Gene regulation in Streptococcus pneumoniae - RePub - Erasmus ...

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Table 2. Oligonucleotide primers used in this study. Name primer Nucleotide sequence 5' - 3' (restriction sites are underlined) Restriction site Cloning CodSacFwd GCGCGCCCGCGGAGGTCGTGCTGGTAAGTCAG SacII CodKpnRv GCGCGCGGTACCGATGATTTTCAGGCCAGATG KpnI CodHindFwdinv TACGAAGCTTTGAGTCTGCGGGGATTATTG HindIII CodPstRvinv ATGCCTGCAGGTGCCATTTTTCACCTCGAA PstI CodKOrv CATATTAGCCCCTTGAACGTAGTC - Cod 5' chrom CATGTCAAGATTGCGGCTAA - pcpA_L1 GTTTCCCAGAAAGACTCTGG pcpA_L2 a CCACTAGTTCTAGAGCGGCGGAGTATCTGCTAGGATTGG pcpA_R1 ATAAGAATACGGATTCGGACG pcpA_R2 a GCGTCAATTCGAGGGGTATCGATGGCTATCGAGTCAATGC pR412_L GCCGCTCTAGAACTAGTGG pR412_R GATACCCCTCGAATTGACGC Overexpression CodY-NheI-H6-Fw b CGGCTAGCCATCACCATCACCATCACGCACATTTATTAGAAAAAACT NheI CodYBamRv CGCGCGGGATCCTTAGTAATCTCTTTTCTT BamHI EMSA codY EMSA fw TGCAAGTCAATAAGGAATTTTCA - codY EMSA rv GCCATTTTTCACCTCGAATT - gdhA EMSA fw CCAAAAACTGAATTGAAAGAATTT - gdhA EMSA rv CTTTAGCAGATGTCATATCGTTCTCC - amiA EMSA fw GACACTTCGAACGACAATTTG - amiA EMSA rv TGACAACCATTATATCACATTATCCA - ilvB EMSA fw CATAAATAAACGTTAAAATAGAAAATTCAG - ilvB EMSA rv CCCTTTCTTTCCTCTTAAAAATAAC - ilvE EMSA fw GAAATGAAGAATCAGTTCTAAGATGG - ilvE EMSA rv TCCCAATCAATCGTTACTGTCA - livJ EMSA fw CCCTTTGTGGGCAATCTTTA - livJ EMSA rv CAAGCGCCACAAACGATA - psaR EMSA fw TGAAAGAAGAGCTATTTTCGTCAT - psaR EMSA rv CTTTGTTTGGGGTCATTCGT - livH EMSA fw TCAACGTCGCCTTGGATTAT - livH EMSA rv CGAGGGTTTTCCCTCACTTT - acuB EMSA fw TTCAGAGCTCTTTTTGCTAGCTT - acuB EMSA rv CCTTGCGGGTCATAAAATCT - asd EMSA fw CCCTAGTCTAGCGACTGGGATT - asd EMSA rv GCGCCGACTACAGCAACT - gapN EMSA fw CGCCTTGACGTAGTGGATTT - gapN EMSA rv TCGGATGATTTCCATTTTCC - aliB EMSA fw TTGAACAATCTTTTAGGAGAACTTGA - aliB EMSA rv CATTTCCAGAACCTCCTGCT - fatD EMSA fw TCCCTCGAAGATATTATTTTATCAGA - fatD EMSA rv TTTCATACCCCGTCCTTTCA - spr0140 EMSA fw GACATTCTATTTAGAACGAGGATTGA - spr0140 EMSA rv ATTCCCCCAGTTCCATTTTT - spr0141 EMSA fw TCTATCAAAATCGCAAATAAGAAA - spr0141 EMSA rv TTCCATTGTTTCTGCAAATTGT - spr0157 EMSA fw AACAATGTTTTAGAAGCAAAGGTG - spr0157 EMSA rv GCTTGCGACATGATAAATACTCC - spr0788 EMSA fw GAGGAAGGCCTTGTCCAGTT - spr0788 EMSA rv CCCATAGAGCAACCTGTCGT - The CodY regulon of S. pneumoniae 95 95
Chapter 4 spr1436 EMSA fw TGCGTAAACTACGTGAGCAA - spr1436 EMSA rv TGACCTGCTTCTGACATTTGA - spr1934 EMSA fw CTCCGGTTGCTTGTCTCAGT - spr1934 EMSA rv AGCAGTCCCTCCACGTGATA - spr1945 EMSA fw TGTGTTTATGGAGAGATGACAATTT - spr1945 EMSA rv AACCGCAGCTGTAGTTAATGA - spr1982 EMSA fw CTTGGTCAGGGTCAAGGAAG - spr1982 EMSA rv TCGCCATAAGTGTGTTCCTG - fake gdhA EMSA fw2 c GAATTGAAAGGGTCTCGAGCTGCTATCTGTTTTTTC - psaR EMSA codY fw2 d GCTATTTTCAATTTTTAGAAAATTTCGTTTTTTC - Footprinting codY FP fw GCAACTTGTCAATAGAAAAGGAA - fake gdhA FP fw GATATTTCCAAGAAAAACGTTCG - RT PCR codYF GATTGCCAGTACCGTTGT - codYR CACGGAGTTCGGAGTAAG - gyrAF TCTTGATTGCGCTAGACC - gyrAR ACGACGAAGACGCATATC - a Overlap with the primers pR412_L and pR412_R in bold. b 6xHis-tag is indicated in bold. c Underlined sequence indicates random nucleotides used to replace the putative CodY-box of the gdhA promoter. d Underlined sequence indicates the CodY-box introduced into the psaR promoter. DNA microarray data analysis Dual-channel array images were acquired with a GeneTac LS IV confocal laser scanner (Genomics Solutions) and analysed with ArrayPro 4.5 software (Media Cybernetics Inc.). Spots were screened visually to identify those of low quality. Slide data were processed using MicroPreP as described (15, 18, 49). Prior to analysis, automatically and manually flagged spots and spots with very low background subtracted signal intensity (5% of the weakest spots (sum of Cy3 and Cy5 net signals)), were filtered out of all datasets. Net signal intensities were calculated using a grid-based background subtraction. A grid-based Lowess transformation was performed for slide normalization, negative and empty values were removed, and outliers were removed by the deviation test. Further analysis was performed using a Cyber-T Student’s t test for paired data (32). For identification of differentially expressed genes, only genes with a minimum of six reliable measurements, a Bayesian pvalue < 0.001, a False Discovery Rate (FDR) < 0.05, and a standard deviation < ratio were included. Since these criteria are purely a statistical measure of differential gene expression and reproducibility across replicates, an additional fold-change cut-off of 2 was applied. 96 96
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Gene regulation in Streptococcus pn
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Cover image by Duve van Boggelen (w
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Promotiecommissie Promotoren: Prof.
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CHAPTER 1 Introduction 7
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Introduction Figure 1. Infections c
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Interestingly, autolysis induced by
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histidine kinase is the sensor prot
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metabolism are often required for v
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egulatory systems, aiming to elucid
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11. Cockeran, R., A. J. Theron, H.
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33. Li, S., S. J. Kelly, E. Lamani,
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53. Romero, P., R. Lopez, and E. Ga
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CHAPTER 2 Regulation of gene expres
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Introduction Streptococcus pneumoni
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Materiaals and Meethods Gene regula
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(Invitrogen), 0.2 mM aminoallyl dUT
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Isolation of pneumococcal RNA durin
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Resultss and Discuussion Gene regul
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Table 1. Genes regulated by RR09 in
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phosphofructokinase, and a fructose
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Figure 4. Expression of rlrA and rr
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correlated well with our in vitro m
Page 46 and 47: Figure 7. Bacterial loads in blood
Page 48 and 49: Acknowledgments This work was suppo
Page 50 and 51: 10. Ibrahim, Y. M., A. R. Kerr, J.
Page 52 and 53: genome sequence of a virulent isola
Page 54 and 55: Gene regulation by RR09 in S. pneum
Page 56 and 57: Gene regulation by RR09 in S. pneum
Page 58 and 59: Table S4. Genes up- and downregulat
Page 60: Table S6. Genes up- and downregulat
Page 63 and 64: Chapter 3 Abstract 62 62 Previous s
Page 65 and 66: Chapter 3 between the wild-type and
Page 67 and 68: Chapter 3 S. pneumoniae TIGR4 by CS
Page 69 and 70: Chapter 3 performance liquid chroma
Page 71 and 72: Chapter 3 Results Transcriptional a
Page 73 and 74: Chapter 3 Table 3. Differentially e
Page 75 and 76: Chapter 3 Figure 2. Colonization mo
Page 77 and 78: Chapter 3 than the D39ΔpsaR-infect
Page 79 and 80: Chapter 3 effect was more severe fo
Page 81 and 82: Chapter 3 in TIGR4ΔpsaR. However,
Page 83 and 84: Chapter 3 References 1. Adrian, P.
Page 85 and 86: Chapter 3 21. Hemsley, C., E. Joyce
Page 87 and 88: Chapter 3 40. McCluskey, J., J. Hin
Page 90 and 91: CHAPTER 4 CodY of Streptococcus pne
Page 92 and 93: Introduction Bacteria encounter var
Page 94 and 95: Materials and Methods Bacterial str
Page 98 and 99: The CodY regulon of S. pneumoniae I
Page 100 and 101: The CodY regulon of S. pneumoniae w
Page 102 and 103: Accession numbers The microarray da
Page 104 and 105: Table 3. Differentially expressed g
Page 106 and 107: Binding of CodY to target promoters
Page 108 and 109: The CodY regulon of S. pneumoniae E
Page 110 and 111: The CodY regulon of S. pneumoniae F
Page 112 and 113: Discussion CodY has been described
Page 114 and 115: levels of adherence in D39. Using a
Page 116 and 117: References The CodY regulon of S. p
Page 118 and 119: Glass. 2001. Genome of the bacteriu
Page 120 and 121: 40. Shivers, R. P., S. S. Dineen, a
Page 122 and 123: CHAPTER 5 Regulation of glutamine a
Page 124 and 125: Introduction Regulation of nitrogen
Page 126 and 127: Materials and Methods Nitrogen Meta
Page 128 and 129: TK136 D39 Δcps; Km R capsule-less
Page 130 and 131: gene from pORI28, was fused to the
Page 132 and 133: Construction of lacZ fusions Chromo
Page 134 and 135: Enzyme assays Cell-free extracts, u
Page 136 and 137: Reverse-transcriptase PCR RNA isola
Page 138 and 139: Table 3. Summary of transcriptome c
Page 140 and 141: effect caused by altered intracellu
Page 142 and 143: examine whether zwf is only transcr
Page 144 and 145: (http://www.bd.com/ds/technicalCent
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H6-GlnR alone at the concentration
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Discussion In this study, we charac
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Acknowledgements TK, JB and HB are
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11. den Hengst, C. D., S. A. van Hi
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Nitrogen Metabolism in S. pneumonia
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52. Wray, L. V., Jr., J. M. Zalieck
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CHAPTER 6 Site-specific contributio
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Introduction GlnR-regulon and pneum
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Materials and Methods Bacterial str
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eferred to as t=0. Bacteriology res
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Results GlnR-regulon and pneumococc
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Figure 3. Colonization model. Bacte
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GlnR-regulon and pneumococcal virul
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Table 2. Differentially expressed g
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Discussion The ability to adequatel
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The microarray data showed that pre
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Acknowledgments WTH is supported by
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12. Ibrahim, Y. M., A. R. Kerr, J.
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33. Zalieckas, J. M., L. V. Wray, J
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CHAPTER 7 Pneumococcal gene regulat
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Introduction Regulation of gene exp
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Gene regulation and metabolism in S
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makes it possible for the pneumococ
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A CcpA homologue exists in the pneu
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double mutant, suggesting direct re
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References Gene regulation and meta
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23. Kerr, A. R., P. V. Adrian, S. E
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45. Sonenshein, A. L. 2005. CodY, a
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CHAPTER 8 Summarizing discussion 20
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that the observed virulence phenoty
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CodY was found to be required for a
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References Summarizing discussion 1
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Summarizing discussion 21. Orihuela
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Samenvatting en discussie Curriculu
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Samenvatting en discussie kunnen ge
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Bevindingen die dit onderbouwen, we
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Curriculum Vitae Curriculum Vitae W
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Dankwoord Jeetje, dat het dan toch
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oekje en ik kijk uit naar de bijbeh
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