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

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Materials and Methods Bacterial strains and media Bacterial strains and plasmids used in this study are listed in Table 1. All pneumococcal strains used in this study were grown in Todd-Hewitt Yeast broth at 37�C or on Colombia base agar supplemented with 5% sheep blood (Biotrading). Pneumococcal strains were maintained in 10% glycerol, 10% skim milk at -80�C. Escherichia coli DH5α (Table 1) was grown in Luria Broth at 37�C while shaking or on Luria Broth agar supplemented with appropriate antibiotics (50 mg/l ampicilin and/or 20 mg/l trimethoprim). Table 1. Bacterial strains and plasmids used in this study. Strains E. coli Antibiotic resistance Reference BL21 (DE3) Stratagene DH5α Stratagene S. pneumoniae D39 wild-type NCTC 7466 D39 ΔcodY trimethoprim this study D39 Δcps kanamycin (7) D39 ΔcpsΔcodY kanamycin; trimethoprim this study D39 ΔcpsΔpcpA spectinomycin; kanamycin this study D39 ΔcpsΔcodYΔpcpA kanamycin; trimethoprim; erythromycin this study Plasmids pBluescript KS+ Stratagene pCR2.1 Invitrogen pET11C New England Biolabs pR412T7 (4) pKOT This study pKOCOD This study Construction of mutant strains The CodY regulon of S. pneumoniae The gene encoding codY (spd1412) was deleted from strain D39 by allelic replacement with the dfr13 cassette conferring trimethoprim resistance (2). To this end, codY with 1000 bp of upstream and downstream flanking sequences was amplified from chromosomal D39 DNA using primer pair CodSacFwd and CodKpnRv (Table 2). This amplicon was cloned into pBlueScript KS+. Coding DNA of codY was deleted from the plasmid by performing an inverse PCR with primer pair CodHindFwdinv and CodPstRvinv, amplifying the codY- flanking sequences and pBlueScript KS+ and introducing HindIII and PstI restriction sites for further cloning. This amplicon was ligated with the dfr13-cassette excised from pKOT with 93 93
Chapter 4 HindIII and PstI to create the knockout construct pKOCOD, and transformed to E. coli DH5α. A 2660-bp linear DNA fragment containing codY-flanking DNA and dfr13 was amplified from pKOCOD using primer pair CodSacFwd and CodKpnRv. This PCR product was used to delete codY from the genome of S. pneumoniae D39 by CSP-1-induced (100 ng/ml) transformation. Transformants were selected on the basis of trimethoprim-resistance and were checked by PCR for recombination at the desired location on the chromosome. Wild-type D39 was subsequently transformed with chromosomal DNA isolated from these transformants to rule out the possibility of any additional mutations on the chromosome. 94 94 The pcpA (spd1965) deletion mutants were constructed by allelic replacement with the spectinomycin-resistance cassette of plasmid pR412T7 as follows. Primers pcpA_L1/pcpA_L2 and pcpA_R1/pcpA_R2 were used to generate PCR products of the left and right flanking regions of pcpA (approximately 500 bp each) (Table 2). These PCR products were fused to the spectinomycin-resistance gene amplified with primers pR412_L and pR412_R by means of overlap extension PCR. The resulting PCR product was transformed to S. pneumoniae D39Δcps and transformants were checked for the presence of the mutation by PCR. Transcriptional profiling of D39ΔcodY Microarray analysis was performed essentially as described (18). In short, 500 ml of THY-broth was inoculated with 10-20 colonies from agar plates, and these cultures were statically grown at 37°C. In all experiments, D39 wild-type and ΔcodY displayed comparable growth characteristics. Samples for RNA isolation were taken when the cultures reached an optical density (OD600) of either 0.1 or 0.2 (early and mid-log growth, respectively). RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (18). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures. Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (18, 48). In all cases, dye-swapping was performed with one of the three biological replicates. Microarrays used in this study were constructed as described (18, 28) and contain PCR amplicons representing 2,087 ORFs of S. pneumoniae TIGR4 and 184 ORFs unique for S. pneumoniae R6, all spotted in duplicate.
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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
Page 44 and 45: correlated well with our in vitro m
Page 46 and 47: Figure 7. Bacterial loads in blood
Page 48 and 49: Acknowledgments This work was suppo
Page 50 and 51: 10. Ibrahim, Y. M., A. R. Kerr, J.
Page 52 and 53: genome sequence of a virulent isola
Page 54 and 55: Gene regulation by RR09 in S. pneum
Page 56 and 57: Gene regulation by RR09 in S. pneum
Page 58 and 59: Table S4. Genes up- and downregulat
Page 60: Table S6. Genes up- and downregulat
Page 63 and 64: Chapter 3 Abstract 62 62 Previous s
Page 65 and 66: Chapter 3 between the wild-type and
Page 67 and 68: Chapter 3 S. pneumoniae TIGR4 by CS
Page 69 and 70: Chapter 3 performance liquid chroma
Page 71 and 72: Chapter 3 Results Transcriptional a
Page 73 and 74: 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 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 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
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(http://www.bd.com/ds/technicalCent
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H6-GlnR alone at the concentration
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Discussion In this study, we charac
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Acknowledgements TK, JB and HB are
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11. den Hengst, C. D., S. A. van Hi
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Nitrogen Metabolism in S. pneumonia
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52. Wray, L. V., Jr., J. M. Zalieck
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CHAPTER 6 Site-specific contributio
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Introduction GlnR-regulon and pneum
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Materials and Methods Bacterial str
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eferred to as t=0. Bacteriology res
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Results GlnR-regulon and pneumococc
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Figure 3. Colonization model. Bacte
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GlnR-regulon and pneumococcal virul
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Table 2. Differentially expressed g
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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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oekje en ik kijk uit naar de bijbeh
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