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

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Introduction Regulation of gene expression in bacteria is crucial for survival, as specific gene products are required during specific conditions, and therefore need to be expressed and regulated accordingly. Especially in bacterial model organisms like Escherichia coli and Bacillus subtilis, regulators of gene expression have been investigated extensively. Similarly, various studies have shown that gene regulatory networks control various cellular processes of the human Gram-positive pathogen Streptococcus pneumoniae, which are essential for different aspects of pneumococcal pathogenesis. Over the last 10 years, attempts to identify the actual genes involved in these processes have been made in several large-scale studies using a signature-tagged mutagenesis approach (4, 16, 27, 36). These studies have demonstrated the impact of a variety of gene products on the different phases of pneumococcal infection such as colonization and invasive disease, many of which (are predicted to) encode transcriptional regulators. Interestingly, several of these regulatory genes were found to contribute to virulence in a tissue-specific manner. Apart from the known virulence genes, genes involved in basal metabolism appear to have a vital role in pneumococcal pathogenesis as well. Studies on bacterial metabolism and production of secondary metabolites have elucidated many basal and conserved metabolic pathways, such as glycolysis, gluconeogenesis, and nitrogen assimilation. Especially for bacteria with industrial relevance, this knowledge has been used to improve their industrial application, for instance flavor improvement by the dairy bacterium Lactococcus lactis, or increased yields of solvents produced by Pseudomonas putida and Clostridium acetobutylicum. For pathogenic bacteria such as S. pneumoniae, knowledge of metabolic pathways is still limited. The ability of these bacteria to adapt to specific niches is likely to be reflected in their metabolic pathways and the regulation thereof. The link between these regulatory pathways and bacterial virulence is nowadays increasingly recognized. Gene regulation and metabolism in S. pneumoniae In this review we discuss regulation of nitrogen acquisition and metabolism in S. pneumoniae, which is predominantly mediated by GlnR and CodY. These regulatory proteins have been studied extensively in Bacillus subtilis and Lactococcus lactis, and in several other Firmicutes. This group also contains pathogenic species for which GlnR and CodY have been linked directly or indirectly to virulence (31, 35, 45). These studies are discussed in detail, and, when applicable, put in the context of pneumococcal GlnR and CodY. In addition, the role of these regulators and their identified targets in pneumococcal virulence are discussed. 185 185
Chapter 7 Nitrogen metabolism 186 186 Nitrogen is an essential building block for virtually every biological molecule. Bacteria utilize nitrogen from their environment and from a variety of molecules, such as free amino acids, peptides, purines, inorganic nitrogen, and gaseous nitrogen. To be able to acquire sufficient nitrogen and nitrogen-containing molecules, bacteria are equipped with sophisticated systems. S. pneumoniae also has the ability to use amino acids as nitrogen source (51). According to early studies, growth media need to contain valine, leucine, isoleucine, arginine, asparagine, histidine, and glutamine (1, 43). Interestingly, auxotrophy for amino acids can vary between strains (1, 5, 25, 43). The vast majority of nitrogen-containing compounds within the cell are derived from the two amino acids glutamine and glutamate. The system dedicated to glutamine and glutamate processing is the so-called GOGAT system. GOGAT has been described in Grampositive bacteria (6, 10, 19, 26), and it regulates the intracellular glutamine/glutamate concentrations via several enzymes such as glutamine synthase (GS or GlnA) and glutamate synthase (encoded by gltAB and gltBD). In addition, glutamine and glutamate can be taken up via an ABC-transporter encoded by glnP and glnQ. In B. subtilis and several other Firmicutes, nitrogen metabolism is regulated by the nitrogen-specific regulators GlnR, TnrA (12, 39, 47, 52, 53), and by the pleiotropic regulator CodY (12, 52). Nitrogen regulation: GlnR Recently, the transcriptional regulator GlnR was shown to regulate several genes involved in glutamine and glutamate uptake and inter-conversion in S. pneumoniae (24 and chapter 5). The gene glnR is located upstream of the gene glnA, and these genes are transcribed as one transcript. In addition to its own operon, pneumococcal GlnR regulates expression of gdhA, and an operon consisting of glnP, glnQ, and zwf. The enzyme GlnA converts glutamate and ammonium in glutamine, while GlnP and GlnQ form a glutamate/glutamine transporter and Zwf functions as a glucose-6-phosphate dehydrogenase (24). The repressing activity of GlnR is dependent on glutamine and ammonium concentrations. Besides this enzymatic function, an important role for GlnA in regulation has also been suggested, since the interaction of GlnA with GlnR is required for the repression of GlnR-targets (24) (Figure 1).
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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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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
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Page 166 and 167: Results GlnR-regulon and pneumococc
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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
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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 188 and 189: Gene regulation and metabolism in S
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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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