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

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The CodY regulon of S. pneumoniae EMSA of the gdhA promoter revealed the presence of two retarded protein-DNA complexes, a major one with lowest mobility seen at intermediate to high concentrations of CodY, and one with intermediate mobility seen with lower concentrations of CodY only in the presence of BCAAs (Fig. 1A). Similar patterns were observed for other promoters tested (not shown). To gain further insight into the binding of CodY to the gdhA promoter region, the sequence matching the L. lactis consensus sequence was replaced with random nucleotides and tested by EMSA (Fig. 1B). The distance of this consensus sequence to the startcodon is 79 bp. Without its putative binding domain, CodY did bind the DNA, but only at high concentrations, i.e., 1000 nM and 2000 nM. Furthermore, only the intermediately shifted band was seen. The major complex might consist of the promoter region to which two (or more) CodY dimers have bound, one to the CodY-box, and one to a secondary binding site (Fig. 1A). This putative secondary binding site would still be present in the mutated gdhA promoter fragment, resulting in an intermediate shift. Next, the upstream sequence of the gene psaR (sp1638), to which CodY is not able to bind under any condition (Fig. 1C), was mutated in such a way that the CodY-box consensus sequence was introduced. Although no CodY binding to this DNA-fragment was observed without BCAAs, a clear shift was observed upon addition of BCAAs (Kd of 400 nM), indicating that this sequence is indeed involved in protein-DNA interaction (Fig. 1D). Only a complex with intermediate mobility was seen, indicative of binding of one CodY dimer to the introduced CodY-box. In comparison to the PgdhA with CodY-box, this intermediate shift occurs at lower CodY concentrations, suggesting that CodY has a higher affinity for the CodY-box than for the putative secondary binding site. DNase 1 Footprinting of the gdhA and codY promoters To determine the CodY binding sites in more detail, we performed DNase I footprint assays. First, H6-CodY was incubated with a 160-bp DNA-fragment containing the promoter region of codY, both without and with the addition of BCAAs (10 mM ILV) or BCAAs (10 mM ILV) and GTP (5 mM). Increased protection from DNase1 was observed with increasing concentrations of H6-CodY (Fig. 2A). This protection was not enhanced by addition of GTP (data not shown) or, surprisingly, BCAAs (Fig. 2A). Three protected regions were identified, one of which contained the putative CodY-box (region I, Fig. 2A). 107 107
Chapter 4 Figure 2. DNase1 Footprinting using H6-CodY. DNase1 footprint of the (A) codY- and (B) gdhA-promotor regions, in the absence or presence of BCAA (10 mM ILV). In each series, successive lanes contained increasing amounts of CodY (0, 2, 5, 10, 20 and 40 μM). Protected regions are marked by vertical bars and underlined in the DNA sequence (bottom). The CodY-boxes are given in bold fonts and the startcodons are given in italic and bold fonts. Additional protected sequences were found to be AT-rich DNA sequences (region II and III, Fig. 2A). Next, the 178-bp region upstream of gdhA was incubated with H6-CodY. Again, we did not observe any effect of addition of BCAA (Fig. 2B) or GTP (data not shown). Three regions were protected from DNase 1 degradation. One large region, similar to that observed in the codY promoter region, contained the putative CodY-box (region I, Fig. 2B). The second region was a T-rich domain (region II, Fig. 2B). The third region was an additional region downstream of region I. At higher CodY concentrations region I and III appeared to form a large protected DNA sequence of approximately 120 bp (Fig. 2B). Apart from the CodY-box, the codY and gdhA promoter regions bear no clear similarities. 108 108
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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
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 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 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
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
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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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