CHARACTERIZATION OF THE ROLE OF THE RPF MOTIF IN MYCOBACTERIOPHAGE TAPE MEASURE PROTEINS by Laura Jane Marinelli Bachelor of Science, Youngstown State University, 2001 Submitted to the Graduate Faculty of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2008 UNIVERSITY OF PITTSBURGH FACULTY OF ARTS AND SCIENCES This dissertation was presented by Laura J. Marinelli It was defended on May 15th, 2008 and approved by Karen Arndt, Ph.D. Associate Professor, Department of Biological Sciences Roger Hendrix, Ph.D. Professor, Department of Biological Sciences Saleem Khan, Ph.D. Professor, Department of Microbiology and Molecular Genetics Valerie Oke, Ph.D. Assistant Professor, Department of Biological Sciences Dissertation Advisor: Graham Hatfull, Ph.D. Professor, Department of Biological Sciences ii Copyright © by Laura J. Marinelli 2008 iii CHARACTERIZATION OF THE ROLE OF THE RPF MOTIF IN MYCOBACTERIOPHAGE TAPE MEASURE PROTEINS Laura J. Marinelli, Ph.D. University of Pittsburgh, 2008 In order to inject their DNA into the bacterial cytoplasm and establish infection, bacteriophages must ensure their genetic material successfully traverses both the bacterial membrane(s) and the layer of peptidoglycan surrounding the host cell. Phages accomplish this in a variety of ways, and some have virion-associated murein hydrolase enzymes that facilitate this process, particularly in conditions where the peptidoglycan is highly cross-linked. Phages that infect the mycobacteria must also contend with these barriers to infection, as well an impermeable layer of mycolic acids that decorates the cell surface; however, the mechanisms by which they do this are mostly unknown. In this regard, three small sequence motifs have been identified within mycobacteriophage tape measure proteins (TMPs) extended molecules that span the tail lumen and determine its length at least two of which have similarity to host proteins with muralytic activity. This suggests that phages may utilize regions of the TMP, which because of its location within the tail might be uniquely primed for host interaction, to facilitate localized peptidoglycan hydrolysis and DNA injection. The focus of this study is the motif found in the TMPs of mycobacteriophages Barnyard and Giles that has identity to a group of bacterial proteins known as resuscitation promoting factors (Rpfs). These Rpf proteins stimulate growth of non-growing bacteria and seem to exert their activity by cleaving inert peptidoglycan in the cell wall. Notably, the Barnyard Rpf Motif is contained within a 70 kDa C-terminal cleavage product of TMP, which appears to be cell wall- iv and/or membrane-associated during infection. Further, mycobacteria expressing TMP fragments containing this motif show aberrant behavior in culture and on solid media, and hybrid proteins in which the Rpf domain of the Micrococcus luteus Rpf protein is replaced with either of the phage motifs have muralytic activity in vivo. A recombineering-based method for generating mutations on lytically replicating mycobacteriophages has been developed and utilized to make multiple mutations in the Giles TMP motifs. Mutant phages infect host cells in late-stationary phase with a reduced efficiency, an observation that further supports a role for these motifs in cell wall hydrolysis during infection. v TABLE OF CONTENTS PREFACE.................................................................................................................................. xvii 1.0 INTRODUCTION........................................................................................................ 1 1.1 BACTERIOPHAGE TAPE MEASURE PROTEINS...................................... 3 1.1.1 The Tape Measure of Bacteriophage λ ....................................................... 4 1.1.2 Tape Measures in Other Bacteriophages.................................................... 6 1.2 THE CELL WALL.............................................................................................. 9 1.2.1 The Gram-Negative Cell Wall ................................................................... 12 1.2.2 The Gram-Positive Cell Wall..................................................................... 13 1.2.3 Mycobacteria and the Mycobacterial Cell Wall ...................................... 16 1.2.4 Cell Wall Modifying Enzymes ................................................................... 21 1.2.5 The Cell Wall as a Barrier ......................................................................... 25 1.3 BACTERIOPHAGE-ASSOCIATED HYDROLASE PROTEINS............... 26 1.3.1 Phages of Gram-Negative Hosts ................................................................ 27 1.3.2 Phages of Gram-Positive Hosts.................................................................. 32 1.4 MYCOBACTERIOPHAGE TAPE MEASURE MOTIFS............................ 34 1.5 BACTERIAL RPF PROTEINS ....................................................................... 39 1.5.1 The Micrococcus luteus Rpf Protein.......................................................... 40 1.5.2 Rpf Proteins in the Mycobacteria.............................................................. 41 vi 1.5.3 Rpf Mechanism of Action........................................................................... 47 1.5.4 Evidence for Rpf-Interacting Proteins...................................................... 49 1.5.5 Distribution of Rpf-Like Proteins.............................................................. 50 1.6 SUMMARY........................................................................................................ 54 2.0 MATERIALS AND METHODS .............................................................................. 56 2.1 BACTERIAL STRAINS AND MEDIA........................................................... 56 2.2 PLASMIDS USED IN THIS STUDY .............................................................. 57 2.2.1 Commercially Available Plasmids............................................................. 57 2.2.2 Plasmids Constructed by Others ............................................................... 58 2.2.3 pLAM Plasmid Constructs......................................................................... 59 2.3 GENERAL CLONING AND DNA MANIPULATION................................. 72 2.3.1 Oligonucleotides and Primers Used in this Study.................................... 72 2.3.2 Transformation and Electroporation........................................................ 76 2.3.3 Sequencing................................................................................................... 77 2.3.4 Site-Directed Mutagenesis.......................................................................... 77 2.3.5 Cosmid Packaging Reactions..................................................................... 77 2.4 PROTEIN EXPRESSION, PURIFICATION AND ANAYLSIS.................. 77 2.4.1 Expression and Purification of the Barnyard TMP Rpf-Like Fragment .. ....................................................................................................................... 77 2.4.2 Expression and Purification of the Barnyard TMP 70 kDa C-Terminal Fragment..................................................................................................................... 79 2.4.3 Production of Polyclonal Antibodies to the Barnyard Rpf Motif........... 79 2.4.4 Immunoblot Analysis.................................................................................. 80 vii 2.4.5 M. smegmatis Cell Fractionation ............................................................... 81 2.5 BACTERIOPHAGE PREPARATION AND ANALYSIS............................. 82 2.5.1 Preparation of Bacteriophage Stocks........................................................ 82 2.5.2 Bacteriophage DNA Isolation .................................................................... 84 2.5.3 Phage Protein Analysis............................................................................... 84 2.5.4 N-Terminal Sequencing.............................................................................. 85 2.5.5 Mass Spectrometry ..................................................................................... 85 2.5.6 Mycobacteriophage Barnyard Liquid Infections..................................... 86 2.5.7 RNA Isolation.............................................................................................. 86 2.5.8 RT-PCR ....................................................................................................... 87 2.6 MICROBIOLOGY............................................................................................ 87 2.6.1 Induction of pBAD/gIII Derivative Constructs in E. coli........................ 87 2.6.2 Induction of pLAM12 Derivative Constructs in M. smegmatis .............. 88 2.7 RECOMBINEERING IN M. SMEGMATIS TO GENERATE BACTERIOPHAGE GILES TMP MUTANTS .............................................................. 89 2.7.1 Mycobacteriophage Mutant Construction and PCR Screening............. 89 2.7.2 Infectivity Assay with Mutant Phages ...................................................... 90 2.8 BUFFERS AND REAGENTS .......................................................................... 92 3.0 GLOBAL ANALYSIS OF MYCOBACTERIOPHAGE TAPE MEASURES AND TAPE MEASURE MOTIFS ...................................................................................................... 95 3.1 INTRODUCTION ............................................................................................