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

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Gene regulation and metabolism in S. pneumoniae In B. subtilis, glutamine synthetase converts glutamate and ammonium to glutamine. Its enzymatic activity is directly inhibited by glutamine. In contrast to GlnR, the gene encoding TnrA is transcribed mono-cistronically. This MerR regulator protein family member binds to specific DNA recognition sites, which, interestingly, overlap with GlnR recognition sites. Sonenshein has recently described a model for regulation by GlnR and TnrA in which the transcriptional regulators TnrA and GlnR, the metabolic enzyme GlnA, and the amino acid glutamine play key roles (47). In this model, feedback-inhibition of GlnA as a result of inhibition by glutamine determines the binding capacity of GlnR and TnrA through direct protein-protein interaction. During nitrogen-limitation, GlnA is enzymatically active, while TnrA binds to its DNA-binding recognition site. Subsequently, TnrA activates or represses genes, while GlnR is not able to bind to the DNA because it cannot dimerize. Conversely, when glutamine is abundantly present, GlnA will be feedback-inhibited as a result of glutamine binding, resulting in inactivation of its enzymatic activity. GlnA will be able to interact with TnrA, which when complexed with GlnA is no longer able to bind to its DNA recognition sequences. In contrast, GlnR in complex with feedback-inhibited GlnA will bind to DNA, thereby repressing gene expression of its targets (47). Recently, supporting evidence for this model was provided by Wray and Fisher (54). Their data suggest that the C-terminal domain of GlnR acts as an auto-inhibitory domain, since truncated versions of GlnR lacking this domain formed significantly more dimers than the full-length proteins. The genomes of L. lactis and S. pneumoniae do not contain a homologue of TnrA. Furthermore, we found that the only target that pneumococcal GlnR shares with B. subtilis GlnR is the glnRA operon (24 and chapter 5). In B. subtilis, GlnR also regulates the expression of tnrA and that of the ureABC operon, genes which are not present in S. pneumoniae (12, 52). Interestingly, the ureABC operon is under strict regulation of GlnR, TnrA and CodY in B.subtilis, with CodY being the major repressor (52). The above suggests that nitrogen regulation in lactic acid bacteria varies considerably from other studied Gram-positive bacteria. For instance, the expression of gdhA is regulated by both GlnR and CodY in S. pneumoniae (17, 24), while this gene is regulated by CcpA, RocR, and AhrC in B. subtilis (3). These differences in nitrogen regulation could be a reflection of the different preferences for particular niches, although the lactic acid bacterial species can inhabit a wide variety of niches. 187 187
Chapter 7 Figure 1. Models of transcriptional regulation by GlnR (R) and CodY (Y). During glutamine, glutamate or ammonium excess, GlnR is in complex with GlnA. This complex binds to the GlnR operator and represses its target genes, e.g. glnPQ. When glutamine, glutamate or ammonium levels decrease, GlnR and GlnA dissociate and transcriptional repression is relieved. GlnA is consequently able to catalyze the conversion of glutamate and ammonium in glutamine. During BCAA excess, CodY is associated with BCAAs, and consequently, binds the promoter of its target genes, e.g. the ami operon, to repress gene expression. When BCAA levels decrease, CodY-repression is relieved and the target genes will be expressed. Pneumococcal GlnR and virulence Recently, we investigated the contribution of the individual genes of the entire pneumococcal GlnR-regulon to in vitro adherence and virulence in mice (18 and chapter 6). In this study, all genes were found to invariably contribute to adherence of S. pneumonia to human pharyngeal epithelial Detroit 562 cells, while in the mouse infection models GlnA was demonstrated to be required for colonization and GlnP for bacterial survival in the lungs (18). Moreover, a glnA-glnP double mutant was fully avirulent. Apparently, glutamine/glutamate metabolic genes play a crucial and site-specific role in pneumococcal virulence (18). Nitrogen regulation: CodY CodY is a pleiotropic transcriptional regulator of many genes. It represses genes that are typically expressed in conditions of nutrient limitation. Recently, we have shown that pneumococcal CodY regulates a variety of genes involved in several cellular processes, such as iron uptake, carbon metabolism, and, potentially, adherence (17 and chapter 4). The most pronounced cellular processes regulated by CodY, however, are nitrogen and amino acid uptake and metabolism (17). All these processes are connected by CodY-regulation, which 188 188
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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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Page 148 and 149: Discussion In this study, we charac
Page 150 and 151: Acknowledgements TK, JB and HB are
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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
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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 218 and 219: Curriculum Vitae Curriculum Vitae W
Page 220 and 221: Dankwoord Jeetje, dat het dan toch
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