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

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H6-GlnR alone at the concentration shown did not bind to PglnR and PglnP (Fig. 6A and B), also not in the presence of glutamine. However, in the presence of H6-GlnA, binding of H6-GlnR to PglnR and PglnP was observed (Fig. 6A and B). At a 5-10 times higher H6- GlnR concentration (2 to 4 μM), a shifted band at the same position could be observed in the absence of H6-GlnA (data not shown), which corresponds with the transcriptional data, showing some weak repression by GlnR in the glnA mutant (Table 3, Fig. 3A). No binding of H6-GlnR was seen with the controls, the promoters without their GlnR operators (Fig. 6A and B). DNAse I footprinting showed that, in the presence of H6-GlnA, H6-GlnR specifically reduces DNAseI sensitivity of the predicted GlnR operator in PglnP (Fig. 6C and Fig. 1). Remarkably, the protected region in PglnR only partially overlapped with the predicted GlnR operator (Fig. 6B and Fig. 1), suggesting that GlnR binds in a different manner to this promoter than to PglnP. In contrast to what would be expected from the expression data, GlnA-dependent binding of GlnR to PglnP and PglnR was only weakly stimulated by the addition of glutamine (Fig. 6A and B). This could be explained by the observation mentioned above, that H6-GlnR seems to be less sensitive to glutamine than the native protein. Addition of the other GlnA substrates glutamate, ammonium, ATP and AMP alone or in combination did not alter the observed GlnA-dependency of the GlnR-DNA interaction at PglnP (Fig. 6A). Thus, although GlnA is required for the binding of GlnR to the GlnR operators in the glnP and glnR promoters, this effect was not modulated by GlnA substrates. GlnA and GlnP contribute to adhesion to pharyngeal epithelial cells Nitrogen Metabolism in S. pneumoniae The crucial first step of pneumococcal virulence is the colonization of the nasopharynx. Therefore, we tested the ability of glnR, glnA, glnRA and glnP mutants to adhere to the human pharyngeal epithelial cell line Detroit 562. These mutants were created in the capsule-less background strain D39 Δcps, as unencapsulated strains tend to show higher levels of adhesion ((34), data not shown). The glnP, glnA and glnRA mutants displayed a significantly decreased adhesion to the pharyngeal epithelial cells compared to the capsule- less wild-type strain (Fig. 7). Thus, both GlnP and GlnA could play a role in colonization of the nasopharynx. However, since most pneumococcal isolates are encapsulated, the actual contribution of these to proteins to virulence remains uncertain. 145 145
Chapter 5 Figure 7. Adhesion of S. pneumoniae strains TK136 (D39 Δcps, WT), TK137 (D39 glnR-stop Δcps, R), TK138 (D39 ΔglnA Δcps, A), TK139 (D39 ΔglnRA Δcps, RA) and TK140 (D39 ΔglnP Δcps, P) to Detroit 562 human pharyngeal epithelial cells. Adhesion is given in % relative to TK136. *p=0.0011, **p
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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
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 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 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 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
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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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