Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2012 Characterization of the Function and Interaction of Proteins Involved in Exopolysaccharide Synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris Angela D. Cefalo Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biology Commons Recommended Citation Cefalo, Angela D., "Characterization of the Function and Interaction of Proteins Involved in Exopolysaccharide Synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris" (2012). All Graduate Theses and Dissertations. 1424. https://digitalcommons.usu.edu/etd/1424 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. CHARACTERIZATION OF THE FUNCTION AND INTERACTION OF PROTEINS INVOLVED IN EXOPOLYSACCHARIDE SYNTHESIS IN STREPTOCOCCUS THERMOPHILUS, STREPTOCOCCUS INIAE, AND LACTOCOCCUS LACTIS SUBSP. CREMORIS by Angela D. Cefalo A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biology Approved: ________________________ ________________________ Dr. Dennis L. Welker Dr. Anne J. Anderson Major Professor Committee Member ________________________ ________________________ Dr. Jeffery R. Broadbent Dr. Jon Takemoto Committee Member Committee Member ________________________ ________________________ Dr. Donald J. McMahon Dr. Mark R. McLellan Committee Member Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2012 Copyright © Angela D. Cefalo 2012 All Rights Reserved iii ABSTRACT Characterization of the Function and Interaction of Proteins Involved in Exopolysaccharide Synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris by Angela D. Cefalo, Doctor of Philosophy Utah State University, 2012 Major Professor: Dr. Dennis L. Welker Department: Biology Amino acid residues that are important for metal binding and catalysis in Gram- positive phosphotyrosine phosphatases were identified in Streptococcus thermophilus Wzh/EpsB proteins. The Wzh protein from S. thermophilus MR-1C was purified after heterologous expression and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine/threonine peptides. The purified Wzh protein was able to remove phosphate from both phosphotyrosine peptides tested and the phosphatase activity of Wzh was dramatically reduced by the presence of the phosphotyrosine phosphatase inhibitor sodium vanadate at concentrations of 1, 5, and 10 mM. Purified Wzh had no activity against the synthetic phosphoserine/threonine peptide. These results established that Wzh functions as a phosphotyrosine iv phosphatase. By using the yeast two-hybrid system, strong intraspecific protein interactions were detected in S. thermophilus MR-1C, Streptococcus iniae 9066, and Lactococcus lactis subsp. cremoris JRF1 between the putative transmembrane activation protein (Wzd, CpsC, and EpsA, respectively) and the putative protein tyrosine kinase (Wze, CpsD, and EpsB, respectively). Weaker protein interactions take place forming a dimer between two identical protein tyrosine kinases and between the protein tyrosine kinase and phosphotyrosine phosphatase (Wzh, CpsB, and EpsC, respectively) in these species. Protein-protein interactions involving a S. thermophilus MR-1C Wzd/Wze fusion protein and Wzd and Wze indicated that these proteins may form multi-protein complexes. All combinations of the S. thermophilus Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation), and CpsU (membrane translocation) proteins were analyzed for protein-protein interactions but no additional interactions were discovered. For each of the intraspecific interactions detected, interspecific interactions were also detected when one protein was from S. iniae and the other was from S. thermophilus. Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. These results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase/phosphatase regulatory system are conserved in the family Streptococcaceae. Interspecific protein-protein interactions v suggest that functional regulatory complexes can be formed in naturally occurring and genetically engineered recombinant strains. (228 pages) vi PUBLIC ABSTRACT Characterization of the Function and Interaction of Proteins Involved in Exopolysaccharide Synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris by Angela D. Cefalo, Doctor of Philosophy Utah State University, 2012 Major Professor: Dr. Dennis L. Welker Department: Biology Many microorganisms produce capsules of repeating sugar units that surround the cell called exopolysaccharides. The synthesis of these capsules is a complex process that involves numerous protein components. A better understanding of the way these proteins function and interact with one another will benefit many industrial processes by aiding in the construction of bacterial strains with enhanced properties and could also lead to new treatment strategies against microbial pathogens in which capsule production is important in their ability to cause disease. In this study, the function of one of the proteins involved in the regulation of capsule synthesis in the dairy starter culture Streptococcus thermophilus MR-1C is clearly established. The research in this study also provides insight into the protein-protein interactions involved in capsule production and their conservation among Gram-positive bacteria in the family Streptococcaceae using the vii dairy starter cultures S. thermophilus MR-1C and Lactococcus lactis subsp. cremoris JRF1, and a commensal strain of the fish pathogen Streptococcus iniae 9066. Experimental data obtained on the ability of the proteins involved in capsule synthesis to interact with counterparts from a different species suggest that the transfer of genes between the streptococci species and to some extent between streptococci and lactococci could form functional regulatory complexes. This would be a necessary requirement for efficient capsule production in starter strains that have been genetically modified to improve their functional characteristics for industrial uses or in naturally occurring recombinant strains. viii ACKNOWLEDGMENTS I would like to thank my major professor, Dr. Dennis L. Welker, for his expertise, support, guidance, patience, and encouragement. Most importantly, thank you for the many papers you edited and for all your efforts to improve my writing skills. I would also like to thank my committee members, Dr. Jeffery R. Broadbent, Dr. Anne J. Anderson, Dr. Jon Takemoto, and Dr. Donald J. McMahon, for their technical advice, editorial activities, and for the use of their lab equipment. I am also grateful to Dr. Joseph K. K. Li for his assistance and advice with the protein purification work, Dr. Joanne E. Hughes for her assistance and advice on using the yeast two-hybrid system, and Beatriz T. Rodriguez-Villalba for technical assistance and preliminary experimentation on the Streptococcus thermophilus MR-1C EPS producing strain. I am obliged to Dr. Melody N. Neely (Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan) for providing us with a Streptococcus iniae 9066 strain and Dr. Ashraf N. Hassan (Dairy Science Department, South Dakota State University, Brookings, South Dakota) for providing a Lactococcus lactis subsp. cremoris JRF1 strain. I am indebted to the biology department, faculty, and staff for all their help and advice during my years at Utah State University. Last, but not least, thank you to my family and friends for their love and support during the difficult times over the last few years. I would especially like to thank my parents, Kate Loader, and Cheryl Francis for their assistance, encouragement, patience, understanding, and their belief that I could finish this dissertation even when I doubted that it was possible. ix This work was supported by research grants from the United States Department of Agriculture (USDA), Dairy Management Inc., and Utah State University Community/ University Research Initiative (CURI). Angela D. Cefalo x CONTENTS Page ABSTRACT ....................................................................................................................... iii PUBLIC ABSTRACT ....................................................................................................... vi ACKNOWLEDGMENTS ............................................................................................... viii LIST OF TABLES .............................................................................................................xv LIST OF FIGURES ......................................................................................................... xvi LIST OF DEFINITIONS ..............................................................................................