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

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Materials and Methods Bacterial strains and media All strains used in this study were constructed in a S. pneumoniae D39 genetic background (NCTC 7466; serotype 2) and are listed in Table 1. The glnAP mutant was constructed by transformation of chromosomal DNA from the glnP deletion mutant to the glnA mutant and selecting for erythromycin resistance (0.25 µg/ml) on GM17 agar plates supplemented with 1% sheep blood (Johnny Rottier, Kloosterzande, The Netherlands). Bacteria were checked for spectinomycin (150 µg/ml) resistance and for the glnA mutation by PCR. Stocks were frozen in 10% glycerol and stored at -80°C until further use. Table 1. Bacterial strains used in this study. Strain Mutation Function D39 / TIGR4 accession# Resistance cassette Reference Δcps cps type 2 capsule locus spd0315-spd0328 kanamycin (26) ΔglnR glnR glutamine synthetase repressor spd0447 / sp0501 none a (17) ΔglnA glnA glutamine synthetase spd0448 / sp0502 spectinomycin (17) ΔglnRA glnR, glnA a , spectinomycin (17) ΔglnP glnP glutamine/glutamate transporter spd1098 / sp1241 spectinomycin (17) ΔglnAP glnA, glnP spectinomycin, erythromycin this study ΔgdhA gdhA glutamate dehydrogenase spd1158 / sp1306 erythromycin (17) a premature stop codon introduced GlnR-regulon and pneumococcal virulence In vitro adherence Adherence of pneumococci to epithelial cells was studied essentially as described previously (4, 17). In short, the human pharyngeal cell line Detroit 562 (ATCC CCL-138) was cultured in RPMI 1640 without phenol red (Invitrogen) supplemented with 1 mM sodium pyruvate and 10% fetal calf serum (FCS). Aliquots of bacteria (grown to mid-log in GM17broth) stored at -80°C were thawed rapidly, harvested by centrifugation, and resuspended in RPMI 1640 medium without phenol red supplemented with 1% FCS to 1x10 7 colony forming units (CFU) per ml. Monolayers of Detroit 562 in 24-well tissue culture plates were washed twice with 1 ml PBS, and 1 ml of bacterial suspension was allowed to adhere for 2h at 37°C in a 5% CO2 atmosphere. Non-adherent bacteria were removed by three washes with 1 ml PBS, after which the Detroit 562 cells were detached by adding 200 µl of trypsin/EDTA and lysed by the addition of 800 µl of ice-cold 0.025% Triton X-100 in PBS. Serial 10-fold 161 161
Chapter 6 dilutions were plated on blood agar plates to count the number of adherent bacteria, and corrected to account for small differences in count in the initial inoculum. All experiments were performed in triplicate and repeated at least three times. Significant differences between wild-type and mutant strains were determined using the Mann Whitney U test (P
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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
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Chapter 3 40. McCluskey, J., J. Hin
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CHAPTER 4 CodY of Streptococcus pne
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Introduction Bacteria encounter var
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Materials and Methods Bacterial str
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Table 2. Oligonucleotide primers us
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The CodY regulon of S. pneumoniae I
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The CodY regulon of S. pneumoniae w
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Accession numbers The microarray da
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Table 3. Differentially expressed g
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Binding of CodY to target promoters
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The CodY regulon of S. pneumoniae E
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The CodY regulon of S. pneumoniae F
Page 112 and 113: Discussion CodY has been described
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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 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 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
Page 188 and 189: Gene regulation and metabolism in S
Page 190 and 191: makes it possible for the pneumococ
Page 192 and 193: A CcpA homologue exists in the pneu
Page 194 and 195: double mutant, suggesting direct re
Page 196 and 197: References Gene regulation and meta
Page 198 and 199: 23. Kerr, A. R., P. V. Adrian, S. E
Page 200 and 201: 45. Sonenshein, A. L. 2005. CodY, a
Page 202 and 203: CHAPTER 8 Summarizing discussion 20
Page 204 and 205: that the observed virulence phenoty
Page 206 and 207: CodY was found to be required for a
Page 208 and 209: References Summarizing discussion 1
Page 210 and 211: 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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