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

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examine whether zwf is only transcribed from PglnP, or also from a possible promoter in the glnQ-zwf intergenic region, the latter was cloned upstream of lacZ in pORI13 and introduced in the RepA + strain D39repA (23) and its glnR mutant. Promoter activity was present in this fragment (~5 Miller units), which was not dependent on GlnR (data not shown). Regulation of zwf by GlnR and GlnA was similar to, but weaker than regulation of glnPQ (Fig. 3B and C), which can be explained by the presence of the second promoter upstream of zwf. Thus, expression of zwf initiates from two promoters, a GlnR-dependent promoter upstream of glnP and a second promoter in the glnQ-zwf intergenic region. Nitrogen Metabolism in S. pneumoniae Figure 4. Expression of gdhA in S. pneumoniae D39 and glnR, glnA and codY mutant derivatives. (A) β- galactosidase activity in strains TK111 (D39nisRK gdhA-lacZ; black bars), TK121 (D39nisRK glnR-stop gdhA- lacZ, white bars) and TK127 (D39nisRK ΔglnA gdhA-lacZ, hatched bars) in the media indicated on the x-axis (see Fig. 2 for abbreviations). (B) GdhA biosynthetic activity in D39 (black bar), TK102 (D39 glnR-stop, white bar), TK104 (D39 ΔglnA hatched bar) and TK strains in GM17Gln. (C) Beta-galactosidase activity in strains TK132 (D39 ΔbgaA::PgdhA-1-lacZ), TK133 (D39 ΔbgaA PgdhA-2-lacZ), TK134 (D39 glnR-stop ΔbgaA::PgdhA-1-lacZ) and TK135 (D39 glnR-stop ΔbgaA::PgdhA-2-lacZ) grown in GM17Gln. Black bars represent D39 background and white bars the glnR mutant background. PgdhA-1, full-length gdhA promoter. PgdhA-2, gdhA promoter without the predicted GlnR operator. (D) GdhA activity in D39, TK102 (D39 glnR- stop), WH101 (D39 ΔcodY) and TK108 (D39 glnR-stop ΔcodY) mutant strains grown in GM17Gln. 141 141
Chapter 5 Regulation of gdhA by GlnR and CodY 142 142 Despite the fully conserved GlnR operator in the gdhA promoter, regulation of gdhA by GlnR and GlnA in GM17Gln and CDM was weaker than regulation of glnPQ and glnRA (Fig. 4A and B). However, expression of an ectopic lacZ fusion to the full-length gdhA promoter (PgdhA-1) and to a truncated version without the GlnR box (PgdhA-2, see also Fig. 1) showed that deletion of the predicted GlnR operator abolished the GlnR-dependent repression of PgdhA (Fig. 4C), demonstrating that the predicted GlnR operator in the gdhA promoter is functional. Interestingly, in the S. pneumoniae R6 genome putative CodY operator sequences are present in the promoter regions of, amongst others, gdhA and zwf (11). To examine whether CodY regulates these genes in S. pneumoniae, the activity of the corresponding enzymes was measured in a codY deletion mutant. No effect of the codY deletion was seen on the activity of Zwf in GM17Gln (data not shown), but activity of GdhA was strongly increased in the codY mutant (Fig. 4D). In a glnRcodY double mutant, GdhA activity was even higher than in the codY mutant, indicating that GlnR and CodY independently repress gdhA in S. pneumoniae (Fig. 4D). glnPQ encodes the main glutamine/glutamate transport operon in S. pneumoniae Of the genes encoding predicted glutamine transporters in the R6 genome (19), glnPQ were the only ones found to be regulated by GlnR. To investigate the role of glnPQ in glutamine metabolism, a deletion of glnP, encoding the permease component of the GlnPQ ABC-transporter, was constructed in D39. Whereas S. pneumoniae D39 is able to grow in CDM containing glutamine (Fig. 5A) or glutamate (23), but not in their absence, the glnP mutant was not able to grow in CDM with either glutamine (Fig. 5A) or glutamate (data not shown). This phenotype could be complemented by in trans expression of glnPQ from a nisin-inducible promoter (Fig. 5A). Moreover, addition of the dipeptide Gly-Gln to the CDM also rescued growth of the glnP mutant (Fig. 5A), while this was not the case with the dipeptide Phe-Gly (data not shown). These data indicate that glnPQ encode the only actively expressed glutamine and glutamate uptake system in S. pneumoniae under these conditions. GlnA activity was increased in the glnP mutant in GM17 (Fig. 5B), although to a lower extent as in the glnR mutant (Fig. 2B). To investigate whether regulation of glnA is affected in the glnP mutant, the effect of casitone as the nitrogen source in the medium was tested. Casitone, an enzymatic (pancreatic) digest of casein, consists of casein-derived peptides and contains no free glutamine and only a very low level of free glutamate
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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
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
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 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 162 and 163: Materials and Methods Bacterial str
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
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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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