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

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Discussion In this study, we characterized the regulation of glutamine and glutamate metabolism mediated by GlnR and GlnA in the human pathogen S. pneumoniae. Previously, GlnR- dependent regulation of nitrogen metabolism has been thoroughly studied in B. subtilis. The only target that B. subtilis GlnR shares with GlnR from S. pneumoniae is glnRA (8). In addition, B. subtilis GlnR is a repressor of the ureABC operon (13, 49) and tnrA (13), which are absent in S. pneumoniae (19). Another difference is that while GlnR is a repressor of glnPQ and gdhA in S. pneumoniae, in B. subtilis the catabolic glutamate dehydrogenase gene rocG is regulated by CcpA, RocR and AhrC (4), and the glnQH glutamine transport operon is activated by TnrA (53). We found that also expression of zwf, encoding a putative glucose-6-phosphate dehydrogenase, is regulated by GlnR. This enzyme catalyzes the first reaction in the pentose phosphate pathway, which provides the cell with NADPH and ribose-5-phosphate, a building block of nucleic acids. As glutamine is a precursor for the synthesis of nucleotides as well, it might be advantageous for S. pneumoniae to coordinate zwf expression with glutamine metabolism. Our data suggest that the regulation by S. pneumoniae GlnR depends on a conserved inverted repeat. The B. subtilis GlnR targets contain 2 copies of the same inverted repeat in their promoter regions (7, 8). S. pneumoniae GlnR resembles B. subtilis TnrA in this respect, as TnrA activates or represses promoters containing only one copy of this repeat (53). The distance between the GlnR operator and the -35 box in PglnR is 7 bp, for PglnP it is 16 bp. GlnR boxes are also present at a distance of 5 to 7 bp from the -35 in the glnR promoters of the S. pneumoniae relatives S. pyogenes, S. agalactiae and S. mutans. Moreover, GlnR operators are located in the glnP promoters of S. pyogenes and S. agalactiae, which, like in S. pneumoniae, have a 16-bp spacing with the -35 sequence. As the spacing in the glnP promoters is 9 to 10 bp longer than in the respective glnR promoters, regulation by GlnR via these operators might be helix-side dependent in these organisms. The GlnR operator in PgdhA confers a less pronounced GlnR-dependent effect than the GlnR operators in PglnP and PglnR, although the inverted repeat is perfectly conserved. The same accounts for the GlnR operator in ParcA. In PglnR and PglnP there is a stretch of A’s immediately upstream of the repeat and a stretch of T’s between the two half-sites. These stretches might explain the more efficient transcriptional repression of PglnP and PglnR than of ParcA and PgdhA, as AT-rich stretches on these positions of B. subtilis PnrgAB enhance TnrA-dependent transcriptional activation (51). Nitrogen Metabolism in S. pneumoniae 147 147
Chapter 5 148 148 Both GlnR and CodY function as a repressor of gdhA in S. pneumoniae, of which CodY seems to be the more important regulator. Furthermore, both regulators control gdhA transcription independently of each other, which is in agreement with the location of their operators, that for CodY lying upstream of the -35 and the GlnR operator downstream of the - 10 in PgdhA. In B. subtilis CodY controls the cellular nutritional and energy status (20, 21). Although GdhA is obviously connected to glutamine metabolism, the observation that gdhA expression is, next to GlnR, also regulated by CodY in S. pneumoniae, might indicate that GdhA is an important control point of the cellular nutritional status in this bacterium. We show that GlnR DNA binding is dependent on GlnA, in contrast to the situation in B. subtilis, where GlnR alone binds with high affinity to its target promoters in the absence of any effectors (8). Since a high concentration of GlnR alone led to a shifted band at the same position as in the presence of GlnA, it is unlikely that GlnR and GlnA bind as a complex to the DNA. It might be that GlnA induces a conformational change or multimerization of GlnR, which increases its DNA binding affinity. Next to GlnA, also GlnP seems important for activity of S. pneumoniae GlnR. Although GlnR and GlnA alone were sufficient for in vitro binding to the glnP and glnR promoters, it could be that in vivo both GlnPQ and GlnA are needed for optimal activity of GlnR. Our results and previous STM screens (18, 28, 36) implicate a role for both GlnP and GlnA in pneumococcal adhesion to human pharyngeal cells, which is a prerequisite to invade the host. Previously, GlnQ was shown to be required for adhesion of S. pyogenes to fibronectin and epithelial cells of the respiratory tract (43). However, it remains to be investigated whether the effect of GlnPQ on adhesion by S. pneumoniae and S. pyogenes is caused by a general effect of distorted glutamine metabolism on e.g. the cell-surface composition or that GlnPQ are directly involved. We are currently analysing glnR, glnA, glnP, and gdhA mutant strains in several in vivo mouse models, to get more insight in the role of glutamate and glutamine metabolism during infection by S. pneumoniae.
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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
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 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 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 190 and 191: makes it possible for the pneumococ
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
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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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