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

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effect caused by altered intracellular glutamine/glutamate levels. Furthermore, a number of other genes of various functions were moderately up-regulated in either the glnR or the glnA mutant. Chromosomal transcriptional lacZ fusions were used to confirm that in both the glnR and the glnA mutant, expression of glnA, glnP, zwf and gdhA was de-repressed (Figures 2A, 3A, 3B, and 4A). In addition, enzymatic activity assays showed that the observed effects on transcription corresponded with altered activities of GlnA, GdhA and Zwf in the glnR and glnA mutants (Figures 2B, 3C, and 4B). No strong effect of the glnR or the glnA mutation on the expression of a chromosomal ParcA-lacZ transcriptional fusion was observed in a range of different media (data not shown). Figure. 2. Expression of glnA in S. pneumoniae D39 (black bars) and its glnR (white bars) and glnA (diagonally hatched bars) mutants in various media. (A) β-galactosidase activity in strains TK110 (D39nisRK glnA-lacZ) and TK120 (D39nisRK glnR-stop glnA-lacZ) in CDM supplemented with 10 mM glutamate (glu10), 0.25 mM glutamine (Gln0.25), 5 mM glutamine (Gln5), 10 mM glutamate and 10 mM NH4Cl (Glu10am10), 5 mM glutamine and 10 mM NH4Cl (Gln5am10), and in GM17 with 0.5 mM glutamine (GM17Gln), which is indicated at the x-axis. (B) GlnA activity in strains D39, TK102 (D39 glnR-stop) and TK103 (D39 ΔglnA) in the media indicated on the x-axis. Nitrogen source-dependent regulation of glnRA and glnPQ by GlnR Nitrogen Metabolism in S. pneumoniae To address the role of GlnA substrates in the observed GlnR-dependent regulation in S. pneumoniae, expression of the chromosomal glnA-lacZ and glnP-lacZ fusions was measured in a chemically defined medium (CDM), to which glutamine, glutamate and ammonium were added in varying amounts (Fig. 2A). In CDM with only glutamate, glnA expression was similar in the wild-type and the glnR mutant. In contrast, glutamine led to 139 139
Chapter 5 repression of glnA expression in the wild-type already at a relatively low concentration (0.25 mM). A higher glutamine concentration did not lead to stronger repression in the wild-type. However, when besides glutamine ammonium was included, glnA expression could be further repressed. The combination of glutamate and ammonium also gave rise to repression of glnA expression in the wild-type. None of the above combinations caused repression of glnA expression in the glnR mutant. GlnA enzymatic activity is regulated in the same way (Fig. 2B). 140 140 Regulation of glnP in response to glutamate, glutamine and ammonium is very similar to that of glnA (Fig. 3A). Derepression of glnP expression is also seen in the glnA mutant, albeit to a somewhat lower extent than in the glnR mutant. This could indicate that, in the absence of GlnA, GlnR is still able to exert a weak repressive effect on the expression of glnP. Figure 3. Expression of glnPQ and zwf in S. pneumoniae D39 (black bars) and glnR (white bars) and glnA (diagonally hatched bars) mutants in various media. (A) Beta-galactosidase activity in strains TK111 (D39nisRK PglnP-lacZ), TK121 (D39nisRK glnR-stop PglnP-lacZ) and TK127 (D39nisRK ΔglnA PglnP-lacZ) in the media as indicated on the x-axis (see Fig. 2 for abbreviations). (B) Βeta-galactosidase activity in strains TK112 (D39nisRK zwf-lacZ), TK123 (D39nisRK glnR-stop zwf-lacZ) and TK130 (D39nisRK ΔglnA zwf-lacZ) in the media indicated on the x-axis. (C) Zwf activity in D39, TK102 (D39 glnR-stop), TK103 (D39 ΔglnA) and TK107 (D39 Δzwf, horizontally hatched bars) in GM17Gln. Regulation of zwf by GlnR and GlnA occurs via the glnP promoter The DNA microarray results showed that zwf, which lies downstream of glnPQ, is also up-regulated in the glnR and glnA mutants. RT-PCR demonstrated that zwf lies on the same transcript as glnPQ and thus forms an operon with these genes (data not shown). To
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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
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Chapter 3 in TIGR4ΔpsaR. However,
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Chapter 3 References 1. Adrian, P.
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Chapter 3 21. Hemsley, C., E. Joyce
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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 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 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.
Page 182 and 183: 33. Zalieckas, J. M., L. V. Wray, J
Page 184 and 185: CHAPTER 7 Pneumococcal gene regulat
Page 186 and 187: Introduction Regulation of gene exp
Page 188 and 189: 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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