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

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Table 3. Summary of transcriptome comparison of S. pneumoniae strains D39 glnR-stop and D39 ΔglnA with D39 wild-type. Gene name a Function b glnR-stop c glnA d SP0092 ABC transporter, substrate-binding protein 1.5 (3.1e-7) NDE e SP0237 Ribosomal protein L17 1.5 (2.0e-8) NDE SP0295 Ribosomal protein S9 1.6 (3.6e-9) 1.6 (4.3e-5) SP454 Hypothetical protein 1.5 (2.2e-4) NDE glnR (SP0501) Nitrogen regulatory protein 5.0 (0.0) 1.7 (3.8e-4) glnA (SP0502) Glutamine synthetase 5.1 (0.0) -2.4 (5.3e-5) SP0922 Carbon-nitrogen hydrolase family protein NDE 1.6 (2.0e-5) SP0964 Dihydroorotate dehydrogenase NDE 1.8 (7.3e-6) SP0965 Endo-beta-N-acetylglucosaminidase -1.6 (2.4e-8) NDE SP1111 Hypothetical protein NDE -3.7 (5.0e-4) glnP(SP1241) Glutamine ABC transport and substrate binding prot. 4.3 (2.8e-15) 2.8 (3.6e-12) glnQ (SP1242) Glutamine ABC transport and substrate binding prot. 4.0 (0.0) 2.5 (8.4e-11) zwf (SP1243) Glucose-6-phosphate dehydrogenase 2.8 (4.7e-15) 1.6 (7.3e-7) SP1275 Carbamoyl-phosphate synthase large subunit 2.4 (1.6e-5) 1.5 (7.9e-7) SP1276 Carbamoyl-phosphate synthase small chain NDE 1.6 (1.4e-8) SP1277 Aspartate carbamoyltransferase 1.8 (1.1e-4) 1.8 (5.0e-7) gdhA (SP1306) NADPH-dependent biosynthetic glutamate dehydrogenase 2.2 (6.9e-12) 2.0 (1.0e-9) SP1354 Ribosomal protein L7/L12 1.8 (3.1e-9) NDE SP1936 Type II restriction-modification system regulatory protein 1.6 (9.3e-6) NDE SP2055 Alcohol dehydrogenase, zinc-containing 1.5 (2.2e-6) NDE SP2060 Pyrrolidone-carboxylate peptidase NDE -1.7 (2.8e-5) SP2063 LysM domain protein, authentic frameshift -1.9 (1.1e-11) -2.3 (6.5e-9) arcA (SP2148) Arginine deiminase 1.6 (4.4e-4) NDE a Gene numbers refer to TIGR4 locus tags. b TIGR annotation. c Ratio’s > 1.5 or < -1.5 (D39 glnR-stop compared to D39) in bold, p-values in parenthesis. d Ratio’s > 1.5 or < -1.5 (D39 ΔglnA compared to D39) in bold, p-values in parentheses. e NDE, not significantly differentially expressed. Nitrogen Metabolism in S. pneumoniae Predicted operators with the highest similarity to the B. subtilis consensus sequence were present in the promoter regions of glnR (spr0443); glnP (spr1120), encoding a 137 137
Chapter 5 glutamine ABC transporter substrate binding protein; gdhA (spr1181) encoding a NADP(H)- specific glutamate dehydrogenase; and arcA (spr1955, spr1956), encoding arginine deiminase (Fig. 1). Re-searching the R6 genome with a weight matrix built from these putative operators did not reveal additional putative GlnR operators. The regulon of GlnR and GlnA in S. pneumoniae To investigate the role of GlnR in S. pneumoniae in more detail, we constructed a glnR mutant in strain D39. To preserve the glnRA operon structure, two consecutive stop-codons were introduced in the beginning of the glnR open reading frame (ORF), specifying amino acids in the middle of the predicted helix-turn-helix DNA-binding motif. DNA microarray analyses were performed of S. pneumoniae D39 wild-type and its isogenic glnR mutant grown in the nitrogen-rich medium GM17, supplemented with 0.5 mg/ml glutamine (GM17Gln). This amino acid is assumed to be a co-repressor of GlnR in B. subtilis (38), and we expected it to also induce repression of GlnR targets in S. pneumoniae. The operons/genes that were most highly up-regulated in the glnR mutant were glnRA, glnPQ, and gdhA (Table 3), all of which have a GlnR operator in their promoter regions (Fig. 1). The arcA gene, which also contains a putative GlnR operator in its promoter, was only weakly upregulated. Remarkably, also zwf, encoding the key enzyme glucose-6-phosphate dehydrogenase of the pentose phosphate pathway, was up-regulated. This gene lies downstream of and in the same orientation as glnPQ. To investigate the influence of GlnA on the expression of the identified GlnR targets, a comparison of the transcriptomes of wild-type D39 with its isogenic glnA mutant, grown in GM17Gln, was performed. This showed that glnR, glnP, glnQ and gdhA were, like in the glnR mutant, also up-regulated in the glnA mutant (Table 3), indicating that GlnA is necessary for the functioning of GlnR. Besides the genes mentioned above, SP2063, encoding a predicted LysM domain containing protein, not directly involved in glutamine metabolism, was 2-fold down-regulated in both the glnR and the glnA mutant. Interestingly, two degenerate GlnR boxes (5’- TGTGACAGAGACCTAACA-3’ and 5’-TGTTATTAGCGTCAACA-3’) are present in the promoter region of this gene. In both the glnR and glnA mutant, genes predicted to encode proteins involved in pyrimidine metabolism (SP1275, SP1276, SP1277, SP0954) were moderately up-regulated, which seems logical as glutamine is a precursor of pyrimidine. However, as no GlnR operator could be identified upstream of any of these genes, the up-regulation is likely to be an indirect 138 138
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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
Page 87 and 88: Chapter 3 40. McCluskey, J., J. Hin
Page 90 and 91: CHAPTER 4 CodY of Streptococcus pne
Page 92 and 93: Introduction Bacteria encounter var
Page 94 and 95: 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 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
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Gene regulation and metabolism in S
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makes it possible for the pneumococ
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A CcpA homologue exists in the pneu
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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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