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

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Introduction Bacteria encounter various environmental cues during their life cycle to which they need to respond appropriately in order to survive. Different niches within a host are likely to provide different nutritional challenges to the microorganism. Such alterations in bacterial gene expression in response to environmental changes are under strict control of transcriptional regulators. CodY is a global nutritional repressor, which is highly conserved in low G+C bacteria and has been investigated extensively in Bacillus subtilis, Lactococcus lactis, Streptococcus pyogenes and very recently in Listeria monocyogenes (3, 16, 33, 35, 39, 42). B. subtilis CodY represses the transcription of over a 100 genes during exponential growth, which are involved in different metabolic pathways and cellular processes, such as peptide uptake, development of genetic competence, branched-chain amino acid biosynthesis, motility, and sugar uptake (35, 38). There is one exception where CodY functions as an activator in B. subtilis: the gene encoding acetate kinase is positively regulated by CodY and the carbon regulator CcpA, possibly ensuring that an overflow pathway for carbon metabolism is active (40). Functional studies have shown that the DNA-binding activity of B. subtilis CodY is enhanced by both GTP and the branched-chain amino acids (BCAAs) isoleucine, leucine, and valine (23, 41). The crystal structure of two fragments of B. subtilis CodY, containing its cofactor and DNA binding domains, revealed that the regulatory protein interacts with DNA as a dimer (31). L. lactis CodY DNA-binding to its recently identified binding consensus is enhanced by BCAAs, but not by GTP (13, 14, 17, 36). Differences between these needs for cofactors of B. subtilis and L. lactis CodY might reflect the physiology of these bacteria. For instance, GTP plays a major role in the development of sporulation in B. subtilis, a process absent from the life cycle of L. lactis. Moreover, in B. subtilis, low levels of GTP induce development of competence by relieve of CodY-repression of comK, a critical competence regulator (20, 38). In contrast, natural transformation has never been observed in L. lactis. Interestingly, the lactococcal genome sequence revealed the presence of orthologues of several genes involved in natural transformation in other bacteria (6, 29), but none of these putative competence genes belong to the lactococcal CodY-regulon (14). The CodY regulon of S. pneumoniae Another process in which GTP plays a major part is the stringent response. This is a response of a bacterium to amino acid starvation, during which the signal molecule ppGpp is accumulated, resulting in a shut down of the synthesis of many rRNAs and tRNAs (21, 42). An essential factor in the accumulation of ppGpp is the ribosome-bound protein RelA, which converts GTP to ppGpp. In S. pyogenes a RelA-independent response to amino acid starvation 91 91
Chapter 4 is found that is, at least in part, regulated by CodY (44, 45). Among the genes repressed during this response are virulence factors such as those encoded by graB, speB, speH (33). Interestingly, CodY induced expression of pel/sagA and mga, genes encoding regulatory proteins that themselves positively affect the expression of numerous other virulence factors. This observation suggests a clear link between nutritional regulation and virulence in S. pyogenes (33). 92 92 Streptococcus pneumoniae (the pneumococcus) is a human pathogen, which causes diseases such as meningitis, pneumonia, and otitis media, in the young, elderly and immunocompromised (5). Pneumococcal disease is preceded by colonization of the nasopharynx, which is asymptomatic. From there it can develop into disease under the appropriate conditions. Analysis of the genomes of S. pneumoniae R6 (19), D39 (30), and TIGR4 (46) revealed that CodY orthologs are present on the chromosomes of these strains (spr1439 for R6, spd1412 for D39, and sp1584 for TIGR4). Here, we report on the physiological role of CodY in S. pneumoniae D39 in global transcription, translation, and DNA-binding. We show that the pneumococcal CodY-regulon consists mainly of genes that are involved in amino acid metabolism, biosynthesis, and uptake. Binding of CodY to its target promoters requires a 15bp recognition site, and is enhanced by BCAAs but not by GTP. Furthermore, we demonstrate that CodY is required for optimal levels of in vitro adherence and colonization of the murine nasopharynx.
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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
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 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 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 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
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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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