TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS

SARJA – SER. D OSA TOM.

MEDICA – ODONTOLOGICA

Molecular Biology, Genetics and Applications of

Yersiniophages

by

Saija Kiljunen

TURUN YLIOPISTO Turku 2006

From the Department of Medical Biochemistry and Molecular Biology, Institute of Biomedicine, University of Turku, Turku, Finland and the Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki Supervised by Professor Mikael Skurnik Department of Bacteriology and Immunology Haartman Institute University of Helsinki Helsinki, Finland Reviewed by

Saija Kiljunen. Molecular biology, genetics and applications of yersiniophages. Department of Medical Biochemistry and Molecular Biology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Annales Universitatis Turkuensis, Medica-Odontologica Series D, Turku, Finland, 2006. ABSTRACT The genus Yersinia in the family Enterobacteriaceae consists of 12 species, three of which are human pathogens. Yersinia enterocolitica and Yersinia pseudotuberculosis cause primarily gastrointestinal infections, whereas Yersinia pestis is the causative agent of plague. Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant and versatile group of organisms on Earth and have a significant impact on microbial ecosystems. Several phages infecting Yersinia are known, but only a few have been characterized in detail. The aim of this thesis was to obtain more information about yersiniophages, with a special interest in understanding factors that determine their host specificity. The phages studied in this thesis were φYeO3-12 and φR1-37, infecting Y. enterocolitica serotype O:3 (YeO3), and Y. pestis phage φA1122. φYeO3-12 and φA1122 are T7 – related members of Podoviridae, whereas φR1-37 belongs to the viral family Myoviridae. These phages utilize different parts of lipopolysaccharide as their receptor. For φYeO3-12 the receptor is the YeO3 O-antigen. In this work, the YeO3 outer core was identified as the receptor for φR1-37 and the core of Y. pestis and Y. pseudotuberculosis as the receptor for φA1122. The basic biological and genetic features of phages φYeO3-12 and φR1-37 were elucidated in this work. φR1-37 was found to be exceptional in having its genome composed of DNA where thymidine is replaced by deoxyuridine. According to the N-terminal amino acid sequences of structural proteins and the partial genomic sequence, no close relatives of φR1-37 have been described. For φYeO3-12, the non-essential regions in the genome were identified and the genes coding for DNA ligase and lysozyme were shown to be evolutionary factors important in adaptation of φYeO3-12 to grow on Yersinia. KEYWORDS: Yersinia, bacteriophage, receptor, lipopolysaccharide, evolution

Saija Kiljunen. Yersiniofagien molekyylibiologia, genetiikka ja sovellutukset. Lääketieteellinen Biokemia ja Molekyylibiologia, Biolääketieteen laitos, Turun yliopisto, Kiinamyllynkatu 10, 20520 Turku. Annales Universitatis Turkuensis, Medica-Odontologica Series D, Turku, 2006. TIIVISTELMÄ Enterobacteriaceae – heimoon kuuluva Yersinia – suku sisältää 12 lajia, joista kolme on ihmiselle patogeenisiä. Yersinia enterocolitica ja Yersinia pseudotuberculosis aiheuttavat suolistoalueen infektioita, kun taas Yersinia pestis on pelätty ruttobakteeri. Bakteriofagit (faagit) ovat bakteereja infektoivia viruksia. Ne ovat maapallon runsaslukuisin ja monimuotoisin eliöryhmä ja ne vaikuttavat huomattavasti mikrobiologisiin ekosysteemeihin. Kirjallisuudessa on kuvattu useita Yersinia – suvun bakteereja infektoivia faageja, mutta näistä vain muutama tunnetaan tarkasti. Tämän väitöskirjatyön tavoitteena oli saada lisää tietoa yersinioiden bakteriofageista, erityisesti tekijöistä jotka vaikuttavat niiden isäntäspesifisyyteen. Tässä työssä tutkitut bakteriofagit olivat Y. enterocolitica serotyyppi O:3:a (YeO3) infektoivat φYeO3-12 ja φR1-37 sekä Y. pestis -spesifinen φA1122. Näistä φYeO3-12 ja φA1122 ovat T7 -sukuisia Podoviridae –heimon jäseniä, kun taas φR1-37 kuuluu virusten heimoon Myoviridae. Nämä faagit käyttävät reseptoreinaan eri kohtia bakteerin lipopolysakkaridista. φYeO3-12:n reseptori on YeO3:n O-antigeeni. Tässä työssä osoitettiin, että φR1-37 käyttää reseptorinaan YeO3:n ulompaa ydinsokeria ja φA1122:n reseptori on Y. pestiksen ja Y. pseurotuberculosiksen ydinsokeri. Väitöskirjatyössä karakterisoitiin faagien φYeO3-12 ja φR1-37 biologisia ja geneettisiä ominaisuuksia. Työssä osoitettiin että φR1-37 on poikkeuksellinen bakteriofagi, jonka genomisessa DNA:ssa tymidiini on korvautunut deoksiuridiinilla. Faagin rakenneproteiinien aminoterminaalisten aminohapposekvenssien ja osittaisen genomisen DNA-sekvenssin perusteella sille ei ole kuvattu läheisiä sukulaisia. Lisäksi työssä identifioitiin φYeO3-12:n genomin alueita jotka eivät ole välttämättömiä faagin elinkyvylle ja osoitettiin, että faagin DNA ligaasia ja lysotsyymiä koodaavat geenit ovat faagin isäntäspesifisyyteen vaikuttavia evoluutiotekijöitä. AVAINSANAT: Yersinia, bakteriofagi, reseptori, lipopolysakkaridi, evoluutio

CONTENTS ABBREVIATIONS 8 LIST OF ORIGINAL PUBLICATIONS 9 1. INTRODUCTION 10 2. REVIEW OF LITERATURE 11 2.1. The genus Yersinia 11 2.1.1. Yersinia enterocolitica 11 2.1.2. Yersinia pseudotuberculosis 12 2.1.3. Yersinia pestis 12 2.1.4. Lipopolysaccharides of Yersinia 13 2.2. Bacteriophages 15 2.2.1. Phage taxonomy 16 2.2.2. Phage genomes 18 2.2.3. Phage evolution 20 2.2.4. The impact of phages on bacterial evolution 20 2.2.5. Phage receptors 21 2.3. Applications of phages 23 2.3.1. Bacteriophage therapy 23 2.3.2. Phages in bacterial diagnostics 24 2.3.3. Other applications 26 2.4. Bacteriophages of Yersinia 26 2.4.1. φYeO3-12 27 2.4.2. φA1122 27 2.4.3. PY54 27 2.4.4. Applications of yersiniophages 28 3. AIMS OF THE PRESENT STUDY 29 4. MATERIALS AND METHODS 30 4.1. Bacterial strains, phages and plasmids 30 4.2. Biological characterization of bacteriophages 35 4.2.1. Host specificity, efficiency of plating, growth curve and fitness-analysis

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4.2.2. Antisera (I) 36 4.2.3. Receptor characterization and adsorption analysis 36 4.2.4. LPS isolation and analysis 36 4.3. Structural characterization of bacteriophages 36 4.3.1. Electron microscopy 36 4.3.2. SDS-PAGE, Western-analysis and N-terminal sequencing of structural proteins

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4.4. Molecular biology techniques 37 4.4.1. General DNA techniques 37 4.4.2. In vitro transposon mutagenesis 37

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4.4.3. mRNA isolation and analysis 37 4.4.4. Analysis of modified nucleotides 38 4.5. Luminescence measurements and the construction of the reporter phage

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4.5.1. Luminescence measurements 38 4.5.2. Construction of the reporter phage 38 5. RESULTS AND DISCUSSION 39 5.1. Characterization of φYeO3-12 39 5.1.1. φYeO3-12 is related to T3 and T7 39 5.1.2. Transposon insertions in the early genomic region of φYeO3-12 cause growth defects on Y. enterocolitica

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5.1.3. φYeO3-12 DNA ligase and lysozyme are needed for growth on Y. enterocolitica

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5.2. Characterization of φR1-37 41 5.2.1. Biological and structural features of φR1-37 41 5.2.2. The φR1-37 receptor 42 5.2.3. The size and composition of the of φR1-37 genome 42 5.2.4. Sequencing of the φR1-37 DNA 43 5.3. Identification of the φA1122 receptor 43 5.3.1. The φA1122 receptor is a LPS core structure present in Y. pestis and Y. pseudotuberculosis but not in Y. enterocolitica

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5.3.2. The φA1122 receptor is blocked by the heterologous expression of Y. enterocolitica O:3 outer core

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5. 4. Reporter phages 45 5.4.1. Construction of the reporter phage by homologous recombina-tion

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5.4.2. Phages φ::lucFF1.2 and φ::lucFF5.3 46 5.4.3. Phage φ::lucFF1 46 6. SUMMARY 48 ACKNOWLEDGEMENTS 49 REFERENCES 50 ORIGINAL PUBLICATIONS 67

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ABBREVIATIONS aa amino acid(s) bp base pair(s) BER base-excision repair pathway CDC Centers for Disease Control and Prevention CFU colony forming unit(s) dA deoxyadenosine dC deoxycytidine dG deoxyguanosine DIG digoxigenin DNA deoxyribonucleic acid DOC deoxycholate ds double stranded dU deoxyuridine dUTP deoxyuridine triphosphate EM electron microscopy EMBOSS The European Molecular Biology Open Software Suit EOP efficiency of plating GFP green fluorescent protein ICTV The International Committee for Taxonomy of Viruses IM inner membrane kb kilo base pair(s) kDa kilodalton(s) Kdo 3-deoxy-D-manno-octulosonic acid LC-MS/MS Liquid Chromatography Mass Spectrometry/Mass Spectrometry LPS lipopolysaccharide mRNA messenger RNA NCBI The National Center for Biotechnology Information OM outer membrane OMP outer membrane protein PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PFGE pulse-field gel electrophoresis PFU plaque forming unit(s) RLU relative light unit(s) RNA ribonucleic acid RNAP RNA polymerase RT room temperature (20oC to 25oC) RT-PCR reverse transcriptase-PCR SDS sodium dodecyl sulphate ss single stranded T thymidine YeO3 Yersinia enterocolitica serotype O:3 YeO8 Yersinia enterocolitica serotype O:8

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LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original publications and some unpublished results. The publications reproduced with the kind permission of the copyright holders will be referred to in the text by the Roman numerals (I-IV).

I. Pajunen, M., Kiljunen, S., and Skurnik, M. (2000) Bacteriophage φYeO3-12 specific for Yersinia enterocolitica serotype O:3 is related to coliphages T3 and T7. J. Bacteriol 182: 5114-5120.

II. Kiljunen, S., Vilen, H., Savilahti, H., and Skurnik, M. (2005) Non-essential genes of the phage φYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3. J. Bacteriol 187: 1405-1414.

III. Kiljunen, S., Hakala, K., Pinta, E., Huttunen, S., Pluta, P:, Gador, A., Lönnberg, H., and Skurnik, M. (2005) Yersiniophage φR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine. Microbiology 151: 4093-4102.

IV. Kiljunen, S., Bengoechea, J. A., Holst, O. and Skurnik, M. Identification of LPS core of Yersinia pestis and Yersinia pseudotuberculosis as the receptor for bacteriophage φA1122. Manuscript.

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1. INTRODUCTION The bacterial genus Yersinia belongs to the family Enterobacteriaceae. Yersiniae are Gram-negative, rod-shaped bacteria that inhabit a wide variety of ecological environments. The genus includes 12 species, of which Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis are pathogenic to humans. The others are considered nonpathogenic or environmental (Bottone, 1997, Carniel et al., 2005, Sprague & Neubauer, 2005, Wauters et al., 1991, Wren, 2003). Bacteriophages or phages are viruses that infect bacteria. They were independently discovered by Frederick Twort in 1915 and Felix d’Herelle in 1917, even though at the time of their discovery, their biological nature was not quite understood (Stone, 2002, Summers, 1999, Summers, 2001). Phages inhabit every possible environment and generally outnumber their bacterial hosts by an order of magnitude. The phage abundance in sea water may be as high as 107 PFU/ml and the global phage population has been estimated to approach 1031, which makes phages the most numerous organisms on Earth (Breitbart & Rohwer, 2005, Pedulla et al., 2003, Weinbauer, 2004). The history of yersiniophages roots back to d’Herelle, who isolated “an antiplague phage” and used it to cure plague patients in 1925 (Stone, 2002, Summers, 1999, Summers, 2001). After that, several Yersinia –specific phages have been isolated, mainly to develop phage typing schemes for Yersinia diagnostics (Baker & Farmer III, 1982, Calvo et al., 1981, Garcia et al., 2003, Kawaoka et al., 1987, Nilehn, 1969, Nunes & Suassuna, 1978). However, not many of these phages have been characterized in detail. In the Skurnik research group, bacteriophages specific for Yersinia lipopolysaccha-ride (LPS) have been isolated and used to study the LPS structure and biosynthesis (Al-Hendy et al., 1991, Skurnik et al., 1995, Skurnik & Zhang, 1996, Zhang & Skurnik, 1994). The present PhD thesis work focused on the characterization of two of these phages, φYeO3-12 and φR1-37, which both infect Y. enterocolitica serotype O:3. This characterization included the study of the basic biological and genetic properties of these phages. For φYeO3-12, a detailed analysis of the function of the phage genome was carried out and the possibility to utilize the phage in bacterial diagnostics was evaluated. In addition, the cell surface receptor for the Y. pestis –specific phage φA1122 was identified.

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2. REVIEW OF LITERATURE 2.1. The genus Yersinia The yersiniae in the family Enterobacteriaceae are Gram-negative coccobacilli. The genus consists of 12 bacterial species: Yersinia aldovae, Yersinia aleksiciae (introduced in 2005), Yersinia bercovieri, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia rohdei and Yersinia rückeri. Out of these, Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis are pathogenic to humans. Y. rückeri is a fish pathogen and the others are considered non-pathogenic or environmental. For Y. aleksiciae, though, the apathogenicity still needs to be confirmed. (Bottone, 1997, Carniel et al., 2005, Sprague & Neubauer, 2005, Wauters et al., 1991, Wren, 2003). 2.1.1. Yersinia enterocolitica Y. enterocolitica causes primarily gastrointestinal infections, which normally ensue after the ingestion of contaminated food or water (Bottone, 1997). In human yersiniosis, the most frequent clinical manifestations are diarrhea, gastro-enteritis, and mesenteric lymphadenitis. These may be followed by sequelae such as reactive arthritis, erythema nodosum, and septicemia (Bottone, 1997, Saken et al., 1994). In Finland there are approx. 500 – 600 laboratory-confirmed Y. enterocolitica cases a year, which makes it the third most common enterobacterial pathogen (Holmström et al., 2004). Y. enterocolitica is also a well recognized cause of transfusion-associated bacteremia. Even though a rare event, the posttransfusional sepsis caused by Y. enterocolitica may have mortality rate as high as 64 % (Bottone, 1997, Leclercq et al., 2005). Y. enterocolitica is a heterogenous species and is separated into six biotypes (1A, 1B, 2, 3, 4, and 5) based on biochemical behavior (Bottone, 1997, Bottone, 1999). Out of these, biotype 1A lacks the Yersinia virulence plasmid pYV (Cornelis et al., 1998, Skurnik, 1985), and has often been considered nonpathogenic. Some biotype 1A strains have, however, been isolated from clinical samples (Tennant et al., 2003), and some of them were recently found to have virulence-associated genes that are related to the insecticidal toxin complex genes of other bacterial species (Tennant et al., 2005). Pathogenic biotypes 1B to 5 rely on pYV and several chromosomally encoded virulence factors for their virulence (Revell & Miller, 2001). Biotype 1B is considered as a high pathogenicity type, since strains belonging to it are lethal in a mouse model and can cause systemic infection in humans. Biotypes 2 to 5 are regarded as low pathogenicity types and can kill mice or cause systemic infection in humans only on condition of iron overload (Bottone, 1997, Bottone, 1999, Wren, 2003). Y. enterocolitica is divided into over 30 serotypes based on differences in O-antigen structures (Wauters et al., 1991). The distribution of Y. enterocolitica

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biotypes and serotypes is shown in Table 1. Clinically, the most significant Y. enterocolitica serotypes are O:3, O:5,27, O:8 and O:9 (Bottone, 1997, Fredriksson-Ahomaa & Korkeala, 2003). Table 1. The distribution of the most commonly encountered Y. enterocolitica biotypes and serotypes.

Biotype Serotype(s) 1A O:5, O:6,30, O:6,31, O:7,8, O:7,13, O:10, O:14, O:16, O:19,8, O:22, O:25, O:37,

O:41,42, O:41,43, O:46, O:47, O:57, nontypeable 1B O:4, O:4,32, O:8, O:13a, O:13b, O:13,7, O:13,8 O:18, O:20, O:21, O:25, O:41,42 2 O:5,27, O:9, O:27 3 O:1,2,3, O:5,27 4 O:3 5 O:2,3

Modified from (Bottone, 1997, Bottone, 1999, Tennant et al., 2003, Wauters et al., 1991). 2.1.2. Yersinia pseudotuberculosis Y. pseudotuberculosis, too, causes gastrointestinal infections, but the disease is slightly different to Y. enterocolitica (Wren, 2003). A typical manifestation is mesenteric lymphadenitis, followed by fever and abdominal pain. Septicemia is a rare outcome of Y. pseudotuberculosis infection, and usually requires an underlying disorder like diabetes, liver disease, or iron overload (Carniel et al., 2005, Ljungberg et al., 1995). Y. pseudotuberculosis infections occur mostly in the northern hemisphere, with about 50 – 200 cases a year in Finland (Holmström et al., 2004, Jalava et al., 2004). Y. pseudotuberculosis is separated into 21 serotypes: O:1 to O:15, of which O:1 and O:2 are divided into subtypes a, b, and c, and O:4 and O:5 into a and b (Bogdanovich et al., 2003, Carniel et al., 2005, Skurnik, 2004). Of these, serotypes O:1a and O:1b are predominantly isolated in Western countries, whereas serotypes O:4b and O:5 are more common in the Far East (Carniel et al., 2005). 2.1.3. Yersinia pestis Y. pestis is the causative agent of bubonic, septicemic, and pneumonic plague (Gage & Kosoy, 2005, Perry & Fetherston, 1997, Titball et al., 2003). The bacterium has a complex zoonotic life cycle, where various mammalian species (primarily rodents) may function as reservoir, and fleas serve as vectors (Perry & Fetherston, 1997, Wren, 2003). Humans usually encounter the disease by being bitten by an infected flea (Jarrett et al., 2004). From the flea bite, the infection rapidly spreads in regional lymph nodes, which enlarge to form a “bubo”. Septicemia usually follows after 2 – 6 days. Primary septicemic plague may occur if the infection spreads in the bloodstream without the formation of the bubo. Occasionally, the infection spreads in lungs to produce a secondary pneumonic plague. This form of plague is extremely contagious and may spread from human

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to human by the airborne route to cause primary pneumonic plague (Perry & Fetherston, 1997, Sebbane et al., 2005, Titball et al., 2003, Zhou et al., 2005). For untreated bubonic plague, the mortality is ca. 60 %, and for untreated septicemic and pneumonic forms it is close to 100 %. Effective antibiotic therapy decreases the mortality to 5 % and 33 % for bubonic and septicemic forms, respectively. For primary pneumonic plague the treatment may decrease the mortality to 10 %, but the medication needs to be started within 24 hours from infection to be efficient (Zhou et al., 2005). According to the World Health Organization, there are annually ca. two thousand plague cases in the world. This number may, however, be largely underestimated due to inadequate diagnostics and reporting in some endemic countries (World Health Organization, 2004). Y. pestis is evolutionary a very new organism and it is believed to have evolved from Y. pseudotuberculosis only 1500 – 20000 years ago (Achtman et al., 1999). The species is classically divided into three biovars, Antiqua, Mediaevalis and Orientalis, according to the ability to ferment glycerol and reduce nitrate (Achtman et al., 2004, Wren, 2003, Zhou et al., 2005). The biovar assignment does not seem to correlate with the virulence. A fourth biovar, Microtus, was recently proposed (Zhou et al., 2004). Strains belonging to this biovar are virulent to small rodents like mice, but avirulent to larger mammals like guinea pigs, rabbits and humans. A more detailed classification of Y. pestis into subspecies is being used in the countries of the Former Soviet Union and Mongolia (Anisimov et al., 2004). 2.1.4. Lipopolysaccharides of Yersinia The outer surface of Gram-negative bacteria is composed of an inner membrane (IM), a periplasmic space and an outer membrane (OM). IM is a bilayer composed of phospholipids, integral proteins and lipoproteins. The periplasm constitutes ca. 10% of the cell volume and contains a peptidoglycan layer and soluble proteins, eg. protein folding and trafficking factors. OM is an asymmetric structure, having phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. In the OM there are integral OM proteins and lipoproteins that participate in solute and protein translocation, pathogenesis and signal transduction (Bond & Sansom, 2004, Bos & Tommassen, 2004, Koebnik et al., 2000, Ruiz et al., 2006). LPS is an amphipathic molecule that is attached to OM by the hydrophobic anchor lipid A (Raetz & Whitfield, 2002, Skurnik & Zhang, 1996). Lipid A is fairly conserved among Gram-negative bacteria, the basic molecule being a disaccharide of two phosphorylated glucosamine residues acylated with five to seven fatty acid chains (Holst, 2003, Raetz & Whitfield, 2002, Skurnik & Toivanen, 1993, Skurnik & Zhang, 1996). The schematic representation of lipid A is shown in Figure 1. The structures of Y. enterocolitica and Y. pestis lipid A have been determined, and shown to vary according to the growth temperature of the bacteria (Holst, 2003, Knirel et al., 2005a, Knirel et al., 2005b). Lipid A, also called endotoxin, is recognized by the innate immune system and is responsible for the endotoxic shock during Gram-negative sepsis (Raetz & Whitfield, 2002).

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The core oligosaccharide is attached to the lipid A via 3-deoxy-D-manno-octulosonic acid (Kdo) (Holst, 2003, Skurnik & Toivanen, 1993). The LPS core structures of Gram-negative bacteria generally contain heptoses, glucose and galactose, and only limited diversity is seen between different species (Bruneteau & Minka, 2003, Raetz & Whitfield, 2002). This illustrates the importance of core oligosaccharide to outer membrane stability and function (Raetz & Whitfield, 2002, Skurnik et al., 1999). The core structures of Y. enterocolitica serotypes O:3, O:8, and O:9 have been determined (Muller-Loennies et al., 1999, Oertelt et al., 2001, Radziejewska-Lebrecht et al., 1994), as well as that of Y. pestis (Vinogradov et al., 2002). For Y. pseudotuberculosis only a partial structure is known (Bruneteau & Minka, 2003). The structure of Y. enterocolitica O:3 LPS core differs from other Yersinia in having inner and outer cores (Figure 1). The outermost structure of smooth LPS is O-polysaccharide or O-antigen. O-antigen structures are hypervariable and they determine the serological specificity within a species (Bruneteau & Minka, 2003, Raetz & Whitfield, 2002). Y. pestis LPS differs from other Yersinia in being rough, i.e. having no O-antigen. This is due to multiple mutations in genes coding for enzymes in O-antigen synthetic pathway (Skurnik et al., 2000). For Y. enterocolitica and Y. pseudotuberculosis O-antigen expression is temperature-regulated, being expressed only at temperatures below 30ºC. Other signals, like bacterial growth phase, iron concentration, pH, and ionic strength, may, however, also affect this regulation. Thus, the O-antigen may be expressed at some stages of mammalian infection (Skurnik & Bengoechea, 2003). Structurally O-antigens may be either hetero- or homopolysaccharides. Heteropolysaccharides are composed of repeating units made of different sugars. Such O-antigens are more common, and all studied Y. pseudotuberculosis and most Y. enterocolitica serotypes fall into this category. Y. enterocolitica serotypes O:3 and O:9 have homopolymeric O-antigens, composed of various number of 6-deoxy-L-altrose and 4-deoxy-4-formamido-D-rhamnose residues, respectively (Figure 1) (Bruneteau & Minka, 2003, Holst, 2003, Raetz & Whitfield, 2002). The outer membrane and LPS as its major constituent has a critical role in the interaction of a Gram-negative bacterium with its environment. The lipid A – core region is important for the membrane integrity. Bacteria with defective core are hypersensitive to compounds such as antimicrobial peptides, detergents, and hydrophobic antibiotics. Such cells are also leaky, and release periplasmic enzymes into the growth medium (Raetz & Whitfield, 2002, Skurnik et al., 1999). The O-antigen part is often involved in bacterial virulence, which is shown e.g. for Y. enterocolitica (Skurnik & Bengoechea, 2003) and Y. pseudotuberculosis (Mecsas et al., 2001). LPS containing at least lipid A and Kdo is essential for Escherichia coli and Salmonella enterica serovar Typhimurium (Galloway & Raetz, 1990, Rick & Osborn, 1977), whereas Y. pestis was recently shown to be viable with LPS containing only lipid A (Tan & Darby, 2005). The only known Gram-negative bacterium that can survive without lipid A is Neisseria meningitidis (Steeghs et al., 1998).

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Hep

P PGlcN GlcN

Kdo

Hep

Hep Hep

Glc

Glc

Glc

Glc

FucNac

Gal

GalNac

GalNac

6d-Alt

n

Lipid A

InnerCore

OuterCore

O-Antigen

A.

Core

AraN

Kdo Sug1

Hep

Hep Hep

Glc

Sug2

GalNac

P PGlcN GlcNAraN

Lipid A

B.

Figure 1. Model of Y. enterocolitica serotype O:3 (A) and Y. pestis (B) LPS. The attachment site of Y. enterocolitica O:3 O-antigen has not been unequivocally determined. GlcN; glucosamine, Kdo; 3-deoxy-D-manno-octulosonic acid, Hep; heptose, Glc; glucose, FucNac; 2-acetamido-2,6-dideoxygalactose, GalNac; 2-acetamido-2-deoxygalactose, Gal; galactose, AraN; 4-amino-4-deoxyarabinose, Sug1; Kdo or D-glycero-D-talo-oct-2-ulosonic acid, Sug2 galactose or D-glycero-D-manno-heptose. Modified from (Frirdich & Whitfield, 2005, Holst, 2003, Knirel et al., 2005b, Skurnik, 2004, Skurnik & Bengoechea, 2003, Skurnik et al., 1995, Skurnik & Zhang, 1996, Vinogradov et al., 2002). 2.2. Bacteriophages Bacteriophages, i.e. the viruses of bacteria, are the most abundant and the most versatile group of organisms on Earth. In environmental samples phages usually outnumber their bacterial hosts three to ten times, and the global phage population has been estimated to approach 1031 (Breitbart & Rohwer, 2005, Hendrix, 2002, Pedulla et al., 2003, Wommack & Colwell, 2000). Phages are active components of their ecosystems and have important roles in food web processes, biogeochemical cycles, gene transfer and prokaryotic diversity (Weinbauer, 2004, Weinbauer & Rassoulzadegan, 2004, Wommack & Colwell, 2000). As an example, phages were recently found to significantly influence the seasonal occurrence of Vibrio cholerae and cholera epidemics in Bangladesh and India (Faruque et al., 2005b, Faruque et al., 2005c). Phages can be divided into lytic and temperate based on their life cycles. The infection by a lytic, or virulent, phage results in the lysis of the bacterial cell and release of a new phage progeny (Ackermann & DuBow, 1987a). The first step of a

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lytic cycle is the adsorption of the phage on the bacterial surface (see Chapter 2.2.5.). The infection then proceeds by the entry of the phage nucleic acid into the bacterial cytoplasm and consequent transcription and translation of phage proteins as well as synthesis of multiple copies of the phage genome takes place. New phage particles are assembled and finally released upon the lysis of the host bacterium. The time until the new phage progeny is beginning to be assembled is called an eclipse period, and the time until the host cell lysis is a latent period. The number of new phage particles released from an infected cell is called a burst size (Ackermann & DuBow, 1987a). The most studied examples of lytic phages are T4 and T7, both infecting E. coli (Mathews et al., 1983, Molineux, 1999). Temperate phages can choose between lytic and lysogenic life cycles (Ackermann & DuBow, 1987a, Little, 2005). In lysogenic, or prophage, state, the phage genome is most often integrated into the bacterial chromosome and replicated as part of it. The expression of lytic genes is repressed, but other genes in the phage genome may be expressed in a state called lysogenic conversion. The expression of these extra genes may benefit the prophage by enhancing the fitness of the lysogenic bacterium, thus indirectly improving the replication of the phage genome ( Brüssow et al., 2004). Examples of lysogenic conversion are toxin genes carried by Pseudomonosa aeruginosa phage φCTX (Nakayama et al., 1999), E. coli O:157:H7 phage 933W (Plunkett et al., 1999), Clostridium botulinum phage c-st (Sakaguchi et al., 2005) and V. cholerae phage CTXφ (Miller, 2003, Waldor & Mekalanos, 1996). Lysogenic conversion and its effects on phage and bacterial evolution will be discussed in more detail in Chapter 2.2.4. Even though most temperate phages acquire the prophage state by integrating their genome into the bacterial chromosome, also other strategies exist. E. coli phage N15 and Y. enterocolitica phage PY54 establish the lysogenic state by residing as a linear plasmid (see Chapter 2.4.3) (Hertwig et al., 2003a, Ravin et al., 2000), and E. coli phage P1 and C. botulinum phage c-st lysogenize their hosts as circular plasmids (Lobocka et al., 2004, Sakaguchi et al., 2005). The choice between lytic and lysogenic cycles, so called lysis-lysogeny decision, is a complex process and understood well for few phages only (Little, 2005), the best-characterized example being E. coli phage λ (Hendrix et al., 1983, Little, 2005 #1129). 2.2.1. Phage taxonomy The International Committee for Taxonomy of Viruses (ICTV) describes viruses as “Elementary biosystems that posses some of the properties of living systems such as having a genome and being able to adapt to a changing environment” (van Regenmortel & Mahy, 2004). Viruses are classified into taxonomic categories based on characteristics like particle morphology, genome type and organization and the strategy of replication. The hierarchy ranks are species, subfamily, family, genus and order. A virus species is defined as “a polyethic class of viruses that constitute a replicating lineage and occupy a particular ecological niche” (Büchen-Osmond, 2003, van Regenmortel & Mahy, 2004). Members of a particular species

16

share a number of properties but do not necessary have any single property in common. A viral family, instead, is “a universal class sharing a number of properties that are both necessary and sufficient for class membership” (van Regenmortel & Mahy, 2004). Bacteriophages are divided into one order and thirteen families (Table 2) (Ackermann, 2003, Ackermann & DuBow, 1987a, Ackermann & DuBow, 1987b). The order Caudovirales (tailed phages) consists of three families, Myoviridae, Siphoviridae, and Podoviridae, and comprises ca. 96 % of known phages. There are six genera in the family Myoviridae, six in the Siphoviridae and three in the Podoviridae (Ackermann, 2003, Ackermann, 2001, Maniloff & Ackermann, 1998). Polyhedral, filamentous and pleomorphic phages are classified into ten families altogether (Table 2), each having one to four genera. The present, morphology-based, taxonomy has faced a major criticism during the past few years (Lawrence et al., 2002, Nelson, 2004). The ICTV taxonomy is dependent on electron microscopic (EM) images and does not take into account the rapidly increasing genomic and proteomic data. However, there are several phages for which there is no EM image available, but whose genomes have been completely sequenced. This specially concerns lysogenic prophages and phages of nonculturable bacteria. It has been estimated that at the moment, half of the phages whose genome has been deposited in databanks are not classified properly by the current taxonomic system (Nelson, 2004). Also, looking only at the phage morphology largely underestimates the diversity of phage genomes and may result in mistakes in classification. A clear example of such a case is Salmonella phage P22, which has been classified as a member of Podoviridae based on its short tail, even though it biologically resembles lambdoid phages in the family Siphoviridae (Vander Byl & Kropinski, 2000). Phage genomes are highly mosaic (Campbell, 2003, Pedulla et al., 2003), and it is now becoming more and more clear that a strictly hierarchical taxonomy can not represent the complex relationships between viral species. There is thus an increasing consensus that in the future, phage classification should be based on genomic data (Lawrence et al., 2002, Nelson, 2004, Proux et al., 2002). New systems for phage taxonomy have been proposed by a number of authors: Rohwer and Edwards (2002) presented a “phage proteome tree”. The authors stated that no single gene, or even a DNA sequence motif, is conserved enough to be used as a base for a taxonomical system. Instead, they used the overall similarity of phage genomes (Rohwer & Edwards, 2002). Lawrence et al. (2002) proposed a system where the highest taxonomic levels, “domains” and “divisions”, would be founded on the nature of genetic material and the possibility for genetic exchange, respectively. The lower levels, “modi”, would be based on phenotypic characters (i.e. common genetic modules) in such a way that most phages would belong to more than one group (Lawrence et al., 2002). Proux et al. (2002) and Chibani-Chennoufi et al. (2004) suggested a classification scheme based on the comparative genomics of structural genes (Chibani-Chennoufi et al., 2004, Proux et al., 2002).

17

A recent proposal came from Chen and Schneider (2005), who used the sequence information of phage promoters and RNA polymerases to build a phylogeny model for T7-like phages (Chen & Schneider, 2005). Most these sequence-based models result in a classification scheme that is fairly well compatible with the current ICTV system. A somewhat different approach is presented by Bamford et al. (2002) who introduced a term “viral self” to describe the fundamental structural principles of the virion, like particle assembly and genome packaging. The authors propose that these conserved structural features could be used to develop a viral phylogeny, which would be based on the ancient viral lineages (Bamford, 2003, Bamford et al., 2002, Benson et al., 1999, Benson et al., 2004, Saren et al., 2005). However, more molecular data is needed before a universal system that would be informative and generally accepted by virologists can be created (Chibani-Chennoufi et al., 2004, Nelson, 2004). 2.2.2. Phage genomes Bacteriophage genomes may be composed of DNA or RNA, which both can be either double-stranded (ds) or single-stranded (ss) (Table 2). Tailed phages, which constitute ca. 96% of known phages, all have dsDNA genomes (Ackermann, 2003, Ackermann & DuBow, 1987a). The sizes of phage dsDNA genomes in databanks range from 2,4 kb of Oenococcus oeni (formerly Leuconostoc oenos) phage L5 that only contains eight genes (accession no. NC003695) to 280 kb of P. aeruginosa phage φKZ (Mesyanzhinov et al., 2002). A number of phages have dsDNA genomes where one of the normal nucleotides is completely substituted by a modified base (Gommers-Ampt & Borst, 1995, Warren, 1980). The most thoroughly studied example is T4 DNA, where cytosine is replaced by 5-hydroxymethylcytosine (Wyatt & Cohen, 1953), which is further glucosylated by α- (70%) or β- (30%) bonds (Lehman & Pratt, 1960). Other examples are Bacillus subtilis phages φe, H1, SPO1, SP8, SP82G, 2C, and φ25, where thymine is substituted by 5-hydroxymethyluracil (Hemphill & Whiteley, 1975) and PBS1 and PBS2, where thymine is replaced by deoxyuridine (Takahashi & Marmur, 1963). In some phages, only a portion of certain base residues is chemically modified (Gommers-Ampt & Borst, 1995, Warren, 1980). These include E. coli phage Mu, where ca. 15% of adenine residues are substituted by N6-carboxymethyladenines (Swinton et al., 1983) and B. subtilis phage SP15, in which 62% of thymine residues are modified to phosphoglucuronated 5-dihydroxypentyluracil (Ehrlich & Ehrlich, 1981). The role of modified nucleotides in phage DNA is in many cases to confer resistance to host or phage encoded nucleases (Gommers-Ampt & Borst, 1995, Warren, 1980), and many restriction endonucleases have been shown to digest modified DNA slowly if at all (Huang et al., 1982). Modified bases may also serve as signals for transcription by phage-encoded RNA polymerases and phage DNA packaging.

18

Tab

le 2

. C

lass

ifica

tion

of b

acte

rioph

ages

O

rder

Fa

mily

Sh

ape

Sche

mat

ic fi

gure

C

hara

cter

istic

s N

ucle

ic a

cid

Exa

mpl

e

Myo

viri

dae

Siph

ovir

idae

Podo

viri

dae

Cau

dovi

rale

s M

yovi

rida

e Ta

iled

Con

tract

ile ta

il ds

DN

A

T4

Si

phov

irid

ae

Taile

d Lo

ng, n

onco

ntra

c-til

e ta

il

dsD

NA

λ

Po

dovi

rida

e Ta

iled

Shor

t tai

l ds

DN

A

T7

19

M

icro

viri

dae

Poly

hedr

al

Smal

l, no

env

elop

e ss

DN

A

φX17

4

Cor

ticov

irid

ae

Poly

hedr

al

Com

plex

cap

sid,

in

tern

al li

pid

mem

-br

ane

dsD

NA

PM

2

Te

ctiv

irid

ae

Poly

hedr

al

Prot

ein

shel

l and

in

ner l

ipop

rote

in

vesi

cle

dsD

NA

PR

D1

Le

vivi

rida

e Po

lyhe

dral

Ic

osah

edra

l cap

sid

ssR

NA

M

S2

C

ysto

viri

dae

Poly

hedr

al

Lipi

d en

velo

pe

dsR

NA

φ6

Inov

irid

ae

Fila

men

tous

Fi

lam

ents

or r

ods

ssD

NA

fd

Lipo

thri

xvir

idae

Fi

lam

ento

us

Rod

s, lip

id e

nve-

lope

ds

DN

A

TTV

1

Ru

divi

rida

e Fi

lam

ento

us

Inov

irid

ae

Lipo

thri

xvir

idae

Rudi

viri

dae

Stra

ight

, rig

id ro

ds

dsD

NA

SI

RV

1

Pl

asm

avir

idae

Pl

eom

orph

ic

No

caps

id, l

ipid

en

velo

pe

dsD

NA

L2

Fu

sello

viri

dae

Pleo

mor

phic

Lem

on-s

hape

d,

lipid

env

elop

e,

spik

es a

t one

pol

e

dsD

NA

SS

V1

Mod

ified

from

(Ack

erm

ann,

200

3, A

cker

man

n &

DuB

ow, 1

987a

), ht

tp://

ww

w.n

cbi.n

lm.n

ih.g

ov/IC

TVdb

/inde

x.ht

m

Mic

rovi

rida

eC

ortic

ovir

idae

Tect

ivir

idae

Levi

viri

dae

Cys

tovi

rida

e

Plas

mav

irid

aeFu

sello

viri

dae

2.2.3. Phage evolution As a group, viruses are believed to be ancient and originate from the time before the divergence of the three domains of life - Archaea, Bacteria, and Eucarya (Hendrix, 1999, Hendrix et al., 2003). Tailed dsDNA phages infect cyanobacteria and other Gram-negative bacteria but have not been described for Archaea, which suggests that the emergence of this virus group succeeded the divergence of Archaea and Bacteria but preceded the split of cyanobacteria from other Gram-negative bacteria. This gives a time estimation of 3–3,5 billion years (Hambly & Suttle, 2005). The molecular mechanisms driving evolution may be either vertical or horizontal. Vertical mechanisms create variation trough mutations (nucleotide exchanges, deletions, and inversions), after which the new variant is inherited to the next generation. Horizontal mechanisms transfer the genetic information (ranging from single genes to complete functional units) within the whole population, and even through species barriers, to form novel combinations ( Brüssow et al., 2004, Woese, 2000). The genomes of modern phages are highly mosaic (Brüssow & Desiere, 2001, Juhala et al., 2000, Pedulla et al., 2003) and according to the present understanding, they constitute a common gene pool that is mixing constitutively by horizontal gene transfer (Campbell, 2003). As stated by the most popular hypothesis on phage evolution, “the modular theory”, each phage genome is composed of multiple functional units, e.g. head genes or tail genes. Each of these units is represented by several alleles, “modules”, which are exchanged by homologous or, less often, non-homologous recombination between different phage genomes ( Brüssow et al., 2004, Hendrix, 2002). Most such recombination events disrupt an essential gene or regulatory function and result into non-viable phage that is rapidly eliminated. Sometimes, however, the recombination produces a viable combination that has a selective advantage over the donor genomes, resulting into a “new” phage. The Pittsburgh phage group has introduced “a moron accretion hypothesis” to explain how phage genomes have gradually increased in size by sequential addition of new genes. The term “moron” (for “more DNA”) describes a genetic element containing a gene preceded by a transcription promoter sequence and followed by a transcription terminator, thus including all the information needed for the gene to be transcribed. Recently added morons are often recognized by a G+C –content that clearly differs from that of the surrounding genes. A moron that benefits its new host then gradually becomes fully integrated into the phage genome and its regulatory circuit (Hendrix, 2002, Hendrix et al., 2003, Hendrix et al., 2000, Juhala et al., 2000). 2.2.4. The impact of phages on bacterial evolution Bacteriophages share a long co-evolution with their hosts and have had a fundamental influence on bacterial evolution and diversification ( Brüssow et al., 2004, Weinbauer & Rassoulzadegan, 2004). The struggle for survival between

20

phage and bacteria has provoked “an arms race”, where bacteria have developed defense mechanisms like restriction enzymes or changes in their phage-receptor molecules. Phages have then circumvented these by chemically modified nucleotides and avoidance of restriction enzyme recognition sequences or mutated adhesins, respectively (Comeau & Krisch, 2005, Weitz et al., 2005). Bacteriophages can promote the bacterial diversity in several ways. In the lytic state they may do it by “killing the winner” – that is, infecting and lysing the most abundant bacterial species, thus allowing less competitive species to co-exist (Weinbauer & Rassoulzadegan, 2004). Phages are also the main mediators in the horizontal evolution of prokaryotes (Canchaya et al., 2003). Phage-mediated gene transfer may occur either via (generalized) transduction or lysogenic conversion. In generalized transduction, the fragments of host DNA are accidentally packaged into phage head and delivered into a new host cell ( Brüssow et al., 2004, Weinbauer & Rassoulzadegan, 2004). Lysogenic conversion is a feature of temperate phages. Since the integration of the phage genome into the bacterial chromosome may be harmful for the bacterium (important genes or regulatory elements may be interrupted and the prophage constitutes a risk of induction to the lytic cycle), the prophage would be rapidly eliminated unless it encodes functions that increase the fitness of the lysogen. Among the commonly encountered lysogenic conversion genes are genes that confer immunity and exclusion to superinfection by similar or closely related phages, or genes whose products enhance the survival of the lysogen in its ecological niche. For pathogenic bacteria this means genes enhancing bacterial virulence, like different toxin genes and genes promoting bacterial adhesion, invasion, or resistance to human serum or phagocytosis ( Brüssow et al., 2004, Canchaya et al., 2003, Wagner & Waldor, 2002). On the evolutional time scale, the nature of prophage genomes in bacterial chromosomes is ephemeral. Mutations and deletions accumulate gradually in the genes coding for phage lytic functions, resulting in a defective prophage that can no longer be induced. Often, most of the original prophage genome is lost, and only the remnants of the phage sequence remain in the bacterial genome. In such cases the prophage origin of the remaining genes may be difficult to interpret ( Brüssow et al., 2004, Canchaya et al., 2003). 2.2.5. Phage receptors The first step in bacteriophage infection is the specific attachment of the phage to the surface components of the host cell. This attachment often takes place in two stages: a temperature-independent reversible step is followed by an irreversible step which is independent on temperature (Lindberg, 1973). The primary adhesin of tailed phages is usually the tail fiber. Extensively studied examples of such adhesins are T4 gp37, the large subunit of the distal tail fiber (Oliver & Crowther, 1981, Tétart et al., 1998), T7 gp17, whose trimers form the tail fibers (Steven et al., 1988) and the tail fiber protein J of λ (Wang et al., 2000). Some short-tailed

21

phages, like Salmonella phages P22 and SP6 and Shigella phage Sf6, have tail spikes instead of fibers (Baxa et al., 1996, Chua et al., 1999, Scholl et al., 2002). Filamentous phages fd, M13 and f1 use their minor capsid protein g3p to attach to E. coli cell (Bennett & Rakonjac, 2006, Lubkowski et al., 1999, Martin & Schmid, 2003) and lipid containing phages PM2 and PRD1 utilize spikes that are located at their vertices (Grahn et al., 1999, Huiskonen et al., 2004, Kivelä et al., 2002). Presumably every structure exposed on the bacterial surface may serve as bacteriophage receptor. For Gram-negative bacteria, this means carbohydrates, outer membrane proteins, or pilus and flagellum structures (Heller, 1992, Koebnik et al., 2000, Lindberg, 1973, Wright et al., 1980). LPS O-antigen is used e.g. by P22 (Baxa et al., 1996), Sf6 (Chua et al., 1999), and V. cholerae phage K139 (Nesper et al., 2000). Of these, both P22 and Sf6 have tail spikes possessing endorhamnosidase activity, which cleaves the O-antigen to facilitate the phage access to the injection site. The core region of LPS serves as receptor for phages like T3 and T4 (Prehm et al., 1976), T7 (Lindberg, 1973) and P. aeruginosa phage φCTX (Yokota et al., 1994). Many clinical isolates of E. coli are covered by a polysaccharide capsule, or K antigen. The structures of K antigens are highly diverse, and more than 80 K serotypes are known (Whitfield & Roberts, 1999). E. coli K antigens are utilized as receptors by several phages, like PK1A to E (Gross et al., 1977), K1F (Scholl & Merril, 2005, Vimr et al., 1984), K5 (Gupta et al., 1982) and K1-5, which is exceptional in having two different tail fiber proteins that allow it to recognize both K1 and K5 type capsules (Scholl et al., 2001). Capsule-specific phages often have capsule-degrading enzymatic activities associated with their tail spikes or fibers, the examples of which are the endosialidases of K1- specific phages (Kwiatkowski et al., 1982, Pelkonen et al., 1989). Except for serving as the receptor for bacteriophages, the polysaccharide capsule may also function as a barrier against phage infection, as shown recently for T7 (Scholl et al., 2005). The outer membrane proteins (OMPs) of Gram-negative bacteria serve to maintain the membrane structural integrity or function in solute and protein translocation. The transport proteins include non-specific porins (for transport of small, hydrophilic solutes), specific porins (for transport of larger molecules, e.g. oligosaccharides or nucleosides), and high affinity transporters (for transport of nutrients that are present in low concentrations). Most these protein groups are also utilized as bacteriophage receptors (Bonhivers et al., 1998, Koebnik et al., 2000). The E. coli major OMP, OmpA, is used by several phages (Morona et al., 1984, Morona et al., 1985, Schwarz et al., 1983), and a ferricrome transporter FhuA is recognized by T1, φ80, and T5 (Bonhivers et al., 1998, Endriss & Braun, 2004, Killmann et al., 1995, Plancon et al., 2002). The most extensively studied bacteriophage, λ, utilizes a maltoporin LamB as its receptor (Randall-Hazelbauer & Schwartz, 1973, Wang et al., 2000). Pili of Gram-negative bacteria are generally utilized as receptors by filamentous phages. E. coli phages fd, M13, and f1 (collectively called Ff phages) recognize the

22

F pilus as their primary receptor, and P. aeruginosa phages Pf1 and Pf3 use the type IV PAK pilus and the conjugative pilus, respectively (Holland et al., 2006, Lubkowski et al., 1999). The mannose-sensitive hemagglutinin type IV pilus serves as receptor for a newly characterized filamentous phage KSF-φ infecting V. cholerae (Faruque et al., 2005a). The utilization of bacterial pili is not, however, merely a feature of filamentous phages, as the broad host-range enveloped phage PRD1, for instance, recognizes the conjugative pilus (Grahn et al., 1999, Olsen et al., 1974). The flagellum can also function as the bacteriophage receptor, as exemplified by phage Χ that infects the motile strains of Escherichia, Salmonella, and Serratia (Samuel et al., 1999) and Aeromonas phage PM3 (Merino et al., 1990). The cell wall of Gram-positive bacteria is composed of multilayered peptidoglycan sacculus (also called murein) decorated with proteins, teichoic acids and neutral polysaccharides. Some strains also contain a surface layer (S-layer) made of paracrystalline protein subunits (Delcour et al., 1999). The phage-host interactions in Gram-positive bacteria are generally less known than those in Gram-negative bacteria. The phage adsorption to Gram-positive cell wall typically occurs via cell wall carbohydrates, often teichoic acids (Dupont et al., 2004, Wendlinger et al., 1996). Carbohydrates have been shown to function as receptors e.g. for Lactococcus lactis phages KH, bIL170 and φ645 (Dupont et al., 2004, Valyasevi et al., 1990) and teichoic acids for B. subtilis phage SP50 (Archibald & Coapes, 1976) and Listeria monocytogenes phages A118 and A500 (Wendlinger et al., 1996). Other structures may, however, also provide phage adsorption for Gram-positive bacteria, as B. subtilis phages PBS1 and SPP1 recognize the flagellum and an integral membrane protein, respectively (Raimondo et al., 1968, Sao-Jose et al., 2004), and Listeria phage A511 the peptidoglycan moiety (Wendlinger et al., 1996). 2.3. Applications of phages 2.3.1. Bacteriophage therapy The early bacteriophage research was largely driven by the desire to use phages to combat bacterial diseases – the phage therapy. Phages were independently discovered by Frederick Twort in 1915 and Felix d’Herelle in 1917, after which d’Herelle dedicated his career in phage therapy research (Stone, 2002, Summers, 1999, Summers, 2001). D’Herelle and other early phage therapy researchers used phages to cure shigellosis, cholerae and staphylococcal infections. The obtained results were often promising, but hampered by improper understanding about the bacteriophage biology. Also the experimental settings used, the crude and inefficient phage preparations and the lack of proper controls generated contradiction. The phage therapy research ceased at the advent of antibiotics in the Western world, but was continued actively in the Former Soviet Union and Eastern

23

Europe, especially Poland (McKinstry & Edgar, 2005, Stone, 2002, Sulakvelidze et al., 2001, Summers, 2001). Along with the increasing incidence of antibiotic-resistant bacteria, the interest in bacteriophage therapy has risen in the Western world again (Merril et al., 2003, Stone, 2002). As potential therapeutic agents, phages have many advances: The host specificity of a particular phage is usually very narrow, which minimizes the side effects of the treatment on the normal flora. The appearance of phage-resistant mutants is not as common as that of antibiotic resistance, and it is relatively easy to isolate a phage variant that is capable in infecting the mutated strain. Also, the development of a new therapeutic phage is cost-effective in comparison with the development of a new antibiotic ( Brüssow, 2005, Matsuzaki et al., 2005, Skurnik & Strauch, 2006). The major concerns of phage therapy include safety issues and pharmacokinetics. As discussed in Chapter 2.2.4., phages may carry genes whose products promote bacterial virulence. This is more a feature of temperate phages, thus only lytic phages whose genome has been sequenced should be used for therapeutic purposes (Brüssow, 2005, Bruttin & Brüssow, 2005). Phage pharmacokinetics is still rather poorly understood and more research in this field needs to be done. Often, in vitro and in vivo situations are substantially different and a phage that lyses its host bacteria in laboratory conditions may fail to do so in mammalian environment (Dabrowska et al., 2005, Kasman et al., 2002, Payne & Jansen, 2003). In recent years, phages have been successfully used e.g. to rescue mice from infection by Enterococcus faecium (Biswas et al., 2002) and Staphylococcus aureus (Matsuzaki et al., 2003). In addition to whole phages, also individual phage enzymes may be used as therapeutic agents, as demonstrated by Schuch et al. (2002) who used a phage lysin to cure mice infected with Bacillus anthracis (Schuch et al., 2002). 2.3.2. Phages in bacterial diagnostics Another field that has found a wide utilization of bacteriophages is bacterial diagnostics. At their best, phage-based detection methods may be rapid, specific, and economical (McKinstry & Edgar, 2005). Phage typing, i.e. studying the ability of a certain phage collection to propagate on a given bacterial isolate, has long been used in bacterial identification and subtyping (Baker & Farmer III, 1982, McLauchlin et al., 1996, Schmieger, 1999). Today, phage typing and other amplification assays are utilized mostly in combination with modern molecular typing methods, even though in developing countries they can provide a good alternative to more expensive diagnostic tools (Alcaide et al., 2003, Gali et al., 2006, Pai et al., 2005, Rodriguez-Calleja et al., 2006, Rybniker et al., 2006, Willshaw et al., 2001). In 1989, Ulitzur and Kuhn patented a detection method based on reporter phages, i.e. phages whose genomes carry reporter genes, like luciferase or green fluorescent protein (GFP). Infection of host bacteria by the reporter phage results in luciferase

24

or GFP expression, which can be monitored with appropriate apparatus (for example, luminometer or fluorescence microscope) (Billard & DuBow, 1998, Ulitzur & Kuhn, 1989, Ulitzur & Kuhn, 2000). Reporter phages have most thoroughly been applied in detection and antibiotic susceptibility testing of mycobacteria (Banaiee et al., 2001, Hazbon et al., 2003, Jacobs et al., 1993, Pai et al., 2005, Pearson et al., 1996, Sarkis et al., 1995), the median sensitivity of the M. tuberculosis detection being 106 CFU/ml (Banaiee et al., 2001). Reporter phages have also been developed for enteric bacteria (Kodikara et al., 1991), Listeria (Loessner et al., 1996, Loessner et al., 1997) and E. coli (Funatsu et al., 2002, Waddell & Poppe, 2000). The Listeria reporter was able to detect 500 to 1000 cells/ml and, after enrichment, one cell per g of salad (Loessner et al., 1996). A slightly different approach, where GFP was fused to capsid proteins of T4 and PP01, was used to detect E. coli K12 and O157:H7, respectively. This method even detected bacterial cells that are in viable but nonculturable state, which is a clear advantage over methods that require phage infection and amplification (Oda et al., 2004, Tanji et al., 2004). A method where phages labeled with fluorescent dyes were used to identify and enumerate bacteria in sea water was introduced by Hennes et al. (1995) (Hennes et al., 1995) and a similar technique has then been applied in the detection of E. coli O157:H7 (Goodridge et al., 1999a, Goodridge et al., 1999b) and Microlunatus phospovorus (Lee et al., 2006). The detection limits in these studies were 102 to 103 cells/ml. In past few years, diagnostic tools where phage-specific identification is combined to modern detection methods have been developed. To shorten the required enrichment time, an immunomagnetic separation step was combined to phage amplification assay in the analysis of Salmonella and E. coli O157:H7 (Favrin et al., 2001, Favrin et al., 2003). A system, where bacteriophage infection was linked to quorum sensing signaling and a bioluminescent bioreporter E. coli cell, was introduced by Ripp et al. and shown to detect E. coli in pure culture at the concentration of 1 CFU/ml (Ripp et al., 2006). Neufeld et al. presented a method where the phage-induced host cell lysis and subsequent release of intracellular enzymes was monitored by the amperometric detection of enzymatic activity, reaching sensitivity of 1 CFU/100 ml (Neufeld et al., 2003). A procedure where the phage amplification was followed by the detection of the phage capsid protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was applied to detect both E. coli alone and E. coli and Salmonella simultaneously. This method sensed ca. 5 × 104 cells in less than two hours (Madonna et al., 2003, Rees & Voorhees, 2005). Recently, Edgar et al. developed a technique where a biotinylation peptide was displayed on T7 major capsid protein and the biotinylation of this ‘reagent phage’ inside the target bacteria was detected by conjugating the phage to streptavidin-coated quantum dots and analyzing the conjugate with flow cytometry or fluorescence microscopy. This method was able to detect and quantify E. coli at ca. 10 CFU/ml in one hour assay time (Edgar et al., 2006).

25

2.3.3. Other applications Phage display, at its simplest, is a technique where peptides, proteins, or antibody fragments are expressed on the surface of phage particles (Willats, 2002). The technology was first developed for filamentous phages in 1985 (Smith, 1985) and is generally used for the selection of molecules that bind specifically to pre-determined targets. Phage display has been widely utilized e.g. in the identification of receptors and their ligands or proteins that modulate receptor activity (Hartley, 2002, Ladner et al., 2004) and isolation of recombinant antibodies (Azzazy & Highsmith, 2002, Petrenko & Vodyanoy, 2003). In addition to filamentous phages, display systems have recently been developed for members of other phage groups, like λ (Ansuini et al., 2002, Gupta et al., 2003), MS2 (van Meerten et al., 2001) and T7 (Danner & Belasco, 2001, Gnanasekar et al., 2004, Sokoloff et al., 2000). The modern molecular biology was largely based on bacteriophage research, and many enzymes nowadays used in in vitro genetic engineering have phage origin. Examples of such enzymes are T4 DNA polymerase, ligase and polynucleotide kinase, which are sold by most (if not all) biotechnology companies. Also heterologous gene expression systems based on T7 RNA polymerase (Studier & Moffatt, 1986) are in common use. A more recent application is recombineering, i.e. the use of enzymatic machinery for generalized recombination encoded by lambdoid phages. These enzymes catalyze homologous recombination between DNA sequences having very short homologies and provide a method for molecular cloning without restriction enzymes or DNA ligase (Thomason et al., 2005). 2.4. Bacteriophages of Yersinia Yersiniophages were first bought to public attention by Felix d’Herelle, who treated plague patients in Alexandria, Egypt, in 1925 with an “antiplague phage” he had isolated few years earlier (Summers, 1999, Summers, 2001). In the following decades, a number of yersiniophages were isolated, primarily for phage typing purposes (Baker & Farmer III, 1982, Calvo et al., 1981, Garcia et al., 2003, Kawaoka et al., 1987, Nilehn, 1969, Nunes & Suassuna, 1978). In these early publications, however, phages were mainly described by their host specificity, and more detailed information about these phages is scarce. The first more thorough characterization of yersiniophages was published in 1982 by Kawaoka et al., who studied morphology, adsorption kinetics and stability of three phages infecting Y. enterocolitica (Kawaoka et al., 1982). Interestingly, already in this paper it was stated that most Y. enterocolitica phages lyse their host cells when grown at 25oC but not at 37 oC, due to smaller amount of phage receptors on the cell surface at 37

oC. According to the present understanding, this indicates phages using LPS O-antigen as their receptors. In 1984, Stevenson and Airdrie reported the isolation and characterization of eight phages infecting Y. rückeri (Stevenson & Airdrie, 1984). A systematic molecular study of temperate yersiniophages was made in 2000 by Popp et al., who characterized the morphology, host range, genome size, DNA

26

homology and protein composition of eight phages (Popp et al., 2000). Still today, the genomic sequences of only three yersiniophages (φYeO3-12, φA1122 and PY54) are known. The main features of these phages are briefly discussed below. 2.4.1. φYeO3-12 φYeO3-12 was isolated from the sewage of Turku, Finland, in 1989 based on its ability to infect Y. enterocolitica serotype O:3 (YeO3) (Al-Hendy et al., 1991). It uses the LPS O-antigen as its receptor and is able to infect and proliferate on E. coli strains expressing the YeO3 O-antigen gene cluster. φYeO3-12 is a lytic phage belonging to Podoviridae and is closely related to E. coli phages T3 and T7 (I). The genome of φYeO3-12 is a 39 600 bp long linear, double-stranded DNA molecule having 58 putative genes that all are transcribed from the same DNA strand (Pajunen et al., 2001). The overall identity between φYeO3-12 and T3 genomes is about 84%, and the genome organization of these two phages is colinear (Pajunen et al., 2002). 2.4.2. φA1122 φA1122 infects most isolates of Y. pestis and is used as a diagnostic agent by the Centers for Disease Control and Prevention (CDC, the U.S.A.) in the identification of Y. pestis. The φA1122 genome is a 37,555 bp long DNA molecule having 51 predicted gene products. The genome is colinear with the genomes of T7, T3 and φYeO3-12 and the genome-wide identities with T7 and T3 are about 89% and 73%, respectively. Even though the general similarity of φA1122 gene products to their counterparts in T7 is much higher than to T3, there is a stretch of 9,188 bp (coding about half of the morphogenetic functions) having 99.8% identity with T3. The genomes of T7, T3, φYeO3-12 and φA1122 are thus mosaics in respect for each other, and it has been proposed that T3 is a host range mutant of a recombinant between two yersiniophages, one resembling φYeO3-12 and the other φA1122 (Garcia et al., 2003). 2.4.3. PY54 PY54 is a temperate phage belonging to Siphoviridae. It was isolated from Y. enterocolitica serotype O:5 and infects Y. enterocolitica strains belonging to the non-pathogenic biogroup 1A and the pathogenic serotype O:5,27 (Popp et al., 2000). The PY54 genome is a linear, 46,339 bp DNA molecule having 67 plausible genes, 55 of which are transcribed rightwards and 12 leftwards on the genetic map (Hertwig et al., 2003b). In the lysogenic state the PY54 genome is not integrated into the bacterial chromosome, instead, it is replicated as a linear plasmid having covalently closed hairpin ends. The phage genome and the plasmid prophage are almost 50% permuted in such a way that the phage cohesive ends are in the middle of the plasmid (Hertwig et al., 2003a). The strategy for PY54 to establish and maintain its prophage state is highly similar to E. coli phage N15, which is closely

27

related to lambdoid phages. However, the sequence similarity of these two phages is fairly limited outside the genomic regions involved in the conversion of the phage DNA into the plasmid prophage and the prophage maintenance and immunity (Hertwig et al., 2003b). 2.4.4. Applications of yersiniophages The early literature about yersiniophages was mainly focused on isolating phage collections and developing schemes for phage typing (see above). The culture-based methods for Yersinia diagnostics are time-consuming and often unreliable (Fredriksson-Ahomaa & Korkeala, 2003, Gomes-Solecki et al., 2005) and phage typing is, in combination with other methods, still used in the diagnostics of Y. pestis (Anisimov et al., 2004, Garcia et al., 2003) and Y. enterocolitica (Soltan-Dallal et al., 2004). After the pioneering work by d’Herelle, not many reports about phage therapy in Yersinia infections have been published, at least in English. Damasko et al. (2005) studied the bactericidal activity of enterocoliticin, a phage tail-like bacteriocin isolated from Y. enterocolitica serotype O:7,8 (Strauch et al., 2001), against Y. enterocolitica in cell culture and mice. In cell culture, enterocoliticin reduced the titer of bacteria adhering to the surface of eukaryotic cells, but had no antibacterial effect against bacteria that had invaded the cells. In the mouse model, enterocoliticin was not able to prevent the Y. enterocolitica infection (Damasko et al., 2005). Somewhat similar results were obtained when Y. enterocolitica –specific phages φYeO3-12 and PY100 were used in phage therapy experiments in mice (Skurnik & Strauch, 2006). In the G. Eliava Institute of Bacteriophage, Tbilisi, Georgia, an active research on yersiniophages that could be used for sanitation, prevention and treatment of the infection is carried out (Darsavelidze et al., 2003). However, only limited amount of data about these studies is available in the Western world, partly because of language restrictions. Yersiniophages have also been utilized as tools in basic microbiology research. Shaw et al. (1983) used an Y. pestis –specific mutant of E. coli phage T6 to follow the internalization of Y. pestis to macrophages (Shaw et al., 1983). In the Skurnik laboratory, phages φYeO3-12 and φR1-37 (III), specific to YeO3 LPS O-antigen and outer core, respectively, and φ80-81, specific to Y. enterocolitica O:8 (YeO8) O-antigen, have had an important role in studying the molecular biology of Yersinia LPS biosynthesis (Al-Hendy et al., 1991, Skurnik et al., 1995, Skurnik & Zhang, 1996, Zhang & Skurnik, 1994).

28

3. AIMS OF THE PRESENT STUDY This PhD thesis work originates from a project to construct a reporter bacteriophage that would be used as a diagnostic tool for detecting Y. enterocolitica serotype O:3 bacteria in food and clinical samples. Phage φYeO3-12 that has a strict host specificity to serotype O:3 strains as it uses the LPS O-antigen as its receptor (Al-Hendy et al., 1991) was chosen as a starting point for the construction of the reporter phage. For this purpose, a detailed molecular genetic analysis of the phage genome became necessary. In the course of the research, the aims of the studies expanded to the molecular, genetic and biological characterization of Y. enterocolitica –specific phages φYeO3-12 and φR1-37 and to the analysis of the receptor of Y. pestis –specific phage φA1122. The detailed aims were: • To characterize the biological properties of φYeO3-12

• To analyze the φYeO3-12 genome and identify the non-essential genes in order to be able to manipulate the phage genetically

• To construct a reporter bacteriophage starting from φYeO3-12 to be used in bacterial diagnostics

• To characterize the biological and genetic features of φR1-37

• To identify the cellular receptor of φA1122

29

4. MATERIALS AND METHODS The detailed description of the materials and methods used in the present study is given in the original publications I-IV. 4.1. Bacterial strains, phages and plasmids The bacteriophages and transposons used in this study are described in Table 3, bacterial strains in Table 4 and plasmids in Table 5. Unless otherwise stated, the Yersinia incubations were done at room temperature (RT) and E. coli was cultured at +37oC. Tryptic soy broth (TSB) medium (Oxoid) and Luria Broth (LB) (Sambrook & Russel, 2001) were used for bacterial liquid cultures. Soft-agar medium included additionally 0.4 % (w/v) agar (Biokar Diagnostics). Luria agar (LA) (Sambrook & Russel, 2001) was used as solid medium for bacteria and lambda agar (Tryptone 10 g l-1, NaCl 2.5 g l-1, agar 15 g l-1) for phage plates. Solid and liquid media were supplemented with antibiotics when required. Large-scale preparations of bacteriophages were purified by glycerol density gradient ultracentrifugation (Sambrook & Russel, 2001). Table 3. Bacteriophages and transposons used in this study Phage/Transposon Description Source

/reference φYeO3-12 Y. enterocolitica serotype O:3 specific, wild type

isolated from sewage (Al-Hendy et al., 1991)

Τ7+ Wild type from F.W. Studier Ian Molineux, Texas

Τ3+ Wild type from F.W. Studier Ian Molineux, Texas

φR1-37 Y. enterocolitica serotype O:3 specific, wild type isolated from sewage

(Skurnik et al., 1995)

φA1122 The reference phage used by the Centers for Disease Control and Prevention to identify Y. pestis

(Garcia et al., 2003)

ΔPK φYeO3-12 with deletion in gene 0.7 I φ::lacZ1 φYeO3-12 with lacZ’ insertion in gene 5.5; 16 237

(TTAAA) II

φ::lacZ2 φYeO3-12 with lacZ’ insertion in gene 0.45; 1 747 (CAGGG)

II

φ::lacZ3 φYeO3-12 with lacZ’ insertion in gene 5.5; 16 269 (GACAG)

II

φ::lacZ4 φYeO3-12 with lacZ’ insertion in gene 1.3; 7 830 (CCGCG)

II

φ::lacZ5 φYeO3-12 with lacZ’ insertion in gene 1.1; 6 475 (ATGGC)

II

φ::lacZ6 φYeO3-12 with lacZ’ insertion in gene 1.3; 7 680 II

30

(φ::lacZ61) (ATGAC) and gene 3.7; 11 300 (φ::lacZ62) (AGTGC)

φ::lacZ7 φYeO3-12 with lacZ’ insertion in gene 4.5; 13 523 (φ::lacZ71) (AGAAG) and gene 5.5-5.7; 16 529 (φ::lacZ72) (TACCT)

II

φ::lacZ8 φYeO3-12 with lacZ’ insertion in gene 1.3; 7 799 (ACATC)

II

φ::lacZ10 φYeO3-12 with lacZ’ insertion in gene 3.5; 11 206 (AACTG)

II

φ::lacZ11 φYeO3-12 with lacZ’ insertion in gene 0.7; 2 772 (GACGC)

II

φ::lacZ12 φYeO3-12 with lacZ’ insertion in gene 1.6; 8 623 (TACTG)

II

φ::lacZ13 φYeO3-12 with lacZ’ insertion in gene 4.3; 13 322 (AAACA)

II

φ::lacZ14 φYeO3-12 with lacZ’ insertion in gene 0,45; 1 682 (ATTGG)

II

φ::lacZ15 φYeO3-12 with lacZ’ insertion in gene 0.7; 2 423 (AGGCT)

II

φ::lacZ16 φYeO3-12 with lacZ’ insertion in gene 3.5; 11 134 (ACCAT)

II

φ::lacZ17 φYeO3-12 with lacZ’ insertion in gene 1.1; 6 528 (GCGGT)

II

φ::lacZ18 φYeO3-12 with lacZ’ Insertion at nt position 888 and deletion of nt 888 to 2449 (genes 0.3-0.7)

II

φΔ888-2449 φYeO3-12 with deletion of nt 888 to 2449 (genes 0.3-0.7)

II

φΔ1681-1741 φYeO3-12 with deletion of nt 1681 to1741 (in gene 0.45)

II

φΔ7801-7823 φYeO3-12 with deletion of nt 7801 to 7823 (in gene 1.3)

II

φΔ11141-11200 φYeO3-12 with deletion of nt 11141 to 11200 (in gene 3.5)

II

φ::lucFF1.2 Like φ::lacZ1; lacZ replaced with lucFF This work φ::lucFF5.3 Like φ::lacZ5; lacZ replaced with lucFF This work φ::lucFF1 φΔ888-2449 with lucFF inserted in between genes 6.3

and 6.5; 17 988 (CTTAA) This work

Transposon lacZ’-Mu(NotI)

Promoterless lacZ’, NotI sites close to transposon ends (Vilen et al., 2003)

Transposon lucFF-Mu

Promoterless lucFF H. Vilen and H. Savilahti, unpublished

For transposon insertion mutants, the interrupted gene and the first base pair after the 5 bp duplication are indicated. The duplicated sequence is shown in parenthesis. Deletion mutants are named by indicating the base pairs that are deleted. For φΔ888-2449, the nucleotide numbering of wt φYeO3-12 is used.

31

Table 4. Bacterial strains used in this study Bacterial strain Characteristics Source

/reference Yersinia enterolitica 6471/76 (YeO3) Serotype O:3; wild-type patient isolate (Skurnik,

1984) 6471/76-c (YeO3-c) Serotype O:3; virulence plasmid-cured derivative

of 6471/76 (Skurnik, 1984)

YeO3-R1 Serotype O:3; spontaneous O-antigen negative derivative of 6471/76-c

(Skurnik et al., 1995)

YeO3-c-OC Serotype O:3. Δ(wzx-wbcQ). Outer core negative derivative of 6471/76-c

(Biedzka-Sarek et al., 2005)

YeO3-OC-R Serotype O:3. Δ(wzx-wbcQ). Outer core and O-antigen negative derivative of 6471/76-c

(Biedzka-Sarek et al., 2005)

8081 (YeO8) Serotype O:8 (Portnoy et al., 1981)

8081-R2 Serotype O:8; O-antigen negative derivative of 8081

(Zhang et al., 1997)

467/73 Serotype O:9. Human stool isolate from patient. Sensitive to φR1-37.

(Skurnik, 1985)

467/73-φR1-37-R Spontaneous phage φR1-37 resistant derivative of 467/73

III

3229 Serotype O:50. Human stool isolate. Sensitive to φR1-37.

(Skurnik & Toivanen, 1991)

3229-φR1-37-R Spontaneous phage φR1-37 resistant derivative of 3229

III

14779/83 Serotype O:5. Human stool isolate. Sensitive to φR1-37.

(Skurnik, 1985, Skurnik & Toivanen, 1991)

14779/83-φR1-37-R Spontaneous phage φR1-37 resistant derivative of 14779/83

III

18425/83 Serotype O:25,26,44. Human stool isolate. Sensitive to φR1-37.

(Skurnik, 1985, Skurnik et al., 1995)

18425/83-φR1-37-R Spontaneous phage φR1-37 resistant derivative of 18425/83

III

Yersinia pseudotuberculosis

PB1 Serotype O:1b (Porat et al., 1995)

1 Serotype O:1a; lcr- (Samuelsson et al., 1974)

43 Serotype O:3; lcr- (Samuelsson et al., 1974)

32 Serotype O:4a; lcr- (Samuelsson et al., 1974)

R708Ly Serotype O:9 reference strain, isolated from mole in Japan. Sensitive to φR1-37.

(Tsubokura & Aleksic, 1995)

32

YPIII Serotype O:3 (Bölin et al., 1982)

R708Ly-R Spontaneous rough derivative of R708Ly III R708Ly-φR1-37-R Spontaneous phage φR1-37 resistant derivative

of R708Ly III

PB1Δwb Serotype O:1b; O-antigen negative derivative of PB1, kmR

IV

PB1Δwb-R4 Spontaneous φA1122 resistant derivative of PB1Δwb

IV

PB1Δwb-R7 Spontaneous φA1122 resistant derivative of PB1Δwb

IV

PB1Δwb-R12 Spontaneous φA1122 resistant derivative of PB1Δwb

IV

Yersinia pestis KIM D27 Lcr+, pgm-, pst+ (Garcia et al.,

1999) KIM D27Nar NalR; Lcr+, pgm-, pst+ III EV76-c Virulence-plasmid cured derivative of EV76 (Ben-Gurion &

Hertman, 1958, Portnoy & Falkow, 1981)

Yersinia frederiksenii IP23047 Serotype O:3 Elisabeth

Carniel, Institut Pasteur

Yersinia mollaretii IP22404 Serotype O:3 Elisabeth

Carniel, Institut Pasteur

Yersinia kristensenii IP22828 Serotype O:3 Elisabeth

Carniel, Institut Pasteur

Yersinia intermedia 821/84 Serotype O:52,54. Human stool isolate (Skurnik &

Toivanen, 1991)

821/84-φR1-37-R Spontaneous phage φR1-37 resistant derivative of 821/84

III

Escherichia coli JM109 F´ traD36 proA+B+ lacIq Δ(lacZ)ΔM15/ Δ(lac-

proAB) glnV44 e14- gyrA96 recA1 relA1 endA1 thi hsdR17

(Yanisch-Perron et al., 1985)

DH10B F- mcrA Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15 ΔlacX74, deoR, recA1 endA1 araΔ139 Δ(ara, leu)7697 galU, galK λ- rpsL nupG λ- tonA

Life Technologies

C600 thi thr leuB tonA lacY supE (Appleyard, 1954)

HB101 F-Δ(gpt-proA)62 leuB6 glnV ara-14 galK2 lacY1 Δ(mcr-mrr) rpsL20 (Strr) xyl-5 mtl-1 recA13

(Boyer & Roulland-Dussoix, 1969)

33

IJ511 ΔlacX74 galK2 galT22 supE44 hsdS Ian Molineux, Texas

IJ512 F42 (F’ lac) derivative of IJ511 Ian Molineux, Texas

IJ855 ara Δ(lac-proAB) thi supD hsrD Ian Molineux, Texas

IJ1419 lacY1 or Δ(lac)6 supE44 galK2 galT22 λ- rfbD1 metB1 mcrA1 hsdR2 rpoC319 (tsnB)

Ian Molineux, Texas

IJ1420 lacY1 or Δ(lac)6 supE44 galK2 galT22 λ- rfbD1 metB1 mcrA1 hsdR2 rpoC320 (BR3)

Ian Molineux, Texas

CJ236 F+LAM-, dut-1, ung-1, thi-1, spoT1, relA1/ pCJ105 (CmR)

E. coli Genetic Stock Center

Sm10λpir thi thr leuB tonA lacY supE recA::RP4-2-Yc::Mu-Km (λpir)

(Simon et al., 1983)

Shigella sonnei IJ286 D2371-48 (Hausmann et

al., 1968) Salmonella enterica serovar Typhimurium

His515 Δ(his-rfb) (Nikaido et al., 1967)

TV-163 rfaL (Beckmann et al., 1964, Subbaiah & Stocker, 1964)

Table 5. Plasmids used in this study Plasmid Characteristics Source

/reference pUC18 ampR, Cloning vector (Yanisch-

Perron et al., 1985)

pAY100 ampR tetR; O-antigen gene cluster of YeO3 cloned in pBR322

(Al-Hendy et al., 1991)

pTM100 clmR tetR; Mobilizable cloning vector (Michiels & Cornelis, 1991)

pAY100.1 ampR; Derivative of pAY100 with the tetracycline-resistance marker inactivated

(Oyston et al., 2003)

pBR322 ampR tetR; Cloning vector (Bolivar et al., 1977, Sutcliffe, 1979)

pRK2013 kmR; Helper-plasmid for conjugation (Ditta et al., 1980)

pBAD33oT clmR; oriT from pTM100 cloned into pBAD33 Pacot-Hiriart et al., unpublished data

pCSS810 clmR kmR; lucFF in E. coli – B. subtilis shuttle vector

(Lampinen et al., 1992)

pRV7 ampR; YeO3 outer core gene cluster cloned in (Skurnik et al.,

34

pBR322 1995) p34S-Km kmR; ampR; Cloning vector (Dennis &

Zylstra, 1998) pCVD442 ampR,kmR, Suicide vector (Donnenberg

& Kaper, 1991)

pBAD33oT-g1.3 clmR; φYeO3-12 gene 1.3 cloned into pBAD33oT

II

pEcligA clmR; E. coli C600 ligA cloned into pTM100 II pg3.5long clmR; φYeO3-12 gene 3.5 cloned into

pBAD33oT II

pprg3.5 clmR; φYeO3-12 promoter 2.5 and gene 3.5 cloned into pBAD33oT

II

pMP300 clmR; reporter plasmid, a pTM100 derivative. The lucFF gene from pCSS810 cloned between φYeO3-12 genes 9 and 10 downstream of φ10 promoter

II

pRV16NP clmR; outer core gene cluster of YeO3 cloned in pTM100

III

pUCwbup ampR; the upstream region of O-antigen gene cluster of YPIII cloned in pUC18

IV

pUCwbdel ampR; the up- and downstream regions of the YPIII O-antigen gene cluster cloned in pUC18

IV

pUCwbGB ampR, kmR; the kanamycin resistance gene of p34S-Km cloned between the up- and downstream regions of the YPIII O-antigen gene cluster in pUCwbdel

IV

pCVDwbGB ampR, kmR; Suicide vector carrying the PvuII fragment of pUCwbGB including the kanamycin resistance gene and the up- and downstream regions of O-antigen gene cluster of YPIII

IV

pMP100 Like pMP300; the orientation of the insert in pTM100 is opposite

This work

pMP200 Like pMP100; the insert contains an additional translation terminator after lucFF

This work

pSK110 clmR; reporter plasmid, a pTM100 derivative. The lucFF gene from pCSS810 was cloned between φYeO3-12 genes 10 and 11.

This work

4.2. Biological characterization of bacteriophages 4.2.1. Host specificity, efficiency of plating, growth curve and fitness-analysis (I, II, III) The host range of phages was determined by pipetting droplets of phage dilutions on bacterial lawn and observing the formation of a clear lysis zone. The efficiency of plating (EOP) was analysed by plating the phage dilution with the bacterial suspension on top agar plates (Sambrook & Russel, 2001).

35

One-step growth curves were determined as described earlier (Birge, 1988). Bacterial cells in mid-exponential growth phase were infected with the phage, samples were taken at time intervals and the PFU/ml was titrated both immediately and after the release of intracellular phages with chloroform. Fitness-analysis was performed as previously described for T7 (Rokyta et al., 2002). The bacteria were infected with the phage and the number of intracellular phages was titrated from samples taken at 0 and 45 minutes. 4.2.2. Antisera (I) Polyclonal antisera against φYeO3-12 was obtained by sequential immunization of three rabbits with the purified phage. The ability of the antisera to recognize φYeO3-12 was tested by enzyme immunoassay (EIA) using microtiter plates (Nunc) coated with the phage. The capability of the strongest antiserum to neutralize the φYeO3-12 infection was tested by incubating the serum and the phage for 10 minutes prior to titrating the PFU/ml. 4.2.3. Receptor characterization and adsorption analysis (III, IV) To characterize the receptor of φR1-37, YeO3-R1 (Table 2) cells were immobilized on nitrocellulose membrane and treated with periodate, Proteinase K or SDS sample buffer. The ability of digoxigenin (DIG) -labeled φR1-37 to recognize the immobilized bacteria was then studied according to the instructions of the DIG Protein Labelling Kit (Roche). For the phage adsorption assay, φA1122 was incubated with the bacteria for five minutes, after which the cells together with the bound phage were centrifuged down and the phage remaining in the supernatant was titrated. The effect of periodate and Proteinase K on the phage receptor was tested by incubating the cells with the reagent for two or three hours, respectively, prior to the adsorption assay. 4.2.4. LPS isolation and analysis (III, IV) LPS was isolated and analysed by deoxycholate-polyacrylamide gel electrophoresis (DOC-PAGE) as described earlier (Skurnik et al., 1995, Zhang & Skurnik, 1994). 4.3. Structural characterization of bacteriophages 4.3.1. Electron microscopy (I, III) The phages were sedimented with ultracentrifuge and stained with phosphotungstic acid. Stained particles were examined with Philips EM300 (I) or JEOL (Tokyo, Japan) JEM-1200 EX (III) electron microscope. For infection samples, YeO3-R1 cells were incubated with φR1-37 for two minutes prior to examination.

36

4.3.2. SDS-PAGE, Western-analysis and N-terminal sequencing of structural proteins (I, III) SDS-PAGE of purified φYeO3-12 and φR1-37 particles was conducted using a Hoefer SE 600 device (Amersham Pharmacia Biotech) according to manufacturers instructions. For Western-analysis, the proteins were blotted to a nitrocellulose membrane and identified with the polyclonal anti-φYeO3-12 serum (above). For the determination of amino-terminal amino acid sequences, the protein bands were blotted to a Problott membrane (Applied Biosystems) and the sequences of the immobilized proteins were determined with an Applied Biosystems 477A pro-tein sequencer equipped with an Applied Biosystems 120A PTH (phenylthiodan-toin)-amino acid analyzer using standard parameters provided by the manufacturer. The sequence data was analyzed with the European Bioinformatics Institute Fasta3 search (http://www.ebi.ac.uk/fasta33/), the National Center for Biotechnology Information (NCBI) BLAST program (www.ncbi.nlm.nih.gov/blast/Blast.cgi) and the European Molecular Biology Open Software Suit (EMBOSS) version 2.6.0. 4.4. Molecular biology techniques 4.4.1. General DNA techniques (I, II, III, IV) Standard DNA techniques were performed (Ausubel et al., 1987, Sambrook & Russel, 2001), and enzymes were used as recommended by the suppliers. Phage DNA was extracted as described for bacteriophage lambda (Sambrook & Russel, 2001). DNA sequencing reactions were performed with ABI PRISMTM BigDyeTM Terminator Cycle Sequencing v2.0 or v3.1 Ready Reaction Kit and analyzed with ABI 377 DNA analyzer as recommended by the manufacturer. The sequence data was analyzed with the Genetics computer Group (GCG) suite of programs (version 10, Accelrys, San Diego, CA), EMBOSS and NCBI BLAST program. For DNA hybridization, the DNA was labelled with DIG using the DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche Molecular Biochemicals). 4.4.2. In vitro transposon mutagenesis (II) The MuA transposase-catalyzed DNA transposition reaction was performed as described previously (Vilen et al., 2003) using lacZ’-Mu(NotI) transposon as donor DNA and φYeO3-12 DNA as target DNA. Mutant phage clones were identified on indicator plates by visual inspection, and transposon insertion sites were localized by restriction analysis, PCR-based analysis and sequence determination. 4.4.3. mRNA isolation and analysis (II) Total RNA from YeO3-c cells infected with wild type φYeO3-12 or its derivatives (Table 1) was isolated with Bio-Rad AurumTM Total RNA Mini Kit, and an extra treatment with RQ1 DNAse (Promega) was performed when necessary. For RT-PCR, the cDNA synthesis was done with Amersham Pharmacia Biotech Ready-To-Go You-Prime First-Strand Beads and the following PCR was done with

37

DyNAzyme DNA polymerase using conditions recommended by the manufacturer. The mRNA and RT-PCR samples were analysed with electrophoresis by standard procedures. 4.4.4. Analysis of modified nucleotides (III) The nucleotide composition of the φR1-37 genome was determined by hydrolyzing the phage DNA to deoxynucleosides by the method of Crain (Crain, 1990) and analyzing the hydrolysate for nucleosides and dinucleotides by LC-MS/MS (PE Sciex API 365 triple quadrupole LC/MS/MS System equipped with two PE Series 200 Micro pumps and a PE Series 200 autosampler). 4.5. Luminescence measurements and the construction of the reporter phage 4.5.1. Luminescence measurements (II) The luminescence was measured from bacterial suspension samples with Luminova 1254 luminometer (Bio-Orbit) using d-luciferin, pH 5.0 (Labsystems) as a substrate. 4.5.2. Construction of the reporter phage To construct the reporter phage by homologous recombination, plasmids where lucFF was cloned in between φYeO3-12 genes 9 and 10 (pMP100 and pMP200) or genes 10 and 11 (pSK110) were made (Table 5) The construction of these plasmids followed the scheme described for pMP300 (II). For homologous recombination to occur, cultures of YeO3-c carrying the plasmids were infected with φYeO3-12. In order to obtain pure reporter phage preparations, a direct cloning approach was utilized. For this purpose, the lacZ’ inserts of transposon mutants φ::lacZ1 and φ::lacZ5 (Table 3, II) were replaced with lucFF gene to generate phages φ::lucFF1.2 and φ::lucFF5.3, respectively (Table 3). For the cloning, standard techniques were used and the recombinant phage DNA was electroporated to bacterial cells like described for transposon mutagenesis (II). The expression of active luciferase during phage infection was verified by measuring the light production and the stability of the reporter phages was studied by plating the lysates on top-agar plates, isolating several plaques and measuring their luminescence production. To confirm the results of stability measurements, DNA was isolated from φ::lucFF1.2 and φ::lucFF5.3 lysates and the genomic region containing the lucFF gene was analysed with PCR. The in vitro transposon mutagenesis approach was utilized to create luciferase reporter starting from the deletion mutant φΔ888-2449 (Table 3, II). The MuA transposon reaction was done using lucFF-Mu (Table 3) as donor and φΔ888-2449 DNA as recipient, otherwise the reaction and the subsequent electroporation and phage plating were performed as described (II). The reporter phage φ::lucFF1 (Table 3) was isolated based on its ability to produce light during the infection of YeO3-c. The ability of φ::lucFF1 to detect YeO3-c cells in pure culture was tested

38

by infecting cells at different concentrations with different PFU of φ::lucFF1 and measuring the luminescence at multiple time points. The stability of the mutant was determined as above. 5. RESULTS AND DISCUSSION 5.1. Characterization of φYeO3-12 φYeO3-12 was isolated from the sewage of Turku, Finland, in 1989 (Al-Hendy et al., 1991). It infects Y. enterocolitica serotype O:3 and other Yersinia strains containing 6-deoxy-L-altrose in their LPS. In addition, it is able to infect and proliferate on E. coli strains expressing the YeO3 O-antigen gene cluster. The spontaneous phage-resistant derivatives of YeO3 have lost the O-antigen. (Al-Hendy et al., 1991, Al-Hendy et al., 1992). 5.1.1. φYeO3-12 is related to T3 and T7 (I) The electron microscopy showed that φYeO3-12 is a member of the family Podoviridae (I, Figure 1). The genome size, ca. 40 kb, was typical for this type of phages (I, Figure 2). As shown by the one-step growth curve (I, Figure 3), the eclipse and latent periods were 15 and 25 minutes, respectively, and the burst size was ca. 140 PFU per infected cell. The antiserum raised against φYeO3-12 was able to inhibit the φYeO3-12 infection (I, Figure 6) and, interestingly, T3 was also neutralised by the serum. T7 infection was not inhibited, which indicated that φYeO3-12 is more closely related to T3 than to T7. This hypothesis was supported by the N- terminal amino acid sequencing of φYeO3-12 structural proteins (I, Table 3) and the growth of φYeO3-12 on bacterial strains that are restrictive to T7 but not to T3. 5.1.2. Transposon insertions in the early genomic region of φYeO3-12 cause growth defects on Y. enterocolitica (II) Even though the φYeO3-12 genome had been sequenced (GenBank accession no. AJ251805) and found to be closely related to T3 and T7 (Pajunen et al., 2002, Pajunen et al., 2001), there were still open reading frames without obvious role. The in vitro transposon mutagenesis was carried out in order to study further the function of the phage genome and, especially, to identify nonessential genes. In this assay, 17 insertion mutants were obtained, in which five insertions were located in the early and 14 in the middle region of the phage genome (II, Figure 1). The transposon mutants having the insertion in the early genomic region, φ::lacZ14, φ::lacZ2, φ::lacZ15, φ::lacZ11 and φ::lacZ18 (Table 3), had lowered EOP on Y. enterocolitica compared with E. coli and other enterobacteria (II, Table 4). For φ::lacZ11, the growth problem was also seen in the fitness-analysis (II,

39

Figure 2). The growth defects were shown to be due to the inserted transposon and not the interruption of the individual genes, since the corresponding deletion mutants plated normally on YeO3-c. The mRNA analysis showed that in the insertion mutants, the expression of gene 1, coding for the phage RNA polymerase (RNAP), was delayed (II, Figure 3), which was considered the probable cause of the growth problems. The effects of the interruption of the early genes 0.45 and 0.7 became visible when a luciferase reporter strain YeO3-c/pMP300, indicating the activity of the phage RNAP, was infected with the aforementioned mutants (II, Figure 4 A, B and C). The gene 0.45 mutants produced slightly more light than the wild type phage, implying that gp0.45 might have a role in the regulation of the phage growth cycle. This protein is unique to φYeO3-12 (Pajunen et al., 2001) and this is the first indication about its role. The functions of gp0.7, on the contrary, are well known, one of which is the shutoff of the host transcription (Hesselbach & Nakada, 1977, Marchand et al., 2001). It is thus not surprising that the mutants having defective gene 0.7 showed an increased RNAP expression. 5.1.3. φYeO3-12 DNA ligase and lysozyme are needed for growth on Y. enterocolitica (II) The transposon mutants having the insertion in the middle region of the φYeO3-12 genome showed heterogenous phenotypes. The interruption of genes 1.1., 1.6 and 5.5. had no obvious effect on the phage phenotype. However, the phage mutants with the transposon integrated in genes 1.3 and 3.5, coding for DNA ligase and lysozyme, respectively, had a clearly slower growth rate on YeO3-c even though they grew normally on E. coli, as indicated by the fitness-analysis (II, Figure 2). The reduction of the growth rate was due to the inactivation of the genes and not the transposon insertion itself, since the corresponding deletion mutants showed a similar phenotype (II, Figure 2) and the slow-growing phenotypes were complemented with the corresponding wild type genes (II, Figure 5). In addition, the growth defects of the gene 1.3 mutants were partially complemented by heterologous expression of E. coli DNA ligase [which is known to complement T7 gp1.3 (Rokyta et al., 2002)] and the expression of the phage lysozyme in trans was able to revert the growth rate of the gene 3.5 deletion mutant to the level comparable with wild type φYeO3-12 (II, Figure 2). In the above-mentioned luciferase reporter assay, the gene 1.3 insertion mutants showed an increase in the RNAP activity (II, Figure 4 D). As the corresponding deletion mutant produced light at a roughly same level than the wild type phage, it is possible that the effect was mainly caused by the transposon insertion itself and not the interruption of the DNA ligase gene. The lysozyme mutants, instead, showed a significant increase in the light production (II, Figure 4 E), indicating that in these mutants the regulation of RNAP activity was lost. This is an expected finding, since T7 lysozyme is known to regulate the phage RNAP activity (Huang et al., 1999, Zhang & Studier, 1997).

40

In conclusion, this part of the study identified genes that are nonessential for φYeO3-12, at least in laboratory conditions. However, the genes coding for DNA ligase and lysozyme were needed for growth on the normal host of the phage, Y. enterocolitica, even though they were not required on E. coli. These genes may thus be considered as evolutionary factors important in adaptation of φYeO3-12 to grow on Yersinia. The molecular mechanisms behind the different requirements for phage growth on E. coli and Yersinia are not, however, understood. 5.2. Characterization of φR1-37 Phage φR1-37 was isolated in 1990 from the incoming sewage of Turku, Finland, using the virulence-plasmid cured, O-antigen negative strain YeO3-R1 (Table 4) as host (Skurnik et al., 1995). The ability of the phage to infect the O-antigen positive, virulence-plasmid negative strain YeO3-c was dependent on growth temperature in such a way that the strain was resistant when grown at 22 oC and sensitive at 37 oC. The virulence-plasmid positive strain YeO3, interestingly, was resistant to phage φR1-37 irrespective of the growth temperature, whereas rough forms of both virulence plasmid positive and negative YeO3 were sensitive at both temperatures. It was thus concluded that the phage receptor is as structure which is blocked by the abundant O-antigen expression or YadA [Yersinia adhesin A, an outer membrane protein which is expressed by the virulence plasmid at 37ºC (Bölin et al., 1982, Bölin et al., 1985, El Tahir & Skurnik, 2001)] combined to less abundant O-antigen. Since φR1-37 resistant mutants were missing the LPS outer core, it was hypothesized that this structure might serve as the phage receptor (Skurnik et al., 1995). Besides the host range and the postulated receptor, no information about φR1-37 was available. The aim of this project was to characterize further the biological, structural and genomic features of this phage. 5.2.1. Biological and structural features of φR1-37 (III) The electron microcopy showed that φR1-37 is a large member of the family Myoviridae (II, Figure 1). In the one-step growth curve (III, Figure 2), the eclipse and latent periods were 40 and 50 minutes, respectively, which were followed by a rise period of 20 minutes. The average burst size of φR1-37 was ca. 80 PFU/infected cell. The presence of φR1-37 related DNA sequences on bacterial chromosomes was tested by hybridizing the DIG-dUTP labelled φR1-37 DNA to the genomic DNA of 128 bacterial strains (not shown). The hybridization produced no positive signals, indicating a strictly lytic life cycle. SDS-PAGE of φR1-37 structural proteins showed four major protein bands, sp69, sp46, sp31 and sp24 (III, Figure 6). Of these, sp46 was the most abundant, which makes it the likely major capsid protein. The N-terminal amino acid sequencing of these proteins revealed that the N-termini of sp69 and sp46 were almost identical,

41

suggesting that sp69 may be the minor capsid protein. The comparison of the amino acid sequences to databanks did not discover significant similarities to any known protein sequences, indicating that close relatives of φR1-37 have not been described. 5.2.2. The φR1-37 receptor (III) There was already an indirect indication that Y. enterocolitica serotype O:3 LPS outer core might serve as the phage receptor (see above), however, it could not be ruled out that the receptor would be composed of some protein structure, either alone or in combination with LPS. To unequivocally determine the φR1-37 receptor, YeO3-R1 cells were immobilized on membranes that were subjected to different treatments. The ability of the phage to bind the immobilized cells was then tested. Since the treatment with Proteinase K had not effect on the phage binding but periodate (which degrades carbohydrates) abolished it (III, Table 2), it was concluded that the receptor is composed of LPS and does not contain protein structures. As the final confirmation, plasmid PRV16NP (Table 5), carrying the gene cluster for YeO3 outer core biosynthesis, was transferred to several φR1-37 resistant Yersinia and E. coli strains. All the strains harboring this plasmid became sensitive to the phage, proving that the YeO3 outer core is the receptor for φR1-37. A somewhat surprising finding was that even though the spontaneous φR1-37 resistant Y. enterocolitica and Y. intermedia derivatives (Table 4) had lost the LPS outer core, two separate phage resistant isolates of Y. pseudotuberculosis serotype O:9 (R708Ly-R and R708Ly-φR1-37-R) did not have the O-antigen (III, Figure 3). It thus seems that the sugar residues forming the YeO3 outer core (shown in Figure 1) are part of O-antigen of Y. pseudotuberculosis O:9. However, the sugar composition and structure of the LPS of this strain are not yet known. 5.2.3. The size and composition of the of φR1-37 genome (III) The size of φR1-37 genome was determined by digesting the phage DNA with restriction enzyme and analyzing the products by pulse-field electrophoresis (PFGE). The analysis gave a size estimation of 270 kb, which is comparable with the 280 kb of P. aeruginosa phage φKZ, the largest bacteriophage genome whose nucleotide sequence has been determined (Mesyanzhinov et al., 2002). While doing the restriction digestions to φR1-37 DNA, we noticed that Acc65I failed to digest the DNA even though its isoschizomer KpnI digested it into > 10 fragments. Since Acc65I is known to be inhibited by DNA methylation, we reasoned that the phage DNA might be chemically modified and set up a study to analyse its chemical composition. For this, the DNA was enzymatically hydrolyzed to nucleosides, which were analysed by LC-MS/MS. The phage genome was found to contain three normal deoxynucleosides dA, dC, and dG (III, Figure 5). However, a highly interesting finding was that instead of T, there was a peak in the

42

chromatogram whose molecular weight and retention time were identical to those of dU. Until this, the only known viruses having dU-DNA were B. subtilis phages PBS1 and PBS2 (Takahashi & Marmur, 1963). The GC-content of the φR1-37 DNA, as estimated from the peak area of the individual nucleosides in the chromatograph, was 36 %. The proportions of individual nucleosides were 31 % for dA, 33 % for dU, 18 % for dG, and 18 % for dC. 5.2.4. Sequencing of the φR1-37 DNA (III, this work) The sequencing of the φR1-37 genome was initiated by cloning fragments of the phage DNA to plasmid pBR322 (Table 5) using E. coli strain CJ236, devoid of dUTPase and uracil-DNA glycosylase (Table 4), as a host. In this strain, the base-excision repair (BER) pathway was inactivated and the dU-DNA remained undamaged (Lindahl et al., 1977, Sung & Mosbaugh, 2003, Taylor & Weiss, 1982). For sequencing, both the plasmid clones and the phage genomic DNA were used as templates. At the moment, ca. 76 kb of the phage genome has been sequenced. The preliminary sequence analysis showed that the GC-content was 33 %, which is in acceptable agreement with the value obtained by the LC-MS/MS. In the sequence analysis, genes coding for structural proteins sp24 and sp46 were identified (accession numbers AJ972879 and AJ972880, respectively). However, it not yet known whether sp46 and sp69 are produced from the same or different genes. Some deduced protein sequences of φR1-37 showed similarity to known proteins, the examples of which are shown in Table 6. An interesting feature was the presence of four stretches of sequence with high similarity to φKZ (Mesyanzhinov et al., 2002). As might be expected, though, most φR1-37 open reading frames (ORFs) produced no hits in databanks. 5.3. Identification of the φA1122 receptor φA1122 is a T7-related bacteriophage that infects most isolates of Y. pestis (Garcia et al., 2003). It is used as a diagnostic phage by CDC and can differentiate Y. pestis and Y. pseudotuberculosis by infecting both strains at 37oC but only Y. pestis at 20oC. In this work, we aimed to characterize the φA1122 receptor by studying the phage adsorption to bacteria with different LPS structures and testing whether the degradation of cell surface proteins or carbohydrates abolishes the phage recognition.

43

Tab

le 6

. Pro

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s sho

win

g si

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to d

educ

ed φ

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rote

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44

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he

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ber,

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eren

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able

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irect

ed R

NA

po

lym

eras

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82 a

a)

65 a

a K

luyv

erom

yces

lact

is

2e

-04

P054

72, (

Wils

on &

Mea

cock

, 19

88)

O

RF

025

(716

aa)

21

0 aa

φK

Z 1e

-06

AF3

9901

1, (M

esya

nzhi

nov

et

al.,

2002

) O

RF

050

(731

aa)

23

0 aa

φK

Z 4e

-10

AF3

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1, (M

esya

nzhi

nov

et

al.,

2002

) O

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RN

A P

olym

eras

e (1

450

aa)

989

aa

φKZ

8e-5

2 A

F399

011,

(Mes

yanz

hino

v et

al

., 20

02)

OR

F 18

2 (6

64 a

a)

194

aa

φKZ

1e-0

9 A

F399

011,

(Mes

yanz

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v et

al

., 20

02)

DN

A p

olym

eras

e I (

875

aa)

240

aa

Sulfo

lobu

s aci

doca

ldar

ius

1e-0

5 P9

5690

, (D

atuk

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t al.,

19

96)

DN

A d

oubl

e-st

rand

bre

ak re

pair

rad5

0 A

TPas

e (8

86 a

a)

228

aa

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lobu

s aci

doca

ldar

ius

5e

-06

O33

600,

(Elie

et a

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997)

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te k

inas

e (1

99 a

a)

59 a

a An

abae

na sp

. (st

rain

PC

C 7

120)

1e-0

5 Q

8Z0I

7, (K

anek

o et

al.,

200

1)

Rev

erse

tran

scrip

tase

(RN

A d

i-re

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olym

eras

e)

168

aa

Con

serv

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omai

n

9e-0

9 pf

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tp://

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ov/S

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6 Pl

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otei

n (9

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) 83

aa

Mag

neto

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-09

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2005

) U

nnam

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in; s

imila

r to

a pu

tativ

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il fib

er p

rote

in o

f a

prop

hage

(493

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383

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Phot

orha

bdus

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ines

cens

su

bsp.

laum

ondi

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-57

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a Onl

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t for

eac

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quen

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pre

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ze o

f the

sim

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dica

tes t

he si

ze o

f the

ded

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φR

1-37

pro

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5.3.1. The φA1122 receptor is a LPS core structure present in Y. pestis and Y. pseudotuberculosis but not in Y. enterocolitica (IV) The adsorption assay showed that the φA1122 adsorption to Y. pseudotuberculosis serotypes O:1a, O:1b, O:3 and O:4a was dependent on temperature in such a way that the phage was able to bind the cells at 37oC but not at 20oC (IV, Figure 1). The rough derivative of Y. pseudotuberculosis O:1b (BP1Δwb), however, adsorbed the phage as efficiently at both temperatures. This indicates that the Y. pseudotuberculosis O-antigen expression sterically blocks the phage receptor. The finding that periodate but not Proteinase K destroys the receptor (IV, Figure 2) implicates that the receptor consists of carbohydrates, most likely, LPS. The essential nature of the receptor, as shown by the result that the phage-resistant derivatives of BP1Δwb still adsorbed the phage (IV, Figure 4), further supported the hypothesis that the LPS core is the phage receptor. The φA1122 receptor was not present in any of Y. enterocolitica strains studied (IV, Figure 3). The core structures of different Yersinia species are rather similar and specially theYeO8 core resembles that of Y. pestis and Y. pseudotuberculosis (IV, Table 2), indicating the highly specific nature of phage-host recognition. Based on the core structures of Y. pestis and YeO8, it might be assumed that the free hydroxyl group in the C2 position of the second heptose is required for the efficient adsorption of φA1122. 5.3.2. The φA1122 receptor is blocked by the heterologous expression of Y. enterocolitica O:3 outer core (IV) To further characterize which outer core sugar residues are needed for the φA1122 adsorption, the potential of Y. enterocolitica serotype O:3 O-antigen and outer core to inhibit the phage binding was studied. The heterologous expression of YeO3 O-antigen on Y. pestis had no effect on φA1122 adsorption, in contrast to outer core which blocked it completely (IV, Figure 6). When expressing the YeO3 outer core in BP1Δwb, two clones with different expression levels were obtained (IV, Figure 7). Of these, the strain expressing the outer core at higher level was completely resistant to φA1122, whereas the less expressing strain adsorbed the phage moderately and was infected with it (IV, Figure 8, not shown). The expression of YeO3 outer core thus inhibited the phage adsorption to both Y. pestis and Y. pseudotuberculosis. In the future, the LPS structure of the over-expressing strain BP1Δwb/pRV16NP#1 will be determined. This will reveal the sugar residues blocked by the outer core, thus indicating the residues required for the phage attachment. 5. 4. Reporter phages The starting point of this PhD project was to construct a reporter bacteriophage by inserting the luciferase gene from Photinus pyralis (firefly) to the genome of

45

φYeO3-12 and to use this reporter in the diagnostics of Y. enterocolitica O:3. In the course of the work, several strategies were utilized to achieve this objective, the most extensively studied examples of which are presented below. 5.4.1. Construction of the reporter phage by homologous recombination The phage lysates resulting from homologous recombination between φYeO3-12 and plasmids pMP100, pMP200 and pSK110 (Table 5) contained phages having lucFF in their genome. This was shown by the production of luminescence during infection of the host cells (not shown). However, it was not possible to isolate the luminescent phages, possibly because of the small number and slower growth of the recombinant phages compared to the wild type φYeO3-12. 5.4.2. Phages φ::lucFF1.2 and φ::lucFF5.3 Phage φYeO3-12 derivatives φ::lacZ1 and φ::lacZ5 (Table 3) were chosen as starting points for constructing the luciferase reporters due to their stability, their ability to grow well on YeO3-c and the intensive blue color that was formed around the plaques on indicator plates (II). It was thought that in these mutants, lacZ’ was inserted in such a genomic location where the insert did not impede the essential funcions of the phage and was expressed with high efficiency. The resulting phages φ::lucFF1.2 and φ::lucFF5.3 (Table 3) produced lumines-cence during infection of YeO3-c (not shown). However, these mutants grew poorly on YeO3-c, as indicated by their very small plaque size on top-agar plates. In addition, the luminescent phenotype of the mutants disappeared during sequential plaque purifications, indicating highly unstable nature. The PCR analysis of the genomic regions containing the inserts confirmed that the lucFF gene was rapidly lost from the genomes of both of these mutants. Since the lacZ’ mutants were stable but the corresponding lucFF mutants were not, it was thought that the insertion of the longer lucFF gene (1.7 kb, compared to 0.45 kb for lacZ’) resulted to genome that was too long to be packaged efficiently, thus leading to instability. 5.4.3. Phage φ::lucFF1 To avoid problems in packaging the elongated genome of the reporter phage, a strategy where the luciferase gene was inserted in the genome of the deletion mutant φΔ888-2449 by MuA catalysed in vitro transposon mutagenesis was followed. After screening 113 candidates, one light-producing mutant was found and named φ::lucFF1. In this reporter, lucFF was inserted between the phage genes 6.3 and 6.5 (Table 3). To measure the sensitivity of φ::lucFF1 in Y. enterocolitica detection, dilutions of YeO3-c culture were infected with different PFU of the reporter phage and the light production was followed at fixed time intervals. An example of these measurements is shown in Figure 2.

46

0 20 40 60 800

1000

2000

3000

4000

5000

6000

RLU

Time/min

Figure 2. The sensitivity of φ::lucFF1 reporter phage in Y. enterocolitica serotype O:3 detection. YeO3-c dilutions were infected with φ::lucFF1 and the light production was followed with luminometer. YeO3-c CFU per sample was 1.2 ×107 ( ), 5.9 ×106 (●), 1.2 ×106 (○), 5.9 ×105 (▼) and 2.4 ×105 (◊). In each sample, there was 2.8 ×107 PFU φ::lucFF1. RLU, relative light units. As the outcome of the sensitivity measurements made, the detection limit of φ::lucFF1 settled to ca. 5×105 CFU. It thus seems that the sensitivity of this reporter is not as high as reported for other reporter phages (see Chapter 2.3.2.) and is not good enough for delicate diagnostic purposes. Moreover, the stability studies indicated that the luciferase gene is lost from the φ::lucFF1 genome with substantial ease (not shown). To conclude, all the attempts to construct a reporter phage from φYeO3-12 resulted in unstable mutants that rapidly lost the inserted luciferase gene. Even though the sensitivity of the present reporters might be improved by e.g. adding a strong phage promoter before the lucFF gene, it is unlikely to obtain a reporter phage that is stable enough for large-scale production.

47

6. SUMMARY φYeO3-12 is a member of Podoviridae that belongs to the T7-group of phages, T3 being its closest relative. The phage has a strict host specificity to Y. enterocolitica serotype O:3 (YeO3). The specificity is determined by the interaction between the tail fiber adhesin and LPS O-antigen, and the phage is able to infect other Enterobacteriaceae carrying the YeO3 O-antigen gene cluster. This work characterized the basic biological properties of φYeO3-12 and identified non-essential regions in the phage genome. The phage genes coding for DNA ligase and lysozyme were shown to be needed for growth on YeO3 but not on E. coli, indicating that they are evolutionary factors contributing to the adaptation of φYeO3-12 to utilize Y. enterocolitica as its host. The information about the non-essential regions of the φYeO3-12 genome was exploited in the construction of a reporter phage that could be exploited in Yersinia diagnostics. The obtained reporter phages were, however, too unstable for large-scale production and utilization. φR1-37 is a Y. enterocolitica O:3 specific phage that uses LPS outer core as its receptor. In this work, φR1-37 was shown to be a large member of Myoviridae. The phage genome is 270 kb dsDNA molecule, in which thymidine is replaced by deoxyuridine. The only other organisms known to have such an exceptional DNA are B. subtilis phages PBS1 and PBS2. The N-terminal amino acid sequences of the φR1-37 structural proteins revealed no significant similarities to known viral proteins, indicating that close relatives of φR1-37 have not been characterized. Interestingly, the deduced protein sequence of the partially sequenced φR1-37 genome showed similarity to four open reading frames of P. aeruginosa phage φKZ. This is a member of Myoviridae with a size comparable to that of φR1-37. φA1122 is a T7-related bacteriophage that infects all but two clinical isolates of Y. pestis. It is used as the reference phage in the Y. pestis diagnostics by the Centers for Disease Control and Prevention. However, the receptor that the phage recognizes on the cell surface was not known. In this work, the LPS core of Y. pestis and Y. pseudotuberculosis was identified as the φA1122 receptor. To conclude, this thesis provides new information about the biology and genetics of three bacteriophages infecting the genus Yersinia. A special attention was paid in the understanding the phage-host recognition and other factors determining the host specificity. In addition, the utilization of φYeO3-12 in bacterial diagnostics was evaluated.

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ACKNOWLEDGEMENTS

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