Figure 4 Qualitative UV assay and mRNA analysis of E. coli R391 mutants KOA, KOB and KOC. (A) AB1157 R391 mutants KOA, KOB and KOC. UV254nm exposure increasing (12 J.m-2) from left to right. (i) From top to bottom, AB1157, AB1157 R391, AB1157 R391 KOA. (ii) AB1157, AB1157 R391 KOB. (iii) AB1157, AB1157 EPZ015666 ic50 R391, AB1157 R391 KOC. (B) SYBR® Safe stained 1% (w/v) agarose gel confirming orf43 mRNA transcription in AB1157

R391 KOA. M, Bioline Hyperladder I DNA marker; 1, AB1157 R391 RNA negative control; 2, AB1157 R391 genomic DNA positive control; 3, AB1157 orf43 cDNA; 4, AB1157 R391 orf43 cDNA; 5, KOA orf43 cDNA; 6, KOB orf43 cDNA; 7, KOC orf43 cDNA; 8, KOB orf20 cDNA. Primers used specific to orf43 generated a 188 bp PCR product. Primers SB525334 supplier used for lane 8 only were specific for the kanamycin resistance gene of ICE R391, orf20, which generated a PCR product of 223 bp. Amplification of orf20 specific cDNA was carried out to show KOB and KOC RNA was not degraded. Lane 1 negative control was DNase treated RNA that was not converted to cDNA. (C) Map of exact locations of KOA, KOB and KOC deletions on ICE R391 genome. The KOA,

KOB and KOC ampicillin resistance cassettes and associated promoter were inserted into the ICE R391 genome in the reverse complement to prevent the ampicillin resistance cassette promoter inducing the transcription of orf43 mRNA. The KOA deletion removed all possible promoters of orf43 in front of the gene and left the last 36 bp specific to the preceding orf42 gene. The KOB deletion removed the

same region as KOA and the 36 bp region. The KOC deletion was a duplicate of KOA with an additional Vildagliptin zeocin resistant orfs90/91 deletion. Site-directed mutagenesis of Orf43 Bioinformatic analysis of orf43 indicated that it belongs to a highly conserved TraV-like family of transfer proteins involved in type IV secretion systems required for conjugation [8]. Site-directed mutagenesis of pBAD33-orf43 was carried out to convert two leucines at a.a. positions 47 and 48 to prolines in the predicted Orf43 protein (GenBank: AAM08037). Insertion of two prolines was expected to disrupt the α-helical transmembrane spanning region of Orf43 by creating a 30° bend [19]. This mutation was found to cause loss of the cytotoxic function of pBAD33-orf43[8] as there was no observable decline in host cell growth rates after induction of the mutant clone compared to the wild type clone [Figure 5A,B]. Since introduction of membrane disruptive mutations abolish the effect, this is suggestive that membrane association is required in addition to over-expression of the Orf43 protein for sensitisation and Cell Cycle inhibitor cytotoxicity associated with this ICE product.