using oligonucleotides #1716 (59- CTT AGA CAG AAA ATA GTT GTT GTT GAG TAA ATA GAT ATT CTG GTC TCA AAG TCG) and #1717 (59 - CAA CAA TCT AGA CCC GTA AGT ATG CTT ATC CCA TG) (XbaI restriction endonuclease site is underlined). The two PCR products were combined by overlapping-end PCR using primers #1714 and #1717, and the resulting product digested with SacI and XbaI and ligated to suicide vector pRE112 [40]. Growth Competition Assays E. coli strain EPI100 carrying plasmids pWEB-TNC (CDI2), pDAL660D1-39 (CdiAEC93), or pDAL879 (CdiAEC93-CTo1EC93 chimera) were grown overnight at 37uC in LB media supplement- ed 100 mg/mL of ampicillin. Overnight cultures were diluted into fresh medium to OD600 of 0.05, and incubated at 37uC with aeration until the culture reached mid-log phase (OD600<0.3). The log-phase inhibitor cultures were then mixed with target E. coli cells – CAG18439 pBR322 (no CdiI), pDAL741 (CdiIEC93), or pDAL867 (CdiIo1EC93) – at an inhibitor to target cell ratio of 10:1. The co-cultures were incubated for 2 hr at 37uC with aeration. Viable target cell counts were determined by serially dilution of the co-cultures into M9 salt solution followed by plating onto LB agar supplemented with 10 mg/mL of tetracycline. To assay CdiIo1 expression in EC93, growth competitions were conducted with streptomycin-resistant EC93 (DL6104) carrying pDAL879 (CdiAEC93-CTo1EC93 chimera) as the inhibitor strain. Target strains were rifampicin-resistant EC93 carrying plasmid pBR322 (no CdiI), and rifampicin-resistant EC93 DcdiA-CTo1 DcdiIo1 cells carrying pBR322 (no CdiI) or pDAL867 (CdiIo1EC93). Growth competitions were conducted as described above except that mid-log phase cells were mixed and co-cultured at an inhibitor to target ratio of 1:1. Viable target cell counts were determined by serial dilution of the co-cultures into M9 salt solution followed by plating onto LB agar supplemented with 150 mg/mL of rifampicin. Quantitative RT-PCR Analysis Total RNA was isolated from wild-type EC93 and EC93 strains deleted for cdiA-CTEC93/cdiIEC93 and cdiA-CTo1EC93/cdiIo1EC93, followed by treatment with RNase-free DNase I (Roche) to remove contaminating chromosomal DNA. RNA (0.5 mg) was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). A control without reverse transcriptase was also prepared to assess chromosomal DNA contamination. Quantitative PCR was carried out on a Bio-Rad MyiQ single-color real-time PCR detection system using SYBR green supermix. The following primers sets were used for amplification: #1700 (59 - GGT GAA GGT GGG CTT ACT CA) and #1701 (59 - TGA TGT GAC AGA GCC AAA GC) for cdiA-CTEC93; #1698 (59 - TGC TAT GTA CTG TAC TTG GTC) and #1699 (59 - TAA AGC CTA TGG GAT TCC T) for cdiIEC93; #1647 (59 - ACT GAC CGC TGA TGA ACT GG) and #1648 (59 - AGT AGC CGC TTG AAC CTG CAC) for cdiA-CTo1EC93; #1649 (59 - TGA ACC CAA CAG TCG CTC TTC) and #1650 (59 - GTC TTC CCC AGC CAG AGG AT) for cdiIo1EC93; #1568 (59 - TCA CCC CAG TCA TGA ATC AC) and #1569 (59 - TGC AAC TCG ACT CCA TGA AG) for 16S rRNA. Thermal cycling conditions were: 95uC for 5 min for polymerase activation and collection of experimental well factors and 40 cycles at 95uC for 10 s; 56uC for 30 s and 72uC for 30s followed by a melting curve (55uC to 95uC) to analyze the end product. Data were analyzed using the iQ5 optical system software (Bio-Rad) and exported to Microsoft Excel and Prism 5.0 for further analysis. For each target gene, a standard curve was generated to assess PCR efficiency (E) allowing the expression level Toxin Effectors of CDI and Rhs Systems (e) to be determined, where (e) = (Etarget)2Ct target/(Eref)2Ct ref [41]. Gene expression was normalized to a 16S rRNA RT-PCR product amplified from the corresponding sample, and the reported values represent the mean 6 SEM from three independent RNA extractions. Supporting Information Figure S1 The EC93 orphan CdiA-CT/CdiI protein pair is related to the CdiA-CT/CdiI proteins from E. coli UPEC 536. A) Pairwise alignment of CdiA-CTo1EC93 and CdiA-CTUPEC536 shows 76% sequence identity. Numbering begins at the Val residue of the conserved VENN motif. B) Alignment of CdiIo1EC93 and CdiIUPEC536 shows 35% sequence identity. (TIF) Figure S2 Orphan cdiA-CT fragments contain varying amounts of cdiA sequence upstream of the region encoding VENN. A) The region II cdi locus from Y. pseudotuberculosis PB1/+. CTo1 to CTo4 designate the orphan cdiA-CToPB1(II) genes numbered according to their position in the locus. Dots above cdiAPB1(II) represent every 500 amino acids of the encoded protein. B) Pairwise alignments of the full-length Y. pseudotuberculosis PB1/+ region II cdiA gene with the linked orphan cdiA-CT sequences. The region where similarity with the full-length cdiAPB1(II) gene begins is shown for each orphan cdiA-CT sequence. Gray shading indicates nucleotide identity, and the numbers correspond to amino acid residues of the full-length CdiAPB1(II) protein. Amino acid sequences are given in one-letter code and asterisks (*) indicate termination codons. C) The nucleotide and predicted amino acid sequences for the VENN- encoding regions are presented. Orphan sequences shaded in gray are identical to that of the full-length cdiAPB1(II) gene. (TIF) Figure S3 Multiple sequence alignment of Y. pseudotuberculosis PB1/+ cdiA-CT nucleotide sequences. The nucleotide sequences encoding orphan cdiA-CTs from the Y. pseudotuberculosis PB1/+ region II cdi locus were aligned to the full-length cdiAPB1(II) gene. Numbering corresponds to the full-length CdiAPB1(II) protein sequence. Black indicates positions of sequence identity with the full cdiAPB1(II) gene. Sequence identity falls off abruptly after the VENN-encoding region (grey). (PDF) Figure S4 VENN-encoding sequences demarcate orphan cdiA- CT fragments. The orphan cdiA-CTo1 of E. coli 254 is related to orphan cdiA-CTo7 of E. coli EC869. The conserved region is shaded gray, and the left and right junctions of the conserved region are presented in detail. Conservation begins at the VENN-encoding regions for cdiA-CTo1254 and cdiA-CTo7EC869 (left junction) and extends through the predicted orphan cdiI genes to the VENN encoding regions of the following orphan cdiA-CT genes (99% sequence identity in 1048 nucleotides). The sequences then diverge immediately after VENN encoding regions cdiA-CTo2254 and cdiA-CTo8EC869. (TIF) Figure S5 The CdiAI-CT of Y. pestis Microtus 91001 is unrelated to CdiA-CTs from other Y. pestis strains. Pairwise alignment of CdiA(I) proteins encoded by the region I cdi loci of Y. pestis Microtus 91001 and Y. pestis CO92. Numbers correspond to amino acid residues of the full-length predicted CdiA(I) proteins. Regions of sequence identity are shaded gray and the VENN peptide motif is boxed. (TIF) PLoS Genetics | www.plosgenetics.org 12 August 2011 | Volume 7 | Issue 8 | e1002217