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

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DNA microarray data analysis Dual-channel array images were acquired with a GeneTac LS IV confocal laser scanner (Genomics Solutions) and analyzed 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 (13, 24, 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. Spots with a signal in one channel and no signal in the other were subjected to an empty-value assignment of 1%, after which 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 (38). For identification of differentially expressed genes, only genes with a minimum of six reliable measurements, a Bayesian P-value < 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. PsaR and pneumococcal virulence Stable isotope labeling in cell culture (SILAC) For SILAC experiments D39 wild-type and psaR-mutant strains were inoculated in THY and grown to mid-log phase. These cultures were used to inoculate CDM, supplemented with both lysine and arginine as the light ( 12 C6 L-lysine, 12 C6 L-arginine; psaR-mutant) or heavy ( 13 C6 L-lysine, 13 C6 L-arginine; wild-type) isotopic counterparts. When these cultures reached an OD600 of 0.2, they were diluted to an OD600 of 0.04 in fresh pre-warmed CDM with the appropriate heavy or light lysine and arginine, and grown to an OD600 of 0.2. This was repeated until the cells were grown in heavy or light lysine and arginine-containing CDM for at least 5-6 generations. After the last generation, the cells were harvested by centrifugation and washed once with PBS. Equal amounts of heavy-labeled wild-type and light-labeled mutant cells were combined and used for mass spectrometry. Bacterial pellets were lysed in lysis buffer (7 M urea, 2 M thiourea, protease inhibitor mix (Roche), pH 8.0). Lysates were subjected to reduction and alkylation using dithiothreitol and iodoacetamide before LysC and trypsin digestion. Peptide mixtures were purified and desalted using C18stage tips. Peptide separation and sequence determination was performed with a nano-high 67 67
Chapter 3 performance liquid chromatography system (Agilent 1100 series, Amstelveen, the Netherlands) connected to a 7-T linear quadrupole ion trap-ion cyclotron resonance Fourier transform mass spectrometer (Thermo Electron, Breda, the Netherlands). Peptides were separated on a 15-cm 100-μm-inner-diameter PicoTip emitter for online electrospray (New Objective, Woburn, MA) packed with 3 μm C18 beads (Reprosil, Dr Maisch GmbH, Ammerbuch-Entringen, Germany) with a 60-minute linear gradient from 2.4 to 40 percent acetonitrile in 0.5% acetic acid at a 300 nl/min flow rate. The four most abundant ions were sequentially isolated and fragmented in the linear ion trap by applying collisionally induced dissociation. Proteins were identified using the MASCOT search engine (Matrix science, London, UK) against the corresponding S. pneumoniae database. MSQuant was used for the quantification and determination of peptide ratios between wild-type and psaR mutant. A protein was considered differentially expressed when the results of 3 technical replicates showed at least a 1.5-fold change in protein abundance and a P-value
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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
Page 18 and 19: egulatory systems, aiming to elucid
Page 20 and 21: 11. Cockeran, R., A. J. Theron, H.
Page 22 and 23: 33. Li, S., S. J. Kelly, E. Lamani,
Page 24 and 25: 53. Romero, P., R. Lopez, and E. Ga
Page 26 and 27: CHAPTER 2 Regulation of gene expres
Page 28 and 29: Introduction Streptococcus pneumoni
Page 30 and 31: Materiaals and Meethods Gene regula
Page 32 and 33: (Invitrogen), 0.2 mM aminoallyl dUT
Page 34 and 35: Isolation of pneumococcal RNA durin
Page 36 and 37: Resultss and Discuussion Gene regul
Page 38 and 39: Table 1. Genes regulated by RR09 in
Page 40 and 41: phosphofructokinase, and a fructose
Page 42 and 43: Figure 4. Expression of rlrA and rr
Page 44 and 45: 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: Chapter 3 S. pneumoniae TIGR4 by CS
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 96 and 97: Table 2. Oligonucleotide primers us
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
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Glass. 2001. Genome of the bacteriu
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40. Shivers, R. P., S. S. Dineen, a
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CHAPTER 5 Regulation of glutamine a
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Introduction Regulation of nitrogen
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Materials and Methods Nitrogen Meta
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TK136 D39 Δcps; Km R capsule-less
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gene from pORI28, was fused to the
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Construction of lacZ fusions Chromo
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Enzyme assays Cell-free extracts, u
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Reverse-transcriptase PCR RNA isola
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Table 3. Summary of transcriptome c
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effect caused by altered intracellu
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examine whether zwf is only transcr
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(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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