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

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Reverse-transcriptase PCR RNA isolation and cDNA synthesis was performed as described above, except that aminoallyl-dUTP was replaced by dTTP during cDNA synthesis. To confirm the absence of DNA contamination, reactions were also carried out without reverse transcriptase. 100 ng cDNA was used for each PCR reaction, and after 20 amplification cycles (30 sec, 95ºC; 30 sec, 52ºC; 60 sec, 72ºC) with primers G6PDH4 and G6PDH5 reactions were analysed on 1% agarose gels. Adhesion assays Nitrogen Metabolism in S. pneumoniae Adhesion of pneumococci to epithelial cells was studied essentially as desribed previously (21). Briefly, the human pharyngeal cell line Detroit 562 (ATCC CCL-138) was cultured in RPMI 1640 without phenol red (Invitrogen) containing 1 mM sodium pyruvate and 10% fetal calf serum (FCS). Aliquots of bacteria, grown to mid-exponential phase in GM17 and stored till use at -80°C, were thawed rapidly, harvested by centrifugation, and resuspended to 1x10 7 CFU/ml in RPMI 1640 medium without phenol red containing 1% FCS. Monolayers of Detroit 562 in 24-well tissue culture plates were washed twice with 1 ml PBS, after which 1 ml of bacterial suspension was allowed to adhere for 2h at 37°C in a 5% CO2 atmosphere. Subsequently, non-adherent bacteria were removed by three washes with 1 ml PBS, and the epithelial cells were detached by treatment with 200 μl of 25% Trypsin, 1 mM EDTA in PBS. Detroit 562 cells were lysed by the addition of 800 μl of ice-cold 0.025% Triton X-100 in PBS, and appropriate dilutions were plated on blood agar plates to count the number of adherent bacteria. This CFU count was first corrected mathematically to account for small differences in count in the initial inoculum, after which data were normalized so that the adhesion of the wild-type strain TK136 was expressed as 100%. Wild type and mutant pneumococci grew comparably in RMPI medium without Detroit 562 cells. All experiments were performed in triplicate and repeated at least three times. Significant differences between wild-type and mutants were calculated by the Mann Whitney t-test (p
Chapter 5 Results Prediction of putative GlnR operators in S. pneumoniae B. subtilis GlnR is known to repress genes that contain two copies of the inverted repeat 5’-TGTNAN7TNACA-3’ in their promoters (7, 8). This repeat is also present in the promoter regions of the Lactobacillus rhamnosus (48) and Bacillus cereus (33) glnRA operons. As the GlnR binding box seems so well conserved between species, we screened the entire genome of S. peumoniae R6 for the presence of putative GlnR operators using Genome2D (3). -42 TGGTGCCCCCAAAAAAGTCAAAATTTTTAAGGAGGAAAATACATG--->arcA Figure. 1. Nucleotide sequences of the promoter regions of the indicated genes/operons of S. pneumoniae R6. Predicted -35 and (extended) -10 core promoter regions are underlined. Putative GlnR operators are boxed. Translational starts are in italics. Numbers indicate the base-positions relative to the translational start. A predicted CodY operator in the gdhA promoter is underlined with a dotted line. Bases in PglnR and PglnP that are in bold were protected in the DNAseI footprinting analyses (Fig. 6B) and vertical arrows below the sequences indicate hypersensitive bases. Horizontal arrows above PglnR (PglnR-1 and PglnR-2), PglnP (PglnP-1 and PglnP-2) and PgdhA (PgdhA-1 and PgdhA-2) indicate the locations of the primers used to make the promoter-truncations as used for Figures 4C and 6A. 136 136 PglnR →PglnR-1 -127 AATTACGATATTGATCGTATTCGTCTCTTTTTAGAGAAAAAAGAAAAATAATGTTACATTTTA ↑ →PglnR-2 -64 TAACATTATCAATTGACGTTTGTCTTTTTTTAGACTATAATAGACAGAAAGAAGGAAATTGTA -1 AATG—->glnRA PglnP →PglnP-1 -142 ATTATTATAGCACTTTTTAATAGCAATTCAAGAAAGAAAAGGAAAAATCAATGTTATATTTTC ↑ →PglnP-2 -79 TGACACGAAAAATATAACAATTTGCCTTTTTTTACTTTTTCTGATATAATGAGAGAATATTCG ↑ ↑ ↑ -16 GAAAAGGAGACTAAAAATG--->glnPQzwf PgdhA -104 GAATTGAAAGAATTTTTAGAAAATTTCTGTTTTTTCTTTACAGTGAAAAAAAATTCTGCTATA ←PgdhA-2 ←PgdhA-1 -41 ATGTCTTATGTAATAAAATATGACAATAAAAGGAGAACGATATGACATCTGCTAAAGA--->gdhA ParcA -168 ATTCCATCCTATGTTATGTATAATAACATAAAGTTGTGCTAAATATCATAATTTATATTATTT -105 TCCGAGAAATTATAGCCATTGACATTACCTAGGAATTGCGTTATAATATACATGTAAGCGGTA
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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.
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 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 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
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
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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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