Escherichia Coli
Total Page：16
File Type：pdf, Size：1020Kb
A Dissertation Entitled Expanded Functionality of the Bacterial Global Regulator Lrp by Benjamin R. Hart Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science _____________________________________ Dr. Robert Blumenthal, Major Advisor _____________________________________ Dr. John David Dignam, Committee Member _____________________________________ Dr. Ivana de la Serna, Committee Member _____________________________________ Dr. R. Mark Wooten, Committee Member _____________________________________ Dr. Isabel Novella, Committee Member ____________________________________ Dr. Patricia Komuniecki, Dean of the College of Graduate Studies The University of Toledo August 2010 Copyright 2010, Benjamin R. Hart This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without expressed permission of the author ii Abstract Predicting gene regulation from genome sequences is an important technique for understanding bacteria that cannot currently be grown in the laboratory. This approach involves extrapolation from a well-characterized bacterium. Several assumptions are made when using this technique; key among these is that sequence-conserved transcription factors, target genes, and binding sites for these transcription factors upstream of the target genes together imply conservation of the regulation. However the level of conservation necessary for accurate predictions has not been defined. Previous studies have illustrated that the Leucine Responsive Regulatory Protein (Lrp) orthologs from Escherichia coli and Proteus mirabilis have only partially- conserved regulatory effects despite 98% overall amino acid sequence identity and complete conservation of the DNA binding helix-turn-helix domain. Studies described here reveal that these regulatory differences are associated with previously-unappreciated but fundamental functional differences between the Lrp orthologs. These studies are particularly important for predicting regulation from genome sequence as Lrp is a global regulator that, in E. coli, directly controls over 200 genes. The first manuscript, prepared for the Journal of Bacteriology, focuses on the amino acid coregulators of Lrp that, for E. coli, were only previously known to include leucine and alanine. This study revealed that methionine, isoleucine, histidine, and iii threonine also have significant coregulatory effects. In addition, modest differences between Lrp orthologs were observed in response to some amino acids. The second manuscript focuses on the role of the N-terminal tail of the Lrp protein. This unstructured tail is a region containing many of the sequence differences between Lrp orthologs.Through the generation of hybrid proteins, this study demonstrates that the N-terminal tails contribute to differences in transcription, and DNA binding between E. coli and P. mirabilis Lrp. Together, these results suggest that even overall sequence identity of 98% is insufficient to allow regulatory extrapolation in the absence of fairly detailed understanding of the regulatory protein. iv To Greg Hart and Susan Habegger, their loving support has always been appreciated and I thank them for all their encouragement. v Acknowledgments This dissertation would not have been possible without the love, support, and help of my family and friends who have helped me along the way. It was through their support that I was able to persist through the challenging times. I wish to thank my major advisor, Dr. Robert Blumenthal, for taking me into his lab and providing me the opportunity to work under his supervision. His guidance, has really helped me develop over the last few years. I would like to extend my gratitude to my committee members, Dr. John David Dignam, Dr. Isabel Novella, Dr. R. Mark Wooten, and Dr. Ivana de la Serna for all their time and advice they have offered. I am especially grateful to Dr. John David Dignam for helping me purify protein and the lengthy discussions regarding protein assembly and the biochemisty involved in this work. I would like to thank my collaborators Dr. Jennifer Hinerman and Dr. Andrew Herr for their work with analytical centrifugation studies. vi I would like to extend my appreciation to Dr. Ronald Viola and the Buenafe Arachea help with and access to the instrument for the Dynamic Light Scattering experiments. I would like to thank the members of the Blumenthal Lab: Dr. Iwona Mruk, Dr. Robert Lintner, Dr. Pankaj Mishra, Dr. Kristen Williams, and Xaiochen Zhao for all their suggestions and advice. I would like to extend my thanks to the secretaries of the Medical Microbiology and Immunology Department especially Sue Payne, Sharron Ellard, Tracy McDaniel, and Tamara Chaimberlin. vii Table of Contents Acknowledgements…………………………………………………………………...…..vi Table of Contents…………………………………………..……………………………viii 1 Literature……………………………………………………………………..……1 1.1 Prediction of transcriptional regulation among bacteria is currently difficult…………………………………………………………………….1 1.1.1 Most regulatory predictions from genome sequences use extrapolation from well-studied bacteria……………………….....1 1.1.2 One of the challenges for regulatory prediction is determining the extent to which conserved sequence indicates conserved function, both among transcription factors and target genes………………..3 1.1.3 An even greater challenge for prediction involves identifying binding sites for transcription factors in the DNA upstream of genes………………………………………………………………5 1.1.4 Challenges to making regulatory predictions are present at the regulatory network and cellular level................................................8 1.1.5 This thesis focuses on the conservation of function in a model transcription factor……………………...…………………………9 viii 1.2 Lrp is a good model transcription factor for studying conservation of function…………………………………………………………………..10 1.2.1 Lrp is a well-studied global regulator in E. coli………………….10 1.2.2 In E. coli, Lrp can have a variety of regulatory effects…………..12 1.2.3 Lrp is widespread, and particularly highly conserved among Enterobacteriaceae……………………………………………….15 1.2.4 Lrp has been structurally characterized………………………….16 1.2.5 Cofactor binding changes the Lrp oligomeric state……………...18 1.2.6 Lrp from E. coli and P. mirabilis have significant functional differences despite 98% sequence identity………………………20 2 Unexpected coregulator range and ortholog-specific differences in the global regulator Lrp of Escherichia coli and Proteus mirabilis………………….……..22 2.1 Abstract…………………………………………………………………..23 2.2 Introduction………………………………………………………………24 2.3 Materials and methods…………………………………………………...29 2.4 Results……………………………………………………………………32 2.5 Discussion………………………………………………………………..47 2.6 References………………………………………………………………..55 3 Recognition of DNA by the Helix-Turn-Helix Global Regulatory Protein Lrp is Modulated by the Amino Terminus……………………………………………...65 3.1 Abstract……………………….………………………………………….67 3.2 Introduction………………………………………………………………68 3.3 Results……………………………………………………………………73 ix 3.4 Discussion………………………………………………………………..88 3.5 Methods……………………………………………………………….….92 3.6 References………………………………………………………………..97 3.7 Supplemental Figures…………………………………………………...104 4 Discussion………………………………………………………………………106 4.1 Lrp orthologs from closely related species have distinct regulatory effects…………………………………………………………………...106 4.1.1 The N-terminus of Lrp is responsible for some of the differences between Lrp function……………………………………..…….107 4.1.2 Comparisons of the N-terminus of Lrp to established regulatory regions offer insights into Lrp function……………………...…108 4.1.3 N-terminus of Lrp is a possible region for fine tuning Lrp regulation without sacrificing important regulatory connections..................................................................................110 4.2 Lrp has a broader range of coregulators than was known……………...111 4.2.1 Amino acid sensitivity is broader than previously expected…...111 4.2.2 Differences within the Lrp RAM domain are associated with differences in Lrp sensitivity to co-regulators…………….……116 4.2.3 Evolutionary dynamics of Lrp. What can we learn from the N and C domains and substitutions that have appeared within these regions?.......................................................................................117 4.3 Summary……………………………………………………………….118 5 References…………………………………………………………………………120 x 1 Literature 1.1 Prediction of transcriptional regulation among bacteria is currently difficult. Prediction of gene regulation across species is a complex task, requiring an in- depth knowledge of evolutionary conservation of transcriptional regulators, binding sites within promoters of target genes and the target genes themselves (Madan Babu, Teichmann et al. 2006; Lintner, Mishra et al. 2008; Lintner, Mishra et al. 2008; Baumbach, Rahmann et al. 2009), as well as structure-function relationships in the regulatory proteins. The basic assumption is that if the target genes, transcription regulators and binding sites are conserved, the regulation of the target gene by the regulator should also be conserved. Determining the presence of a target gene and regulator are relatively straightforward since bacterial “parts lists” (consisting of genes, open reading frames, and regulatory elements) can readily be determined (VanBogelen, Greis et al. 1999; Mao, Su et al. 2006; Powell and Hutchison 2006). Major challenges remain, however, especially in making the connections between parts in the regulatory network