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

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TK136 D39 Δcps; Km R capsule-less derivative This work TK137 TK102 Δcps; Km R capsule-less derivative This work TK138 TK103 Δcps; Km R capsule-less derivative This work TK139 TK104 Δcps; Km R capsule-less derivative This work TK140 TK106 Δcps; Km R capsule-less derivative This work L. lactis E. coli NZ9000 MG1363ΔpepN::nisRK (27) LL108 MG1363 RepA + (multi-copy); Cm R (29) EC1000 Km R ; MC1000 derivative carrying a single Plasmids copy of the pWV01 repA gene in glgB pORI13 Em R ; ori + repA - ; promoterless lacZ, for single-copy chromosomal lacZ fusions. pORI280 Em R ; ori + repA - ; deletion derivative of pWV01; constitutive lacZ expression from P32 promoter pORI28 * Em R ; ori + repA - ; deletion derivative of pWV01 (30)( (30) (37) (30) pORI38 * Spec R ; ori + repA - ; deletion derivative of pWV01 (30) pNZ8048 Cm R ; Nisin-inducible PnisA (9) pNG8048E Cm R Em R ; Nisin-inducible PnisA, pNZ8048 derivative containing em R gene to facilitate cloning pPP2 Amp R Tet R ; promoter-less lacZ. For replacement of bgaA (spr0565) with promoter-lacZ fusions. Derivative of pPP1. pORI28spec1 * pORI28 containing spec R gene from pORI38 This work pTK8 pORI13 ’glnA-lacZ This work pTK9 pORI13 PglnP-lacZ This work pTK10 pORI13 ’gdhA-lacZ This work pTK11 pORI13 ’zwf-lacZ This work pTK12 pORI13 ParcA-lacZ This work pTK13 pPP2 PgdhA-1-lacZ This work pTK14 pPP2 PgdhA-2-lacZ This work pTK15 pNG8048E carrying His6-glnA downstream of PnisA This work pTK16 pNG8048E carrying His6-glnR downstream of PnisA This work pTK17 pNG8048E carrying glnPQ downstream of PnisA This work pTK18 pORI28spec1 containing 886 bp downstream of glnA This work pTK19 pORI28spec1 Δzwf This work pTK20 pORI280, containing glnR with stop mutations (K20(AAG)�stop(TAG) and L21(TTG)�stop (TAG)) Nitrogen Metabolism in S. pneumoniae Laboratory collection (25) This work 127 127
Chapter 5 128 128 pTK21 pORI13 Pzwf-lacZ This work pTK22 pORI28spec1 containing 883 bp upstream sequence of glnR This work pTK23 pNG8048E carrying glnR downstream of PnisA This work Trmp R , trimethoprim-resistance; Spec R , spectinomycin-resistance; Em R , erythromycin resistance; Tet R , tetracycline resistance; Km R , kanamycin-resistance; Cm R , chloramphenicol resistance. * Plasmid sequences are available at http://molgen.biol.rug.nl/publication/glnRAspn_data. The glnA deletion strain (TK103) was generated by allelic-replacement mutagenesis, removing 1300 bp of the glnA ORF: A PCR fragment, generated with primers Spec_pORI38- Fp and Spec_pORI38-Rp on the spectinomycin resistance gene from pORI38, was cloned into the HindIII site of pORI28 in the same orientation as the erythromycin gene on this vector, yielding pORI28spec1. Next, the 3’ flanking region of glnA, amplified with primer pair glnA_R6-3/glnA_R6-4 (886 bp), was cloned into the NcoI/BglII sites of pORI28spec1, giving pTK18. pTK18 was cut with NdeI/AatII and a PCR fragment generated with primers glnA_R6-1/glnA_R6-2 (808 bp), which was digested with the same enzymes, was ligated to it. This ligation mixture was used to generate a PCR product with primers glnA_R6-1 and glnA_R6-4, which was transformed to S. pneumoniae D39. Spectinomycin resistant clones were examined for the presence of the glnA deletion by PCR and Southern blotting. The zwf deletion mutant (TK107), removing 1416 bp of the zwf ORF, was constructed in a similar way as the glnA mutant, using primers G6PDH-4/G6PDH-5 (660 bp) and G6PDH-6/G6PDH-7 (610 bp). To construct the glnRA mutant (TK104), the upstream part of glnR, amplified with primer pair glnR_R6-1/glnR_R6-2 (883 bp), was cloned into the XbaI/BamHI sites of pORI28spec1, giving pTK22. pTK22 was used together with pTK18, which contains the glnA_R6-3/glnA_R6-4 PCR product cloned into the NcoI/BglII sites of pORI28spec1, as a template in a PCR reaction with primers glnR_R6-1 and glnA_R6-4. In this way a PCR product was obtained containing the spectinomycin resistance gene flanked by the upstream and downstream sequence of glnRA. The resulting PCR product was transformed to D39. The deletion was confirmed by PCR and Southern blotting. L. lactis 108 was used as the cloning host for plasmid pTK19. All other construct were made in E. coli EC1000. To construct the glnP deletion mutant (TK106), removing 2080 bp of the glnP ORF, a PCR fragment, generated with primer pair Ery-rev/Ery-for on the erythromycin resistance
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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
Page 77 and 78: 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 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 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
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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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