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

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makes it possible for the pneumococcus to adequately react to external signals. The specific signals pneumococcal CodY recognizes are the branched-chain amino acids (BCAA) isoleucine, leucine, and valine, leading to an orchestrated change in gene expression in anticipation of a changing environment (Figure 1). CodY was first described in B. subtilis (44), and has been studied extensively in this organism. B. subtilis CodY regulates the expression of over a hundred genes, varying from genes involved in sporulation, genetic competence, motility, and chemotaxis, but its role is most pronounced in nitrogen metabolism (32). In B. subtilis, CodY reacts on intracellular GTP concentrations and on branched-chain amino acids, and these molecules enhance binding of CodY to its DNA-binding box (38, 42). B. subtilis CodY carries a helix-turn-helix and a GTP-binding motif at the carboxy-terminal half of the protein. Strikingly, in L. lactis, Streptococcus mutants and S. pneumoniae, the GTP-binding motif is present, but CodY did not seem to respond to GTP, as in vitro binding assays did not show any enhancement of protein-DNA interactions (8, 17, 28). The GTP-binding motifs of L. lactis, S. mutants and S. pneumoniae do show multiple amino acid residue substitutions, which may explain the observed unresponsiveness to GTP (38, 44). The difference of CodY-mediated repression in response to GTP between this group of bacteria and B. subtilis might reflect the importance of GTP-sensing in B. subtilis: GTP plays a crucial role in the physiology of B. subtilis, as pivotal species-specific processes such as sporulation take place when the cellular levels of GTP decrease (29, 33). In contrast, L. lactis, S. mutants, and S. pneumoniae lack a functional sporulation machinery. Stringent response and CodY Gene regulation and metabolism in S. pneumoniae In B. subtilis, CodY is clearly linked to the so-called stringent response. The stringent response is a reaction during amino acid limitation in which ribosomal RNA synthesis is shut down and a ribosome-bound protein called RelA is activated. RelA is a GTP pyrophosphokinase that converts GTP to (p)ppGpp, a molecule which is often referred to as the alarmone (for review see 22, 37). During the stringent response the GTP concentration is lowered by two processes: production of (p)ppGpp out of GTP, and the inhibition of IMP dehydrogenase (20, 33). Recently, Lemos et al. have shown that in S. mutants, CodY- regulation and the stringent response (and the effect of RelA activation) are closely linked (28). The introduction of an additional codY mutation in S. mutans ΔrelAPQ fully restored growth in medium lacking leucine or valine, demonstrating that the growth-defective phenotype of ΔrelAPQ was directly linked to CodY. Moreover, artificially lowering GTP 189 189
Chapter 7 levels did not change expression of CodY targets in S. mutans, indicating once more that S. mutans CodY, like CodY of S. pneumoniae and L. lactis, is not responsive to GTP. However, the exact molecular link between the (p)ppGpp-mediated stringent response and CodY in lactic acid bacteria remains unclear. CodY and intracellular signaling of peptides 190 190 The first described target of CodY is the dipeptide permease of B. subtilis encoded by dppABCDE (40). Similarly, the L. lactis opp gene, coding for the oligopeptide permease Opp, is regulated by CodY (8, 9, 15). Uptake of oligopeptides in S. pneumoniae is mediated through an ABC transporter, the Ami/Ali permease, which is encoded by amiACDEF, aliA, and aliB (2). Interestingly, the Ami/Ali permease has been shown to be involved in adherence, by either direct interaction to human receptors or modulating pneumococcal adhesins (7). Moreover, the Ami/Ali permease is required for successful colonization of the nasopharynx of mice, but not for invasive infection (23). Claverys and co-workers hypothesized that peptide uptake by the Ami/Ali permease results in an intracellular response (5). In their model, uptake of peptides modifies the intracellular amino acid pool, which will activate a global regulatory protein, which in turn will activate (or repress) gene expression. Interestingly, this route also included a direct branch towards the stringent response, i.e., the response to low peptide or amino acid pools. Recently, we showed that the genes encoding the Ami/Ali permease are part of the pneumococcal CodY-regulon (17). This suggests that the expression of the ami operon and ali genes is regulated in response to amino acid availability, BCAAs in particular, conferring a negative feed-back. Direct molecular links with the stringent response have as yet not been identified, and therefore, direct molecular signaling between these systems seems unlikely. CodY and carbon metabolism In B. subtilis, CodY also plays a profound role in carbon metabolism. For instance, the gene encoding acetate kinase (ackA) is activated by CodY. This activation ensures that the cell has at least one pathway operational to generate ATP during nutrient excess (41). In a recent review by Sonenshein, an extensive regulatory scheme has been described in which CcpA and CodY act as master regulators of carbon flow in B. subtilis (46). In addition, GlnR and TnrA also play a role in balancing the flow of metabolites, but their roles are more specific for nitrogen metabolism.
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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
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CHAPTER 4 CodY of Streptococcus pne
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Introduction Bacteria encounter var
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Materials and Methods Bacterial str
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Table 2. Oligonucleotide primers us
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The CodY regulon of S. pneumoniae I
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The CodY regulon of S. pneumoniae w
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Accession numbers The microarray da
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Table 3. Differentially expressed g
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Binding of CodY to target promoters
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The CodY regulon of S. pneumoniae E
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The CodY regulon of S. pneumoniae F
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Discussion CodY has been described
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levels of adherence in D39. Using a
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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
Page 140 and 141: effect caused by altered intracellu
Page 142 and 143: examine whether zwf is only transcr
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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
Page 192 and 193: A CcpA homologue exists in the pneu
Page 194 and 195: double mutant, suggesting direct re
Page 196 and 197: References Gene regulation and meta
Page 198 and 199: 23. Kerr, A. R., P. V. Adrian, S. E
Page 200 and 201: 45. Sonenshein, A. L. 2005. CodY, a
Page 202 and 203: CHAPTER 8 Summarizing discussion 20
Page 204 and 205: that the observed virulence phenoty
Page 206 and 207: CodY was found to be required for a
Page 208 and 209: References Summarizing discussion 1
Page 210 and 211: Summarizing discussion 21. Orihuela
Page 212 and 213: Samenvatting en discussie Curriculu
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Page 216 and 217: Bevindingen die dit onderbouwen, we
Page 218 and 219: Curriculum Vitae Curriculum Vitae W
Page 220 and 221: Dankwoord Jeetje, dat het dan toch
Page 222: oekje en ik kijk uit naar de bijbeh
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