pej605 pej414 containing 81 bp downstream and 579 bp This study

Transcription

1 SUPPLEMENTARY DATA Table S. Details of plasmids used in this study. Plasmid Description Reference or source pfm8 Protein expression vector based on pet5b containing (0) His-tagged lexa. pcr 4-TOPO Cloning vector Invitrogen pej44 Integrating mycobacterial lacz transcriptional reporter (53) vector. pej602 pej44 containing 73 bp downstream and 283 bp This study upstream of annotated start site of Rv000c made using primers Rv000cprF and Rv000cprR. pej603 pej44 containing 240 bp downstream and 64 bp This study upstream of annotated start site of Rv378c made using primers Rv378prF and Rv378prR. pej604 pej44 containing 56 bp downstream and 284 bp This study upstream of annotated start site of Rv3074 made using primers Rv3074prF and Rv3074prR. pej605 pej44 containing 8 bp downstream and 579 bp This study upstream of annotated start site of Rv3395c made using primers Rv3395cprF and Rv3395cprR. pej607 pej602 containing A-4G and T+4C mutations of SOS This study box by SDM with primers Rv000cSDMF and Rv000cSDMR pej608 pej603 containing A-4G and T+4C mutations of SOS This study box by SDM with primers Rv378SDMF and Rv378SDMR pej609 pej604 containing A-4G and T+4C mutations of SOS This study box by SDM with primers Rv3074SDMF and Rv3074SDMR pej60 pej605 containing A-4G and T+4C mutations of SOS This study box by SDM with primers Rv3395cSDMF and Rv3395cSDMR pej634 pej44 containing 36 bp downstream and 20 bp This study upstream of annotated start site of Rv200 made using primers Rv200prF and Rv200prR pej635 pej634 containing A-4G and T+4C mutations of SOS This study box by SDM with primers Rv200SDMF and Rv200SDMR pej636 pej634 containing A-4G and T+4C mutations of SOS This study box 2 by SDM with primers Rv200SDM2F and Rv200SDM2R. pej637 pej634 mutation of SOS box and 2 This study

2 2 Table S2. Primers used in this study. Primer Sequence 5-3 Rv3395cprF GCTCTAGAGTGGTGACAGATCCGCCCCGTAAT Rv3395cprR GCAAGCTTCAAAGCCATCTGCCGTCGTAGTGA Rv378cprF ATTCTAGAGGATTCCCAGCAGCACCAC Rv378cprR GTAAGCTTAGCCGCGGCCGATTTGAC Rv3074prF GCTCTAGACGCTCCGGCTGGTGGTGGTA Rv3074prR TTGATATCTGCTCCTTCGGCGGCTCTGC Rv000cprF GCTCTAGACGGCCCCGCTGTGATGTAGT Rv000cprR CTGATATCGCTGTCTTCGCTCGTTGTGCTC Rv200prF GCTCTAGACCTCCGGGCTCGCAATCACAAAC Rv200prR GCAAGCTTACGGCCGCGCAGTCCCAGAAGTC Rv3395cSDMF GTTTACTGACTTCGAGCATATTCTCGAACAGGAGGC Rv3395cSDMR GCCTCCTGTTCGAGAATATGCTCGAAGTCAGTAAAC Rv378SDMF CTGCTATACTCGAGCACATGCTCGATTTAGTGAG Rv378SDMR CTCACTAAATCGAGCATGTGCTCGAGTATAGCAG Rv3074SDMF CTGCTATTCTCGAGCACATGCTCGAGACATTGAC Rv3074SDMR GTCAATGTCTCGAGCATGTGCTCGAGAATAGCAG Rv000cSDMF CGCCATCGAGCGAATGCGCGATAAGCTTG Rv000cSDMR CAAGCTTATCGCGCATTCGCTCGATGGCG Rv200SDMF CTACTAGTATCGCGCACATGCTCGAGATATCGC Rv200SDMR GCGATATCTCGAGCATGTGCGCGATACTAGTAG Rv200SDM2F CACTGTTCGAGCCGTCGCTCGCCGCAG Rv200SDM2R CTGCGGCGAGCGACGGCTCGAACAGTG Rv586R AATCTCCCGGCGCAACA Rv588down CAACCTCAAGCCGCTCACTAA MTS2823probe GCACCCACGCGGAGTCATAGCCACGATAACGGCAGAAGC CTGTCTC RACE primers Rv3395cGSP CCGGGGTGATCTCATA Rv3395cGSP2 GCCGCAAGCCGCGTCGACA Rv3395cGSP3 CTTTTTGACGGGCACGGGCCAC Rv3395cGSP4 CCCCCGGACACCTTCTCGGACAGCAAA Rv3395cGSP5 GAGAGGCTGCCTTGTGCAATCGCGTAAAACCACC Rv378cGSP CAATGCGGACATGTCG Rv378cGSP2 AGCTCGGCGTCGAGTGCGCG Rv378cGSP3 GCAGGCCGACTCCCGCACGAT Rv3074GSP CGTCGAGCCAGGGCAATCTCACT Rv3074GSP2 GCAACAGGGCGGTCAGATACGTCAT Rv3074GSP3 TTGGCCGCGGGAGCGCCCGTCGCA Rv3074GSP4 CCGGTCCGCGACATCCAGACATGCGCTTT Rv000cGSP CCCATCACCAATAGAT Rv000cGSP2 GTCGCAACGATGGACCGCGGCCAC Rv000cGSP3 GGGTGGCGCCGAGGCTGACGATC Rv000cGSP4 GCGGATGCGGCGGATCTTCGATGGTCA Rv000cGSP5 TACATCTGACGGCGCTCGGCTCGCC RV200GSP GACGTGGATGACCACAC RV200GSP2 CGTTGCCGGCACCGGAGGG RV200GSP3 CCAGGGTGCGCGGGTCATCCT RV200GSP4 GCCGCGCACCAAGATGCCCGGCCAAAA RV200GSP5 TTTGCCGTGGCTGATAGTCAACGCCGCAGCC qrt-pcr primers sigaqrtf TCGGTTCGCGCCTACCT

3 3 sigaqrtr TGGCTAGCTCGACCTCTTCCT 6srRNAqRTF AAGAAGCACCGGCCAACTAC 6srRNARTR TCGCTCCTCAGCGTCAGTTA Rv3074SOSqRTF CCGGCGCCACCAGTT Rv3074SOSqRTR CGGTCAATGTCTCGAACATGTG Rv3776SOSqRTF GGCCTGTTGCCTTATGTCAAG Rv3776SOSqRTR CGACCACGTTGACTCGTTGAT dnae2sosqrtf GGTGCCGGCGTTCGA dnae2sosqrtr AGGAAGCTGTACGCCGAATG dnae2qrtf ACATCGAGTCGGATCAGCG dnae2qrtr GTGATGACGTTGGCGACCT MTS082qRTF ATCCGTGTTCCAACAGAGTGAA MTS082qRTR CGGCGCTCGCTCATTC MTS2823qRTF GGCGCCCAGCATGGT MTS2823qRTR CATCTGCTGTTCGCAATTACG Rv3074qRTF ACTACGCCAAACAAGCCCC Rv3074qRTR GGCTGTGTGCGTGTGATTTT uvraqrtf CGCGAGCAGCGGTTCT uvraqrtr CGCCGTAGGGCGAGTTG whib2qrtf TGAGGCCAAGAAGATTTGCA whib2qrtr CGGAAAGACCACCCCAGAT fbiaqrtf TGCGAAACCCATGTAGTGATCA fbiaqrtr GCACCCACCACTCCTGAAA Rv057qRTF CGACGGGCGCACTGA Rv057qRTR TCATCCGCACGCATTGC

4 Table S3. Potential LexA binding sites for each individual replicate identified by ChIP-seq. Peak# a Coordinates b Height cut-off analysis c BayesPeak analysis d e f

5 a Peak ID# as in Table, binding peaks chosen for further analysis if found in at least two replicates by peak height cut-off analysis and at least one replicate by BayesPeak analysis and after manual confirmation. b Genome coordinates encompassing total peak region. c Height cut-off analysis determined as any region where the normalised number of reads goes above mean standard deviations for over 50 bases, cut-off values 9.88, 2.96, for replicates, 2 and 3 respectively. d BayesPeak algorithm performed on replicates and 2 only using a bin size of 20 bases and PP threshold of 0.. e This region contains the ribosomal operon. f This region contains PPE54. 5

6 SUPPLEMENTARY FIGURES 2.0 Enrichment dnae2 Rv3074SOS Rv3776SOS dnae2sos siga Figure S. Quantitative PCR to validate ChIP protocol. Quantity of siga, dnae2, Rv3074SOS and Rv3776SOS and dnae2sos regions recovered from α-lexa immunoprecipitated (IP) DNA samples were analysed by quantitative PCR using primers sigaqrtf and sigaqrtr, dnae2qrtf and dnae2qrtr, Rv374SOSqRTF and Rv374SOSqRTR, Rv3776SOSqRTF and Rv3776SOSqRTR or dnae2sosqrtf and dnae2sosqrtr. Enrichment was defined and the quantity recovered from the IP sample divided by the quantity recovered from the input control. Data is mean + standard deviation from two independent replicates. 6

7 Figure S2. Genome coverage plots. Boxplots and genome coverage plot of the number of reads mapped to each base normalised to the total number of reads of anti-lexa IP and input control for each individual replicate. The position of the ribosomal operon is indicated (rrna). 7