THE ROLE OF FERRIC IRON UPTAKE REGULATOR (FUR) PROTEIN ON IRON REGULATION IN COXIELLA BURNETII

  THE ROLE OF FERRIC IRON UPTAKE REGULATOR (FUR) PROTEIN ON IRON REGULATION IN COXIELLA BURNETII      A Senior Honors Thesis by  MARY JEANELL WILSON  Submitted to the Office of Honors Programs & Academic Scholarships  University Texas A&M In partial fulfillment of the requirements of the UNIVERSITY UNDERGRADUATE RESEARCH FELLOWS April 2006 Major: Biochemistry  THE ROLE OF FERRIC IRON UPTAKE REGULATOR (FUR) ON IRON REGULATION IN COXIELLA BURNETII A Senior Honors Thesis by MARY JEANELL WILSON Submitted to the Office of Honors Programs & Academic Scholarships Texas A&M University In partial fulfillment for the designation of UNIVERSITY UNDERGRADUATE RESEARCH FELLOWS Approved as to style and content by: - James E Samuel (Fellows Advisor) Edward A Funkhouser (Executive Director) April 2006 Major: Biochemistry iii ABSTRACT  The Role of Ferric Iron Uptake Regulator (Fur) Protein in Iron Regulation in Coxiella burnetii (April 2006) Mary Jeanell Wilson Department of Biochemistry Texas A&M University Fellows Advisor: James E Samuel Department of Microbial and Molecular Pathogenesis Coxiella burnetii is a gram negative, obligate intracellular bacterium It is the etiologic agent of Q fever in a variety of species including livestock, humans and arthropods The bacterium infects the monocytes of its host and is encapsulated in the phagolysosome, an acidic vacuole meant to kill the bacterium, where it survives and replicates C burnetii must be able to acquire all the nutrients necessary for survival within this acidic environment In all but one species of bacteria, iron has been shown as necessary for replication as is serves as a cofactor for many cellular processes However, iron concentration must be maintained as a delicate balance Too little iron and replication is impeded, while too much iron initiates the iv production of oxygen radicals which are fatal to the cell Ferric iron Uptake Regulator (Fur) is responsible for the regulation of iron acquisition genes in many gram negative bacteria Fur acts as a transcriptional repressor of iron regulated genes These genes have a sequence within their promoter region called the “Fur box” that binds to the Fur protein when the protein is also bound to its co-repressor, Fe2+ In E coli, Fur has been found responsible for the regulation of over 30 genes Previous work showed that C burnetii has a functional fur gene We hypothesize that C burnetii genes that contain promoters with a highly conserved consensus sequence are part of a Fur regulon Our goal is to characterize this regulon Nineteen putative Fur boxes were identified in C burnetii Fifteen of these were cloned into the pBlue plasmid expressing beta-galactosidase These plasmids were then co-transformed with a plasmid expressing Fur into an E.coli fur deletion strain The β-galactosidase assay was then used to test promoter activity Eleven of these promoters were evaluated Three promoters, for open reading frames CBU0970, CBU0769 and feoB were found to be repressed in the presence of iron We predict that although a Fur regulon is present in C burnetii, it includes only a limited set of genes DEDICATION v I would like to dedicate this work to my family for all their support and diversion throughout the course of my efforts ACKNOWLEDGEMENTS I would like to acknowledge Dr Samuel for his willingness and guidance as my advisor and I would like to especially acknowledge Heather Briggs for teaching me vi all the techniques needed for the following work as well as thank her for her encouragement and troubleshooting expertise Thank you to all of the Samuel Lab for your support and laughter! This research was performed while on appointment as a U.S Department of Homeland Security (DHS) Scholar under the DHS Scholarship and Fellowship Program, a program administered by the Oak Ridge Institute for Science and Education (ORISE) for DHS through an interagency agreement with the U.S Department of Energy (DOE) ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-AC05-00OR22750 All opinions expressed in this paper are the author's and not necessarily reflect the policies and views of DHS, DOE, or ORISE." TABLE OF CONTENTS Page ABSTRACT………………………………………………………… iii DEDICATION……………………………………… …….……… v ACKNOWLEDGEMENTS……………………………………… … vi TABLE OF CONTENTS………………………………………… … vii vii LIST OF FIGURES……………………………………………… … LIST OF TABLES…………………………………………………… INTRODUCTION……………………………………………… Coxiella burnetii: Life in the Monocyte…………………… Iron & Iron Regulation… ……………………… …… Summary & Objectives:… …………… … ……… MATERIALS & METHODS………………………………………… RESULTS & DISCUSSION……………….………………………… Cloning Results……………………………………… Beta-Galactosidase Assay Results…………………… Real Time PCR Results……………………………… Conclusions…………………………………………… Future Work………………………………………… REFERENCES……………………………………………………… CURRICULUM VITA…………………………………………….… ix x 10 18 18 23 35 39 40 42 44 viii LIST OF FIGURES FIGURE Page Schematic of Fur-Fe2+ Transcriptional Repression Fur Box Consensus Sequence 10 C burnetii Fur Complementation of an E coli fur deletion 20 CBU0970 and CBU0968 Fur Boxes 21 Comparison of C burnetii putative Fur Boxes to the E coli 22 consensus sequence Methods Schematic 24 Percent Repression of lacZ in heterologous expression systems 27 Real Time PCR Results 38 LIST OF TABLES TABLE Putative Fur Box Primers List of all plasmids used in Cloning and Beta-Galactosidase Assays Beta-Galactosidase Assay Results in Miller Units Page 13 15 26 ix 30 CBU0769 is a glutamine aminotransferase and has a mismatch of base pairs as compared to the feoB Fur Box In the presence of iron, BN4020 pMH15 p769 Fur Box had an activity of 215 ± 53 Miller units by beta-galactosidase assay, and an activity of 593 ± 37 Miller units in the presence of deferoxamine The p value for this data was 0.001, a highly significant difference Therefore, lacZ was shown to be repressed 64% in the presence of iron and E coli fur In the presence of iron, BN4020 pNP104 p769 Fur Box had an activity of 512 ± 38 Miller units by Beta-galactosidase Assay, and an activity of 715 ± 65 Miller units in the presence of deferoxamine The p value for this data was 0.007, a significant value Therefore, lacZ was shown to be repressed 28% in the presence of iron and C burnetii Fur Though both Fur proteins showed significant repression of the promoter, E coli Fur exhibits much tighter control over the promoter than C burnetii Fur CBU0216 In the presence of iron, BN4020 pMH15 p216 Fur Box had an activity of 326 ± 29 Miller units by beta-galactosidase Assay, and an activity of 397 ± 48 Miller units in the presence of deferoxamine The p value for this data was 0.162, not a significant value We concluded that lacZ was not repressed by E coli fur In the 31 presence of iron, BN4020 pNP104 p216 Fur Box had an activity of 952 ± 121 Miller units by beta-galactosidase assay, and an activity of 1163 ± 216 Miller units in the presence of deferoxamine The p value for this data was 0.076 Therefore, lacZ was not shown to be repressed by either Fur CBU0280 This protein has been annotated as a DNA-damage inducible protein In the presence of iron, BN4020 pMH15 p280 Fur Box had an activity of 1806 ± 193 Miller units by beta-galactosidase assay, and an activity of 1846 ± 44 Miller units in the presence of deferoxamine The p value for this data was 0.730, not a significant value Therefore, we conclude that lacZ was not repressed by E coli fur Therefore, lacZ was not shown to be repressed by either Fur CBU0395 This protein has been annotated as a putative lipoprotein In the presence of iron, BN4020 pMH15 p395 Fur Box had an activity of 391 ± 65 Miller units by betagalactosidase assay, and an activity of 394 ± 58 Miller units in the presence of deferoxamine The p value for this data was 0.911, not a significant value Therefore, we 32 conclude that lacZ was not repressed by E coli fur A heterologous system containing C burnetii Fur has yet to be successfully made with p395 Fur Box CBU0432 This protein has been annotated as a member of the major facilitator transporter family In the presence of iron, BN4020 pMH15 p432 Fur Box had an activity of 803 ± 25 Miller units by Beta-galactosidase Assay, and an activity of 1232 ± 23 Miller units in the presence of deferoxamine The p value for this sample was 0.004, a significant value Therefore, we conclude that lacZ was repressed by E coli fur A heterologous system containing C burnetii Fur has yet to be successfully made with p432 Fur Box CBU0968 In the presence of iron, BN4020 pMH15 p968 Fur Box had an activity of 464 ± 43 Miller units by Beta-galactosidase Assay, and an activity of 843 ± 22 Miller units in the presence of deferoxamine The p value for this sample was 0.008, a significant value Therefore, we conclude that lacZ was repressed 45% by E coli Fur A heterologous system containing C burnetii Fur has yet to be successfully made with p968 Fur Box CBU1106 33 In the presence of iron, BN4020 pMH15 p1106 Fur Box had an activity of 88 ± 10 Miller units by beta-galactosidase assay, and an activity of 120 ± 21 Miller units in the presence of deferoxamine The p value for this data was 0.038, not a significant value Therefore, we conclude that lacZ CBU1106 promoter was not repressed by E coli fur In the presence of iron, BN4020 pNP104 p1106 Fur Box had an activity of 185 ± 11 Miller units by beta-galactosidase assay, and an activity of 183 ± 28 Miller units in the presence of deferoxamine The p value for this data was 0.628, again not a significant value Therefore, the lacZ was not shown to be repressed by C burnetii Fur either This promoter has consistently shown very low activity similar to that of the negative control This may be due to the fact that this is a weak promoter CBU2012 In the presence of iron, BN4020 pMH15 p2012 Fur Box had an activity of 215 ± 35 Miller units by beta-galactosidase assay, and an activity of 251 ± 36 Miller units in the presence of deferoxamine The p value for this data was 0.080, not a significant value Therefore, we conclude that lacZ was not repressed by E coli fur In the presence of iron, BN4020 pNP104 p2012 Fur Box had an activity of 253 ± 36 Miller units by beta-galactosidase assay, and an activity of 216 ± 53 Miller units in the presence 34 of deferoxamine The p value for this sample was 0.076, again not a significant value Therefore, the lacZ was not shown to be repressed by either Fur CBU1493 In the presence of iron, BN4020 pMH15 p1493 Fur Box had an activity of 203 ± 18 Miller units by beta-galactosidase assay, and an activity of 249 ± 17 Miller units in the presence of deferoxamine The p value for this data was 0.148, not a significant value Therefore, we conclude that lacZ was not repressed by E coli Fur A heterologous system containing C burnetii Fur has yet to be successfully made with p1493 Fur Box CBU1556 In the presence of iron, BN4020 pMH15 p1556 Fur Box had an activity of 268 ± Miller units by beta-galactosidase assay, and an activity of 345 ± 15 Miller units in the presence of deferoxamine The p value for this sample was 0.007, a significantdifference Therefore, we conclude that lacZ was 22% repressed by E coli Fur A heterologous system containing C burnetii Fur has yet to be successfully made with p1556 Fur Box CBU1695 35 In the presence of iron, BN4020 pMH15 p1695 Fur Box had an activity of 1112 ± 85 Miller units by Beta-galactosidase Assay, and an activity of 1479 ± 121 Miller units in the presence of deferoxamine The p value for this sample was 0.017, a significant value Therefore, we conclude that lacZ was repressed by E coli Fur A heterologous system containing C burnetii Fur has yet to be successfully made with p1695 Fur Box Our pNP104 data is lacking because after transformation, we were unable to subculture some of the samples It is possible that light sensitive tetracycline became degraded during the course of the experiment At this time we have been unable to revise the protocol to allow for construction of the remaining promoter systems Real-Time PCR Real-time PCR was employed to quantitate expression of the putative FurFe regulated genes under varying levels of iron Many of the promoters containing putative Fur Boxes were in front of hypothetical genes active transcription is unknown This screening assay was intended to identify genes that were members of the Fur regulon of C burnetii and to support data from the heterologous systems Values are represented as fold changes 10uM of iron was considered normal growth conditions within the phagolysosome Therefore, 0uM would be iron 36 deficient and 50uM would be an iron rich environment For each gene, the amount of transcript present in the metabolically active bacteria at 0uM and 50uM were compared to 10uM Therefore, iron derepression would occur when RNA levels at 0uM are significantly higher than RNA levels at normal iron concentration (10uM) while RNA levels are significantly lower at 50uM This correlates to a positive fold induction at 0uM and a negative fold change at 50uM Significant values were determined as having +2 fold change at 0uM The negative control was com1 (constituitively transcribed gene) Com1 values were the “normalizer” for all samples to correct for fluctuations in total RNA from sample to sample since transcription was not so affected by iron concentration Real-time assays were attempted several times; however, none of these trials were successful The problem appeared to be with the RNA extraction and DNase treatment protocols The samples have consistently been contaminated with DNA This is a problem because the Real-time quantification will give the number of copies of RNA transcripts as well as the number of genomic DNA copies are present This contamination would skew the data Figure shows an attempt using a contaminated sample Subsequent studies in the lab have corrected this protocol and demonstrate clear 37 iron dependent transcriptional regulation of feoB expression in axenic media Our positive control, feoB, was not induced (fold change >+2) in the absence of iron Because we know that feoB is significantly induced by the removal of iron, we concluded that this data is incorrect and must be repeated 38 Figure Real Time PCR Results Real Time PCR was used to quantitate changes in RNA transcript levels of putative iron regulated genes under varying concentrations of iron Transcript levels are shown as fold changes Significant values are >+2 for samples without iron and