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

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Materials and Methods Nitrogen Metabolism in S. pneumoniae Strains, media and growth conditions Strains used in this study are listed in Table 1 and were stored in 10% glycerol at - 80ºC. S. pneumoniae was grown essentially as described (23): on plates in a flame-pot, giving an elevated CO2 concentration, or in liquid medium as standing cultures. L. lactis and E. coli were grown as described previously (23). Kanamycin and tetracycline were used in concentrations of 500 μg/ml and 2.5 μg/ml for S. pneumoniae, respectively. Ampicillin was used in a concentration of 100 μg/ml for E. coli. Chemically defined medium with a final pH of 6.4 was composed as described (23), except that sodium-citrate was used in the buffer instead of ammonium-citrate and that glutamine was omitted from the amino acid mixture. Glutamine, glutamate and ammonium were added as specified in the Results section. Induction of gene expression with nisin was performed as described, using a stock solution of nisaplin, containing 20 mg/ml nisin (23). DNA isolation and manipulation Primers used in this study are listed in Table 2. Primers were based on the genome sequence of strain S. pneumoniae R6 (19). Unless otherwise indicated, chromosomal DNA of S. pneumoniae D39 was used as a template for PCR amplification. All DNA manipulations were done as described (23). Construction of glnR, glnA, glnRA, zwf and glnP mutants of S. pneumoniae The glnR-stop mutant (TK102) was constructed using plasmid pORI280 as follows. Primer glnR-stop 1 with two point mutations, leading to two premature stop codons at codonpositions 20 and 21 in the glnR reading frame, was used in combination with primer glnR_R6-1 to PCR amplify a fragment comprising the upstream part and the beginning of glnR. A second PCR product, comprising the rest of the glnR gene and part of the downstream sequence, was produced with primers glnR-stop 2 and glnR-3. These PCR products were complementary by 20-bp covering the position of the stop codons and were used as a template in a PCR reaction with primers glnR_R6-1 and glnR-3. The resulting product was cloned as an XbaI, BglII fragment in pORI280, giving plasmid pTK20. pTK20 was used to introduce the mutations into the chromosome of S. pneumoniae D39 as described (23), giving strain TK102. The mutations led to the disappearance of a HincII site, on the basis of which the proper mutant could be identified. The mutations were further verified by DNA sequencing. 125 125
Chapter 5 Table 1. Strains and plasmids used in this study. Strain/plasmid Description Reference or source S. pneumoniae 126 126 D39 Serotype 2 strain, cps2 (2), lab. P. Hermans D39repA D39 ΔbgaA::repA; Trmp R (23) D39nisRK D39 ΔbgaA::nisRK; Trmp R (23) TK100 D39nisRK ΔglnA; Spec R This work TK102 D39 glnR-stop, contains two stop mutations in the start of glnR This work TK103 D39 ΔglnA; Spec R This work TK104 D39 ΔglnRA; Spec R This work TK105 TK102 ΔbgaA::nisRK; Trmp R This work TK106 D39 ΔglnP; Em R This work TK107 D39 Δzwf; Spec R This work TK108 D39 ΔgdhA; Em R This work TK109 TK102 ΔbgaA::repA; Trmp R This work WH101 D39 ΔcodY; Trmp R This work TK108 TK102 ΔcodY; Trmp R This work TK109 TK106 ΔbgaA::nisRK; Trmp R This work TK110 D39nisRK glnA-lacZ; Em R This work TK111 D39nisRK PglnP-lacZ; Em R This work TK112 D39nisRK gdhA-lacZ; Em R This work TK113 D39nisRK zwf-lacZ; Em R This work TK114 D39nisRK ParcA-lacZ; Em R This work TK120 TK105 glnA-lacZ; Em R This work TK121 TK105 PglnP-lacZ; Em R This work TK122 TK105 gdhA-lacZ; Em R This work TK123 TK105 zwf-lacZ; Em R This work TK125 TK105 ParcA-lacZ; Em R This work TK126 TK100 glnA-lacZ; Em R This work TK127 TK100 PglnP-lacZ; Em R This work TK129 TK100 gdhA-lacZ; Em R This work TK130 TK100 zwf-lacZ; Em R This work TK131 TK100 ParcA-lacZ; Em R This work TK132 D39 ΔbgaA::PgdhA1-lacZ; Tet R This work TK133 D39 ΔbgaA::PgdhA1-lacZ; Tet R This work TK134 TK102 ΔbgaA::PgdhA2-lacZ; Tet R This work TK135 TK102 ΔbgaA::PgdhA2-lacZ; Tet R This work
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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
Page 75 and 76: 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 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 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
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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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