Additional file 1: of Induction of the immunoprotective coat of Yersinia pestis at body temperature is mediated by the Caf1R transcription factor

Table S1 List of constructs used in this study. Table S2 List of primers used in this study. Table S3 Thermal cycling conditions for RT-PCR. Table S4 Thermal cycling conditions for PCR. Table S5 Thermal cycling conditions for 5′ RACE. Figure S1 Schematic of primer design. A diagram of the caf1 operon present in the plasmids used in this study is shown, with arrows corresponding to the forward and reverse primers and placed in the approximate position where they bind. Forward primers are shown on top of the genes and reverse primers shown beneath. (A) Primers used for RT-PCR are shown in red for caf1R, green for caf1M, orange for caf1A and blue for caf1. (B) Primers used for detecting the presence of intergenic regions in cDNA are shown: purple for intergenic region I, olive for intergenic region II and dark blue for intergenic region III. Figure S2 Schematic of the P1, P2 and P3 regions. The sequence of intergenic region I (INT1), located between caf1R and caf1M is shown, highlighted in green, with the P1, P2 and P3 regions aligned and highlighted in red, yellow and orange respectively. The ATG nucleotides corresponding to the start codon of caf1M are labelled with blue triangles. Figure S3 Analysis of Caf1 content in the flocculent layer (A) Image of E. coli cultures containing the pT7-COP plasmid grown at 35 °C for the amounts of time stated, and centrifuged in capillary tubes to visualise the flocculent layer height. (B) SDS-PAGE analysis of the flocculent layers of the cultures from (A). (C) Graph of Caf1 band intensities in arbitrary units, obtained by densitometry of the gel shown in (B). Figure S4 Diagram depicting 5’RACE experimental design. Part of the caf1 operon is shown, with caf1A in green, intergenic region III in orange and caf1 in cyan. The location of the gene specific primer binding site at the 3′ end of caf1 is highlighted. Using this primer, cDNA was synthesised and sequenced using the 5’RACE method. Predicted individual transcript sequences are depicted as red lines, where the length of the line represents the length of the sequence read. Only the promoter in the P2 region (responsible for transcription of the polycistronic mRNA) is present, and so the polymerase synthesises cDNA until it dissociates from the DNA. This means the majority of sequence reads continue through intergenic region III into caf1A, terminating at different positions. Figure S5 Sequences obtained by 5′ RACE analysis of caf1 transcripts. The partial coding sequence of caf1A, followed by intergenic region III and the coding sequence of caf1 is shown (caf1 operon, COP) aligned to sequence data obtained from 8 separate 5′ RACE reactions. Regions of similarity are bounded by blue boxes with red text, and regions of complete conservation highlighted in red with white text. The regions corresponding to caf1A, intergenic region III and caf1 are underlined in green, orange and cyan respectively. (PDF 1619 kb)