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

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adjacent genes, encoding a MerR family transcriptional regulator (sp1856) and czcD (sp1857), a Zn 2+ -efflux pump (Table 3). Four genes were downregulated: guaC, encoding GMP reductase (sp1249), ply, encoding pneumolysin (sp1923), and two genes encoding the B and C component of a putative cellobiose-specific PTS (sp2022 and sp2023). The complete set of differentially expressed genes in TIGR4∆psaR is given in Table 3. Proteome analysis of ΔpsaR in D39 and TIGR4 To examine if the observed PsaR-mediated differences in gene expression corresponded with changes in protein expression, we performed SILAC for both wild-type strains and their psaR mutants. To this end, wild-type and isogenic ΔpsaR counterparts were cultured in the presence of stable isotope-labeled (heavy, 13 C6) or normal (light, 12 C6) L-lysine and L-arginine, respectively. Equal amounts of bacteria were mixed and analyzed by mass spectrometry, after which changes in protein expression between wild-type and isogenic ΔpsaR strains were derived from the corresponding heavy to light peptide ratios. In D39ΔpsaR, nine proteins were found to be more abundant than in the wild-type (Table 3). Most pronounced were FtsY, PsaA, SufS, Cps2G, CysK, and PrtA, all of which displayed expression levels at least eight times higher in the psaR-mutant than in the wildtype. No proteins were identified that were less abundant in D39ΔpsaR (Table 3). In TIGR4ΔpsaR, six proteins were more abundant than in the wild-type. Most pronounced were PcpA, PsaA, and RrgB, all with at least a four-fold increase in expression in the psaR-mutant (Table 3). Three proteins were less abundant in TIGR4ΔpsaR, i.e., an ABCtransporter of a putative bacteriocin system (sp0148), TrxB, and ZmpB (Table 3). Figure 1. The number of genes differentially expressed in D39ΔpsaR and TIGR4ΔpsaR. PsaR and pneumococcal virulence 71 71
Chapter 3 Table 3. Differentially expressed genes/proteins in ΔpsaR of D39 and/or TIGR4. Ratios are given as log2- transformed expression of ΔpsaR / wild-type. 72 72 Identifier Gene Annotation (www.kegg.com) Microarray SILAC TIGR4 D39 TIGR4 D39 TIGR4 D39 Upregulated sp0232 spd0215 infA translation initiation factor IF-1 2.4 sp0303 spd0277 bglA 6-phospho-beta-glucosidase 1.1 sp0306 spd0280 transcriptional regulator, putative 1.2 sp0308 spd0281 PTS system, cellobiose-specific IIA component 1.5 sp0310 spd0283 PTS system, cellobiose-specific IIC component 1.2 - spd0322 cps2G glycosyl transferase, group 1 family protein 4.5 sp0459 spd0420 pflB formate acetyltransferase 0.8 sp0461 - rlrA transcriptional regulator 2.7 sp0462 - rrgA cell wall surface anchor family protein 3.1 sp0463 - rrgB cell wall surface anchor family protein 3.7 2.0 sp0464 - rrgC cell wall surface anchor family protein 3.7 sp0467 - sortase SrtC, putative 2.5 sp0468 - sortase SrtD, putative 2.6 sp0641 spd0558 prtA serine protease, subtilase family PrtA 3.6 3.0 3.9 sp0869 spd0764 sufS cysteine desulfurases, SufS subfamily protein 5.0 sp1244 spd1101 ftsY signal recognition particle-docking protein FtsY 7.6 sp1517 spd1345 greA transcription elongation factor GreA 2.9 sp1531 spd1360 hypothetical protein 2.7 sp1636 spd1448 Rrf2 family protein 1.3 sp1637 spd1449 hypothetical protein 1.4 1.2 sp1647 spd1460 pepO endopeptidase O 0.6 sp1648 spd1461 psaB manganese ABC transporter, ATP-binding protein 3.6 3.3 sp1649 spd1462 psaC manganese ABC transporter permease 2.8 2.4 sp1650 spd1463 psaA ABC transporter, substrate binding lipoprotein 5.4 5.1 sp1805 spd1591 hypothetical protein 0.6 sp1856 spd1637 transcriptional regulator, MerR family 1.2 sp1857 spd1638 czcD cation efflux system protein 1.7 sp2136 spd1965 pcpA choline binding protein PcpA 4.0 4.6 7.4 sp2210 spd2037 cysK cysteine synthase A 4.0 Downregulated sp0112 spd0109 polar amino acid transport system substrate-binding protein -1.2 sp0138 spd0141 hypothetical protein -1.7 sp0139 spd0142 hypothetical protein -1.8 sp0140 spd0143 ugd UDP-glucose/GDP-mannose dehydrogenase -1.7 sp0141 spd0144 mutR positive transcriptional regulator of MutA -1.2 sp0142 spd0145 hypothetical protein -1.0 sp0143 spd0146 hypothetical protein -1.9 sp0144 spd0147 hypothetical protein -1.5 sp0145 spd0148 hypothetical protein -2.0 sp0146 spd0149 hypothetical protein -1.7 sp0148 spd0150 ABC transporter, substrate-binding protein -2.1 sp0524 spd0466 BlpT protein, fusion -1.5 sp0525 spd0467 blpS BlpS protein -1.4 sp0526 spd0468 blpR response regulator BlpR (TCS13) -1.2 sp0527 spd0469 blpH sensor histidine kinase BlpH, putative (TCS13) -1.1 sp0529 spd0471 blpB BlpC ABC transporter -1.5 sp0530 spd0472 blpA BlpA, pseudogene -1.5 sp0533 spd0046 blpK bacteriocin BlpU (highly similar to sp0533) -1.5 sp0541 spd0046 blpO bacteriocin BlpO (5' highly similar to sp0541) -1.5 sp0545 spd0473 blpY immunity protein BlpY -2.4 sp0546 spd0474 blpZ BlpZ protein, fusion -1.2 sp0547 spd0475 hypothetical protein -2.4 sp0664 spd0577 zmpB zinc metalloprotease ZmpB, putative -0.8
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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.
Page 22 and 23: 33. Li, S., S. J. Kelly, E. Lamani,
Page 24 and 25: 53. Romero, P., R. Lopez, and E. Ga
Page 26 and 27: CHAPTER 2 Regulation of gene expres
Page 28 and 29: Introduction Streptococcus pneumoni
Page 30 and 31: Materiaals and Meethods Gene regula
Page 32 and 33: (Invitrogen), 0.2 mM aminoallyl dUT
Page 34 and 35: Isolation of pneumococcal RNA durin
Page 36 and 37: Resultss and Discuussion Gene regul
Page 38 and 39: Table 1. Genes regulated by RR09 in
Page 40 and 41: phosphofructokinase, and a fructose
Page 42 and 43: 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: Chapter 3 Results Transcriptional a
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
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CHAPTER 5 Regulation of glutamine a
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Introduction Regulation of nitrogen
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Materials and Methods Nitrogen Meta
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TK136 D39 Δcps; Km R capsule-less
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gene from pORI28, was fused to the
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Construction of lacZ fusions Chromo
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Enzyme assays Cell-free extracts, u
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Reverse-transcriptase PCR RNA isola
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Table 3. Summary of transcriptome c
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effect caused by altered intracellu
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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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