were recovered in 1.0 mL of LB media for 2 hr without selection, then 20 mL of the cell suspension was plated onto LB-agar supplemented with ampicillin (150 mg/mL), tetracycline (10 mg/mL) and 0.4% L-arabinose. E. coli DsspB cells were also transformed in the same manner with arabinose- inducible rhs-CT/rhsI(DAS) constructs to confirm that growth inhibition was dependent upon RhsI-DAS degradation. EC93 cdiA-CT/cdiI Deletions The main cdiA-CTEC93 region and cdiIEC93 gene were deleted from rifampicin-resistant E. coli strain EC93 (DL3852) using allelic exchange as described [4,40]. Sequence upstream of cdiA-CTEC93 was amplified using oligonucleotides #1683 (59 - CAA CAA GAG CTC GAA CAT CCT GGC ATG AGC G) and #1684 (59 - CAG CGA GTT ATT CTC AAC AAC AAC TA CGA GTT CCT ACC TGC CTG) (SacI restriction endonuclease site is underlined). Sequence downstream of cdiIEC93 (including the cdiIEC93 stop codon) was amplified using oligonucleotides #1685 (59 - CAG GCA GGT AGG AAC TCG tag TTG TTG TTG AGA ATA ACT CGC TG) and #1686 (59 - CAA CAA TCT AGA CCC GAC TTT GAG ACC AGA ATA TC) (XbaI restriction endonuclease site is underlined). The two PCR products were combined by overlapping-end PCR using primers #1683 and #1686, and the resulting product digested with SacI and XbaI and ligated to suicide vector pRE112 [40]. The orphan cdiA-CToEC93/cdiIoEC93 module was deleted from rifampicin-resistant EC93 in a similar manner. Sequence upstream of cdiA-CTo1EC93 was amplified using oligonucleotides #1714 (59 - CAA CAA GAG CTC GTG AAG GTG GGC TTA CTC AG) and #1715 (59 - CGA CTT TGA GAC CAG AAT ATC TAT TTA CTC AAC AAC AAC TAT TTT CTG TCT AAG) (SacI restriction endonuclease site is underlined). Sequence downstream of cdiIo1EC93 (including the cdiIo1EC93 stop codon) was amplified PLoS Genetics | www.plosgenetics.org 11 August 2011 | Volume 7 | Issue 8 | e1002217