P1 BACTERIOPHAGE AND TOL SYSTEM MUTANTS
Cari L. Smerk
A Thesis
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
August 2007
Committee:
Ray A. Larsen, Advisor
Tami C. Steveson
Paul A. Moore
Lee A. Meserve
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ABSTRACT
Dr. Ray A. Larsen, Advisor
The integrity of the outer membrane of Gram negative bacteria is dependent upon proteins of the Tol system, which transduce cytoplasmic-membrane derived energy to as yet unidentified outer membrane targets (Vianney et al., 1996). Mutations affecting the Tol system of Escherichia coli render the cells resistant to a bacteriophage called P1 by blocking the phage maturation process in some way. This does not involve outer membrane interactions, as a mutant in the energy transucer (TolA) retained wild type levels of phage sensitivity. Conversely, mutations affecting the energy harvesting complex component, TolQ, were resistant to lysis by bacteriophage P1. Further characterization of specific Tol system mutants suggested that phage maturation was not coupled to energy transduction, nor to infection of the cells by the phage.
Quantification of the number of phage produced by strains lacking this protein also suggests that the maturation of P1 phage requires conditions influenced by TolQ. This study aims to identify the role that the TolQ protein plays in the phage maturation process. Strains of cells were inoculated with bacteriophage P1 and the resulting production by the phage of viable progeny were determined using one step growth curves (Ellis and Delbruck, 1938). Strains that were lacking the TolQ protein rendered P1 unable to produce the characteristic burst of progeny phage after a single generation of phage. E. coli strains containing the paralogous ExbB were also unable to produce viable phage progeny in the absence of TolQ, suggesting that this role in phage maturation is unique to the TolQ protein. This role is also independent of the energy harvesting function of TolQ, as a strain containing an energetically inactive TolQ protein with a
iii point mutation are able to produce enough viable progeny in one generation of phage to constitute a burst. This data suggests that there is some unique, undetermined function of TolQ that is parasitized by the P1 bacteriophage in order to mature and produce viable, infectious progeny.
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ACKNOWLEDGEMENTS
The fulfillment of the requirements for my Masters degree could not have been made without the love, help, and contributions of many people along the way. I would like to take this opportunity to thank those people who have supported me throughout this process.
I would first like to thank my advisor, Dr. Ray Larsen. Without your continuous guidance throughout this entire project, from start to finish, the significance of this research may never have been recognized. I would also like to thank you for your help writing this document.
Without your seemingly endless knowledge of research that others have done, we may never have seen the culmination of this project either. I would also like to thank my entire committee,
Dr. Tami Steveson, Dr. Lee Meserve, and Dr. Paul Moore for their support and collective guidance over the past two years (some of you much longer than that). The time and knowledge you have all provided has given me a great deal of help along the way, and inspired me to always look for the bigger picture. I would also like to thank Dr. George Bullerjahn for his support and assistance throughout this project. Though he was not a member of the committee, his scientific experience and input helped to work out some of the protocols I needed to develop for this project.
My sincerest thanks go out to my family—my parents, Jerry and Linda Byrd; my brothers, Collin, Chad, and Tony Smerk; and my grandparents, Dan and Kay Klonk. Without the constant love and support of my family, I would have never lasted six years at BGSU and become the person that I am today, both academically and personally. I am so grateful to all of you for being there for me when I needed advice, support, or just the unconditional love that you have always given me. Thank you, Mom and Dad, for attending my defense, one of the most
v important academic moments in my life thus far. It meant more to me than you will know to have both of you sitting in the audience and waiting to hear what happened afterwards.
I never would have survived the endless frustration of failed experiment after failed experiment if not for the humor and wisdom provided by my labmates. Dr. Kerry Brinkman and
Dr. Kimberly Keller have been a part of my research experience from day one, and without the love, advice, and lunches had at B-dubs, this degree would have never come to fruition.
To all of the friends that I have made while in Bowling Green, Ohio, I thank you. To Tori
Comstock, Luann Carpenter, and Chad Green, I appreciate most of all that you all listened to all the complaints over the last four summers we have worked together. Though I am moving on to bigger and (maybe) better employment ventures, I hope that we will remain friends. To Erica
Hertzfeld, even though we never get to sit and talk, when we do, I know that you will be an ear to listen and a shoulder to cry on, if need be.
This masters thesis was funded by the National Science Foundation (MCB-0315983) and the Center for Biomolecular Sciences at Bowling Green State University, and Dr. Ray Larsen.
Thank you to BGSU, the BGSU Department of Biological Sciences, and to the Graduate College for allowing me to perform and complete this research project.
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TABLE OF CONTENTS
INTRODUCTION TO BACTERIOPHAGE
CHAPTER ONE: INTRODUCTION …………………………………………………………..1
CHAPTER TWO: SPECIFIC AIM ONE
VERIFY THE ESSENTIAL ROLE OF THE TOLQ PROTEIN IN THE
P1 LYTIC CYCLE…………………………………………………………….……………...…12
Introduction……………………………………………………………………………...12
Materials and Methods…………………………………………………………………..13
Results……………………………………………………………………………………17
Discussion………………………………………………………………………………..26
CHAPTER TWO: SPECIFIC AIM TWO
USE MORE SENSITIVE AND SPECIFIC ASSAYS TO CONFIRM THE
ROLE OF TOLQ AND VARIOUS REGIONS OF THE TOLQ PROTEIN
IN THE PRODUCTION OF VIABLE P1……………………………………….………………30
Introduction………………………………………………………………………………30
Materials and Methods…………………………………………………………………...31
Results……………………………………………………………………………………33
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Discussion………………………………………………………………………………..40
CHAPTER FOUR: BROADER IMPLICATIONS, FUTURE
DIRECTIONS, AND CHARACTERIZATION OF P1 AND
THE ROLE OF TOLQ IN ITS LYTIC CYCLE……………………………………………...... 43
REFERENCES…………………………………………………………………………………..50
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LIST OF FIGURES
Figure Page
1 The Tol system of Escherichia coli. …………………...………………………….. 8
2 Growth profile of wild type bacterial cells (W3110) incubated with
P1 at various dilutions. …………………………………………………………… 20
3 Growth profile for tolQRA deletion strain (RA1017) incubated with
P1 at various dilutions……………………………………………………………… 21
4 Preliminary growth curve showing RA1004 (W3110 tolR::cm)…………………… 22
5 Growth profile of an E. coli tolA- strain (RA1009) incubated with P1……………. 23
6 Growth profile of TPS66 (tolQ-G181D)…………………………………………… 24
7 Example of a one step growth curve for Yersinia enterocolitica phage φYeO3-12… 31
8 One step growth curve of the wild type strain, W3110……………………………… 34
9 One step growth curve of RA1051 (ΔtolQRA, exbBD)……………………………… 35
10 One step growth curve of RA1035 (ΔtolQR)………………………………………… 36
11 One step growth curve of TPS66 (tolQG181D)……………………………………… 38
12 One step growth curve of RA1034 (ΔtolR, exbBD)……………………………… 39
13 One step growth curve of RA1044 (ΔtolR, exbB)………………………………… 40
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LIST OF TABLES
Table Page
1 Host range of P1……………………………………………… 5
2 Description of strains used in all experiments, including relative
genotypic deletions from the chromosome and phenotypic protein
expression………….………………… ………………………...... 13
3 Results of colicin and phage assays indicate the level of activity
as compared to the wild type strain for the TolA system (ColA)
and the TonB system (φ80)……………………………………………… 19
4 Phage and cell quantification……………………………………………. 26
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CHAPTER ONE
INTRODUCTION
Viruses are infectious agents that are obligate intracellular parasites, taking over a host cell in order to replicate. Bacteriophages are viruses that infect hosts of the kingdoms Bacteria and Archaea. Consistent with the differences between their hosts, bacteriophages are very distinct from viruses of eukaryotes. Within bacteriophages, different life cycles, morphologies, and host ranges exist, and most bacteria have bacteriophages that are capable of infecting them.
Despite their number and diversity, very few bacteriophages have been studied in great detail. Those that have interested researchers, however, have provided many insights into fundamental processes in nature. Since their discovery in the early 1900s, researchers have found that bacteriophages make many contributions to various aspects of biology, from playing a key role in the ecological impact of nutrient cycling, to their evolutionary influence on bacterial genomes (Chennoufi et al., 2004). From an ecological perspective, bacteriophages appear to play a substantial role in the growth and evolution of bacteria in their natural habitats, and it is likely that selection for phage resistance, as well as the development of anti-phage defenses, has shaped the bacterial properties we see today (Campbell, 1994). Bacteriophages have also been presented as a potential substitute for antibiotics, and in recent years, “phage therapy” has grown in popularity. In the years following their discovery, phage research focused mainly on the idea of using bacteriophages to combat bacterial disease, however, their most direct application to medical bacteriology has been to use them as reagents for typing bacterial strains (Campbell,
1994; Summers, 2005). This importance in the medical field has played a crucial role in the advancement of the knowledge gained about phages in the past century.
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A select few bacteriophages have played an integral part of the development of molecular biology. Early work with the Escherichia coli phage T4 provided a fundamental understanding of the nature of mutations and mechanisms of genetic recombination (Benzer, 1955; Benzer,
1959). Subsequently, T4 became a model for studying viral self-assembly (Eiserling, 1983).
Viable T4 assembly occurs via three separate pathways that ultimately converge to connect the tail fibers, the tail, and the icosahedral capsid together (Kuhn et al., 1987). The assembly of the capsid of this particular bacteriophage is preceeded by the formation of a prohead containing a shell and a protein core (Kuhn et al., 1987). The gene expression of bacteriophage T4 involved in this prohead formation, as well as assembly and subsequent release from the cell, requires gene products of both T4 and its host (Kao and Snyder, 1988). The host genes involved in T4 assembly have been sequenced and identified, and these genes are required for T4 to produce viable progeny. The specific host protein involved with T4 head assembly is an inner membrane protein, and T4 head scaffoldings assemble on the inner face of the inner membrane of E. coli
(Kao and Snyder, 1988). The scaffolding core structure, however, represents an intermediate step, and is proteolytically removed once the entire prohead is formed (Kuhn et. al, 1987). Kuhn et al, have also shown further evidence of this by the ability of this phage to use a strain of E. coli that expresses the necessary genes at higher temperatures to allow the naked cores of T4 to assemble when given the proper host signals.
Another, more detailed example of genetic regulation in bacteriophage comes from work with another E. coli phage, lambda. Lambda has been the model system for the regulation of gene expression in bacteriophages, particularly in terms of how it regulates the process of its lysogeny. At any given time, each cell, regardless of the type of cell, expresses only a specific subset of its genes, and the expression of those genes is regulated (Ptashne et al., 1976). The
3 unique aspects of lambda’s lysogeny and lytic cycle, including the various genes expressed at different times during its replication, have been rigorously characterized for many years. Like all phages, lambda requires its host machinery to replicate and produce progeny virus, however, once its genome is injected into a host cell, lambda can enter one of two pathways: a lytic cycle
(where the genome circularizes and begins replicating), or a lysogenic cycle (where the lambda genome is integrated into the host chromosome and expresses only those genes whose products function in keeping the phage in a cryptic state) (Ptashne et. al, 1980). This prophage form of lambda recombines into the host genome via an attP attachment site on the phage chromosome that is homologous to the attB site in the bacterial DNA, and requires only a single phage gene product, int (Ptashne et al., 1976). During the resultant lysogeny, the phage is replicated along with the host chromosome, and lies dormant, awaiting the proper signals to begin its lysis.
Genetic manipulations of lambda and related phage provide a means for exploiting lysogeny to introduce short stretches of DNA into the E. coli genome, a process known as specialized transduction. Other phage can provide for a more random replacement of large blocks of host genome by foreign DNA. This process, called generalized transduction, was first noted for the Salmonella phage, P22 (Zinder and Lederberg, 1952). Shortly after this observation, a similar process was noted in E. coli, mediated by a phage called P1 (Lennox,
1955). P1 is a very large bacteriophage whose genome sequence has only recently been determined (Lobocka et al., 2004). The 93,601 bp genome of P1 encodes at least 117 genes, with
49 of these genes having no identified homologues in other organisms (Lobocka et al., 2004).
Because this phage is able to package large blocks of nonspecific DNA into its capsid, P1 has been the main vehicle for generalized transductions (the movement of selected genes between bacterial strains) in E. coli (Miller, 1972). This manipulation of the bacteriophage for laboratory
4 use has led to relatively in-depth studies of this bacteriophage over the past fifty years. The method by which phages such as P1 package their DNA into their capsid can best be described as “processive headful packaging”, meaning that DNA is packaged into the prohead until no more can be inserted (Yarmolinsky and Sterling, 1988). P1 is capable of inserting approximately
100 kb of DNA into the mature prohead, though its genome is somewhat smaller than that. The assembly of the phage head allows for a specific amount of DNA to be packaged and then cut at pac sites to stop insertion of the DNA into the phage head. This “headful” packaging method allows for P1 to be less specific about the DNA that progeny phage will contain, and therefore makes it especially useful in generalized transductions because it is more likely that host DNA will accidentally be included in the capsid.
P1 can infect a taxonomically wide range of bacteria (Lobocka et al., 2004; and see Table
1). The wide host range of P1 reflects its use of a common target, the terminal glucose residue in the core region of the lipopolysaccharide (LPS) molecules in the outer membrane of the gram negative bacterial cell as a bacteriophage receptor for adsorption onto cells. The use of this widespread molecule for host recognition was first demonstrated by Franklin in 1969 (as referenced in Yarmolinsky and Sterling, 1988) using a K12 strain of E. coli with a defective galU gene. This mutation prevents the addition of the terminal glucose residue to the lipopolysaccharide core of the bacterial outer membrane, one consequence of which is resistance to P1. P1 binding to glucose in this context is also calcium (Ca2+) dependent. Note, that while
P1 can bind to and inject DNA into a wide range of bacterial species, the range of taxa in which this results in the production of viable progeny is more restricted (Table 1), indicating that additional specific host factors beyond that of a surface receptor are essential for P1 development.
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Table 1: Host Range of P1. (Adapted from Yarmolinsky and Sterling, 1988).
Taxonomic Grouping* Bacterium P1 DNA Injection P1 Phage Production
γ Escherichia coli,wild type + + Shigella dysenteriae + + Salmonella typhimurium + + Klebsiella pneumoniae + + Serratia marcescens + (-) + Enterobacter aerogenes + + Yersinia pestis + - Proteus vulgaris + + α Agrobacterium tumefaciens + - β Alcaligenes faecalis + - δ Myxococcus xanthus + - Bacteroidetes Flavobacterium spp. + -
* “γ", "α", "β"and “δ” refer to the respective divisions of the phylum Proteobacteria. Bacteriodetes is a related phylum containing a variety of bacterial species best known for their roles in the environment.
Following phage adsorption, P1 DNA enters the host cell via an as yet unknown mechanism, and circularizes almost immediately, providing protection from degradation by host nucleases. This circular DNA can then enter into one of two pathways: either a lysogenic or dormant prophage path, where it is maintained as a low copy number plasmid via specific partitioning mechanism (Abeles et. al., 1985) and a plasmid addiction system (Hazan et. al.,
2001); or a lytic path, where the DNA is replicated, packaged into a capsid, and the progeny virus released by cell lysis. Assembly of the virion inside the host cell is initiated by proteins under the control of an lpa operon, and the head and tail fiber proteins are capable of assembling independently, and ultimately attach at the base plate prior to cell lysis (Streiff et al, 1987). This
6 lpa operon involves some of the early genes on the chromosome. The various head proteins appear to be proteolytically processed, and this virus exhibits head size variation from infectious particles with large heads (P1B), non-viable progeny with small heads (P1S), and less commonly, minute heads (P1M) (Walker and Walker, 1983). Upon complete assembly of the infective virion, approximately one hour after the initiation of infection, three proteins are produced under the control of the lpa operon: the endolysin, an enyzme that degrades the cell wall; the holin, a protein that forms holes in the cytoplasmic membrane of the host to provide endolysin access to the peptidoglycan layer; and the antiholin, a protein that regulates the timing of cell lysis to ensure proper assembly of the new infective virion (Young, 1992; Wang et al.,
2000).
As first demonstrated with T4, and later seen with lambda, virus assembly appears to occur in association with the inner surface of the host membrane. While this process has not been studied for P1, early studies indicated that the cytoplasmic membrane Tol system of E. coli plays a distinct role in the lytic cycle of P1 (Sun and Webster, 1986). Certain Tol system mutants do not support plaque formation by P1, but still allow for generalized transductions, indicating that phage attachment and DNA injection proceeds normally (Sun and Webster, 1986). Further, sequence analysis of mutants suggested that TolQ was the only member of this system essential for P1 development (Sun and Webster 1987). Finally, some mutations that disrupt traditional
Tol-dependent functions (see Germon et al., 2001) do not alter the ability of these cells to plaque
P1. This suggests that the contribution of the Tol system to the P1 lytic cycle was independent of the energy transduction function of the system. Thus, mutations in the Tol system that do disrupt
P1 development might to represent the loss of a specific trait exploited by the phage, rather than a global change resulting from a disruption of a Tol-mediated energy-dependent process.
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The specific function of the Tol system of E. coli and other gram negative bacteria is largely unknown, though several studies indicate that the Tol system is involved in energy transduction and other processes that are important in the maintenance of the outer membrane
(Lloubes et al., 2001; Lazzaroni et al., 2002). Gram negative bacteria have a unique feature in their cell walls: the possession of an inner and an outer membrane. These two membranes are layers of the same cell wall have very different compositions, as well as functions. The inner, cytoplasmic membrane (CM) is a standard phospholipid bilayer that can establish and maintain an ion electrochemical gradient, and is therefore independently energized (Kadner, 1996).
Separated from the cytoplasmic membrane by a thin layer of peptidoglycan and an aqueous compartment (the periplasmic space) (Oliver, 1996) is a second membrane. This outer membrane
(OM) serves as a permeability barrier that confers a protection against the outside environment
(Nikaido, 1996).
Unlike the CM, the OM does not maintain a significant ion electrochemical potential, nor does it have direct access to the phosphate-based energy carriers such as ATP, however, energy dependent processes still occur here (Postle and Kadner, 2003), fueled by proton motive force from the CM. This energy is harnessed by the TonB system to drive the transport of iron siderophores, cobalmin, and perhaps other ligands across the OM. Similarly, this energy is also coupled to the maintenance of OM integrity, by a set of proteins collectively known as the Tol system. These proteins form a complex to harvest the energy from the electrochemical gradient in the CM and deliver it to other proteins that mediate these energy-dependent processes at the outer membrane (reviewed in Lazzaroni et al., 2002; Postle and Kadner, 2003; Weiner, 2005).
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A
P1 PB
ybgC tolQ tolR tolA tolB pal ybg F
B
Figure 1. The Tol system of Escherichia coli.
A. The tol system gene cluster, organized from two promoters, P1 and PB (Figure adapted from Vianney et al., 1996).
B. The products of the Tol gene cluster and their spatial distributions in the cell envelope are depicted. OmpA, OmpF, and Lpp (encoded elsewhere in the genome) represent a larger suite of OM-associated proteins and also interact with the Tol system components.
The Tol system is comprised of five core proteins: the energy transducer protein, TolA; the energy harvesting complex of TolR and TolQ; and the OM proteins TolB and Pal, through which at least some Tol dependent processes are mediated (Figure 1, adapted from Vianney et al., 1996). These proteins are organized into a single gene cluster on the E. coli chromosome, and are transcribed from two individual promoters, P1 and PB (Vianney et al., 1996). TolA, the energy transducer, contains one transmembrane domain, with its carboxy terminal extending far into the periplasm (Levengood et al., 1991). TolA receives energy from a multi-protein energy
9 harvesting complex, comprised of TolQ and TolR. The smaller of these proteins, TolR, has a single amino-terminally located transmembrane domain (Muller et al., 1993). The larger of these two proteins, TolQ, has three transmembrane domains, with a topology that places its amino terminal in the periplasmic space (Vianney et al., 1994). While the specific OM targets of the
Tol proteins remain unclear, the energy transduction component appears to be involved in maintaining the barrier function of the OM. This has been illustrated in cells bearing mutations in the Tol system, which exhibit membrane blebbing, hypersensitivity to bile salts and detergents, and leakage of periplasmic proteins into the external environment (Lazzaroni et al., 1989;
Bernadac et al., 1998). Recently, the Tol system has also been implicated in mediating the invagination of the OM during cell division (Gerding et. al., 2007), disruption of which would be consistent with this phenotype. The proteins of this energy harvesting complex, TolQ and TolR, are homologous to ExbB and ExbD (Eick-Helmerich and Braun, 1989), respectively, the protein components that make up the energy harvesting complex of the well-studied TonB system (Held and Postle, 2002). While the Tol and TonB systems energize distinct processes at the OM, the large degree of conservation between these two energy harvesting complexes is sufficient to allow one set, with diminished efficiency, to substitute for the other (Braun and Herrmann,
1993).
The energy transduction systems of E. coli are parasitized by a variety of agents, including filamentous bacteriophages and colicins. Filamentous bacteriophage exploit the Tol system to gain entry into the cell (Russel et al., 1988). These single-stranded DNA phage, including f1, fd, and M13, bind to the F pilus used in cell conjugation. Following phage attachment, the pilus is retracted by depolymerization into the membrane, and is believed to bring the phage into contact with the outer membrane (Russel et al., 1988). The Tol system
10 appears to be required for the efficient transport of the DNA of these phage into the cell, with a greater than 500-fold reduction in transduction efficiency for strains containing mutations in tolQ, tolR, or tolA (Russel et al., 1988). While in the absence of an F pilus these phage can still infect cells (at a reduced efficiency), the ability of these phage to infect cells lacking the F pilus remains dependent upon the Tol system (Click and Webster, 1997). These phage, however, assemble within the cell and bud through the cell envelope in a continuous release fashion, without lysing the host cell or interfering with cell division, leading to high phage yields (Russel,
1991).
Colicins are bacterial toxins that are produced by certain strains of E. coli and are active against other E. coli strains and related species (reviewed in Braun et al., 1994). These toxins kill cells through various methods, including depolarization of the cytoplasmic membrane, cytotoxic activity against cytoplasmic nucleic acids (DNase and RNase activity), and perforation of cell membranes. One specific class of colicins (Group A) requires the Tol system to energize their passage across the OM. Because these colicins cannot normally access their targets in the absence of a functional Tol system, sensitivity of the cells to these colicins provides an indicator of Tol system activity (reviewed in Lazzaroni et al., 2002).
Hypothesis & Specific Aims:
The objective of this research project has been to ultimately determine the mechanism by which P1 uses the proteins of the Tol system to assemble and release its progeny. Preliminary evidence indicates that P1 maturation involves the energy transducing Tol system, though it is not dependent upon the energy transduction function of this system. Further, mutant studies suggest only one component of the system, TolQ, is actually required for the process. This study
11 aimed to confirm and extend these observations, and identify the specific protein or specific protein motifs that are essential to P1 maturation. My approach has been to test various isogeneic strains for sensitivity based on the ability of P1 bacteriophage to produce viable progeny from these various host strains. Based on these observations, I submit the following hypothesis:
Hypothesis: TolQ is the main protein involved in the maturation of P1 and the
production of viable progeny phage.
To test this hypothesis, I developed the following Specific Aims:
1. Verify the essential role of the TolQ protein in the bacteriophage P1 maturation cycle.
2. Use more sensitive and specific assays to confirm the role of TolQ and various
regions of the TolQ protein in the production of viable P1.
These specific aims will be addressed in the chapters that follow.
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CHAPTER TWO
SPECIFIC AIM ONE: VERIFY THE ESSENTIAL ROLE OF THE
TOLQ PROTEIN IN THE P1 LYTIC CYCLE.
Introduction:
While early experiments suggested the possibility that the P1 lytic cycle was TolQ dependent (Sun and Webster, 1986), the significance of this was not recognized, and thus the observation has remained obscure. We were reminded of these findings during routine generalized transduction experiments using the non-lysogenic P1 derivative, P1vir, a strain containing a mutation preventing it from entering the lysogenic cycle, and thus it remains a lytic phage. To test a new E. coli strain created in the laboratory to determine if the phenotype reflected the genotype we had hoped to achieve, a P1 sensitivity assay was performed using methods adapted from the procedure for generalized transductions to produce phage lysates
(Miller, 1972). We had assumed that P1 required a complete and functional Tol system to effectively lyse cells. Thus we predicted a bacterial strain in which the tolR gene was insertionally inactivated would be consequently resistant to lysis by P1 phage. As detailed in the results section, this was surprisingly not the case. With the confirmation of this observation, I then examined other strains carrying specific known mutations in the Tol system to dissect which components of the Tol system were important for the production of P1 virions and which ones were not.
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Materials and Methods:
Bacterial strains. The major bacterial strains used are summarized in Table 2. The strain
W3110 was described by Hill and Harnish (1981), and serves as the wild type standard. The strain RA1017 is a derivative of W3110, the construction of which is described by Larsen et al.
(2007). All of the remaining strains are W3110 derivatives, made in the Larsen lab and to be described elsewhere, except TPS66, a GM1 derivative isolated in the Webster laboratory (Sun and Webster, 1986).
Table 2: Description of strains used in all experiments, including relative genotypic deletions from
the chromosome and phenotypic protein expression.
Strain Relative Genotype Phenotype W3110 Wild Type ExbB, ExbD,TolQ,TolR, TolA RA1017 ΔtolQRA, ΔexbBD No energy transduction RA1004 ΔtolR::cm, ΔexbBD TolQ, TolA RA1009 ΔtolA ExbB, ExbD, TolQ, TolR RA1034 ΔtolR, ΔexbBD TolQ, TolA RA1035 ΔtolQR ExbB, ExbD, TolA RA1044 ΔexbB, ΔtolR TolQ, ExbD RA1051 ΔexbBD,ΔtolQR TolA TPS66 tolQ::G181D ExbB, ExbD, TolR, TolA
Phage and colicins. The P1kcvir bacteriophage from which stock preparations were made was purchased from American Type Cultural Collection (ATCC; Manassas, VA). The φ80 phage was received from the Postle laboratory (Department of Molecular Biology, The
14
Pennsylvania State University; College Park, PA). The colicins were prepared from E. coli strains containing plasmids obtained from the Pugsley laboratory (Institut Pasteur; Paris, France) and described previously (Pugsley and Reeves, 1977).
Media. Cultures of bacterial cells were subcultured from overnight cultures and grown in
5 ml of Luria Bertani broth (LB) as previously described (Larsen et al., 1994) that was supplemented with 5 mM calcium and 0.2% glucose to aid in phage binding and cell growth, where appropriate. The LB referred to hereafter as “supplemented LB” was amended in this manner. To plate samples for the one step growth curve, phage and cell quantifications, and phage titrations, T-Top agar was used, supplemented in the same manner as the LB broth. The samples were plated onto tryptone (T) plates without supplementation (Miller, 1972). Samples from colicin and phage spot titer assays were plated onto LB plates (Larsen et al., 1994).
Bacteriophage preparations. The stock preparation of P1kcvir, hereafter referred to as
“P1”, was prepared using the methods for producing phage lysates for P1 generalized transductions (Miller, 1972). An overnight culture of the wild type strain (W3110) was subcultured 1:100 into five tubes containing 5 ml of supplemented LB broth. The cultures were grown for 1 hour with shaking at 37ºC, and culture turbidity was measured by absorbance of light at 550 nm in the Spec20 spectrophotometer with a path length of 1.5 cm. After 1 hr, 100 µl of various dilutions of a previously prepared P1 stock were added to the bacterial cultures, and culture turbidity was measured. As the cultures continued, lysis was evident as a decrease in absorbance, with the culture that grew to the highest absorbance prior to lysis (incubated with the
10-4 dilution of P1) chosen for the P1 stock preparation (data not shown). To extract the P1 progeny from the cells and prevent any further infection by P1, 50 µl of chloroform was added to lyse the cells, and the mixture was vortexed. Cell debris were removed by centrifugation at
15
10,000 x g for 5 min. Supernatants were recovered and these stock P1 lysate preparations were stored at 4ºC.
To determine the concentrations of the P1 preparations, lysates were tittered as follows.
Overnight cultures of W3110 were subcultured 1:100 in 5 ml supplemented LB broth and incubated with shaking at 37ºC. These cultures were allowed to grow until they reached exponential phase (as measured by culture turbidity reaching an absorbance of light at 550 nm of approximately 0.35), at which time P1 was serially diluted in λCa2+ buffer (Miller, 1972) and hundred-fold increments (10-2, 10-4, 10-6, and 10-8) were added to 100 µl of this exponential phase culture of wild type cells and 500 µl of T-broth supplemented with 5 mM CaCl2 and 0.2% glucose. After a 10 min incubation period, a 3 ml aliquot of similarly supplemented T-top agar
(Miller, 1972) was added and the resultant mixture was poured onto T plates, then incubated overnight at 37ºC. Plaques were then counted and plaque forming units (PFU)/ml determined.
Colicin and phage assays. Colicin and φ80 phage spot titer assays were performed to determine phenotypes of the strains used in the experiments. Group A colicins were used to evaluate the phenotype of the Tol system, and φ80 phage was used to evaluate the TonB system activity in these strains. The colicin and phage assays were performed as described in Larsen et al.(2003). The cells were grown as overnight cultures in 5 ml of LB broth, then subcultured 1:50 and allowed to grow until they reached exponential phase, as measured by absorbance of light at
550 nm. At that time, 100 µl of the cells were plated in 3 ml T-top onto T plates and allowed to solidify. A concentrated stock of Colicin A was diluted five-fold in T broth and 10 µl aliquots of each dilution were added (out to 5-8) to the surface of the solidified top agar. The φ80 phage was diluted ten-fold in λCa2+ buffer from a stock preparation of 2x1011 PFU/ml, and plates similarly inoculated. Plates were incubated overnight at 37ºC, then scored as the level of dilution that
16 produced an evident zone of killing in the lawn of bacteria. All assays were performed in triplicate.
Growth Profiles. Procedures modified from the methods for producing phage lysates for generalized transductions (Miller, 1972) were used in these experiments. Overnight LB cultures of individual bacterial strains were subcultured 1:100 in supplemented LB. The cultures were then grown at 37ºC with shaking for 1 hr before 100 µl of a dilution of P1 was added (10-1, 10-2,
10-3 in λ Ca2+ buffer from an original stock containing 3.5-6.5x109 PFU/ml). Bacterial cell density was monitored over time by the light absorbance at 550nm (using the Spec20 spectrophotometer with a path length of 1.5cm).
Phage and cell quantification experiments. Wild type E. coli cells and an isogeneic strain of cells with a tolQRA- and exbBD- genotype (RA1017) were grown in an overnight culture of 5 ml LB broth, and subcutltured 1:100 into 5 ml of fresh calcium and glucose supplemented LB. The cultures were grown for one hour to an approximate A550 of 0.1, then inoculated with 6.5x10-6 PFU/ml of bacteriophage P1, providing a starting MOI of approximately 1 bacteriophage per cell. Samples were taken for both cell and phage quantification at t=0, 1, 2, and 3 hours. Each sample was divided into two aliquots. To the cell sample, 100 µl of 1M sodium citrate was added to prevent any further phage binding and therefore further phage replication and cell death by chelating all available calcium in the medium. To the phage sample, 50 µl chloroform was added to solubilize the membranes of the cells and lyse them, preventing any phage from replicating from that point on.
To count the number of bacteriophage produced by each strain, the t=0, t=1, and t=2 samples were diluted 1:10 in λCa2+ buffer, respectively, the t=3 samples were diluted 1:100 in
17 the same media, and the t=4 samples were diluted 1:1000 in the same media to allow for countable numbers. A mixture of 100 µl of the phage samples and100 µl of the wild type cells in
500 µl supplemented T broth was incubated for 10 min and plated in 3 ml T-Top, also supplemented with calcium and glucose. These mixes were plated onto T plates in triplicate, and incubated overnight at 37ºC. The plaques were counted and data recorded. The bacterial cells were plated in a similar manner, with the cells diluted in T broth. The cells were plated in 3 ml
T-Top onto LB plates, with no incubation period. Respective samples were counted the next day for CFU in the case of cell samples and PFU in the case of phage samples.
Results:
The colicin and phage assays performed were used to determine the activity of the TolA system (ColA) and the TonB system (φ80). These colicin and phage assays are vital for determining and characterizing the relative phenotypes of the strains to be used in present and future experiments (Table 3). The colicin assays for W3110 (wild type strain) represented a wild type level of activity to Colicin A, as this strain contains complete TolA and TonB systems.
Strains lacking the TolA system, or components of the TolA system, exhibited resistance (as indicated by an “R” in Table 3) to Colicin A. The scores in the table represent the highest dilution at which zones of killing were observed for each of the strains. The strains with the genotypes ΔtolA (RA1009), ΔtolQRA, ΔexbBD (RA1017); ΔtolR, ΔexbBD (RA1034); ΔexbB,
ΔtolR (RA1044); and ΔexbBD,ΔtolQR (RA1051) exhibited a phenotype that indicated no energy transduction by the TolA protein had occurred in these strains, and therefore exhibited resistance to the group A colicin. The strains containing the ΔtolR::cm, ΔexbBD (RA1004); ΔtolQR
(RA1035), and tolQ::G181D (TPS66) genotypes exhibited some evidence of energy transduction
18
(with scores of 5-2, 5-2, and 5-1, respectively), most likely as a result of cross-talk from the TonB system.
The φ80 spot titers indicated the activity of the TonB system. Again, W3110 represented wild type sensitivity, as it contained a complete and functional TonB system. RA1009, RA1035, and TPS66 also contained a complete TonB system, and also exhibited wild type levels of energy transduction, as evidenced by their sensitivity to this phage (Table 3). RA1004, RA1017,
RA1034, RA1044, and RA1051 were all lacking some components of the TonB system, and exhibited full resistance to infection by this phage, as indicated by uniform growth of the bacterial lawn throughout the plate.
19
Table 3: Results of colicin and phage assays indicate the level of activity as compared to the wild
type strain for the TolA system (ColA) and the TonB system (φ80).
Strain Col A* φ80*
W3110 (wild type) 7, 6, 6 7, 7, 7
RA1017 (ΔtolQRA, exbBD) R, R, R** R, R, R
RA1004 (tolR::cm, ΔexbBD) 2, 2, 2 R, R, R
RA1009 (ΔtolA) R, R, R 7, 7, 7
RA1034 (ΔtolR, exbBD) R, R, R R, R, R
RA1035 (ΔtolQR) 2, 2, 2 7, 8, 8
RA1044 (ΔexbB, tolR) R, R, R R, R, R
RA1051 (ΔtolQR, exbBD) R, R, R R, R, R
TPS66 (tolQ—G181D)*** 1, 1, 1 7, 6, 7
*The colicins were diluted five-fold in T broth, the phage preparation was diluted ten-fold in λCa2+ buffer, and the numbers indicate the highest dilution at which zones of killing were observed. W3110 indicated wild type sensitivity, and strains lacking a Tol system, or components of the Tol system exhibited resistance to the colicin A. Similarly, strains lacking TonB system exhibited resistance to the φ 80 phage.
**The score of “R” represents a complete resistance to the 10-1 dilution of the φ 80 phage and the 5-1 dilution of the colicin A, indicating a complete lack of system activity.
***TPS66 showed a very grainy lawn, with possible contamination surrounding the spots where both the colicin A and the φ 80 phage were spotted onto the plates. The zones of killing surrounding the aliquots were scored as the highest dilution at which killing was observed.
20
Growth profiles of the wild type bacterial cells formed sigmoidal curves when absorbance was plotted against time of culture growth as the cultures grew to saturation (Figure
2). When E. coli cells with a wild type Tol system were inoculated with P1 at relatively low multiplicities of infection (MOI), growth was evident as increased absorbance of light by the culture, however, absorbance ultimately declined sharply, indicative of cell lysis (Figure 2). The higher the concentration of the phage inoculum, the sooner this lysis became apparent.
W3110 Growth Profile
1.4
1.2 W3110 0.1 P1 1 0.01 P1 0.001 P1 0.8 A550 0.6
0.4
0.2
0 0 50 100 150 200 250 Time (min)
Figure 2: Growth profile of wild type bacterial cells (W3110) incubated with P1 at various dilutions. Cell density is depicted on the y axis as light absorbance at 550 nm (A550). Lysis is evident as the absorbance peaks and then begins to decline over time. Time of culture growth is depicted in minutes on the x axis. Inoculation of P1 occurred at t=60 min. For reference, the 10-3 dilution of P1 is the equivalent of 3.5-6.5x105 PFU/ml.
Conversely, when a strain in which the tolQRA genes had been deleted from the bacterial chromosome (RA1017) and bacterial cells were inoculated with P1 at low MOI, this lysis was not observed, and the cultures grew to saturation. Interestingly, at a high MOI, the apparent growth of the tolQRA- strain was delayed for several hours, but ultimately, the culture grew to saturation (Figure 3), indicating complete resistance to infection by P1 bacteriophage.
21
! tolQRA (RA1017) Growth Profile
1.4 RA1017
1.2 0.1 P1 0.01 P1 0.001 P1 1
0.8 A550 0.6
0.4
0.2
0 0 50 100 150 200 250 300 Time (min)
Figure 3: Growth profile for tolQRA deletion strain (RA1017) incubated with P1 at various dilutions, as described above and in the text. This strain lacking a Tol system is resistant to lysis by bacteriophage P1, however, at very high MOI, a delay in culture development is observed. The cell density is displayed on the y axis as the absorbance of light at 550 nm (A550) and the time of culture development displayed on the x axis in minutes. Inoculation of the bacterial cultures with the dilutions of P1 occurred at t=60 min. For reference, the 10-3 dilution of P1 is the equivalent of 3.5-6.5x105 PFU/ml.
Based on the assumption that a complete Tol system was necessary for P1 to produce viable progeny phage, a strain carrying an insertional inactivation of the tolR gene should have exhibited resistance to lysis by bacteriophage P1. Surprisingly, however, this was not the case.
When incubated with a low MOI of P1, the tolR::cm strain (RA1004) exhibited a decline in the absorbance of light at 550 nm, indicative of cell lysis, similar to that of the wild type (Figure 4).
As higher concentrations of the P1 inoculum were added, this lysis was evident sooner. The cell density, displayed on the y axis as absorbance of light at 550 nm, began to decline at a culture development time (displayed on the x axis) of t=210 minutes for the 10-2 inoculum of P1, which was equivalent to 3.5-6.5x106 PFU/ml.
22
tolR::cm (RA1004) Growth Profile
0.6
0.5 RA1004 0.1 P1 0.01 P1 0.4 0.001 P1
0.3 A550
0.2
0.1
0 0 50 100 150 200 250 Time (min)
Figure 4: Preliminary growth curve showing RA1004 (W3110 tolR::cm). This E. coli strain with an insertionally inactivated tolR gene exhibited sensitivity to lysis by various dilutions of bacteriophage P1. Inoculation with various amounts of P1vir occurred at 60 min, displayed as time of culture development on the x axis, and culture density [the -2 absorption of light at 550 nm (A550)] is displayed on the y axis. The 10 inoculum of P1 is equivalent to 3.5-6.5x106 PFU/ml.
With the information gained from the tolR gene inactivation mutation (RA1004), the individual components of the Tol system were examined individually. P1 was found to be capable of producing enough viable progeny phage in strains lacking the energy transducer protein, TolA, (RA1009) for the culture to exhibit lysis (Figure 5). Even at a relatively low MOI, the bacterial culture exhibits lysis simlar to that of the wild type. The 10-3 dilution of P1 is equivalent to 3.5-6.5x106 PFU/ml, and this titration of phage is sufficient for bacterial cell lysis to be observed as a decrease in absorbance of light at 550 nm.
23
! tolA (RA1009) Growth Profile
1.4
1.2 RA1009 0.1 P1 1 0.01 P1 0.001 P1 0.8 A550 0.6
0.4
0.2
0 0 50 100 150 200 250 300 Time (min)
Figure 5: Growth profile of an E. coli tolA- strain (RA1009) incubated with P1. At a relatively low MOI, RA1009 exhibits lysis similar to that of the wild type when incubated with tenfold dilutions of P1, as evidenced by the decreased absorbance of light at 550 nm (displayed on the y axis) over time (in minutes, displayed on the x axis). Inoculation of bacterial cells occurred at 60 minutes. The 10-3 dilution of P1 is equivalent to 3.5-6.5x105 PFU/ml.
Based on a reduced efficiency of its energy transduction as evidenced by decreased sensitivity to Group A colicins, (Table 3) the Tol system was not functional when either the energy transducer protein, TolA, or part of the energy harvesting complex, TolR, were removed.
Together, the observations regarding TolR and TolA suggested that while the removal of either of these proteins inactivated the energy transduction function of this system, it did not affect the ability of P1 to produce viable progeny. Together, these observations suggested that energy transduction and formation of viable P1 progeny are independent events.
Further evidence for the observation that P1 maturation and energy transduction are independent events was gained from the growth characterization of a strain carrying a point mutation in TolQ (TPS66—G181D) in which a conserved glycyl residue was substituted with an
24 aspartyl residue at position 181. This mutation inhibited energy transduction [as evidenced by resistance to Group A colicins (Table 3)], but did not appear to hinder the maturation of P1.
Bacterial cultures incubated with low phage MOIs grew, then lysed in a manner similar to that observed for cells with a wild type Tol system (Figure 6, as compared to Figure 2).
tolQ G181D (TPS66) Growth Profile
1.2
1 TPS66 0.1 P1 0.01 P1 0.8 0.001 P1
0.6 A550
0.4
0.2
0 0 50 100 150 200 250 300 Time (min)
Figure 6: Growth profile of TPS66 (tolQ-G181D). This strain, unexpectedly, was sensitive to lysis by P1 out to dilutions of 10-3, (equivalent to 3.5-6.5x106 PFU/ml) as evidenced by the decreased absorbance of light (displayed on the y axis) over time (in minutes, depicted on the x axis). Inoculation with various dilutions of P1 occurred at 60 min.
The above observations led to the hypothesis that TolQ, one of the energy harvesting proteins, was an essential protein involved in the maturation of bacteriophage P1. These growth profiles, or “killing assays”, were important in the initial observations of sensitivity of various E. coli strains to lysis by P1, but did not provide rigorous data to show if, and, more importantly, how much viable progeny were produced. In order to make more accurate and quantitative observations regarding the hypothesis that TolQ is essential for the maturation of bacteriophage
25
P1, a more sensitive assay was needed that measured the production of phage from a given E. coli strain.
Based on the killing assays previously performed, the wild type strain and the strain lacking the TolA and TonB system (RA1017) were examined for viable progeny phage production. The phage and cell quantification experiments assayed the number of bacteriophage produced over time for a given E. coli strain, as well as the number of cells remaining in the culture after phage replication. We expected to observe an increase in phage production over time when P1 was incubated with the wild type bacterial cells, and a corresponding decline in bacterial cell number. In the E. coli strain lacking the Tol system proteins as well as ExbB and
ExbD, I expected to see the opposite—a decline in the number of phage present and an increase in the number of bacterial cells in the culture over time. The results confirmed expectations
(Table 4), and allowed for several generations of P1 progeny as well as cells to be observed and quantified. The colony forming units (CFU) are indicated in the left column for W3110 and
RA1017, and the plaque forming units (PFU) are indicated in the right column. The timepoints correspond to hours of bacterial culture growth, with each integer representing one hour.
Timepoint 0 was taken immediately following infection with P1 bacteriophage.
26
Timepoint Cells-CFU/ml Timepoint Phage-PFU/ml
W3110-0 3910 W3110-0 2080
W3110-1 6280 W3110-1 18240
W3110-2 400 W3110-2 TNTC
W3110-3 800 W3110-3 TNTC
RA1017-0 12 RA1017-0 3330
RA1017-1 3740 RA1017-1 5040
RA1017-2 54800 RA1017-2 800*
RA1017-3 1317000 RA1017-3 2400*
Table 4: Phage and cell quantification. The colony forming units are indicated in the second column for both W3110 and RA1017. Over time, the number of viable cells in the wild type culture declines, while the number of viable cells in the RA1017 (ΔtolQRA, exbBD) increases over time. The number of PFU/ml are indicated in the last column for both strains as well. The number of viable progeny phage produced from the wild type strain increases over time and at 2 hrs, the plates show large zones of clearing where the cells were unable to grow. When incubated with RA1017, the number of PFU/ml decreased over time, as expected.
*The asterisks indicate two plates which may have been switched during the experiment, and based on the expected results, this was most likely the case.
Discussion:
Initial observations indicated that the TolQ protein is essential for P1 to mature and produce viable progeny (Sun and Webster, 1986). Together, the observations from early experiments with P1 and the preliminary experiments performed in this study suggested that at least the TolQ protein is responsible, either alone or as part of the intact Tol system, for the
27 proper development and maturation of viable bacteriophage P1. When incubated with P1 phage at a very low MOI, E. coli strains lacking the TolR and TolA proteins were capable of producing enough bacteriophage for the whole culture to exhibit lysis similar to the lysis exhibited by the wild type strain as measured by the absorbance at 550 nm (Figures 4 and 5). While these proteins were required for the role that the Tol system plays in energy transduction (Table 3), they were not required for the maturation of P1 bacteriophage. This suggested that energy transduction and production of viable progeny P1 are independent of one another. Additional evidence supporting this hypothesis was obtained from the strain carrying a point mutation in tolQ that eliminates its role in harvesting energy (Table 3), but does not appear to hinder the production of viable P1 progeny (Figure 6). The higher the concentration of phage in each of these cases, the sooner lysis is observed as a decline in absorbance of light at 550 nm.
A strain that lacks a Tol system (RA1017) was also examined, and growth profiles, as well as phage quantification, showed that viable progeny production did not increase substantially over time. The cell quantification also showed that very few cells remained after the initial infection by P1 (Figure 2 and Table 4). Bacterial culture development was delayed in this strain, however, as evidenced by a slower growth to saturation (Figure 2), suggesting that P1
DNA injection proceeded normally. In these cells, however, P1 was unable to produce enough viable, mature particles to maintain the cycle of infection, and the cells that were not initially eliminated by the phage adsorption continued to grow after the phage were depleted from the media.
These observations led to further questions about the role of TolQ in the lytic cycle of P1.
The growth profiles suggested that while TolQ may be required for the maturation of infective virions, it was not involved in P1 DNA injection. The delay in culture development, as well as
28 the inability to recover much of the phage that was inoculated into the bacterial culture, indicated the possibility that once phage DNA is injected, many of the cells initially infected were still lysed by some phage process occurring inside the cell, but if the phage particles were produced, they were defective in some way, and were not capable of maintaining the cycle of infection.
This is consistent with observations from generalized transductions, because even though P1 cannot form plaques on strains lacking TolQ, these strains are still capable of being transduced
(for example, in constructing the strain RA1017; Larsen et al., 2007).
Because of its significant homology to ExbB (Eick-Helmerich and Braun, 1989), the possibility of ExbB substituting this role in the absence of TolQ, as it does in energy dependent processes at a reduced efficiency, also needed to be investigated to determine if this role is in fact unique to the TolQ protein. The growth curves provided an initial means of determining the sensitivity of various strains to lysis by P1 bacteriophage, but the assays were qualitative in nature and were not useful in determining the amount of bacteriophage, if any, that was produced by these strains. The quantification studies performed with strains lacking the entire Tol system, as well as the homologous ExbB and ExbD proteins, provided an initial opportunity to visualize and quantify the number of bacteriophage produced from a given strain of cells. While these experiments showed an increase in the number of viable phage progeny over a long period of time in the wild type cells, this procedure showed the phage particle production of an entire bacterial culture over several generations of P1. These assays did not provide a means to quantify the production of P1 from either a single cell or a single infection. Thus, an alternative approach was needed.
To more specifically examine the effects of Tol system mutations on the P1 development cycle, I wanted to determine the ability of individual cells to produce viable progeny virus. A
29 more sensitive method to determine the number of phage produced during a single generation of an infection is the one step growth curve, which had not previously been performed for P1 bacteriophage. Thus, adapting this strategy to P1 is the subject of my second aim.
30
CHAPTER THREE
SPECIFIC AIM TWO: USE MORE SENSITIVE AND SPECIFIC ASSAYS TO
CONFIRM THE ROLE OF TOLQ AND VARIOUS REGIONS OF THE TOLQ
PROTEIN IN THE PRODUCTION OF VIABLE P1.
Introduction:
One step growth curves were pioneered by Emory Ellis and Max Delbruck in the late
1930s (Ellis and Delbruck, 1938). These methods have been slightly modified with the advancement of technology and scientific technique, but the basic protocol and principle remains the same: to determine the number of infective bacteriophage produced in a single generation by an available number of host cells in a culture. This procedure has been adapted to fit several uses, including the titration of a bacteriophage preparation to determine the number of viable phage in a stock solution (Ellis and Delbruck, 1938).
Within the literature, an accurate procedure developed specifically for P1 could not be found, however, the procedure for a one step growth curve has been specialized for several other bacteriophage, including lambdoid phages like φ80 (http://www-micro.msb.le.ac.uk/LabWork/ grow/grow2.htm) and the Yersinia enterocolitica bacteriophage, φYeO3-12, which is related to the E. coli bacteriophages T3 and T7 (Pajunen et. al., 2000; see Figure 7). These procedures clearly illustrate the one step growth curve as a very accurate means of determining not only the generation time of these particular bacteriophages, which is the time that it takes for one entire generation of the bacteriophage to lyse their host cells, but also the burst size—the number of bacteriophage produced by a single cell in an infected culture during one generation of
31 bacteriophage. These pieces of information are important with regard to the replication cycle of
P1 and the influence that specific proteins, such as the TolQ protein, may have on this bacteriophage.
Figure 7: Example of a one step growth curve for the Yersinia enterocolitica phage φYeO3- 12. The latent period and eclipse period are illustrated with arrows, and the open boxes indicate the chloroform treated cells, intentionally lysed to recover phage particles prior to bacterial cell lysis by the phage. The burst size is also illustrated (as indicated by arrows) in this diagram. (Pajunen et. al., 2000). The y axis depicts the PFU/cell that are formed over time, which is depicted in minutes post-infection on the x axis.
Materials and Methods:
Culture media, strains and phage. The media used in these experiments are detailed in the Materials and Methods section of Chapter 2. The bacterial strains used in this aim are the
32 same as those described in Table 2, and the P1 preparations originated and were prepared as detailed in Chapter 2.
One step growth curves. The particular procedure used for my experiments was adapted from a website that walks an individual through the process of a one step growth curve, and illustrates what the graphs of the bacteriophage generations should look like if the experiment is done correctly (http://www-micro.msb.le.ac.uk/LabWork/grow/grow2.htm). The cells were subcultured 1:50 from a 5 ml overnight culture of supplemented LB into fresh supplemented LB, and allowed to grow until they entered exponential phase, as measured by culture turbidity in the spectrophotometer. At an A550=0.1, 1 ml of this culture was removed and infected with the non- lysogenic bacteriophage P1 at a MOI of 10. To accomplish this MOI, the phage preparation was diluted ten-fold in λCa2+ buffer, and 580 µl of the dilution was added to the 1 ml of the culture.
7 The approximate number of E. coli CFU present in a culture at an A550=0.1 was 2x10 cells/ml
(data not shown). The titration of the phage preparation used was approximately 3.5x109
PFU/ml. The high MOI ensured the infection of as many bacterial cells as possible, even though infection by one P1 phage particle confers immunity to any further phage infection (Yarmolinsky and Sterling, 1988). The mix was then incubated for 1 min at 37ºC and the cells were centrifuged for 5 min at 10,000 x g. This centrifugation step is the critical step that Ellis and Delbruck ultimately discovered, as it synchronizes the infection of all the cells. Any bacteriophage that have not already infected a host cell at this point remain suspended in the supernatant by
Brownian motion, and are then removed as the supernatant is aspirated. The infected cells are then resuspended in 1 ml of fresh LB medium, free of calcium and glucose, and this 1 ml is added back to 19 ml of LB broth lacking calcium and glucose and mixed by gently swirling the flask (into a total volume of 20 ml LB). A 1 ml sample is removed at this time, for a timepoint of
33 t=0, and 50 µl chloroform is added to lyse the cells. This sampling technique is repeated at fifteen minute intervals to 150 min. The procedures used for the preliminary phage and bacterial cell quantification were also used as guidelines to begin the development of these protocols.
Phage counts. To plate these samples, the timepoints corresponding to the known generation time of P1 (timepoints 4-10) were diluted 1000-fold in λCa2+ buffer to allow for countable numbers of bacteriophage, while timepoints 0-3 were assayed as undiluted samples. A wild type culture W3110 was grown to exponential phase, and 100 µl of the appropriate dilution of each sample was incubated with 100 µl of these wild type cells in 500 µl of supplemented T- broth, then allowed to incubate for 10 min at 37ºC. 3 ml of supplemented T-top was vortexed into the culture tube, and poured onto a T plate, then incubated overnight at 37ºC. The PFU were then counted. This procedure was modified and refined through several trials prior to gaining countable numbers of plaques and well-grown lawns of wild type cells (data not shown).
Results:
Once the protocol was developed for these experiments using the wild type strain, and countable numbers were achieved via dilutions, the wild type strain exhibited a generation time for P1 of approximately 60 min, with a burst size of approximately 83 PFU/cell (Figure 8). This burst size is evident as the difference between the amount of phage per cell initially and the amount of phage released and recovered after the eclipse period. After the 60 min eclipse period and the initial burst, the graph declines below the number of phage released from the initial burst.
This most likely reflects the bacteriophage that were released during the initial burst infecting other bacterial cells that had not yet lysed and remained growing and dividing in the culture. This second eclipse period ends at t=105 min, and a second, much smaller burst is observed, and this
34 is indicative of the bacterial cells that were infected from the first generation of P1 releasing their progeny. This is repeated once more on the graph illustrated here, as the graph peaks a second time, and again declines and peaks for a third time. The PFU/cell are depicted on the y axis of the graph, and the time of culture development in minutes is depicted on the x axis.
Wild Type (W3110) One Step Growth Curve
1.00E+04
1.00E+03
1.00E+02
1.00E+01 PFU/cell 1.00E+00
1.00E-01
1.00E-02 -15 0 15 30 45 60 75 90 105 120 135 150 Time (min)
Figure 8: One step growth curve of the wild type strain, W3110. PFU/cell are depicted on the y axis on a logarithmic scale, and the time of culture growth in minutes is depicted on the x axis. The graph illustrates a burst size of 80 PFU/cell during a generation time of 60 minutes. After the initial burst, the graph declines to indicate a second eclipse period of the bacteriophage into bacterial cells that have not yet lysed. This is seen as a second, much smaller burst at t=105 min.
When P1 was incubated with a strain lacking all the proteins from both energy transduction systems (RA1051—ΔexbBD, tolQRA) a significant burst was not detectable. The number of bacteriophage that remained in the culture were equivalent to the background phage that may have remained from the supernatant that was aspirated from the pellet in the early steps of the growth curve protocol (Figure 9). The inability of this bacterial strain to produce viable progeny phage is illustrated as the graph declines into an eclipse period of the bacteriophage that
35 does not recover into a burst, as is seen with the wild type bacterial cells. These results were consistent with the results previously obtained with this strain (Figure 3) that contains none of the proteins from the TolA system on the bacterial chromosome.
! tolQR, exbBD (RA1051) One Step Growth Curve
1.00E+01 1.00E+00
1.00E-01 1.00E-02 1.00E-03 1.00E-04
1.00E-05 PFU/cell 1.00E-06 1.00E-07
1.00E-08
1.00E-09 1.00E-10 -15 0 15 30 45 60 75 90 105 120 135 150 Time (min)
Figure 9: One step growth curve of RA1051 (ΔtolQRA, exbBD). The graph depicts the PFU/cell on the y axis on a logarithmic scale and the time of culture development in minutes on the x axis. The lack of a peak near the 60 minute generation time indicates that no viable progeny phage were produced from this strain. The very small number of phage present could be the remaining, free phage from the supernatant that were not efficiently aspirated.
In order to ensure that absence of the paralagous TonB system did not have an effect on the ability of P1 to mature and produce viable progeny, a bacterial strain that was lacking only the Tol system, carrying a genotype on the chromosome of ΔtolQR (RA1035) was also tested for
P1 production. In this strain, a similar pattern was observed, with P1 being unable to produce enough viable progeny to be recognized as a burst (Figure 10). The graph displays the phage entering into eclipse period, with no viable progeny recovered after the predicted generation
36 time. The PFU/cell are displayed on a logarithmic scale on the y axis of the graph, and the time of culture development is displayed in minutes on the x axis. The prior phage and colicin assays indicate TonB system activity in this strain (Table 3), as ExbB is capable of energizing TonB.
The one step growth curve of RA1035 supports the idea that the role that TolQ plays in P1 maturation is a unique feature of this protein, because ExbB, even with 79.1% sequence similarity to TolQ (Eick-Helmerich and Bruan, 1989), did not substitute its role in this process.
! tolQR (RA1035) One Step Growth Curve
1.00E+01 1.00E+00
1.00E-01 1.00E-02 1.00E-03 1.00E-04
1.00E-05 PFU/cell 1.00E-06 1.00E-07 1.00E-08
1.00E-09 1.00E-10 -15 0 15 30 45 60 75 90 105 120 135 150 Time (min)
Figure 10: One step growth curve of RA1035 (ΔtolQR). The intact TonB system did not enable P1 to produce viable progeny in the absence of TolQ. No visible burst was observed during the expected generation time in this strain, as indicated by PFU/cell depicted on a logarithmic scale on the y axis. The time of culture development is depicted on the x axis in minutes. The phage is inoculated and enters into the eclipse period, without recoverable phage produced.
In addition to strains lacking the Tol system, TPS66, a strain that contains a point mutation in tolQ of a conserved glycyl residue to an aspartic acid residue at position 181 (tolQ—
G181D) was assayed for viable P1 progeny production. The TolQ protein in this strain was ineffective in energy transduction for the TolA energy transducer, as evidenced by colicin assays
37
(Table 3). While P1 was capable of infecting an entire culture via several replication cycles and causing lysis of cells at the whole culture level (Figure 6), the burst size of one generation of P1 when produced on this strain was much less than that of the wild type (Figure 11). The burst indicated at t=60 min was consistent with the generation time of P1 when incubated in wild type cells. The burst size of approximately 0.4 PFU/cell was substantially less than that of P1 when grown in the wild type bacterial cells, but the number of bacteriophage present after this burst remained fairly constant. This bacterial strain did not exhibit several generations of P1 in the growth curve as the wild type does. The wild type bacterial strain may have had more bacteriophage particles that remained suspended in the supernatant to further infect cells that were not lysed by the first generation than the TPS66. More cells may have also been available to be infected after the first replication cycle as well. Because TPS66 was a strain that was examined later in the experimental procedures, the technique of aspirating supernatant may have been slightly more precise than when these assays were performed on the wild type cells. The observations regarding this bacterial strain support the hypothesis that P1 maturation is not coupled to the energy transduction function of the Tol system, and that some other aspect of the
TolQ function in the cell is involved in the production of viable P1 progeny.
38
tolQ::G181D (TPS66) One Step Growth Curve
1.00E+02
1.00E+01
1.00E+00
1.00E-01
1.00E-02 PFU/cell
1.00E-03
1.00E-04
1.00E-05 -15 0 15 30 45 60 75 90 105 120 135 150
Time (min)
Figure 11: One step growth curve of TPS66 (tolQ::G181D). The E. coli strain containing an energetically inactive TolQ with a point mutation to an aspartyl residue of the conserved glycyl residue at position 181 does not exhibit the characteristic burst of P1 like the wild type. However, the phage is still capable of producing enough viable progeny in a 60 minute generation time to illustrate a substantial burst of approximately 0.4 PFU/cell. The PFU/cell are indicated on the y axis on a logarithmic scale, and the time of culture development is indicated on the x axis in minutes.
Curiously, in a bacterial strain containing only the TolQ protein, RA1034, a significant burst was not observable at the known generation time (Figure 12). The PFU/cell are represented on a logarithmic scale on the y axis, and the time of culture development is represented in minutes on the x axis. However, at t=105 min, a hundred-fold increase of viable progeny phage present was seen. The phage produced at this time by this strain appeared to remain at a relatively constant titer throughout the time course of this experiment, suggesting that this burst was indicative of a longer generation time exhibited by P1 when incubated in this bacterial strain. This behavior indicated that while the TolQ protein was required for phage maturation, some aspect of TolQ that is required by P1 to produce its viable progeny was not distinguishable
39 when TolQ is the only protein present in the system. Initially, we believed that this may be a result of TolQ protein instability when it is not in a complex with another protein, improper membrane insertion, or absence of the specific function of which P1 takes advantage to produce its progeny.
! tolR, exbBD (RA1034) One Step Growth Curve
1.00E+02
1.00E+01
1.00E+00
1.00E-01
1.00E-02
PFU/cell 1.00E-03
1.00E-04
1.00E-05
1.00E-06 -15 0 15 30 45 60 75 90 105 120 135 150 Time (min)
Figure 12: One step growth curve of RA1034 (ΔtolR, exbBD). This strain does not exhibit a substantial burst that is characteristic of P1 during the expected generation time. The PFU/cell are depicted on a logarithmic scale on the y axis, and the time of culture development is indicated on the x axis in minutes. At t=105 min, a100-fold increase of PFU/cell is observed, though this was not regarded as a burst. This may be a result of a small amount of bacteriophage produced during an increased generation time with the predicted TolQ instability.
Surprisingly, in another strain containing TolQ and ExbD (RA1044), a significant burst was also not observed (Figure 13). ExbD is the protein in the TonB system that is homologous to the TolR protein (Eick-Helmerich and Braun, 1989), with which TolQ complexes to harvest energy at the cytoplasmic membrane (Vianney et al., 1996). The PFU/cell are depicted on the y axis on a logarithmic scale, and the time of culture development in minutes is depicted on the x axis. At t=60 minutes, there was a very small number of phage observed, and this number was
40 almost equivalent to background phage quantities. At t=75 minutes, the number of phage produced by this strain of cells began to increase, with a more substantial increase at t=135 min.
These results may indicate TolQ instability in the absence of TolR, improper insertion of TolQ into the CM, or that the specific function of TolQ of which P1 takes advantage to produce its progeny is inhibited, similar to the strain containing only the TolQ protein (Figure 12).
! tolR, exbB (RA1044) One Step Growth Curve
1.00E+02
1.00E+01
1.00E+00
1.00E-01
1.00E-02
PFU/cell 1.00E-03
1.00E-04
1.00E-05
1.00E-06 -15 0 15 30 45 60 75 90 105 120 135 150 Time (min)
Figure 13: One step growth curve of RA1044 (ΔtolR, exbB). The PFU/cell are depicted on a logarithmic scale on the y axis and the time of culture development is depicted in minutes on the x axis. In a strain that contains only TolQ and ExbD, the protein that is homologous to the natural energy harvesting partner of TolQ, a characteristic burst is not observed. This may be a result of TolQ instability, improper insertion into the CM, or the absence of the function of which P1 takes advantage in the absence of the natural partner of TolQ.
Discussion:
The results obtained from these experiments support the hypothesis that the TolQ protein was required, at least in some capacity, for P1 to produce viable progeny. The wild type bacterial cells, W3110, produced a burst size of 83 PFU/cell, which was consistent with what others have
41 stated in the literature (Lobocka et al., 2004). This burst occured after a 60 minute generation time of P1 (Figure 8), which was also consistent with the previous literature.
As expected, bacterial strains that are completely lacking a Tol system (RA1035 and
RA1051) produced no visible burst when incubated with P1 at an MOI of 10 phage/cell (Figures
9 and 10). These bacterial strains both exhibited no recovery of phage particles beyond that of background phage after the phage enter into the eclipse period. The absence of the TonB system also did not affect the ability of P1 to produce viable progeny, as illustrated by a strain lacking only the Tol system. RA1035 has an intact TonB system, and the characteristic burst of P1 was not exhibited by this strain (Figure 10). The observations from this bacterial strain supported the idea of a unique role that TolQ plays in the replication cycle of P1, as ExbB cannot substitute this role in the absence of TolQ as it can in some capacity for processes requiring energy transduction.
Similarly, another strain that contains an energetically inactive TolQ protein (TPS66) supported the maturation of P1 to a lesser extent, as the lack of energy transduction was only partially inhibitory, if related at all, to the production of viable progeny phage (Figure 11). The presence of a burst, albeit a very small burst of only 0.4 PFU/cell, together with the information gained from growth profiles and phage quantification studies (Chapter 2, Figure 6 and Table 4), indicated that the energy transduction function of TolQ and the role that TolQ plays in the production of viable P1 progeny may be independent of one another.
Based on the observations from the RA1034 one step growth curves, TolQ alone was not sufficient to produce viable progeny (Figure 12). While an increase in phage titer is seen at t=105 min, a much longer generation time for P1, enough viable progeny were not produced by P1
42 when incubated in this strain to constitute a burst size characteristic of that of P1, and this may be a result of various aspects of the phenotype of this particular strain. Because TolQ was the only protein present from either of the energy transduction systems in E. coli, it may be unstable in the membrane or not properly inserted into the membrane, and therefore not be capable of interacting with P1 as it normally would. Also, in the absence of an energy harvesting partner, the specific function of which P1 takes advantage may not have been present, thereby inhibiting the maturation of viable P1 phage particles.
A doctoral student in the lab created several chimeric bacterial strains using the protein complexes in the TolA and TonB systems (Brinkman and Larsen, 2007). These strains contained mixed chimeras that paired the ExbB protein with TolR, and the TolQ protein with ExbD. These chimeric protein complexes were then evaluated using colicin spot titer assays, φ80 adsorption assays, and radioactive iron transport assays to determine their ability to transduce energy to the
TonB protein in these combinations to fuel TonB energy-dependent processes at the outer membrane (Brinkman, 2007). In the bacterial strain containing TolQ and ExbD (RA1044), a partner is provided with which TolQ is capable of interacting. These experiments found that this combination of proteins is not sufficient to support energy transduction at a level comparable to that of wild type, or capable of performing the necessary functions of the cell (Brinkman and
Larsen, 2007). This chimeric combination of TolQ and ExbD was also unable to produce viable progeny P1 at a level of efficiency comparable to the E. coli wild type strain (Figure 14). These observations also supported the hypothesis that the energy transduction function of TolQ and the role that TolQ plays in the production of viable P1 progeny phage are independent of one another.
43
Together, the observations of the one step growth curves performed in the present study suggest that while TolQ alone may not be sufficient to support the complete maturation of P1 bacteriophage, it is a necessary component of the lytic cycle of this phage.
44
CHAPTER FOUR:
BROADER IMPLICATIONS, FUTURE DIRECTIONS, AND
CHARACTERIZATION OF P1 AND THE ROLE OF
TOLQ IN ITS LYTIC CYCLE
The role that TolQ plays in the lytic cycle of P1 bacteriophage is still undetermined. It has been established that the TolQ protein exhibits some significant activity in the production of viable progeny phage as illustrated by the inability of P1 to produce infectious progeny in its absence, but the exact role and its requirements by P1 are still unclear. This is also a unique role to the TolQ protein, as the paralogous ExbB protein, which shares a 79.1% amino acid sequence similarity with TolQ (Eick-Helmerich and Braun, 1989) and can substitute, to some extent, for
TolQ in the energization of TolA (Braun and Hermann, 1993) has not been shown to substitute the function of TolQ during the lysis by P1. After confirming the necessity of TolQ to the lytic cycle of P1, more rigorous characterization of its exact role is necessary, and subsequently, characterization of the replication of P1 bacteriophage.
Bacteriophages provide a useful model for the study of viruses that infect eukaryotic cells. The field of genetics is growing rapidly, and with the discovery of each new disease, another, more sophisticated approach to researching that disease is needed. Bacteriophage share many structural and architectural features with eukaryotic viruses (see Benson et al., 2004 for a recent review). One particularly interesting relationship is evident between the E. coli phage T4 and human herpesvirus. For T4, the E. coli genes involved in bacteriophage assembly have been sequenced and identified, and these genes are required in order for T4 to produce viable progeny.
45
The specific host protein involved with T4 head assembly is an inner membrane protein, and T4 head scaffoldings assemble on the inner face of the inner membrane of E. coli (Kao and Snyder,
1988). The scaffolding core structure, however, is an intermediate step, and is proteolytically removed once the entire prohead is formed (Kuhn, et. al, 1987). Double stranded DNA phages, like T4, assemble their heads in a similar fashion: the T4 procapsid precursor is formed and undergoes cleavage of the precursor proteins in order for the virion particle to mature (Kuhn et al.,1987). For example, T4 shows a high degree of homology in its prohead cleavage protease to that of herpesvirus (Cheng et al., 2004). The gene expression of bacteriophage T4 involved in this prohead formation, as well as assembly and subsequent release from the cell, requires gene products of both T4 and its host (Kao and Snyder, 1988), however, these interactions for herpesvirus are still under investigation. Bacteriophages then become especially integral to the viral research community because if their replication cycles can be well-characterized similar to those of T4 and lambda, the procedures applied to bacteriophages can then be used to characterize, potentially even treat, the viruses of eukaryotic cells. With the complete genome sequences of many organisms now available for comparison through GenBank, the genes and resulting protein gene products of multiple bacteriophages can be compared to those of eukaryotic viruses.
The characterization of the assembly of P1 bacteriophage will provide useful insight into the replication cycles of similar eukaryotic viruses and the proteins with which they interact at the cellular membrane, much like that of lambda and T4. Determining the degree to which P1 has been influenced by evolution and the proteins expressed by the host may further its use as a tool of molecular genetics, mainly as a vehicle for generalized transductions. P1 also infects a variety of hosts, and this wide host range is attributed to the use of a terminal glucose residue in
46 the outer core of the LPS of its bacterial host. The host range of P1 and on the tolQ sequences that are available allow for comparisons to be made between the TolQ proteins that support P1 maturation and those that do not.The tolQ gene from species that not only support P1, but are also closely related to E. coli can be cloned into plasmids and transformed into E. coli strains to determine if they will support energy transduction in the cell. Based on these comparisons, we can predict specific motifs and features that are essential to the production of viable P1 phage progeny. This information can tell us which regions of the protein are involved specifically in
P1 head assembly and particle formation, and thus provide insight into the complete replication cycle of this mysterious phage, and while many aspects of the molecular biology of bacteriophage have been rigorously studied, as well as the assembly and reproduction of those phage, many of the contributions of the host cell to these replication cycles remain obscure.
Several approaches can be taken to determine how TolQ and other proteins are involved in P1 assembly, maturation, and cell lysis. The genome of E. coli, as well as that of several other hosts of P1 are now available, and the amino acid sequences of the TolQ proteins present in these bacteria can be compared to determine the subtle differences that may occur in these proteins that allow for the maturation of infectious P1 particles or inhibit their production. After careful sequence analysis of the different proteins, investigations of the specific regions of TolQ involved in the maturation of P1 can begin. Specific regions in this protein that are necessary can be determined by site directed mutagenesis, and one can alter the TolQ protein of E. coli such that P1 cannot produce mature, infectious particles. A researcher can then begin to identify and characterize mutations that knock out the ability of TolQ to support the maturation of P1, but do not lose their ability to support energy transduction. The sequences of these proteins can then be used to determine the necessary regions of the protein, and subsequently applied to eukaryotic
47 organisms. Eukaryotic organisms that contain similar molecules in their cell membranes or proteins on the inner surface of their membrane can then potentially use P1 as a vector for genetic engineering, similar to the way it is used in generalized transductions in bacterial cells.
Genetic testing and genetic engineering have become popular topics of concern in the medical field, as well. Genetic engineering strategies often require permanent modifications of the target genome, and poor control over the position of the DNA introduced into the recipient genome may result the undesirable mutagenesis of genes necessary to the function of the organism (Groth et al., 2000). Bacteriophages can provide a much more specific mechanism for the alteration of genes by site specific integration into the host chromosomes by way of plasmids.
Similar to the lambda prophage, the Streptomyces phage, φC31, is capable of integrating into its host chromosome via two homologous sites of recombination—the attP site on the phage chromosome and the attB site on the bacterial chromosome (Groth et al., 2000). This integration, however, is unidirectional and is capable of recognizing more compact recognition sites, and requires accessory factors for the excision of the phage from its host chromosome, which makes it especially useful as a recombinase in studies of genetic engineering, even in mammalian cells.
With further, more detailed characterization of host proteins and enzymatic activity in its lytic cycle, P1 has the potential, because of its ability to package such a large amount of DNA into its prohead, to become an extremely useful tool in the world of eukaryotic genetics as it has played such a significant role in the field of microbiology.
The development of vaccines for potentially fatal human and companion animal diseases has been an area of interest to the medical and scientific community for many years. Most recently, a vaccination that confers immunity to retroviruses, specifically human immunodeficiency virus (HIV), has become an area of significant research. Bacteriophage T4
48 has become a focal point of the development of this vaccine as well. Because of the expression of a particular protein, a nonessential highly antigenic outer capsid protein called Hoc, in over
150 copies in its mature capsid, a preliminary system has been created that allows for the expression of three HIV antigens on the surface of T4 (Sathaliyawala et al., 2006). This system was shown to elicit a high immunogenic response in mice, and these model animals generated highly specific antibodies to the HIV antigens displayed on the surface of the bacteriophage.
Because of the wide host range of P1, if the lytic cycle and assembly of mature virus particles are characterized, this antigenic introduction technique can be refined and specified for P1, and made potentially useful as a system for introducing vaccines against other bacteriophage and eukaryotic viruses.
While the involvement of the Tol system of E. coli in the P1 lytic cycle remains obscure, the role of the Tol system in the host cell is to energize the energy dependent processes that occur at the outer membrane (Vianney et al., 1996). This system is highly conserved among
Gram negative bacteria (Sturgis, 2001), but similar systems have not been explored in eukaryotic organisms. Upon the characterization of the replication cycle of bacteriophage P1, the involvement of TolQ and the other components of the Tol system can be identified and potentially compared to similar proteins or protein systems in eukaryotic organisms that viruses may exploit to mature in their hosts.
Another approach to determine the involvement of TolQ in the P1 lytic cycle is to examine and characterize the proteins that are expressed by P1 during lysis. Experiments currently being performed in the lab are examining the size of the proteins that are produced by
P1, ran against standards and known proteins expressed by E. coli on an SDS-PAGE gel, and determining which of the proteins that are visible may be involved in the lysis of the host cell by
49
P1. These genes encoding these proteins can then be cloned into plasmids and overexpressed in the host cells to determine their precise involvement with host proteins. These proteins can also be in vivo cross-linking experiments to determine if it associates with TolQ at the cytoplasmic membrane during its assembly.
A more visual approach to examining the lytic cycle of P1 also includes the use of transmission electron microscopy. During transmission electron microscopy (TEM), the specific association of P1 with either the membrane or specific proteins in the membrane can be visualized as a snapshot of what is happening at a given moment during the replication cycle.
When P1 is visualized in association with different proteins and portions of the cell membrane, a clearer picture of the exact mode of replication under which this phage reproduces will be available, as well as the assembly of the phage capsid and attachment of the tail to the capsid.
Bacteriophages have played a significant role in the history and development of microbiology. From the definition of the random nature of mutations (Luria and Delbruck, 1943) to the identification of nucleic acids as the genetic blueprint (Hershey and Chase, 1952), and the definition of a gene and how recombination occurs (Benzer, 1955; Benzer, 1959), bacteriophage have had substantial influence in the discovery of many of the processes we know today. With the advent of new diseases, new cures and new genetic approaches are necessary. Many bacteriophages associate themselves with the inner surface of the membrane of their host cells, and while the lytic cycle of P1 has yet to be determined or characterized in great detail, the TolQ protein is involved to some degree in the maturation of a mature virion. The use of P1 in the laboratory has made this bacteriophage one of great interest. The detailed characterization of the infection, assembly, and lytic components of this phage will increase the value of this phage as a
50 genetic tool, as well as yield new information that can be applied to the viruses that infect eukaryotic cells.
While P1 is only a small portion of the broader spectrum of viruses and bacteriophages,
P1 serves as a very simple model for a very complex system. The characterization of the lytic cycle of P1 bacteriophage will provide new tools, procedures, and information that can be applied to a much broader range of viruses, and has the potential to serve as a model for disease control and prevention.
51
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