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

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Construction of lacZ fusions Chromosomal transcriptional lacZ fusions were constructed with the integration plasmid pORI13 as described (23, 37). For lacZ fusions to glnA, gdhA and zwf, 600-to 800-bp fragments of the 3’ ends of the genes were PCR amplified using primer pairs R6_glnA- 5/R6_glnA-6, R6_gdhA-4/R6_gdhA-5 and R6_G6PDH-1/R6_G6PDH-2, respectively. These fragments were digested and cloned into the XbaI/EcoRI sites of PORI13, giving pTK8, pTK10, pTK11, respectively. The constructs were introduced into S. pneumoniae D39nisRK, and D39nisRK containing either the glnA (TK100) or glnR (TK105) mutation and clones were checked by PCR. Analogously, pTK9 and pTK12 were constructed with primers R6_PglnP- 1/R6_PglnP-2 and R6_arcA-3/R6_arcA-4. These plasmids were used to generate chromosomal lacZ fusions to the glnP and arcA promoters. The glnQ-zwf intergenic region was cloned into pORI13 using primers Pg6pdh-1/ Pg6pdh-2, giving pTK21. The lacZ fusions to the gdhA promoter were constructed in pPP2 with primer pair PgdhA-2/PgdhA-4, giving a PCR product comprising the full-length promoter (PgdhA-1), and primer pair PgdhA-3/PgdhA-4, resulting in a PCR product without the predicted GlnR operator (PgdhA-2), using E. coli EC1000 as the cloning host. The constructs were introduced in S. pneumoniae strains D39 and TK102. Nitrogen Metabolism in S. pneumoniae Construction of overexpression constructs The glnR, glnA and glnPQ genes were PCR amplified with primer pairs glnR-9his/glnR_R6-10, glnA-his/glnA_R6-8 and glnP-OX1/glnP-OX2, respectively, and cloned into the NcoI/XbaI sites of pNG8048E, giving pTK16, pTK15 and pTK17. In addition, the native glnR gene was cloned into pNG8048E using primers glnR-_R6-9/glnR_R6-10, giving pTK23. Purification of GlnR and GlnA Overexpression of N-terminally 6xHis-tagged GlnR and GlnA (H6-GlnR and H6- GlnA) was achieved with the nisin-inducible system (NICE) in strain L. lactis NZ9000 (27). Expression was induced with nisin in 1 L cultures at an OD600 of 0.6, using a 10 -7 dilution (2 ng/ml) of nisaplin, which was prepared as described (23). After two hours of induction, cells were harvested and resuspended in 10 ml buffer A (0.25 M NaCl, 10 mM MgCl2, 20 mM Tris-HCl, pH 8, 10% glycerol, 1 mM β-mercaptoethanol) with 1 mg/ml lysozyme and 1 tablet of protease inhibitor cocktail (Complete Mini, Roche). After 20 min of incubation on ice, cells were disrupted by shaking 5 times 1 min with 400 mg glass-beads (75-150 μm, Fischer 131 131
Chapter 5 Scientific BV) per ml of cell suspension in a Biospec Mini-BeadBeater-8 (Biospec Products) and cell debris was removed by centrifugation. 1 ml Ni-NTA beads (Qiagen), pre-equilibrated in buffer A, was added to the cell lysate and protein binding was allowed for 1h at 4ºC, with continuous gentle shaking. Beads were washed 10 times with buffer A containing 20 mM imidazole after which H6-GlnR and H6-GlnA were eluted with buffer A containing 250 mM imidazole and subsequently with buffer A containing 350 mM imidazole. H6-GlnA was dialysed against 2,000 times excess of buffer B (20 mM Tris-HCl, pH 8.5, 10% glycerol, 1 mM β-mercapto-ethanol) for 6 hrs at 4ºC. Since H6-GlnR precipitated during dialysis, imidazole was removed by means of a PD-10 desalting column (Amersham Biosciences), using buffer C (20 mM Tris-HCl, pH 7.5, 10% glycerol, 10 mM MgCl2, 1 mM EDTA, 1 mM β-mercapto-ethanol), in which way precipation was not observed. Purified fractions contained >95% pure protein of the expected size with a concentration of between 0.2 and 1 mg protein/ml. Electrophoretic mobility shift assays and DNAseI footprinting EMSA’s were performed essentially as described previously (10). PCR products of PglnR with and without the predicted GlnR operator were made with primer pairs glnR_R6- 2/PglnR-2 (PglnR-1, 146 bp) and glnR_R6-2/PglnR-3 (PglnR-2, 84 bp), respectively. In the same way, PCR products spanning PglnP were generated with primer pairs PglnPQ- 1/PglnPQ-2 (PglnP-1, 190 bp) and PglnPQ-2/PglnPQ-3 (PglnP-2, 131 bp), respectively. The binding buffer was composed of 20 mM Tris-HCl, pH 8.0, 50 mM MgCl2, 1 mM dithiotreitol (DTT), 8.7% (w/v) glycerol, 62.5 mM KCl, 25 µg/ml bovine serum albumin, 50 µg/ml poly(dI-dC) and 3000-5000 cpm of [γ- 32 P]ATP-labeled PCR product. Glutamine, glutamate, ammonium and purified H6-GlnR and H6-GlnA were added as specified in the Results section. Reactions (20 µl) were incubated for 20 min at 25ºC after which they were run on a 6% poly-acrylamide gel for 75 min at 90V. DNAseI Footprinting was done essentially as described (10). 150.000 cpm of [γ- 32 P]ATP-labeled PCR products of the glnR and glnP promoters, made with primer pairs R6_PglnR_FP/R6_glnR-7 (244 bp) and R6_glnP-1/R6_glnP-GFP1 (235 bp), respectively, were used as probes in 40 μl binding buffer (EMSA) containing 5 mM glutamine and purified H6-GlnR and H6-GlnA as specified in the Results section. 132 132
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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
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Figure 7. Bacterial loads in blood
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Acknowledgments This work was suppo
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10. Ibrahim, Y. M., A. R. Kerr, J.
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genome sequence of a virulent isola
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Gene regulation by RR09 in S. pneum
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Gene regulation by RR09 in S. pneum
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Table S4. Genes up- and downregulat
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Table S6. Genes up- and downregulat
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Chapter 3 Abstract 62 62 Previous s
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Chapter 3 between the wild-type and
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Chapter 3 S. pneumoniae TIGR4 by CS
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Chapter 3 performance liquid chroma
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Chapter 3 Results Transcriptional a
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Chapter 3 Table 3. Differentially e
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Chapter 3 Figure 2. Colonization mo
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Chapter 3 than the D39ΔpsaR-infect
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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
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 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
Page 146 and 147: H6-GlnR alone at the concentration
Page 148 and 149: Discussion In this study, we charac
Page 150 and 151: Acknowledgements TK, JB and HB are
Page 152 and 153: 11. den Hengst, C. D., S. A. van Hi
Page 154 and 155: Nitrogen Metabolism in S. pneumonia
Page 156 and 157: 52. Wray, L. V., Jr., J. M. Zalieck
Page 158 and 159: CHAPTER 6 Site-specific contributio
Page 160 and 161: Introduction GlnR-regulon and pneum
Page 162 and 163: Materials and Methods Bacterial str
Page 164 and 165: eferred to as t=0. Bacteriology res
Page 166 and 167: Results GlnR-regulon and pneumococc
Page 168 and 169: Figure 3. Colonization model. Bacte
Page 170 and 171: GlnR-regulon and pneumococcal virul
Page 172 and 173: Table 2. Differentially expressed g
Page 174 and 175: Discussion The ability to adequatel
Page 176 and 177: The microarray data showed that pre
Page 178 and 179: Acknowledgments WTH is supported by
Page 180 and 181: 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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