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

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gene from pORI28, was fused to the flanking regions of glnP, which were PCR amplified with primer pairs glnPKO-1/glnPKO-2 (628 bp) and glnPKO-3/glnPKO-4 (610 bp), by means of overlap-extension PCR (42). The resulting PCR product was transformed to S. pneumoniae D39, and clones were checked for the presence of the mutation by PCR. In the same way a deletion mutant of gdhA, removing 1311 bp of the gdhA ORF, was constructed in D39 using primer pairs gdhAKO-1/gdhAKO-2 (479 bp) and gdhAKO-3/gdhAKO-4 (498 bp). Construction of capsule-less derivatives of D39 and its glnA, glnR, glnRA and glnP mutants was done as described (35), using primers PE21 and FI4. Mutants were checked by PCR and appearance. In addition, they adhere several orders of magnitude better than the encapsulated mutants. Nitrogen Metabolism in S. pneumoniae Table 2. Oligonucleotide primers used in this study. * Stop-mutations are indicated in bold. Overlap of primers glnR-stop 1 and 2 is in italics. # 6xHis-tag in bold. $ Extra codon for lysine in italics. ** Overlap with emR gene from pORI28 in bold. Name Nucleotide sequence (5’ to 3’); Restriction restriction enzyme sites underlined site Ery-for GCATGCATCGATTAGATCTC - Ery-rev TAACGATTATGCCGATAACT - Spec_pORI38-Fp CCCCAAGCTTCTAATCAAAATAGTGAGGAGG HindIII Spec_pORI38-Rp CCCCAAGCTTACTAAACGAAATAAACGC HindIII R6_glnA-5 TGCTCTAGACTTTCTTCTATATTAGTATTAGTAAAGGTC XbaI R6_glnA-6 CGGAATTCGGTATTGCTGGATCAGGTATGCAC EcoRI R6_PglnP-1 CGGAATTCCGAAAGTCACTTGTTTTATCC EcoRI R6_PglnP-2 TGCTCTAGAAGGACATCTGAATGTTCCAGC XbaI R6_gdhA-4 CGGAATTCCGATGAAAACGGTATCGACTTCG EcoRI R6_gdhA-5 TGCTCTAGACTTAAACAATACCTTGTGCAATCATAGC XbaI R6_G6PDH-1 CGGAATTCGCTCTGGTAAGATTGATGG EcoRI R6_G6PDH-2 TGCTCTAGATTTTTATTCTAAGCGACCATC XbaI R6_arcA-3 TGCTCTAGACCGAGAAGACCTGAATTGGATGTG XbaI R6_arcA-4 CGGAATTCGGTAGGCTCAGGATTTTTCTCC EcoRI Pg6pdh-1 CGGAATTCGAAAGAGTTCTTAGATAAGG EcoRI Pg6pdh-2 TGCTCTAGACGAAAATTGTAACAATAACC XbaI glnP-OX1 CGAGCCATCATGAAGAAAAAATTTCTAGCATTTTTGC RcaI glnP-OX2 TGCTCTAGAGTAGAAAAACTGCAAGGAAATCC XbaI glnR_R6-9 GCCATCATGAAGGAAAAAGAATTTCGCC RcaI glnR-9-his #$ CGAGCCATCATGAAACATCATCATCATCATCATAAGGAAAA AGAATTTCGCCG RcaI glnR_R6-10 GCTCTAGAGTTTTCTCCTTAATCTATGAC XbaI 129 129
Chapter 5 glnA-his # GGCGGTCTCTCATGCATCATCATCATCATCATCCAATCACAG 130 130 CTGCAGATATTCG glnA_R6-8 GCTCTAGATGTGACTGTTTACCCACAG XbaI glnA_R6-1 GAATTCCATATGGGAAATAGTATCGGTGGAC NdeI glnA_R6-2 CGGCGACGTCGCAGCTGTGATTGGCATAAG AatII glnA_R6-3 CATGCCATGGGGAAATTGATAATTATTTAGACC NcoI glnA_R6-4 GAAGATCTCTAGACGTATCTATATACC BglII glnR_R6-1 TGCTCTAGAGGTGGTGACTCAGCTGCCGC XbaI glnR_R6-2 CGGGATCCCCTTCATTTACAATTTCCTTC BamHI glnR-stop 1 * CGGTCTACTACATAACACTGCCGATAGGAAAAACAGCC - glnR-stop 2 * CAGTGTTATGTAGTAGACCGATCTATCGGCGCGTCAG - glnR-3 GAAGATCTACTTCAAGTGTTGGGTCCCC BglII glnPKO-1 AAAAAGTGCCAAGCCTAGAC - glnPKO-2 ** GAGATCTAATCGATGCATGCTACCTAATGAGAAAATTGGG - glnPKO-3 ** AGTTATCGGCATAATCGTTACGAAACGCTTAGAAAAGAGG - glnPKO-4 TAATCATGGTCATGCCTTGC - gdhAKO-1 GATCGATTTGCCCTGTTCTTG - gdhAKO-2 ** GAGATCTAATCGATGCATGCGCAGATGTCATATCGTTCTCC - gdhAKO-3 ** AGTTATCGGCATAATCGTTACTATGATTGCACAAGGTATTG - gdhAKO-4 AAGGAGTAGAGATGGCTATAG - G6PDH-4 TGCTCTAGACCAACACTTCAACCTCTTCCC XbaI G6PDH-5 CGGGATCCCCGAAAATTGTAACAATAACC BamHI G6PDH-6 CATGCCATGGTCACCTATCGTCAAGATGGTCG NcoI G6PDH-7 GAAGATCTGGCCCACCGCTACAAACAAGC BglII PgdhA-2 TGCTCTAGATCTTTAGCAGATGTCATATC XbaI PgdhA-3 TGCTCTAGACATAAGACATTATAGCAG XbaI PgdhA-4 CGGAATTCGATATTTCCAAGAAAAACGTTCG EcoRI PE21 CTGGAACAACCATGACCTCCCTCG - FI4 CGCTGAACTTTTGTAGTTGCTTGGTCAAC - R6_glnP-1 CGGAATTCCATTTTTGAAGCTTGGAAGTC Footprinting R6_glnR-7 GCTCTAGACTGCCGATAGGAAAAACAGCC Footprinting R6_PglnR_FP TTGTACGTGTTTGTGCGTG Footprinting R6_glnP-GFP1 CGGAATTCCATTTTTAGTCTCCTTTTCCG Footprinting PglnR-2 CGGAATTCCGATATTGATCGTATTCGTC EMSA PglnR-3 CGGAATTCATTATCAATTGACGTTTGTC EMSA PglnPQ-1 CGGAATTCGGCACTTTTTAATAGCAATTCAAG EMSA PglnPQ-2 TGCTCTAGAGGAATAAAATTAGCAAAAATGC EMSA PglnPQ-3 CGGAATTCGCGAAAAATATAACAATTTGCC EMSA BsaI
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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
Page 79 and 80: Chapter 3 effect was more severe fo
Page 81 and 82: Chapter 3 in TIGR4ΔpsaR. However,
Page 83 and 84: Chapter 3 References 1. Adrian, P.
Page 85 and 86: 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 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 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
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12. Ibrahim, Y. M., A. R. Kerr, J.
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33. Zalieckas, J. M., L. V. Wray, J
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CHAPTER 7 Pneumococcal gene regulat
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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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