Biocontrol of Cronobacter Spp. Using Bacteriophage in Infant Formula

Biocontrol of Cronobacter spp. using Bacteriophage in Infant Formula

Reza Abbasifar

A Thesis presented to The University of Guelph

In partial fulfillment of requirements for the degree of Doctor of Philosophy in Food Science

Guelph, Ontario, Canada © Reza Abbasifar, May, 2013

ABSTRACT

Biocontrol of Cronobacter spp. using Bacteriophage in Infant Formula

Reza Abbasifar Advisor: Dr. Mansel W. Griffiths

University of Guelph, 2013 Co-Advisor: Dr. Parviz M. Sabour

The purpose of this research was to explore the potential application of lytic phages to control Cronobacter spp. in infant formula. More than two hundred and fifty phages were isolated from various environmental samples against different strains of

Cronobacter spp. Selected phages were characterized by morphology, host range, and cross infectivity. The genomes of five novel Cronobacter phages [vB_CsaM_GAP31

(GAP31), vB_CsaM_GAP32 (GAP32), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161

(GAP161), vB_CsaP_GAP227 (GAP227)] were sequenced. Phage GAP32 possess the second largest phage genome sequenced to date, and it is proposed that GAP32 belongs to a new genus of “Gap32likeviruses”. Phages GAP52 and GAP227 are the first C. sakazakii podoviruses whose genomes have been sequenced. None of the sequenced genomes showed homology to virulent or lysogenic genes. In addition, in vivo administration of phage GAP161 in the hemolymph of Galleria mellonella larvae showed no negative effects on the wellbeing of the larvae and could effectively prevent

Cronobacter infection in the larvae. A cocktail of five phages was highly effective for biocontrol of three Cronobacter sakazakii strains present as a mixed culture in both broth media and contaminated reconstituted infant formula. This phage cocktail could be

iii potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models.

ACKNOWLEDGMENTS

I am grateful to my advisor, Dr. Mansel W. Griffiths, for his great advice, support and help through my PhD program. I would also like to express my appreciation to my

Co-Advisor, Dr. Parviz M. Sabour for his valuable advice, suggestion, and encoragement that patiently gave me during this long process. I am thankful to my Advisory Committee

Member, Dr. Andrew M. Kropinski, who was greatly helpful in my thesis. This thesis would not be completed without my advisors’ help and suggestions.

Also, I would like to thank Dr. Hans-W. Ackermann, Dr. Brian Dunphy, Dr. John

H.E. Nash, Dr. Jim Chambers, Dr. Rob Lavigne, Dr. Roger Johnson, Dr. Elizabeth

(Betty) Kutter, Dr. Keith Warriner, Dr. Kimberly Seed, Joanne MacKinnon, Erika

Lingohr, Dr. Haifeng Wang, Dr. Franco Pagotto, Dr. Roger Stephan, Dr. Andrew Chibeu,

Dr. Angela M. Tellez, Ann Blake, and William (Bill) Lachowsky for their helpful assistance and suggestions in this project.

My sincerest appreciation goes to my family for their unlimited and nonstop support during all the moments of my study. My father and mother have supported me all my life and have continued it while I was doing my thesis although I was far from them.

My dearest wife, Argentina Alanis Villa, has supported me continuously with love, caring and courage in school and in other aspects of life. Thanks to my brothers, Dr. Arash and

Mehdi for their help and being there for me.

I am thankful to The University of Guelph for providing the opportunity to gain my PhD degree and for financial support from NSERC and DFO.

My gratitude goes to all my colleagues in CRIFS and The Department of Food

Science.

TABLE OF CONTENTS

Abstract……………………………………………………………………………….….ii Acknowledgements……………………………………………………………………...iv Table of Contents………………………………………………………………………..vi List of Tables……………………………………………………………………………..x List of Figures…………………………………………………………………………..xii List of Table in Appendix……………………………………………………………...xiv

Chapter 1. Introduction ...……………………………………………………………….1 1.1. Research Introduction ………………………………………………………..1 1.2. Cronobacter: A food-borne pathogen ………………………………………..2 1.2.1. Epidemiology ……………………………………………………..5 1.2.2. Physiology ………………………………………………………...9 1.2.3. Detection ………………………………………………………...10 1.2.4. Control …………………………………………………………..14 1.3. Overview of Bacteriophage ………………………………………………...18 1.3.1. Discovery of Bacteriophage ……………………………………..18 1.3.2. Biology of Bacteriophage ……………………………………….19 1.3.3. Taxonomy of Bacteriophage …………………………………….21 1.3.4. Lysogenic and Lytic Cycle of Bacteriophage …………………...25 1.4. Biocontrol of Food-borne Pathogens Using Bacteriophage ………………..30 1.4.1. Phage Applications in Food ……………………………………..30 1.4.2. Phage Application as Biocontrol Agent …………………………42 1.4.3. Phage Therapy ...………………………………………………..45 1.5. Research Objectives ………………………………………………………...47

Chapter 2. Isolation and Characterization of Lytic Cronobacter Bacteriophages…49 2.1. Abstract ……………………………………………………………………..49 2.2. Introduction …………………………………………………………………49

vii

2.3. Material and Methods ………………………………………………………51 2.3.1. Bacteria and Bacteriophages ……………………………………...51 2.3.2. Isolation of Bacteriophages ……………………………………….52 2.3.3. Purification of Bacteriophages ……………………………………53 2.3.4. Propagation of Bacteriophages …………………………………...54 2.3.5. Host Range Determination………………………………………...54 2.3.6. Transmission Electron Microscopy (TEM) ………………………55 2.3.7. Cross Infectivity …………………………………………………..56 2.4. Results ………………………………………………………………………57 2.4.1. Isolation of Bacteriophages …………………………...…………..57 2.4.2. Host Range ………………………………………………………..57 2.4.3. Characterization of the Selected Phages ………………………….60 2.4.3.1. Morphology ……………………………………………..60 2.4.3.2. Cross Infectivity ………………………………………...65 2.5. Discussion …………………………………………………………………..67

Chapter 3. Sequencing and Genome Analysis of Cronobacter phage GAP32 …...…72 3.1. Abstract ……………………………………………………………………..72 3.2. Introduction …………………………………………………………………73 3.3. Material and Methods ………………………………………………………74 3.3.1. Bacteria and Bacteriophage ………………………………………74 3.3.2. Phage Purification, DNA Isolation and Sequencing ……………...74 3.3.3. Bioinformatic Analysis …………………………………………...76 3.3.4. Proteomic Analysis ……………………………………………….77 3.3.5. Genome Sequence ………………………………………………...77 3.4. Results ………………………………………………………………………77 3.5. Discussion …………………………………………………………………..80

Chapter 4. Sequencing and Genome Analysis of Cronobacter phages GAP31, GAP52, GAP161 and GAP227…………………………………………………………82 4.1. Abstract ……………………………………………………………………..82

viii

4.2. Introduction …………………………………………………………………83 4.3. Material and Methods ………………………………………………………84 4.3.1. Bacteria and Bacteriophages ……………………………………...84 4.3.2. Phage Purification, DNA Isolation and Sequencing ……………...85 4.3.3. Bioinformatic Analysis …………………………………………...86 4.3.4. Genome Sequences ……………………………………………….87 4.4. Results ………………………………………………………………………87 4.4.1. Features of the Genome of Phage GAP31 ………………………..87 4.4.2. Features of the Genome of Phage GAP52 ………………………..90 4.4.3. Features of the Genome of Phage GAP161 ………………………93 4.4.4. Features of the Genome of Phage GAP227 ………………………95 4.5 Discussion ………………………………………………………………..….97

Chapter 5. Efficiency of Bacteriophage Therapy Against Cronobacter sakazakii in Galleria mellonella (Greater Wax Moth) Larvae...…………………..……………99 5.1. Abstract …………………………………………………………..…………99 5.2. Introduction ………………………………………………………………..100 5.3. Materials and Methods …………………………………………………….103 5.3.1. Bacterial Strains and Culture Conditions ………………………..103 5.3.2. Bacteriophage Isolation and Characterization …………………..104 5.3.3. Efficiency of Bacteriophage Therapy Against C. sakazakii in G. mellonella Larvae …...…………….……………………..……...105 5.3.4. Statistical Analysis ………………………………………………108 5.4. Results ……………………………………………………………………..108 5.4.1. Mortality of C. sakazakii in G. mellonella Larvae ………………108 5.4.2. Bacteriophage and Its Persistence in G. mellonella Larvae …………………………………………………………………………..109 5.4.3. Efficiency of Bacteriophage Therapy Against C. sakazakii in G. mellonella Larvae ………………………………………………………110 5.5. Discussion …………………………………………………………………112

Chapter 6. Use of Cocktail of Five Phages to Control C. sakazakii in Broth Media and in Infant Formula………………………………………………………………...116 6.1. Abstract ……………………………………………………………………116 6.2. Introduction ………………………………………………………………..117 6.3. Material and Methods ……………………………………………………..122 6.3.1. Bacteria and Bacteriophages …………………………………….122 6.3.2. Host Range Determination ………………………………………123 6.3.3. Effect of the Phage Cocktail on C. sakazakii Strains in Broth and in Reconstituted Infant Formula ………………………………….………124 6.3.4. Statistical Analysis ………………………………………………127 6.4. Results ……………………………………………………………………..128 6.4.1. Host Range ………………………………………………………128 6.4.2. Effect of the Phage Cocktail on the Mixture of C. sakazakii Strains in Broth ………………………………………………………………...129 6.4.3. Effect of the Phage Cocktail on the Mixture of C. sakazakii Strains in Reconstituted Infant Formula ……………………………………….133 6.4.4. The Correlation Between Luminescence and Plate Count …...…138 6.5. Discussion …………………………………………………………………141

Chapter 7. Conclusions and Future Work ...………………………………………..151 7.1. Thesis Summary and General Conclusion ………………………………...151 7.2. Future Work ……………………………………………………………….158

References……………………………………………………………………………...159

Appendix ………………………………………………………………………………197

LIST OF TABLES

Table 1.1. Global outbreaks and sporadic cases of Cronobacter infections…..………………………………………………………………………………4

Table 1.2. Classification and basic properties of bacteriophages...……………….…………………………………………………………24

Table 2.1. Cronobacter strains used for isolation and propagation of phages ……….....52

Table 2.2. Bacterial strains used to examine the cross infectivity of the selected phages……………………………………………………………………………………56

Table 2.4. The host range of 11 selected Cronobacter phages against 21 Cronobacter strains based on the spot test (16 h incubation at 30ºC) …………………………………59

Table 2.5. Selected bacteriophages and the susceptible bacterial hosts used for propagation ……………………………………………………………………………...60

Table 2.6. Dimensions, head shape, and related phages for the selected bacteriophages..…………………………………………………………..………………65

Table 2.7. Cross infectivity of the selected phages to other bacterial species in Enterobacteriaceae and 2 strains of Lactobacillus ………………………….…………..66

Table 4.1. General features of the genome of phages GAP31, GAP52, GAP161, and GAP227 and their related phages based on proteomic analysis using CoreGenes..………………………………………………………………………………88

Table 5.1. Least Square Means (LSM) of the survival of G. mellonella larvae following challenge with C. sakazakii HPB 3253 and/or phage GAP161 administration ………………………………………………………………………...... ……………..111

Table 6.1. lux+ strains of Cronobacter (amp::lux) that were used for artificially contaminating of infant formula and TSB ………………………………..……………122

Table 6.2. Selected bacteriophages and the susceptible bacterial hosts used for propagation and titration …………………………………………………..…………...123

Table 6.3. Approximate analysis of Similac Advance Neosure infant formula used in the experiment as declared by the manufacturer ……………………………………….125

Table 6.4. The host range of five selected Cronobacter phages against luminescent strains of Cronobacter (amp::lux) based on the spot test (16 h incubation at 30ºC)..……...…………………………………………………………………………...128

Table 6.5. Effect of phage cocktail on the growth of different concentrations of the mixture of three C. sakazakii strains in TSB ……………………………..……………133

Table 6.6. Effect of phage cocktail on the growth of different concentrations of three C. sakazakii in RIF ………………………………………………………………………..137

Table 6.7. Effect of phage cocktail on the growth of the mixture of three bioluminescent C. sakazakii strains (initial concentration of 105 CFU/ml) in RIF and TSB ……………………………………………………………………………….………….141

xii

LIST OF FIGURES

Figure 1.1. Morphotypes of prokaryotic viruses of the three families Myoviridae, Siphoviridae and Podoviridae within the order of Caudovirales..…………………………………………...……………………………….25

Figure 1.2. Lysogenic and lytic cycles of bacteriophages ………………..……………27

Figure 2.1. Transmission electron micrographs of negatively stained (UA) phages GAP31, GAP32, GAP33, GAP72, GAP136, and GAP161 that belong to Myoviridae family ………………………………………………………………………….………...63

Figure 2.2. Transmission electron micrographs of negatively stained (UA) of phages GAP52, GAP184, GAP186, GAP188, and GAP227 that belong to Podoviridae family………………………………………………………………………….…………64

Figure 3.1. The map of the linear genome of phage vB_CsaM_GAP32 in comparison to phage vB_KleM-RaK2 created with CGview using TBLASX.……………………………………….………………………………………..79

Figure 4.1. The map of the linear genome of phage vB_CsaM_GAP31 in comparison to Salmonella phage PVP-SE1 created with CGview using TBLASX…………………………………………………..……………………………..89

Figure 4.2. The map of the linear genome of phage vB_CsaP_GAP52 in comparison to Salmonella phage 7-11 created with CGview using TBLASX…………………………………………………………………………………92

Figure 4.3. The map of the linear genome of phage vB_CsaM_GAP161 in comparison to coliphages RB16 created with CGview using TBLASX…………………………………………………………………………………94

Figure 4.4. The map of the linear genome of phage vB_CsaP_GAP227 in comparison to Yersinia phages φR8-01 created with CGview using TBLASX…………………………………………………………………..……………..96

Figure 5.1. Mortality rates of different Cronobacter strains compared with SM buffer or no injection in G. mellonella larvae 24 h p.i. at 37ºC (~4.0 × 107 CFU)……………………………………………………………………………………109

Figure 5.2. Electron micrograph of phage vB_CsaM_GAP161 negatively stained with uranyl acetate ………………………………………………………………..…………110

xiii

Figure 5.3. G. mellonella larvae infected by C. sakazakii HPB 3253.…….………………………………………………………………………………111

Figure 5.4. Survival of G. mellonella larvae infected with C. sakazakii and treated with phage GAP161 at different times prior to and after infection ………………………….112

Figure 6.1. Effect of the phage cocktail on the growth of different concentrations of the mixture of the three C. sakazakii strains in liquid medium (TSB) ………………..…...131

Figure 6.2. Effect of the phage cocktail on the growth of different concentrations of the mixture of the three C. sakazakii strains in RIF ……………………………….……….136

Figure 6.3. Effect of phage cocktail on the growth of the mixture of three C. sakazakii strains (initial concentration of 105 CFU/ml) in RIF and TSB ……………..………….140

xiv

LIST OF TABLES IN APPENDIX

APPENDIX 1. Host range of isolated phages

Table 2.3. The host range of isolated Cronobacter phages against 21 Cronobacter strains based on the spot test (16 h incubation at 30ºC) …………………………...…………..197

APPENDIX 2. Genome of phage GAP32

Table 3.1. General features of ORFs in the DNA of phage GAP32 and homology to proteins in the databases ………………………………………………….……………211

Table 3.2. Proteomic and HHpred analysis of phage GAP32………..……..………….241

APPENDIX 3. Genome of phages GAP31, GAP52, GAP161, and GAP227

Table 4.2. General features of ORFs in the DNA of phage GAP31 and homology to proteins in the databases ………………………………………………….……………250

Table 4.3. General features of ORFs in the DNA of phage GAP52 and homology to proteins in the databases ……………………………………………………….………262

Table 4.4. General features of ORFs in the DNA of phage GAP161 and homology to proteins in the databases …………………………………………………….…………273

Table 4.5. General features of ORFs in the DNA of phage GAP227 and homology to proteins in the databases ………………………………………………….……………301

Chapter 1. INTRODUCTION

1.1. RESEARCH INTRODUCTION

The members of the genus of Cronobacter, belonging to the family of

Enterobacteriaceae, are emerging opportunistic pathogens, which can cause rare but life- threatening cases of neonatal meningitis, necrotizing enterocolitis (NEC) and sepsis

(Iversen & Forsythe, 2003). Powdered infant formula (PIF; a non-sterile product) has been epidemiologically linked as a vehicle for Cronobacter infections in infants (Drudy et al., 2006; Van Acker et al., 2001). Although breast-feeding is highly recommended, there are infants who cannot use their mother’s milk, such as infants of HIV positive mothers or infants with lactose intolerance, and they have to use infant formulae. The contamination of PIF with Cronobacter spp. is a significant concern for health organizations and infant formula manufacturers. During the past years, several outbreaks of Cronobacter infections in infants have occurred which has led to increasing numbers of studies on this pathogen (Farmer III, 2008). Since these bacteria do not survive the pasteurization process (Nazarowec-White & Farber, 1997a), contamination must occur at various points following pasteurization and during the manufacture of PIF. Also, this pathogen can tolerate desiccation and can survive in dry conditions for a long period of time in PIF (Breeuwer et al., 2003; Edelson-Mammel & Buchanan, 2004). Since the level of contamination is usually low in PIF (<1 CFU/100g) (FAO/WHO, 2004; FAO/WHO,

2006; Muytjens, Roelofs-Willemse, & Jaspar, 1988; Nazarowec-White & Farber, 1997a), outgrowth of Cronobacter spp. after rehydration and preparation of formula before use is crucial. In addition, the pathogen is relatively resistant to acidic environments (Edelson-

Mammel, Porteous, & Buchanan, 2006) and is protected by infant formula matrices, allowing its survival in the light acid environment of the immature neonatal stomach subsequently leading to intestinal colonization. It is therefore very important to develop effective methods to control the bacterium in rehydrated formula and to prevent infection and colonization of infants with the pathogen. Application of bacteriophage for biocontrol of Cronobacter spp. is a method that may achieve this goal.

1.2. CRONOBACTER: A FOOD-BORNE PATHOGEN

Cronobacter spp. are Gram-negative bacteria that belong to the family

Enterobacteriaceae, which originally were called “yellow pigmented Enterobacter cloacae”, then “Enterobacter sakazakii”, and now they are recognized as belonging to a new genus, Cronobacter (Forsythe, 2005; Gurtler & Beuchat, 2005; Iversen et al.,

2007a,b). Cronobacter spp. are ubiquitous having been isolated from various types of food such as cheese, eggs, ground meat, rice, various dry food ingredients (e.g. lactose, lecithin, starch, herbs and spices), water, vegetables (e.g. lettuce and tomato), milk powder, casein and caseinate, dried infant foods and PIF (Gurtler, Kornacki, & Beuchat,

2005; Iversen & Forsythe, 2004; Jung & Park, 2006; Mullane et al., 2006). Cronobacter spp. have been also isolated from food production (chocolate, cereal, potato flour, pasta, spice and PIF factories) and other environmental samples (air in a hospital, clinical materials, rats, soil, sediment, wetlands and crude oil) (Iversen & Forsythe, 2003;

Kandhai et al., 2004).

Cronobacter spp. are occasional contaminants of PIF that can cause in premature and full-term neonates (Iversen & Forsythe, 2003) a rare, but life-threatening form of

meningitis, bacteraemia, necrotizing enterocolitis (NEC) and necrotizing meningoencephalitis. Infant mortality from Cronobacter meningitis is 40-80%, with death often occurring within hours of infection (Muytjens et al., 1983). Epileptic activity has been reported in one-third of the cases of neonatal Cronobacter meningitis, with physiological responses including grunting, bulging fontanelles, convulsions, twitching and an increase in cranial circumference.

Epidemiologic investigations have linked PIF as well as equipment and utensils used to prepare rehydrated formulae in hospitals, to outbreaks of Cronobacter infection in infants and neonates (Gurtler, Kornacki, & Beuchat, 2005). In 1988, Muytjens et al.

(Muytjens, Roelofs-Willemse, & Jaspar, 1988) detected Cronobacters (E. sakazakii) in 20 of 141 (14.2%) samples of infant formula from 13 of 35 countries (including six samples from Canada) (Muytjens, Roelofs-Willemse, & Jaspar, 1988). In the Canadian samples this pathogen was found at levels of less than 1 CFU/100 g of dried-infant formula

(FAO/WHO, 2004; FAO/WHO, 2006; Muytjens, Roelofs-Willemse, & Jaspar, 1988;

Nazarowec-White & Farber, 1997a).

Although the incidence of outbreaks and cases of neonatal Cronobacter infections are low (Gurtler, Kornacki, & Beuchat, 2005), at least 76 cases of neonatal infections, 19 deaths in infants and children, and nine adult cases of Cronobacter infections have been reported worldwide between 1958 and 2003 (Table 1.1) (Iversen & Forsythe, 2003). In

1990 and 1991, three incidents of neonatal meningitis caused by Cronobacter were reported in Canadian hospitals, and in 2007 a case of Cronobacter meningitis was reported in a 17-day-old infant in Canada, which was associated with powdered infant formula (Nazarowec-White & Farber, 1997a; Pagotto & Farber, 2009). In 2004,

FAO/WHO (FAO/WHO, 2004) ranked Cronobacter with Salmonella enterica in the

category A (“clear evidence of causality”) of microorganisms in PIF, and recommended

appropriate control measures.

Table 1.1. Global outbreaks and sporadic cases of Cronobacter infections [Adapted and updated from (Iversen & Forsythe, 2003; Mullane et al., 2006; Nazarowec-White & Farber, 1997a)].

Number Year of Location of cases Cases Source References outbreak (deaths) 1958 England 2 (2) O UN (Urmenyi & Franklin, 1961) 1958 Denmark 1 (1) S UN (Joker, Norholm, & Siboni, 1965) 1958 Georgia 1 (0) S UN (Monroe & Tift, 1979) 1958 Oklahoma 1 (1) S UN (Adamson & Rodgers, 1981) 1958 Indiana 1 (0) S UN (Kleiman et al., 1981) Suspected 1977–81 Denmark 8 (6) O (Muytjens et al., 1983) to be PIF 1977–81 Greece 1 (1) S NS (Postupa & Aldova, 1984) 1984 Greece 11 (4) O UN (Arseni et al., 1987) 1984 Missouri 1 (0) S UN (Naqvi, Maxwell, & Dunkle, 1985) 1984 Massachusetts 2 (1) O UN (Willis & Robinson, 1988) 1986–87 Iceland 3 (1) O PIF (Biering et al., 1989) PIF, 1988 Tennessee 4 (0) O (Simmons et al., 1989) blender 1988 Maryland 1 (0) S PIF (Noriega et al., 1990) 1988 Ohio 1 (0) S NS (Gallagher & Ball, 1991) Ontario, 1990-91 2 (?) O NS (Nazarowec-White & Farber, 1997a) Canada 1994 France 13 (3) O PIF (Caubilla-Barron et al., 2007) 1998 Belgium 12 (2) O PIF (van Acker et al., 2001) PIF, 1999–00 Jerusalem 2 (0) O (Bar-Oz et al., 2001) blender 2001 Tennessee 11 (1) O PIF (Himelright et al., 2002) 2002 Belgium 1 (1) S PIF http://www.cfsan.fda.gov/dms/inf-ltr3.html 2004 New Zealand 5 (1) O PIF http://www.nzfsa.govt.nz http://www.hpa.org.uk/cdr/archives/ 2004 France 4 (2) O PIF archive04/news/news5204.htm#pregestimil 2007 Canada 1 (1) S PIF (Pagotto & Farber, 2009) 2007 India 2 (1) O PIF (Ray et al., 2007) (Centers for Disease Control and Prevention 2008 New Mexico 2 (1) O PIF (CDC), 2009) USA (Florida, Illinois, http://www.fda.gov/NewsEvents/PublicHealt 2011 4 (2) S PIF Missouri, & hFocus/ucm285401.htm Oklahoma) S: Sporadic; O: Outbreak; NS: Not Specified; UN: Unknown

It has been revealed that pasteurization at 68ºC for 16s of bovine whole milk resulted in a 5-log reduction of the Cronobacter population (Nazarowec-White & Farber,

1999). Although manufacturers apply pasteurization during PIF production, there are still reports of Cronobacter contamination in this product, and procedures of prevention of post-pasteurization contamination have not been well-defined. Interestingly, it has been shown that this bacterium can survive spray drying (160ºC) (Arku et al., 2008). Two main ways of contamination have been suggested by which Cronobacter spp. may enter rehydrated PIF: intrinsic contamination of PIF (through contaminated ingredients or from the processing environment); and/or external contamination (contamination during rehydration and handling) (Mullane et al., 2006). Therefore, the urgency to develop novel methods to control this pathogen in PIF is essential. One of the potentially useful and promising methods to control food pathogens such as Cronobacter contamination in PIF is biocontrol by using bacteriophages.

1.2.1. Epidemiology

Cronobacter spp. can cause disease in all age groups, but certain individuals are likely to experience higher illness rates (FAO/WHO, 2006). As reported by FAO/WHO

(FAO/WHO, 2004), a survey in 2002 estimated that the annual rate of invasive

Cronobacter infection was 1/100,000 infants (i.e. children <12 months of age), whereas the rate among low-birth weight (<2,500 g) neonates (<28 days) was 8.7/100,000, and this rate increased to 9.4/100,000 among very low-birth weight (<1,500 g) neonates. It has been stated that a Cronobacter infection rate of 1/10,660 is achieved among very low-birth-weight infants and confirms that this infection is rare in these infants (Stoll et

al., 2004). Health Canada in 2002 (Pagotto et al., 2009) emphasized that contributing factors resulting in an increased risk of infection in neonates, are immunosuppression, premature birth and low birth weight (Gurtler, Kornacki, & Beuchat, 2005; Pagotto et al.,

2009).

Although the majority of Cronobacter cases are reported in adults (FAO/WHO,

2008), the major groups at risk of serious illness are neonates and infants in whom there is a high mortality rate due to NEC, septicemia, and meningitis. Only three species of

Cronobacter (C. sakazakii, C. malonaticus, and C. turicensis) have been linked to infections in neonates (Joseph et al., 2012b). In older age groups, Cronobacter mainly cause bacteremia, urosepsis and wound infections. It seems that strains of C. malonaticus are more associated with infections in adult than in neonates (Joseph & Forsythe, 2011;

Joseph & Forsythe, 2012).

Urmenyi & Franklin were the first to associate this organism with neonatal deaths in 1958 (Urmenyi & Franklin, 1961). In 2002, the International Commission on

Microbiological Specification for Foods described the organism as a “Severe hazard for restricted populations, life threatening or substantial chronic sequels of long duration”.

Subsequently it has been allocated the same ranking as more familiar food and waterborne pathogens such as Listeria monocytogenes, Clostridium botulinum and

Cryptosporidium parvum (Forsythe, 2005).

In England, Wales and Northern Ireland the estimated incidence of Cronobacter bloodstream infection among mixed ages was 0.09-0.12/100,000 between 2001 and 2003.

Consequently, adults and children >12 months were assumed to be at lower risk than infants for invasive infection with Cronobacter spp. (FAO/WHO, 2006).

Although it seems that there are two distinct infant risk groups (premature infants that develop bacteraemia after 1 month of age, and term infants that develop meningitis during the neonatal period) the difference in timing of infection may be related to differences in timing of exposure to Cronobacter rather than differences in susceptibility

(FAO/WHO, 2006).

In addition, Cronobacter meningitis tends to develop in infants during the neonatal period, which may reflect the neonates’ immunologic or neurovascular condition

(Schuchat et al., 1997). Cronobacter bacteraemia tends to develop in premature infants after the neonatal period and before 2 months of age. However, immunocompromised infants have developed bloodstream infections as late as age 10 months and previously healthy infants have also developed invasive disease outside the neonatal period.

Infections have occurred in both hospital and outpatient situations but usually older infants live at home and infections in such infants may be more likely to be under- reported (FAO/WHO, 2006).

Cronobacter and Salmonella enterica are well-established causes of illness in infants (e.g. systemic infection, NEC, and severe diarrhea), and they have been found in

PIF (FAO/WHO, 2004). The presence of Cronobacter in powdered infant formula (and its association with illness in infants) is more likely than for other members of the

Enterobacteriaceae or other Enterobacter. The joint FAO/WHO (2006) workshop on

Cronobacter spp. and Salmonella in powdered infant formula recommended that in the case of the infants that are not breastfed, caregivers, particularly of infants at high risk, should be regularly reminded that “PIF is not a sterile product and can be contaminated

with pathogens that can cause serious illness”. It is hoped that by providing this information the risk of infection decreases (FAO/WHO, 2006).

In one study conducted in a powdered milk protein manufacturing facility during a 10-month period, thirty Cronobacter isolates were characterized by pulsed-field gel electrophoresis (PFGE) (Mullane et al., 2008). These Cronobacters were isolated from air filters (80%), the environment (including production vacuum sample and samples from swabs of dryer outlet and dehumidifier air intake) (10%), and powdered product (10%).

These results reveal the importance of proper installation and maintenance of air filters to limit the distribution of microorganisms into processing sites.

Hein and colleagues (2009) sampled a milk powder processing line for the presence of Enterobacteriaceae and Cronobacter over an 11-month period. The

Enterobacteriaceae-positive samples were mostly recovered from non-product samples from the processing line (86.5%) (including swabs from the drying tower, screw conveyers, and explosion chamber, underneath the outlet of the packaging machine, air filters, and waste water after the automated cleaning-in-place (CIP) procedure), and the raw milk concentrate before pasteurization (78.2%). Cronobacters were isolated from the pre-final (drying tower), prepackaged, and packaged final products with a prevalence of

14.3%, 3.8%, and 2.1%, respectively. A comparison of the PFGE profiles of isolates from different sampling sites showed that the supplied air could be a potential source for extrinsic Cronobacter contamination. Moreover, PFGE profiles revealed that hygiene measures such as CIP process followed by heat treatment were inefficient to eliminate

Cronobacter completely from all areas of the processing line (Hein et al., 2009).

1.2.2. Physiology

Cronobacter spp. are Gram-negative, motile, non-sporeforming, facultatively anaerobic, α-glucosidase-positive, straight rod-shaped bacteria, which belong to the family Enterobacteriaceae. These bacteria initially were called “yellow pigmented

Enterobacter cloacae”, and Farmer et al. in 1977 changed the name to “Enterobacter sakazakii”, in honor of the Japanese bacteriologist Riichi Sakazaki, and later in 1980 they were designated as a new species (Forsythe, 2005; Gurtler & Beuchat, 2005). At that time

15 biogroups were described and it was suggested that they could represent multiple species. In 2007, a taxonomic reclassification of the pathogen in the new “Cronobacter” genus consisting of five species was proposed (Iversen et al., 2007a,b). Subsequently another species was added (Iversen et al., 2008). The original six species (C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis and C. genomospecies 1) were comprised of 16 biogroups. In 2012, a new species (C. condimenti) was identified

(Joseph et al., 2012a), and the original C. genomospecies 1 was replaced by C. universalis.

Six names previously have been used for Cronobacter including the Urmenyi and

Franklin bacillus, yellow coliform, yellow Enterobacter, pigmented cloacae A organism, yellow-pigmented E. cloacae, and Enterobacter sakazakii (Gurtler, Kornacki, & Beuchat,

2005).

Traditionally it was considered that the major differences between E. sakazakii and other Enterobacter species were its inability to ferment D-sorbitol and its ability to produce an extracellular deoxyribonuclease (Farmer III et al., 1980). Muytjens et al.

(1984) mentioned that two major differences between Cronobacter spp. and the other

Enterobacter species are the presence of α-glucosidase and the absence of phosphoamidase in Cronobacter, and that the α-glucosidase reaction is the most important (Muytjens, Vanderrosvanderepe, & Vandruten, 1984). However, some strains of E. sakazakii are able to ferment D-sorbitol (Heuvelink et al., 2003).

1.2.3. Detection

The main challenge for Powdered Infant Formula (PIF) manufacturers is to follow a consistent standard detection method for the presence of Cronobacter. Currently, PIF is tested for coliforms as general indicators of hygienic manufacturing practice, and

Salmonella is the only specific member of the Enterobacteriaceae that has an associated microbiological criterion (less than 1 cell in 25g PIF, multiple samples taken for each production batch). In 2004, FAO recommended that coliform counts in PIF should be <3

CFU/g (FAO/WHO, 2004). Effective control measures for Salmonella are well established in PIF manufacturing plants (Iversen, Caubilla-Barron, & Forsythe, 2004).

However, Cronobacter cells have been isolated from samples that pass the quality control tests for both coliform and Salmonella.

As a consequence of cases caused by the presence of Cronobacter in PIF, the

European Food Safety Authority has recommended the introduction of a Performance

Objective (PO) for PIF and follow-on formula. It is necessary to establish a very low level of Cronobacter and Salmonella (e.g. absence in 1, 10 or 100 kg) in PIF products. To verify the PO, the presence of Enterobacteriaceae in the environment and the product is monitored (Mullane et al., 2006). Cronobacter spp., E. cloacae, Citrobacter koseri,

Citrobacter freundii, Pantoea agglomerans and Escherichia vulneris have all been

isolated from reconstituted PIF by using an enrichment method (Iversen, Caubilla-

Barron, & Forsythe, 2004). However, specific methods of detection may be required for each of these organisms (Mullane et al., 2006).

To protect the safety of the product, applying standardized detection methods is essential. Also it is important to facilitate the comparison of data, which are useful to evaluate the presence and occurrence of Cronobacter spp. in the environment and in the raw material used in the manufacture of PIF (Mullane et al., 2006).

Conventional methods. Muytjens et al. (1988) developed the first detection method for Cronobacter species, and later the US Food and Drug Administration introduced a protocol for isolation and enumeration of Cronobacter from powdered infant formula based on Muytjens’ method (FDA, 2002; Muytjens, Roelofs-Willemse, & Jaspar,

1988). In this method, the pre-enrichment is done by addition of sterile water to PIF, which is then incubated overnight at 36°C, followed by another overnight incubation in

Enterobacteriaceae-enrichment broth. After plating on Violet Red Bile Glucose agar

(VRBG) by direct spreading and also direct streaking, followed by overnight incubation at 36°C, presumptive Cronobacter colonies are subcultured onto a Trypticase Soy Agar

(TSA) plate, and incubated at 25°C for 48-72 hours. Any yellow-pigmented colonies from the TSA plates are isolated and their identity confirmed by means of the API 20E biochemical identification system. Obviously, this method is very time consuming.

The International Dairy Federation and the International Organization for

Standardization (ISO 2009) in 2006 introduced a method (ISO⁄TS 22964) to detect

Cronobacter species from milk-based powdered formula (ISO/IDF, 2006). Recently, the

US FDA protocol has been modified to detect Cronobacter species by combining real-

time PCR, chromogenic agars (R&F E. sakazakii chromogenic agar), and RAPID ID 32E biochemical tests, and this method provides results within 24 to 48 h (Chen et al., 2012), which shortens the detection process by 5 days from the original protocol.

In all these methods, the PIF samples are pre-enriched for different periods (from

6 h to 24 h), followed by selective enrichment and then isolation from selective agars. A selective agar and/or a real-time PCR assay is used to confirm the identity of the presumptive positive colonies, and biochemical or molecular characterization is used for final identification.

Based on the α-glucosidase enzyme marker (Iversen, Caubilla-Barron, &

Forsythe, 2004; Muytjens, Vanderrosvanderepe, & Vandruten, 1984) and β- cellobiosidase activity that all Cronobacter strains possess (Restaino et al., 2006), various

Cronobacter selective media have been developed; including, Druggan-Fosythe-Iversen agar (Iversen, Caubilla-Barron, & Forsythe, 2004), Leuschner–Bew agar (Leuschner,

Baird, Donald, & Cox, 2004) Oh-Kang agar (Oh & Kang, 2004), HiCromeTM

Cronobacter spp. agar (Sigma-Aldrich, Switzerland), and ESPM agar (Restaino et al.,

2006).

However, the accuracy and reliability of some of these media have been questioned, as they do not support adequate growth of all Cronobacter (Iversen &

Forsythe, 2007). Furthermore, other related species that usually can be found in the same ecological environment (e.g. Enterobacter pulveris, Enterobacter helveticus, and

Enterobacter turicensis) can grow on these media. Thus, improved selective media are needed for isolation and identification of Cronobacter strains.

Immunologic methods. Immuno-based assays such as enzyme-linked immunosorbent assay (ELISA) and the VITEK® immunodiagnostic assay system

(VIDAS®, bio-Merieux Vitek Inc., Hazelwood, MO, USA) have been developed for rapid detection of several food-borne pathogens such as Escherichia coli O157:H7, Salmonella,

Campylobacter jejuni, Listeria spp., and Staphylococcus spp. enterotoxins. So far, the

VIDAS® method for Salmonella has been approved by the European Microbiological

Method and Health Canada, and there is ongoing research on a VIDAS®-based method for Cronobacter with promising preliminary results (Yan et al., 2012).

Molecular methods. During the past seven years, several molecular assays based on real-time PCR have been designed to detect Cronobacter strains (Drudy et al., 2006;

Kothary et al., 2007; Malorny & Wagner, 2005; Nair & Venkitanarayanan, 2006; Seo &

Brackett, 2005; Stoop et al., 2009; Zhou et al., 2008). These methods use different target genes such as the 16S rRNA gene or 16S-23S rDNA intergenic region, or the dnaG, ompA, gluA (1,6 α-glucosidase) or zpx (zinc-containing methalloprotease) genes. Until

2009, PCR-based methods could not detect the newly described species, and a study is in progress by Stoop et al. to improve the method (Stoop et al., 2009; Yan et al., 2012).

In addition, other molecular methods like subtyping have been applied to detect this pathogen. In 2007, pulsed-field gel electrophoresis (PFGE) was used to distinguish and trace Cronobacter species in a PIF manufacturing plant (Mullane et al., 2007), which allowed the authors to suggest more efficient measures to control and decrease

Cronobacter in the PIF production area. Moreover, in 2009 the first comparative genomic analysis of Cronobacter genus using microarray techniques was conducted (Healy et al.,

2010), which recognized species-specific genes as candidate markers for a molecular- based method for detection.

A recent molecular technique used for typing of bacteria is multilocus sequence typing (MLST), which can be helpful for the typing of bacterial pathogens. Baldwin et al

(2009) were the first to develop a MLST scheme of seven loci (atpD, fusA, glnS, gltB, gyrB, infB, and ppsA) for Cronobacter spp. and reported reliable identification and discrimination of C. sakazakii and C. malonaticus strains, and demonstrated the utility of

MLST over 16S rDNA sequence analysis, as the latter was unable to distinguish between these two species (Baldwin et al., 2009). Further studies have shown that MLST can be used to differentiate all seven species in the genus of Cronobacter (Czerwicka et al.,

2010; Hamby et al., 2011; Joseph & Forsythe, 2011; Joseph et al., 2012a,b; Kucerova et al., 2010). In contrast to other methods, MLST is reliablely able to recognize the whole

Cronobacter genus including two new species; C. universalis and C. condimenti.

By using the MLST, it has been revealed that the majority of neonatal meningitis cases reported over half a century in six countries are attributed to the ST4 clonal lineage of C. sakazakii (Joseph & Forsythe, 2011). Therefore, clonal complex 4 is identified as a genetic signature for C. sakazakii neonatal meningitis. The MLST database for

Cronobacters is currently composed of more than 530 profiled strains that are accessible at http://www.pubMLST.org/cronobacter (Joseph & Forsythe, 2012).

1.2.4. Control

Cronobacter spp. are sensitive to antimicrobial agents, or at least show intermediate sensitivity to numerous antibiotics such as aminoglycosides,

acylureidopenicillins, ampicillin, aztreonam, carbapenems, antifolates, cephalosporins, chloramphenicol, nitrofurantoin, tetracyclines, ticarcillin, quinolones, and several β- lactams (Farmer III et al., 1980; Hawkins, Lissner, & Sanford, 1991; Muytjens &

Vanderrosvanderepe, 1986; Willis & Robinson, 1988). Among the Enterobacteriaceae, almost all species have a resistance phenotype to rifampicin, linocosamides, glycopeptides, fusidic acid, and streptogramins, which might be due to their outer membrane that prevents uptake of these antimicrobials (Stock & Wiedemann, 2002). In addition, Cronobacter spp. are less susceptible to benzylpenicillin, clindamycin, oxacillin, and some macrolides, and some strains are less sensitive to fosfomycin as well

(Stock & Wiedemann, 2002).

Kim et al. (2007) have investigated the efficacy of 13 different disinfectants commonly used in clinics, food environments and day-cares for cleaning surfaces and controlling Cronobacter spp. (Kim, Ryu, & Beuchat, 2007). All the disinfectants studied could reduce the population of planktonic cells to undetectable levels (<0.30 log CFU/ml) in less than 5 minutes. However, when bacterial cells in infant formula were dried onto a solid surface (e.g., stainless steel) the disinfectants were less effective, and the cell reduction was not significant in biofilms.

It is believed that Cronobacter infections are significantly related to extrinsic contamination of materials and surfaces with these pathogens. Redmond and Griffith

(2009) evaluated the efficacy of recommended methods for the decontamination of PIF- feeding bottles. The results revealed that good hygiene along with using hypochlorite or heat treatments (according to the suggested guidelines) led to the effective decontamination of utensils used for feeding PIF (Redmond & Griffith, 2009).

In addition, bacteriophages have been applied to prevent the growth of

Cronobacter in infant formula (Kim, Klumpp, & Loessner, 2007; Zuber et al., 2008). The findings suggested that bacteriophages could reduce the number of Cronobacter cells through lysis. Thus, because of the high specificity of bacteriophages, their application in infant formula may reduce Cronobacter counts in rehydrated PIF and result in a product that would not have an adverse effect on the microbiome of the infant’s gut when consumed. These studies are reviewed in more detail below in the bacteriophage section.

The effect of bioactive molecules (antimicrobial peptides) produced by

Lactobacillus acidophilus on pathogenic bacteria was also assessed (Hayes et al., 2006).

Two peptides (caseicins A and B) showed antimicrobial activity against Cronobacter spp.

Because these bioactive molecules were derived from milk proteins, they may have a bioprotective role in dairy foods like PIF. In another study, a sodium caseinate fermentate was tested against Cronobacter spp. in reconstituted PIF (Hayes et al., 2009), and at final concentrations of 3.33% (wt/vol), the bacterial level was reduced from ~ 106 to 0 CFU/ml in 1 hour.

It has been demonstrated that breast milk contains probiotic bacteria, including

Bifidobacterium spp. (e.g. B. longum and B. animalis) and Lactobacillus spp. (e.g. L. rhamnosus, L. acidophilus, L. fermentum) (Gueimonde et al., 2007; Martin et al., 2005).

As a result, the inclusion of probiotics in infant formula has been suggested in order to promote intestinal microbiota in infants that increase resistance to gastrointestinal pathogens. Probiotics in infant formula can help to develop healthy gut microbiota and may prevent pathogen growth by competitive exclusion (Collado, Isolauri, & Salminen,

2008). In addition, probiotics can produce anti-microbial compounds (e.g. organic acids,

H2O2 and/or bacteriocins) and improve the function of the intestinal barrier by reducing intestinal permeability and increasing mucin production (Lara-Villoslada et al., 2007).

Currently, infant and follow-up formulae supplemented with probiotics are marketed in several countries, in order to mimic some of the beneficial effects of breast milk and enhance the resemblance of infant formula to human milk. Different species of

Bifidobacterium and Lactobacillus have been successfully used as probiotic bacteria in infant formula. For instance, Weizman et al. have revealed that infants fed a formula supplemented with B. lactis or L. reuteri in a 12-week period had fewer and shorter episodes of diarrhea, compared with a control group fed with no probiotic formula

(Weizman, Asli, & Alsheikh, 2005).

Osaili et al. (2008) have studied Bifidobacterium breve, which is used to supplement infant formulae in many countries, and is claimed to inhibit pathogenic bacteria in the gut of formula-fed infants (Osaili et al., 2008). The effect of B. breve was studied on a cocktail of five isolates of Cronobacter in rehydrated infant formula at 4 to

45ºC, and 2-8 h of storage. Although at 12 and 20ºC the presence of B. breve reduced

Cronobacter growth in the rehydrated formula after 8 h of storage, this probiotic led to an increase in growth of Cronobacter isolates at 37ºC within 2 h. In addition, B. breve was not able to reduce the pathogen load at temperatures higher than 30ºC, and Cronobacter showed an inhibitory effect on B. breve at these temperatures. The authors suggested that selection of probiotics to supplement infant formulae should involve a more competitive probiotic with negative effects on the growth and/or survival of Cronobacter spp. to reduce the health risk of consuming contaminated infant formulae.

In other work, Collado and colleagues have studied the effects of five probiotic strains and their combinations on one Cronobacter isolate (Collado, Isolauri, &

Salminen, 2008). They concluded that specific probiotics, especially Lactobacillus rhamnosus and L. paracasei, reduced adhesion of Cronobacter to mucosal cells. They suggested selection of specific probiotic strains and their combinations in infant formula as a helpful way to counteract Cronobacter contamination and reduce its infection risk.

Wakabayashi and coworkers have studied the susceptibility of four isolates of

Cronobacter and two strains of E. coli to bovine lactoferrin (bLF), lactoferricin B (LFcin

B), and other antimicrobial peptides in Bacto Peptone broth (Wakabayashi, Yamauchi, &

Takase, 2008). LFcin B showed bactericidal activity, and was the most effective agent against both pathogens.

1.3. OVERVIEW OF BACTERIOPHAGE

1.3.1. Discovery of Bacteriophage

In 1914, a British pathologist, Frederick William Twort, who was working on smallpox vaccines contaminated with Staphylococcus, saw small clear areas in the colonies of contaminant bacteria during culture (Ackermann & DuBow, 1987; Sambrook

& Russell, 2001). He realized that a substance caused bacterial growth inhibition in those areas. In his publication in 1915, Twort called that substance “Bacteriolytic Agent”. The agent could pass through porcelain filters and needed bacteria to grow. Two years later a

French-Canadian bacteriologist, Félix Hubert D’Hérelle, who was not aware of Twort’s work and was working at the Pasteur Institute in Paris on an epidemic of dysentery, presented a paper on an invisible microorganism and an obligate parasite of living

bacteria, which could kill bacteria, and he called it “Le Bactériphage” (Ackermann &

DuBow, 1987; Summers, 2005).

Bacteriophages, which are also called phages, are viruses that infect bacteria.

They are obligate intracellular parasites, and are not able to metabolize (Hagens &

Loessner, 2007). They multiply within their specific host, disrupt bacterial metabolism and cause lysis. The history of the discovery of phages starts even before Twort and

D’Hérelle. In 1896, the British bacteriologist Ernest Hankin reported that the waters of the Ganges and Jumna rivers in India had an antibacterial effect on Vibrio cholerae, and drinking the water of these rivers prevented spread of cholera epidemics. He suggested an unidentified substance that could pass through fine porcelain filters and was heat sensitive, as the cause of this phenomenon. Two years later, the Russian bacteriologist,

Nikolay Fyodorovich Gamaleya, reported similar observations while studying Bacillus subtilis (Deresinski, 2009).

1.3.2. Biology of Bacteriophage

Phages are the largest group of viruses, utilize species in the Bacteria and Archaea as hosts and are 20 to 200 nm in length (Ackermann & DuBow, 1987). On earth, phages are the most abundant form of life, and their estimated population is 1031 in the biosphere

(Kutter & Sulakvelidze, 2005). A phage resembles a complex spaceship carrying its genome from one susceptible bacterial cell to another in which it can produce more phages. Each phage particle (virion) consists of a nucleic acid genome (DNA or RNA) that is wrapped with a protein or lipoprotein coat, or capsid (Guttman, Raya, & Kutter,

2005). The capsid has important roles in the life cycle of phage including shielding the

genome against DNA-degradation (e.g. enzymes) until the phage finds the right host, and assists delivery (uptake) of the genome into the cytoplasm of the now-infected host (Gill

& Abedon, 2003). The capsid holds a single copy of the genome, and in addition some subunits of capsid proteins are involved in the genome packaging, adsorbtion to the host cell, and injecting the genome into the host (Maloy & Maloy, 1994). The nucleocapsid is composed of the combined nucleic acid and capsid. Every bacterium has a group of phages capable of infecting it. Phages are usually strain or species specific, though some will lyse members of different genera. Similar to all viruses, phages are absolute parasites

(Guttman, Raya, & Kutter, 2005).

The genome of the majority of studied phages is double-stranded DNA (dsDNA), however, there are small groups of phages with single-stranded DNA (ssDNA), ssRNA, or dsRNA (Ackermann, 2006). The genome size of phages varies from very small (e.g. E. coli phage R17, only 4 genes and ~3600 bp) to very large, for example Bacillus megaterium phage G, which possesses a genome of more than 490 kb that encodes 684 genes (Birge, 1994; Pedulla et al., 2003).

Most phages have tails, which are complex hollow tubes attached to the phage head. The genome passes through the tail during infection. The tail size varies in different phages, while some phages do not possess a tail. The tail of some phages is surrounded by a contractile sheath, which contracts when the phage infects the host. At the distal end of the tail, the more complex phages (e.g. T4) have a structure called a base plate, to which one or more tail fibers are attached. Tail proteins at the free end of the fibers are able to recognize molecules on the surface of bacterial cell walls, which limits their

ability to attach to non-specific cells. The baseplates and tail fibers are not present in all phages (Ackermann & DuBow, 1987; Kutter & Sulakvelidze, 2005).

Most of the tailed phages do not have lipid in their structure; however, about 30% of them are inactivated by lipid solvents such as acetone, chloroform, ether, and toluene

(Ackermann, 1999). Phages have different sensitivities to ultraviolet (UV) light with ssDNA being most sensitive. Most tailed phages are resistant to pH of 5 to 9, but are inactivated by heating at 60°C for 30 min (Ackermann, 2007). Apparently, large head phages of the Myoviridae family are more sensitive to freezing and thawing than other types. Although lyophilization or immersion in liquid nitrogen after addition of 15-50% glycerol are the best methods to preserve tailed phages, these conditions quickly inactivate some phages. As an alternative preservation method, storage at 4°C is adequate for many phages (Kutter & Sulakvelidze, 2005; Puapermpoonsiri, Ford, & van der Walle,

2010).

1.3.3. Taxonomy of Bacteriophage

By 1918 Félix d’Hérelle assumed that there was just one phage, which had numerous races, and he called it the “Bacteriophagum intestinale” (Ackermann, 2005).

However, by isolation of phages against numerous bacterial species in many environments, the single phage theory became hard to accept. Early classification was conducted by means of serology, particle size (determined by filtration), stability and host range, and later by using electron microscopy to determine morphological types of bacteriophages. In 1962 a classification based on the type of nucleic acid (DNA or RNA), the shape of phage capsid, presence or absence of an envelope, and number of capsomers,

was proposed by Lwoff, Home, and Tournier (LHT); which lead to the development of the LHT system (Ackermann, 2005). In 1965, the Provisional Committee on

Nomenclature of Viruses (PCNV) adopted this system. The PCNV one year later became the International Committee for Nomenclature of Viruses (ICNV), which was changed to

International Committee on Taxonomy of Viruses (ICTV) in 1973 (Matthews, 1983). The overall purpose of classification is to concentrate information, condense and classify data, and simplify complex circumstances by creating groups. The ICTV applies all of the available criteria for classification and has agreed to the “polythetic species concept”, which means that a set of properties describe a species, however, not all of the properties may be present in a given member (Ackermann, 2005).

In a survey published in 2007, about 5,568 bacterial viruses had been examined by electron microscopy (Ackermann, 2007). That makes bacteriophages the largest category of viruses studied by that method, and also shows the huge amount of hard work performed to characterize phages. Around 96.2% of the tested phages were tailed and just

23.7% of them were polyhedral, filamentous, or pleomorphic (PFP) (Ackermann, 2007).

Non-tailed phages that have been reported belong to 17 families or “floating genera”, which mostly represent small families of viruses with unusual hosts like Mycoplasmas and Archaea. Phages were reported in 10 archeal and 144 eubacterial genera, with phages specific for members of the Enterobacteriaceae having the most observations at 906

(Ackermann, 2007).

Currently, bacteriophages are classified by ICTV into 1 order, 13 families, and 31 genera. Families are mainly distinguished by the type of phage nucleic acid and general morphology of the virion. For classification, about 40 criteria are used, but there are not

universal criteria for genera and species (Ackermann, 2006). The DNA content and composition, dimensions and fine structure, and physiology of tailed phages vary greatly.

For instance, DNA sizes differ from 17 to about 500 kb and tail lengths vary from 10 to

800 nm (Ackermann, 2006). Tailed phages belong to the order Caudovirales and distinct tail morphologies of the phages define three main families of bacteriophages as:

Myoviridae (long, contractile tails; 24.5%), Siphoviridae (long, non-contractile tails;

61%) and Podoviridae (short, non-contractile tails; 14%) (Figure 1.1) (Ackermann, 1996;

Ackermann, 2001; Ackermann, 2003; Ackermann, 2007; Maniloff & Ackermann, 1998;

Ackermann, 2009b).

In case of tailless phages, ten families have been described so far with few members in each family. To distinguish the families, the following criteria can be used: shape (spherical, lemon-shaped, rods, or pleiomorphic), being enveloped in a lipid coat, containing single- or double-stranded DNA or RNA genomes that are segmented or not; and being released by lysis of the host cell or being repeatedly extruded from the surface of the cell (Guttman, Raya, & Kutter, 2005). Table 1.2 shows the classification and basic properties of different categories of bacteriophages.

The three families of tailed phages include 18 genera that are named after their respective type viruses. The genera are classified according to a set of partially overlapping criteria regarding the genome and deoxyribonucleic acid (DNA) packaging, such as the presence of pac sites and cohesive ends, circular permutations and terminal repeats, terminal proteins, DNA or ribonucleic acid (RNA) polymerases, or inclusion of uncommon DNA bases into the phage genome (Maniloff & Ackermann, 1998). In each family, tailed phages could be divided into three morphotypes based on heads of phages:

isometric, moderately elongated or very long (Ackermann, 2007). More tailed phages are being identified, and tailed phage taxonomy is still in its infancy and most likely more phage genera and species will be identifed in the future. Currently, there are six, eight, and four genera in Myoviridae, Siphoviridae, and Podoviridae families, respectively

(Ackermann, 2007; Fauquet & Fargette, 2005). In 2009, the proteomic analysis of 102 myoviruses showed that the Myoviridae family should contain three new subfamilies of

Peduovirinae, Teequatrovirinae and Spounavirinae and eight new independent genera

(Lavigne et al., 2009a). In addition, based on their life cycle, phages are classified into two categories of virulent and temperate (Guttman, Raya, & Kutter, 2005).

Table 1.2. Classification and basic properties of bacteriophages (Ackermann, 2006).

Symmetry Nucleic acid Order and families Particulars

Binary (tailed) DNA, ds, L Caudovirales Myoviridae Tail contractile Siphoviridae Tail long, noncontractile Podoviridae Tail short Cubic DNA, ss, C Microviridae ds, C, T Corticoviridae Complex capsid, lipids ds, L Tectiviridae Internal lipoprotein vesicle RNA, ss, L Leviridae ds, L, S Cystoviridae Envelope, lipids Helical DNA, ss, C Inoviridae Filaments or rods ds, L Lipothrixviridae Envelope, lipids ds, L Rudiviridae Resembles TMV Pleomorphic DNA, ds, C, T Plasmaviridae Envelope, lipids, no capsid ds, C, T Fuselloviridae Spindle-shaped, no capsid C, circular; L, linear; S, segmented; T, superhelical; ss, single-stranded; ds, double-stranded.

Figure 1.1. Morphotypes of prokaryotic viruses of the three families Myoviridae, Siphoviridae and Podoviridae within the order of Caudovirales. They possess an isocahedral head with a tail, which is long and contractile, long and non-contractile, or short and non-contractile, respectively. Phages of such as Corticoviridae, Plasmaviridae, Inoviridae, Ampullaviridae, Bicaudaviridae, and Cystoviridae are polyhedral, filamentous, or pleomorphic in morphology (Ackermann, 2009b).

1.3.4. Lysogenic and Lytic Cycle of Bacteriophages

As stated above, phages are also classified into two categories of virulent and temperate based on their life cycle. In the virulent cycle, phages propagate just by lysis of the host cell. They inject their genome after absorption to the surface of the cell, and the genome takes control of the majority of the bacterial cell metabolism, and directs it to

produce more phages. Within minutes or hours, the host cell gets lysed and new phages are released. On the other hand, when a cell is infected by a temperate phage, the phage has an alternative way of reproduction. Sometimes, infecting phage goes through the lytic cycle, which lyses the cell and liberates many new phages. Alternatively, temperate phage may choose a lysogenic pathway, in which instead of propagation the genome of the phage enters a dormant state called prophage. The prophage genome usually recombines with the genome of the host cell; however, it may remain as a plasmid in the cell. Prophage can stay in this state forever, and its genome is replicated when the cell reproduces; therefore, all daughter cells contain the prophages. Occasionally, the prophages leave the dormant state and start the lytic cycle and lyse the host. Thus, the cells that carry prophages are called lysogenized or lysogenic (Guttman, Raya, & Kutter,

2005).

The preference between the two life cycles depends on the relative expression rates of phage repressor that is encoded by cI gene, which promotes lysogeny, and cro protein that can turn off the repressor gene expression and start the lytic cycle (Campbell,

1994). Figure 1.2 shows the life cycle of bacteriophages.

Figure 1.2. Lysogenic and lytic cycles of bacteriophages.

The lytic cycle of phages can be explained in the following five sequential steps:

Adsorption and Infection. To start infecting their host cell, tailed phages use specialized adsorption structures (e.g. fibers or spikes) to bind to specific surface molecules (receptors) on the target cell. Only if the specific receptor sites for the phage exist on the cell surface will the replication proceed. There are many types of molecules on the surface of the host cell that could act as specific phage receptors, which include proteins that have important functional roles in the bacterial cell. The type of the receptor varies for different hosts and not only exist on the cell wall but also could be located on capsules, flagella, pili, or possibly the plasma membrane (Lindberg, 1973). Some

environmental factors may be required to enhance the adsorption. The most frequently required cofactors are Ca++ ions, followed by Mg++ ions (Ackermann & DuBow, 1987;

Kutter & Sulakvelidze, 2005). Phage diffusion rate and bacterial density are among the factors that control the possibility of phage attack on the host cell. Furthermore, the adsorption of phage to its host depends on chemical and physical interaction between phage and bacteria (Gill & Abedon, 2003).

Injection and Penetration. With most phages only their nucleic acid enters into the host and the phage shell remains outside. In case of T4 phage, when the phage contacts the receptors on the host cell, conformational changes are initiated in the phage structure, which end in the contraction of the tail sheath that pushes the hollow inner tube into the cell. The baseplate is anchored to the receptors on the cell surface by short-tail fibers. Meanwhile, the shape of the baseplate shifts from a hexagon to a star-shaped structure. The whole structure of the tail then shrinks and widens, which brings the internal tail tube in contact with the outer membrane of the host cell, and phage enzymes that are located at the tip of the tail degrade the cell wall. When the outer and inner membranes of the cell are punctured by the tail tube, the phage DNA is injected through the tail tube into the cytoplasm of the host cell (Kostyuchenko et al., 2005). The contraction of the phage tail can be triggered by a number of factors or agents including formalin, H2O2, alcohol, Cd(CN3), urea, changes in pH, freezing and thawing, or sonication (Ackermann & DuBow, 1987). Members of the Siphoviridae like phage λ do not contain a contractile tail. In the case of E. coli filamentous DNA phages, as they are uncoated, they enter the cell by being drawn into the inner membrane of the cell

envelope. The coat protein of the phage dissociates into subunits, which remain in the membrane, and the DNA is released intracellularly (Gottesman & Oppenheim, 1994) .

Latent Period. The time interval from infection to the release of new phages is the latent period, and the rise period is when phage titres increase to the maximum. The

“burst size” is the number of new phages per infected cell (Kutter & Sulakvelidze, 2005).

Immediately after the phage genome enters the host cell, the early genes are expressed to produce proteins needed to replicate the phage genome and to modify the cellular machinery in such a way as to subvert the synthetic capacity of the cell in order to reproduce the phage. The products of the early genes are usually not found in the completed phage. Early proteins include enzymes to repair the hole in the bacterial cell wall, nucleases to degrade the host DNA into precursors of phage DNA, and phage DNA- specific polymerase to copy and replicate the viral DNA. Although the host cell still has the ability to generate energy and synthesize proteins, the virus subverts these abilities.

Each copy of the de novo synthesized DNA can be used to transcribe and translate a second set of proteins, which are called the late proteins that build the capsomeres and the various components of the tail assembly for the new phages. One of the late proteins is lysozyme, which is packaged in the phage tail, and also during the last step will be used by the phage to escape from the host cell (Birge, 1994; Kutter & Sulakvelidze, 2005).

Morphogenesis or Maturation. During this stage, the components of the new phage are assembled into virions. Assembly occurs with the help of specific enzymes or spontaneously. The DNA is packaged into procapsids, which are preassembled protein shells. Complex interactions between specific scaffolding protein and the major head structural proteins are involved in the assembly of most phages. The head expands and

becomes more stable before or after the packaging due to the increase of internal volume required for the DNA. In case of tailed phages, separate pathways are used to assemble the head and tails, and then they are joined together (Ackermann & DuBow, 1987;

Guttman, Raya, & Kutter, 2005; Maloy & Maloy, 1994).

Lysis or Release. Lysis liberates the new phages. Lysozyme or lysin enzme lyses the cell wall, liberating infectious phages, which can infect new host cells, and start a new cycle over again (Ackermann, 2003; Guttman, Raya, & Kutter, 2005). By lysis of the host cell, new phages are released into the surrounding medium. The number of progeny phages depends on the phage type and the physiology of the host cell. There are two proteins that the tailed phages use to lyse the host cell: i) lysin, which enzymatically degrades the peptidoglycan in the cell wall, and ii) holin, which is able to assemble pores in the inner membrane to help lysin reach the peptidoglycan layer. By disruption of the cell membrane and cell wall by these enzymes, the cell bursts and phages are released into the surrounding medium. In the case of the tailless phages, a variety of lysis- precipitating proteins are encoded, which can sabotage the host peptidoglycan-processing enzymes through different mechanisms (Gottesman & Oppenheim, 1994; Guttman, Raya,

& Kutter, 2005; Maloy & Maloy, 1994).

1.4. BIOCONTROL OF FOOD-BORNE PATHOGENS USING BACTERIOPHAGE

1.4.1. Phage Applications in Food

Microbial food-borne diseases are a major cause for concern worldwide, and it is estimated that approximately 72% of the deaths in the USA associated with food-borne pathogens are due to bacteria (Mead et al., 1999). Bacteriophages are the natural enemies

of bacteria, and an old adage “the enemy of my enemy is my friend” describes the concept for their use as biocontrol agents. Therefore, phages could be used to control problematic bacteria, such as spoilage and pathogenic bacteria. However, these applications of phages require an in-depth understanding of the epidemiology of the pathogen and identification of the critical points in the processing cycle where using phage would be most efficient (Stone, 2002), as well as complete knowledge of the candidate phage. Because phages are very host-specific and only can infect specific species or even strains, they can be used for biocontrol of undesired bacteria without known interference of the natural microbiota or the cultures used in fermented products.

In addition, phages or phage-derived proteins have been also used to detect pathogens in food or production environments, which offers a quick and specific identification of as few as 10 viable cells per gram of food samples (Brovko, Anany, & Griffiths, 2012;

Kodikara, Crew, & Stewart, 1991; Loessner, Rudolf, & Scherer, 1997). The increasing concerns of antimicrobial resistance have renewed interest in the use of phage, particularly in the food industry. This has led to a novel biotechnology to control bacterial contamination in food, called biocontrol (Hagens & Loessner, 2007; Rees & Dodd,

2006).

Lytic phages can attach to unfavorable bacteria, reproduce in them, lyse the bacterial cell and release new phage that can infect more bacteria. This could be beneficial for food safety applications. On the other hand, temperate phages can remain dormant within the host cell, and are able to transfer genes from one bacterium to another, which potentially allows the development of more virulent and resistant pathogens (lysogenic conversions) (Greer, 2005; Hagens & Loessner, 2010; Kutter &

Sulakvelidze, 2005). There are reports on pathogenicity associated with lysogenic phages.

For instance, in the integrative phage CTXФ of Vibrio cholerae, ctxA and ctxB genes encode the cholera toxin, CTX (Waldor & Mekalanos, 1996). Another good example is

Shiga-like toxin (ST) producing E. coli, an important food-borne pathogen, which is known for a phage-dependent virulence phenotype. Genes stx1 and stx2 encoding the toxin in many cases are located on temperate phages integrated into the bacterial host genomes (O'Brien et al., 1984). Consequently, the possible lysogenic conversion of temperate phages decreases their beneficial application in biocontrol. In addition, usually temperate phages have narrower host ranges than virulent phages (Hagens & Loessner,

2010). Virulent (strictly lytic) phages, therefore, are the practical choice for food safety applications (Greer, 2005).

Three possible areas of phage application have been proposed: i) to decrease the intestinal carriage of food-borne pathogens in agriculturally beneficial livestock; ii) sanitize raw food, or food-contact surfaces of food processing plants; and iii) remove pathogens on ready-to-eat (RTE) foods, or in the production area of RTE foods

(Sulakvelidze & Barrow, 2005).

Pathogen contamination in RTE foods is potentially a more serious issue than contamination of raw food as the latter are usually further processed (cooked) before consumption. One of the first applications of phage in RTE foods was with fresh-cut fruits and vegetables (Leverentz et al., 2001). Direct application of Salmonella Enteritidis phages (5 × 106 PFU/melon slices) to artificially contaminated fruit was able to reduce

Salmonella on melon slices more effectively than application of commonly used chemical

sanitizers. The efficacy of phage applied to apple slices was lower, which probably was due to the more acidic pH of apples that could affect the phage.

The majority of research on phage biocontrol has been conducted in liquids and generally high concentrations of pure target bacteria were applied (Hagens & Loessner,

2010). In liquids, it is more likely for phages to contact, attach to and infect the host bacterium because of the thermal, motion-driven particle diffusion and mixing due to either fluid flow or active bacterial motility (Hagens & Loessner, 2010; Murray &

Jackson, 1992). Two major obstacles exist for the application of phage in food. Most targeted foods are solids rather than liquids. In addition, due to the expected high hygiene standards, usually bacterial contamination is present at very low levels (Hagens &

Loessner, 2010). It is unlikely that low numbers of phages infect low numbers of bacteria, simply because of the low probability that phages and bacteria will encounter one another. Thus, to ensure sufficient and rapid infection of targeted bacterial cells that are present in low numbers in food, it is important to have a significantly greater number of phages (threshold of ~ 1 × 108 PFU/ml). When the critical threshold number of phages is present so that they cover the entire matrix of the targeted food, the concentration of bacterial host will not be a limiting factor (Hagens & Loessner, 2010). This claim has been verified in a study in which Salmonella phage P7 was incubated with its host bacterium at 24°C for up to 2 h in LB broth at different ratio of phage and host cell, and the surviving cells were counted (Bigwood, Hudson, & Billington, 2009). It seemed that

Salmonella was inactivated by P7 independently of the cell concentration, with the bacteria being almost complete inactivated when the phage concentration was around 5 ×

108 PFU/ml.

Although, there is no universal agreement on the minimum required bacterial density as a prerequisite for successful phage biocontrol (Kasman et al., 2002), some researchers support the idea that a threshold level of bacterial cells must be attained to allow phage propagation. For example, studies on the control of spoilage bacteria on meat surfaces suggested that phages could be effective when the host cells were as few as 46

CFU/cm2 (Greer, 1983).

Incubation temperature is another important factor to be considered (Hagens &

Loessner, 2010). Temperatures higher than normal storage conditions, which support good growth of the undesired contaminants, as well as recommended storage temperatures should be tested to evaluate the efficacy of phages to control target pathogens. For instance, Anany and colleagues (2011) have investigated the efficiency of their phage cocktails under different storage temperatures (25, 10, or 4°C) and packaging conditions (aerobic, modified atmosphere packaging, or vacuum). Phage cocktails against

L. monocytogenes and E. coli O157:H7, which were immobilized on positively charged cellulose membranes, were able to control these pathogens effectively in RTE and raw meat at the temperatures and in atmospheres tested (Anany et al, 2011).

Another factor to consider is the ratio of phages to host cells or multiplicity of infection “MOI” (O'Flynn et al., 2004). To calculate MOI the number of phages that infect bacterial cells has to be considered, not the number of adsorbed phages to the cell or, number of added phages to the food (Kasman et al., 2002). As a more descriptive term, ratio of PFU/CFU was suggested for food applications to take into account the fact that physical barriers may prevent or slow the adsorption of phage (Bigwood, Hudson, &

Billington, 2009; Whichard, Sriranganathan, & Pierson, 2003). Many factors can affect

the exact concentration of phage that needs to be used in a particular application; including micro-structure of the surface that can affect phage diffusion rates and accessibility of target bacteria, the amount of available fluid that affects phage diffusion, and the target reduction levels that are required (Hagens & Loessner, 2010). This suggests that if very high concentrations of phage are applied, food-borne pathogens might be eliminated without the need for bacterial growth and replication during the phage application (Strauch, Hammerl, & Hertwig, 2007).

In some phage applications targetting food-borne pathogens, the inactivation of bacteria may be the result of “lysis from without” (Delbruck, 1940). This phenomenon occurs when the host cell adsorbs numerous phage particles, and lyses without phage being replicated. In case of coliphage T4, a lysozyme on the base plate mediates lysis from without (Abedon, 1999). Once more than 100 phages were adsorbed on the host cell, the membrane swelled and bulged 5-10 min after infection. In the end, cytoplasmic contents may escape through the holes that are formed (Tarahovsky, Ivanitsky, &

Khusainov, 1994).

The studies of phage application in contaminated food mostly focus on reduction of pathogen loads and to achieve a more effective pathogen control the right phage in the right place and in the right concentration must be applied. Finding an effective phage is the starting point for the use of phage for food safety applications. There are some basic requirements for the phages intended for these purposes to asure their efficacy and safety.

Phage DNA should be screened for any possible genes that encode virulence factors such as toxins. Thus, the complete genome sequence must be known (Hagens & Loessner,

2010). In addition, “generalized transduction” should be checked when selecting a phage.

This phenomenon is a process during which instead of phage DNA, host DNA is packaged into phage heads. This might introduce new genes into the recipient bacterium

(Ikeda & Tomizawa, 1965). In several pathogens, it has been shown that virulence- associated genome regions were distributed via transduced DNA (Cheetham & Katz,

1995). Therefore, phages that are unable to transduce non-viral (i.e. bacterial) DNA should be used for biocontrol or phage therapy.

Moreover, suitable phage candidates should have a broad host range, being able to infect large numbers of the target species and/or genera (Hagens & Loessner, 2010). In some bacterial species there are many sub-types, all of which require to be controlled, so narrow host range phages may be problematic for biocontrol purposes. Thus, a

“Goldilocks” (not too narrow and not too broad) host range is necessary for a phage to be effective. A perfect example is phage Felix O1 that can lyse 96-99.5% of Salmonella serovars. The problem of narrow host range, however, can be solved by using phage cocktails (McIntyre et al., 2007). Another important factor that should be considered is the stability of the phage at different storage and application conditions (Strauch,

Hammerl, & Hertwig, 2007). It is definitely important to check the durability of phages in the environment where they are to be used. This requires extensive characterization of the phages (Gill & Hyman, 2010). Other standard criteria for a good phage candidate for biocontrol of pathogens in food are the capacity for large-scale commercial production and the ability to be propagated in non-pathogenic hosts (Hagens & Loessner, 2010). To treat food products, not only large volumes of phage lysates with a high level of infective phages are needed, but also, it is necessary to remove endotoxins and undesirable cell

debris, which requires that phage preparations should be purified to prevent any adverse effect due to oral consumption (Gill & Hyman, 2010).

Infection of mammalian cells by phages is unlikely, as they are bacteria-specific viruses, and there is evidence that shows their oral consumption is entirely harmless to humans. The safety of phages was first demonstrated by d’Herelle who along with several members of des Enfants-Malades hospital in Paris ingested them before giving them to patients. In addition, he injected his family and his colleagues to evaluate the safety of phage therapy (D'Herelle, 1926). In a toxicity study on rats, L. monocytogenes phages were administered orally at a daily dose of 2 × 1012 PFU/kg body weight and no abnormal signs in morbidity, mortality, or following histological examinations were observed (Carlton et al., 2005). In another study, human drinking water was supplemented with E. coli T4 phages, and provided to human volunteers with no ill effects (Bruttin & Brüssow, 2005). The applied phages could infect commensal E. coli strains in vitro, but apparently E. coli located in the gut of the human volunteers were only slightly affected by the phage. This could be due to the localization of the commensal E. coli in niches where they are not easily accessible for phages (Hagens &

Loessner, 2010). Even HIV positive and other immunocompromized individuals as well as healthy volunteers who received purified phages (e.g. ФX174) intravenously (IV) did not develop any apparent side effects (Atterbury, 2009). In addition, in Eastern European countries (particularly Poland and the former Soviet Union) thousands of patients have received very successful phage therapy for several diseases (Kutter et al., 2010). Not only were the phages given orally or superficially, but also they were administered IV,

intramuscularly (IM), and even injected into the carotid artery and pericardium

(Ackermann & DuBow, 1987; Kutateladze & Adamia, 2008).

Phages are ubiquitous and natural components of the microflora in the environment. They can be found in all habitats where their bacterial hosts exist, and they have commonly been isolated from water, food, soil, and sewage (Kutter & Sulakvelidze,

2005). Freshwater environments have up to 109 phages per milliliter, and marine surface waters contain up to 107 phage-like-particles per milliliter. In terrestrial ecosystems, such as topsoil, similar numbers have been reported (Rohwer & Edwards, 2002). Phages can be isolated from farms to retail outlets and are amazingly stable in all environments

(Greer, 2005). Because they have been detected in many foods such as lettuce, mushrooms, oysters, mussels, turkey, chicken, yoghurt, cheese, buttermilk, beef and pork, it can be assumed that phages are ingested daily by humans (Hudson et al., 2005). In a study on fresh pork, chicken, ground beef, lettuce, raw vegetables, mushrooms, chicken pie, and delicatessen food, E. coli phages were isolated at titers up to 104 PFU/gram

(Allwood et al., 2004). Campylobacter phages, also, have been reported at levels of 4 ×

106 PFU/gm in chicken (Atterbury et al., 2003), and Brochothrix thermosphacta phages have been isolated from beef (Greer, 1983; Lu et al., 2003). High levels of phages that infect the fermentation flora have been found in fermented foods. In commercial cabbage

(Sauerkraut) fermentation plants, for instance, twenty-six different phages have been reported (Lu et al., 2003). Propionibacterium freudenreichii phages have been isolated at

7 to 14 × 105 PFU/gm in Swiss Emmental cheese (Gautier et al., 1995). In Argentinean dairy plants, sixty-one phages against Lactobacillus delbrueckii subsp. bulgaricus and

Streptococcus thermophilus were isolated at levels up to 109 PFU/ml (Suarez et al.,

2002).

In addition, many people consider phages as “green” and environmentally friendly

(Fox, 2005), and so have also been considered as a potential natural alternative to chemical preservatives (McIntyre et al., 2007). Although phages have been, are and will be a permanent part of human nutrition, convincing consumers regarding addition of viruses to their foods will be the most critical hurdle that must be overcome to allow their widespread use in the food and agriculture industries (Strauch, Hammerl, & Hertwig,

2007).

In order to apply phages as biocontrol agents in food, strategies need to be optimized to be the most economical, most convenient, and least invasive for the processing facilities. Different methods for the industrial application of phages in food have been reviewed by Hagens and Loessner (Hagens & Loessner, 2010). Phages can be used at different and multiple points during food processing, and by applying phages at the locations where bacterial contamination is most recent, killing efficiency is enhanced, and the possibility of bacterial evolution to phage resistance is decreased. In the classic

“farm to fork” approach, using phage at all stages of production throughout the entire food chain could be valuable (Garcia et al., 2008). Methods like spraying, dipping or as a liquid may not be the ideal choice for phage application, because these methods are wasteful and could potentially result in inactivation of the phage particles due to the inclusion of materials such as bleach within the wash fluid. Furthermore, if the washing fluids could support the growth of bacteria, these organisms might evolve for phage resistance. Dilution of phages and evolution of bacterial resistance are the two major

concerns when phages are added directly to a batch of food. The first concern can be resolved by adding a higher concentration of phages or by immobilization (Anany et al.,

2011), and the development of resistance can be overcome by disinfecting the equipment regularly with a highly efficient disinfectant (Hagens & Loessner, 2010).

Several features of phages make them ideal for use as an alternative to antibiotics, namely: phages attack specific bacteria with no undesirable effect on the natural microbiota; phages have no severe side effects on humans and animals; phages can be reproduced easily and at low cost; they self-replicate with no need to repeat the dosing.

However, there are some disadvantages such as limited host range, the potential evolution of resistant bacterial mutants, and the risk of transducing virulence from one bacterial strain to another. Moreover, the efficiency of phage biocontrol depends on the chance of phage and bacteria being in the same place at the same time. The fact that most research to date has involved experiments conducted with artificially inoculated foods far removed from real commercial environments is another important issue to be considered (Greer,

2005; Hagens & Loessner, 2010; Hanlon, 2007).

Overall, the advantages of phage applications in food exceed their disadvantages.

For example, spontaneously produced phage-resistant mutants may not have a significant influence on the efficacy of the treatment, and use of phages with broad host range and applying phage cocktails could overcome the complex mechanisms of bacterial phage resistance (Hagens & Loessner, 2010). Using higher concentrations of the phages may also increase the chance of contact between phages and bacteria (Garcia et al., 2008;

Greer, 2005). The positive scientific results obtained, in addition to several governmental approvals of phage as biocontrol agent in foods, have encouraged many companies in

Europe and North America to carry out studies in this area and develop commercial products (Garcia et al., 2008). ListShield™ (formerly LMP-102) is an example of a commercial phage mixture (Intralytix Inc., U.S.A) against L. monocytogenes, which is used in ready-to-eat meat products with no impact on their color, odor, taste, or quality

(www.intralytix.com). The US FDA has approved this product as a safe food additive for use on meat and poultry ready-to-eat products before packaging (www.fda.gov/).

LISTEXTM P100 (www.ebifoodsafety.com) consists of a single lytic Listeria phage, and was the first phage product that US FDA recognized as GRAS (generally recognized as safe). There are also phage products for E. coli and Salmonella (www.omnilytics.com), which are approved for use in cattle and chicken production, respectively, where they are applied as sprays on the animals before slaughter to reduce transfer of these pathogens to meat products (Sulakvelidze & Barrow, 2005). The same company has developed a phage product to treat bacterial-spot diseases in tomato and pepper caused by

Pseudomonas putida (www.omnilytics.com), which has been approve by the US

Environmental Protection Agency (EPA) (Balogh et al., 2010). In addition, another phage product against E. coli O157:H7 has been approved by the US EPA for use as a wash or spray to be applied to cattle hides before slaughter (Hagens & Loessner, 2010). More studies on phages will lead to the development of more and better phage products.

The companies that commercially produce phages as biocontrol agents should consider some safety issues. Phage preparations after purification, for example, must be evaluated to be free of the pathogen, toxins and/or virulence factors. Additionally, it is reasonable to have a system to monitor the development of phage resistant bacterial cells, to ensure the efficacy of the product. Genome analysis and bioinformatic evaluation is

another important consideration, which helps to ensure the absence of virulence genes and any possible genes that could mutate the virulent phages to adopt a temperate life cycle and lysogenize a pathogen by horizontal gene transfer. As well, it should also be considered that microflora on the surface of a food may be altered by the phages and, therefore, intensive studies on the changes in the composition of the microflora should be conducted (Strauch, Hammerl, & Hertwig, 2007).

In conclusion, we should remember that it is not reasonable to assume that phage will completely replace standard cleaning procedures and addition of preservatives to food products. Instead, phages should be used as adjuncts to other means of bacterial control (Garcia et al., 2008; Leverentz et al., 2003; Martinez et al., 2008; Roy et al.,

1993).

1.4.2. Phage Application as a Biocontrol Agent

Using phages during the pre-harvest and post-harvest phases of food production to decrease bacterial pathogens is a promising method to achieve a safer food supply

(Strauch, Hammerl, & Hertwig, 2007). Applying lytic phages to disinfect surfaces in food plants (e.g. ready-to-eat products), decontaminate carcasses, or eliminate zoonotic pathogens from living animals have been successful (Hagens & Loessner, 2010; Strauch,

Hammerl, & Hertwig, 2007). Although, phages are less active in the real food than in liquid medium, their application to control food-borne pathogens like E. coli O157:H7,

Salmonella and L. monocytogenes has been beneficial (Carlton et al., 2005; Greer, 2005;

Hagens & Loessner, 2007; O'Flynn et al., 2004). Indeed, the approval of the use of phage

as food additives by the US FDA in 2006 has also increased the interest for research on new applications for these natural killers of bacterial pathogens (FDA, 2006).

The use of phages in combination with other antibacterials to contorol food-borne pathogens has been studied. For instance, it was found that Listeria phage alone could reduce Listeria counts by 3 log units on stainless steel and polypropylene surfaces (Roy et al., 1993). However, when phage was applied in combination with a quaternary ammonium compound (QUATAL) at 40 ppm, the reduction was 5 log units. The Listeria phage remained active in the presence of QUATAL at concentrations up to 50 ppm. In other studies, cocktails of Listeria phages alone and in combination with nisin were evaluated on honeydew melon and apple slices (Leverentz et al., 2003; Leverentz et al.,

2004). In honeydew melon, phages alone could reduce Listeria counts by between 2 - 4.6 log units, and addition of nisin showed a cumulative effect. In one study, Listeria phage

LH7 showed no significant effect on two strains of L. monocytogenes in beef samples that were artificially inoculated and stored for 4 weeks at 4°C (Dykes & Moorhead, 2002).

However, a later analysis of the data suggested that the negative results might have been due to an insufficient phage concentration which was ~ 0.5 - 1 phage/cm2 (Hagens &

Loessner, 2010).

There are several studies describing the use of phage against Salmonella. For example, Salmonella Typhimurium PT160 inoculated onto raw and cooked beef in the presence of different phages titers was significantly reduced by 2-3 and >5.9 log units at

5°C at 24°C, respectively (Bigwood et al., 2008). In contrast, a study that evaluated two broad host range Salmonella phages (SSP5 and SSP6) against Salmonella Oranienburg in vitro and on artificially contaminated alfalfa seeds revealed that lysis during in vitro

treatment was incomplete and also, in treated seed the reduction of the population of the viable Salmonella was not significant (Kocharunchitt, Ross, & McNeil, 2009). The reason might be due to the application of phage at a low MOI of around 70.

Another pathogen that interests researchers for phage biocontrol in food is E. coli.

In a study, E. coli O157:H7 was experimentally inoculated on beef surfaces and then treated by a cocktail of three phages. After an hour of incubation at 37°C, the results showed that E. coli O157:H7 was eliminated from seven of nine samples, and the other two samples had very low cell numbers. In addition, a similar experiment in broth culture revealed a 5 log reduction in the bacterial count upon addition of phage (O'Flynn et al.,

2004). This indicates the possible effect of food matrices on the effectiveness of phage on bacteria, and also that studies in broth cultures have limited value for phage applications in food matrices (Rees & Dodd, 2006). Moreover, Anany and coworkers (2011) have investigated the efficacy of immobilized phages against E. coli O157:H7. Their phage cocktail was able to control the pathogen effectively in raw meat (Anany et al., 2011).

Two studies have evaluated phage biocontrol in infant formula. In the first study, the efficiency of two Cronobacter phages (ESP 1-3 and ESP 732-1) against this pathogen was investigated in reconstituted infant formula as well as in ½ × Brain Heart Infusion

(BHI) broth at 12, 24, and 37°C (Kim, Klumpp, & Loessner, 2007). The bacterial concentration was 102 CFU/ml and phages were added at 107, 108, or 109 PFU/ml. The growth inhibition produced by the phages was concentration-dependent, and phages present at the highest concentration resulted in the greatest growth inhibition regardless of incubation temperature, with the maximum effect in infant formula being obtained with phage ESP 732-1. Both phages were most effective at 24°C.

In the other publication, a cocktail of five phages against 40 Cronobacter strains was added to experimentally-contaminated infant formula, and BHI broth (Zuber et al.,

2008). Reconstituted infant formula was inoculated with individual Cronobacter strains at 102 CFU/ml, and treated with the phage cocktail at 108 PFU/ml (MOI of 106). The outgrowth of 90% of the 40 tested Cronobacter strains was prevented at 30°C. However, the phage cocktail did not prevent bacterial growth when applied at MOIs of 102 or 104.

1.4.3. Phage Therapy

Shortly after the discovery of phage, Félix d’Hérelle used them to treat bacterial infections. In 1919, he isolated Salmonella Gallinarum from a fowl typhoid outbreak in chickens in France. He also isolated a phage from the same samples, and he tested the efficacy of phage in preventing and treating S. Gallinarum infections in chickens with promising results (Sulakvelidze & Barrow, 2005). Thus, d’Hérelle was encouraged to focus on application of phages as therapeutic agents. In addition, he treated Shigella dysentery in rabbits with success. He then extended the use of phage to the treatment of bacillary dysentery in humans (Summers, 2001). Phage therapy flourished and became the main point of interest of antimicrobial clinical studies in the pre-antibiotics era. The discovery of antibiotics in 1935, the successful application of antibiotics against bacterial infections during and following the Second World War, their cheap mass production, and the conflicting results obtained caused a decline in interest in phage therapy. As a result, there was a gap of about three decades before interest in phage applications and research was renewed (Merril, Scholl, & Adhya, 2003; Summers, 2001). Another reason for discontinuing phage research was the lack of convincing theoretical explanations for the

perceived failure of phage therapy (Merril, Scholl, & Adhya, 2003). However, studies of phage therapy and its application in medicine did continue in Eastern Europe and the

Soviet Union. In this region, phage therapy is still practiced to treat bacterial infections in humans, and is also applied as a complement to conventional antibiotics (Kutter et al.,

2010). Nowadays the emergence of antibiotic resistant bacteria has impacted our world dramatically. It was estimated, for instance, that the number of people killed by methicillin-resistant Staphylococcus aureus (MRSA) was more than by HIV in the USA in 2005 (Klevens et al., 2007). Therefore, there is increased concern that we are entering the post-antibiotic era, and the interest in phage therapy has been renewed. Consideration of phages as alternative control agents has been rekindled mainly because of their high specificity and effectiveness to kill bacterial pathogens without affecting the host commensal microbiota.

In the early 1980s, Smith and Huggins reported the results of an experiment, which caused researchers to reconsider the value of phage therapy. They showed that a cocktail of two phages could protect calves and piglets against enteropathogenic strains of E. coli. The calves and piglets that were treated with phages had a much lower titre of

E. coli in their GI tract than untreated groups (Smith & Huggins, 1982). Their study revealed that for successful phage therapy and biocontrol, applying phage cocktails against the target bacterium may be necessary for success (Strauch, Hammerl, & Hertwig,

2007).

Phage biocontrol of pathogens including Salmonella, Campylobacter and Listeria in the food industry is recognized in the US and Europe (Burrowes et al., 2011;

Goodridge & Bisha, 2011; Kropinski, 2006) and may be particularly useful to control

Cronobacter because of its intrinsic antibiotic resistance (Lai, 2001).

Several studies have confirmed the efficacy of phage therapy in animal models for the treatment of bacterial pathogens such as Pseudomonas aeruginosa (Hagens et al.,

2004; McVay, Velasquez, & Fralick, 2007; Wang et al., 2006), E. coli (Smith & Huggins,

1982; Smith, Huggins, & Shaw, 1987), S. aureus (Capparelli et al., 2007; Fedhila et al.,

2006), Klebsiella pneumoniae (Vinodkumar, Neelagund, & Kalsurmath, 2005), and

Campylobacter jejuni (Loc Carrillo et al., 2005).

There is only one clinical study on phage therapy against Cronobacter. In that study, Cronobacter turicensis was injected directly into the urinary tract of mice, which were simultaneously treated by administering Cronobacter-specific phages, and kidneys and bladder were examined after 24 hours. The authors reported that their phage therapy against Cronobacter resulted in a 70% reduction in incidence of the organism in the kidney, and prevented ascending renal infection due to urinary tract infection in this murine model (Tothova et al., 2011).

1.5. RESEARCH OBJECTIVES

Based on the above description, the purpose of this research is to explore the potential application of isolated lytic phages to control Cronobacter spp. in infant formula. The objectives of this doctoral project were:

1. Isolation and characterization of effective and stable lytic phages against

Cronobacter spp. from different environmental samples.

2. The complete genome analysis of selected phages to ensure their safety and

lytic activity in order to ensure their safe application in infant formula.

3. Study the efficiency of isolated bacteriophages against Cronobacter sakazakii

in vivo using Galleria mellonella (Greater Wax Moth) larvae as a pre-

screening model.

4. Investigate the efficiency of using the selected phages as biocontrol agents for

Cronobacter sakazakii in liquid media and in food.

In the next chapters, the efforts undertaken in the present thesis towards the application of isolated lytic phages to control Cronobacter spp. in infant formula are described. Chapter 2 describes the isolation and characterization of lytic phages against

Cronobacter spp. The most potent and stable phages with broad host range were selected and characterized. One of those isolated phages was found to be unique and, to the best of our knowledge, possesses the second largest phage genome sequenced. The genome of five selected phages were sequenced, and bioinformatic studies performed on these phages. Virulence genes were not detected in the genome of these phages and hence they can be applied safely in food. This will be discussed in Chapters 3 and 4. In Chapter 5, the efficacy of the selected phage against Cronobacter sakazakii was established in vivo.

Galleria mellonella (Greater Wax Moth) larvae were used as a whole animal model in this experiment. Chapter 6 presents the evaluation of a cocktail of five phages to control

Cronobacter sakazakii in broth and in infant formula.

Chapter 2. ISOLATION AND CHARCTERIZATION OF LYTIC

CRONOBACTER BACTERIOPHAGES

2.1. ABSTRACT

Two hundred and fifty two phages, which were active against twenty-one

Cronobacter strains, were isolated from various environmental samples. Using the spot test, these phage isolates showed varying host ranges from broad to limited to just one strain. Eleven phages with the broadest host ranges and strongest lytic activities were selected for further characterization. Based on their morphology from transmission electron microscopy (TEM) images, six phages belonged to the Myoviridae family and five were members of the family Podoviridae. From these eleven phages, five were selected for complete genome sequence and proteomic analysis and evaluation for their lytic activity against C. sakazakii in infant formula.

2.2. INTRODUCTION

Although various procedures are applied to ensure hygiene and to meet the sanitation standards in food processing facilities, there are still reported outbreaks of food-borne diseases (Scallan et al., 2011), with substantial economic and health impact globally (Mead et al., 1999; Scharff, 2012). Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. The mortality rate of Cronobacter infections in neonates and infants can approach 80%, with antibiotics not having a dramatic affect on clinical

outcomes (Drudy et al., 2006; Lai, 2001; Norberg et al., 2012; Ray et al., 2007). Almost all patients surviving infections of the central nervous system (CNS) experience delays in mental and physical development (Lai, 2001). In addition to its effects on neonates and infants, this nosocomial pathogen has been reported to produce rare instances of infections such as urosepsis, bacteremia and pneumonia in adults, especially in the elderly (Emery & Weymouth, 1997; Hawkins, Lissner, & Sanford, 1991; Jiménez &

Giménez, 1982; Lai, 2001; Ongradi, 2002; Pribyl et al., 1985; See, Than, & Tang, 2007).

Consideration of bacteriophages (phages) as alternative control agents has been rekindled because of their high specificity and efficacy for killing bacterial pathogens without affecting the host commensal microbiota. For food safety applications, phages with strong lytic activity are the best choice due to their ability to attack and lyse cells of their bacterial hosts, which leads to the release of more phage particles that are able to infect more bacterial cells (Greer, 2005). Phages exist in all natural environments, where bacteria can be found, including food, and phages specific for bacterial pathogens are abundant in environments like soil and sewage (Kutter & Sulakvelidze, 2005). Phage biocontrol of pathogens including Salmonella, Campylobacter and Listeria in the food industry is recognized in the US and Europe (Burrowes et al., 2011; Goodridge & Bisha,

2011; Kropinski, 2006) and may be particularly useful to control Cronobacter because of its intrinsic antibiotic resistance (Lai, 2001).

Determining host range, lysis strength, and TEM imaging are important steps to identify, differentiate and characterize a phage before its possible application to control food-borne pathogens (Hagens & Loessner, 2010). Although isolation, characterization and determination of the host range are time-consuming and laborious, they are critical

steps of the process to identify phages suitable for use as biocontrol agents. The objective of this part of the study was to isolate and characterize lytic phages against Cronobacter spp.

2.3. MATERIALS AND METHODS

2.3.1. Bacteria and Bacteriophages

Twenty-one Cronobacter strains (including 14 C. sakazakii strains) used in this study were obtained from the Canadian Research Institute for Food Safety (CRIFS)

Culture Collection at the University of Guelph, Franco Pagotto (Public Health Agency of

Canada, Ottawa, ON) and Roger Stephan (Institute for Food Safety and Hygiene, Zurich,

Switzerland). Cronobacter muytjensii 51329 was purchased from the ATCC (Manassas,

VA, USA) (Table 2.1). To grow Cronobacter strains and to isolate and propagate the phages, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), and Tryptose Top Agarose

(TSB + 0.5% agarose) (Fisher Scientific, Ottawa, ON) were used. Pure cultures were obtained from frozen stocks kept at -80°C, and maintained at 4°C on TSA. To maintain cell viability, cultures were re-streaked biweekly.

Table 2.1. Cronobacter strains used for isolation and propagation of phages.

Cronobacter strain Identification number Origin of Isolation 1 C. sakazakii HPB 2855 Clinical Powdered Infant Formula 2 C. sakazakii HPB 2870 - Company A 3 C. sakazakii HPB 2871 Food Powdered Infant Formula 4 C. sakazakii HPB 2876 - Company B 5 C. sakazakii HPB 3253 Environmental (hospital) 6 C. malonaticus HPB 3263 Clinical, stool 7 C. sakazakii HPB 3199 Environment of food manufacturing facility ATCC 51329 8 C. muytjensii Unknown (Type Strain) 9 C. sakazakii HPB 3290 Clinical, CSF 10 C. dublinensis HPB 3169 Medicinal herb 11 C. malonaticus HPB 3267 Powdered Infant Formula 12 C. muytjensii HPB 3270 Powdered Infant Formula 13 C. universalis HPB 3287 Powdered Infant Formula 14 C. sakazakii 130/3 Fruit Powder 15 C. sakazakii 236/04 Fruit Powder 16 C. sakazakii 324/04 Fruit Powder 17 C. sakazakii 354/03 Fruit Powder 18 C. sakazakii 974/03 Fruit Powder 19 C. sakazakii 1084/04 Fruit Powder 20 C. sakazakii 1103/03 Fruit Powder 21 C. malonaticus 1154/04 Fruit Powder

2.3.2. Isolation of Bacteriophages

Lytic bacteriophages against C. sakazakii were isolated from 23 samples of untreated sewage (from a local wastewater treatment plant, Guelph, ON, Canada), fresh cattle manure and soil (Dairy Barn, University of Guelph, Guelph), and water (Fraser

River, False Creek, Burrard Inlet and Strait of Georgia; Vancouver, BC, Canada) using the method previously described (Jamalludeen et al., 2007). Samples were centrifuged(5000 × g for 15 min) and the supernatant was filtered through a 0.45 µm filter (Corning Inc., NY, USA). Twenty milliliters of Cronobacter culture incubated for 8 h at 37ºC in Tryptic Soy Broth were added to 200 ml of filtered supernatant, together with 20 ml of bacteriophage broth and 20 ml of TSB containing 2 mM CaCl2. This mixture was incubated at 37ºC overnight, centrifuged at 5000 × g for 15 min at 4ºC

(Beckman Avanti J-20 XPI, Beckman Coulter Inc., Mississauga, ON, Canada) and the supernatant was treated by adding 1% v/v chloroform, and stored at 4°C. Bacteriophage broth was prepared as previously described (Jamalludeen et al., 2007) by adding 100 g peptone (Difco Laboratories, Detroit, MI, USA), 30 g beef extract (Difco), 50 g yeast extract (Fisher), 25 g NaCl (Fisher), and 80 g potassium dihydrogen phosphate (BDH

Laboratory, Toronto, ON, Canada) to one liter of distilled water.

For isolation of the phages, the overlay method (Kropinski et al., 2009) was carried out. Briefly, 100 µl of phage isolate and an equal volume of Cronobacter 6-hour culture were added to 4 ml of molten (47ºC) top agarose (5 g low-melt agarose/l TSB containing 2 mM CaCl2) and mixed. The mixture was poured immediately onto TSA plates and allowed to solidify for 15 min on a level surface, before incubation for ~16 h at

30°C. After incubation, the plates were examined for the presence of plaques, and single plaques were picked by using sterile plastic Pasteur pipettes, and each plaque was eluted in 1 ml of SM buffer (NaCl 100 mM, 50 mM Tris-HCl, pH 7.5, 0.002% (w/v) gelatin, 8 mM MgSO4•7H2O) containing 1 drop of chloroform. The tubes containing plaques and

SM buffer were held at room temperature for 4 h to let the phages diffuse out of the top agarose, before being stored at 4ºC.

2.3.3. Purification of Bacteriophages

The purification of isolated phages was conducted using ten-fold serial dilutions of phage isolates and the top agar overlay method (Kropinski et al., 2009) as described above. Single separate plaques of varying sizes and different morphology from different

Cronobacter strains, were picked from the overlay plates and placed separately in 1 ml

SM buffer containing one drop of chloroform, and left for 4 h at room temperature to allow the phages to diffuse into the buffer before being refrigerated. This process

(dilution, overlay plating, incubation, and picking single separate plaques) was repeated three times for each phage suspension to achieve purified phages. The phage suspensions were kept at 4°C until propagation.

2.3.4. Propagation of Bacteriophages

To prepare phage stocks, selected phages were propagated by the top agar overlay method (Kropinski et al., 2009) as described above. Ten plates were prepared for each phage. After incubation, 3 ml of SM buffer were added and kept at 4°C overnight before scraping off the top layer of the top agarose from all plates using sterile glass rods. For each phage, all the scraped top agarose layers were transferred into 50 ml tubes, and the remaining agarose and phages were washed off from TSA plates by adding 1 ml of SM buffer to each plate, which was added into the same 50 ml tube. The tubes were centrifuged at 6000 × g for 20 min at 4oC, and the supernatant was transferred to another tube and treated with chloroform. For each phage stock, the titre was determined by preparing 10-fold serial dilutions and tested using the overlay method described previously. The phage lysate was refrigerated at 4°C.

2.3.5. Host Range Determination

The host range of all the isolated phages using the available Cronobacter strains

(Canadian Research Institute for Food Safety (CRIFS) Culture Collection, University of

Guelph) was determined by spot test. A 6-8 h culture of each Cronobacter strain was

inoculated on TSA plates with sterile cotton swabs and allowed to dry. Three drops of 20

µl of each phage sample were spotted onto the plate and allowed to dry. Plates were examined for lysis after 16 h incubation at 30ºC, for the presence of plaques. The results were recorded as 3 (very clear zone of complete lysis on the bacterial lawn), 2

(clear/turbid zone of lysis), 1 (turbid lysis) and 0 (no lysis). In order to reach the goal of this project, which is to control Cronobacter in infant formula, the phages with the strongest lytic activity (score 3 in lysis) and the widest host range were selected. To do so, phages were given a lytic score using the sum of the numbers that were designated to their plaque clarity on all Cronobacter strains.

2.3.6. Transmission Electron Microscopy (TEM)

Transmission Electron Microscopy (TEM) was applied to examine the morphology of the isolated phages. To prepare the samples for TEM, 1 ml of phage stocks with high titre was centrifuged at 16000 × g for 1 h at 4°C (Beckman J-20 centrifuge, Beckman Coulter Inc., Mississauga, ON, Canada), and the pellet was washed twice using SM buffer. The supernatant was discarded and the pellet was gently resuspended in 30 µl of SM buffer. Five microliters of these phage preparations were applied onto 300-mesh copper grids coated with formvar and allowed to stand for 2 min at room temperature. The extra liquid was drawn off by blotting with filter paper and the remaining phages were negatively stained by 2% uranyl acetate (UA) for 30s and then the excess liquid was again removed with filter paper. The stained phage samples were examined in a LEO 912AB electron microscope (Energy filtered TEM, EFTEM, LEO

912ab model operated at 100 kv, Zeiss, Germany). Also, selected phages were sent to

Laval University for further electron microscopic examination of 2% UA and 1% phosphotungstic acid (PT) stained phages (Ackermann, 2009a).

2.3.7. Cross Infectivity

To determine the ability of the selected phages to infect different species other than Cronobacter, 20 µl of each phage were spotted over a lawn of different bacterial species among the Enterobacteriaceae (Table 2.2). Also, the infectivity of the phages on two strains of Lactobacillus were tested to find their effect on possible probiotics present in infant formula, which are added to help develop healthy gut microbiota, inhibit pathogens by competitive exclusion, and produce anti-microbial compounds (Collado,

Isolauri, & Salminen, 2008; Lara-Villoslada et al., 2007). After incubation at 30°C for 16 h, the development of plaques in the plates was examined. Phages were tested in triplicate. Lactobacillus strains were incubated under anaerobic conditions.

Table 2.2. Bacterial strains used to examine the cross infectivity of the selected phages.

Genus Species/Serovar Identification number 1 Enterobacter helveticus 1129/04 2 Enterobacter helveticus 1159/04 3 Enterobacter cloacae 13047 (ATCC) 4 Enterobacter cloacae 35030 (ATCC) 5 Escherichia coli K21 6 Escherichia coli 11229 (ATCC) 7 Shigella flexneri C909 8 Klebsiella pneumoniae C1191 9 Hafnia alvei C1194 10 Salmonella enterica, subsp. enterica, C435 serovar Typhimurium 11 Serratia odorifera C1168 12 Yersinia enterocolitica LJH408 13 Lactobacillus paracasei C1268 14 Lactobacillus rhamnosus C1017

2.4. RESULTS

2.4.1. Isolation of Bacteriophages

Two hundred and fifty two phages with different plaque morphologies and variably active against 21 Cronobacter strains (Table 2.3 in Appendix 1) were isolated from the collected environmental samples (untreated sewage, fresh cattle manure, soil and river water). All isolated phages were subjected to host range determination using the spot test method, although it was expected that some of these phages were probably identical based on very similar plaque morphologies.

2.4.2. Host Range

The lysates of the isolated Cronobacter phages were tested against 21 strains of

Cronobacter (including 14 C. sakazakii). The results were assigned values from 0 to 3, based on the clearness of the plaque (0: no lysis; 3: very clear zone of complete lysis).

Table 2.3 (Appendix 1) shows the host range of all 252 isolated phages. The sum of lytic strength of the phage isolate against 21 Cronobacter strains was calculated and recorded as the lytic score for each phage. The lytic scores vary from 1 to 57. Phage GAP31 was able to inhibit completely the growth of 18 out of 21 (85.7%) Cronobacter strains tested, and showed the broadest host range and strongest lytic activity. Only one strain (C. universalis 3287) was not infected by phage GAP31. On the other hand, thirty-four phage isolates exhibited a narrow host range with a lytic score of 1, which indicates that these phages could just lyse one Cronobacter strain weakly, and were unable to infect the other

20 strains. In addition, the sum of numbers of the lytic activity of all phage isolates

against each Cronobacter strain (sensitivity score) shows the sensitivity of that strain to the phages (Table 2.3). Cronobacter muytjensii 3270 gave the highest lytic score (361) and therefore is the most susceptible bacterial strain to the isolated phages, and C. universalis 3287 is the least susceptible bacterial host with a score of 35.

Based on the total lytic score of the phages against all Cronobacter strains and the lytic strength of each phage against individual bacteria, GAP31 along with ten other phages with the broadest host range and the strongest lytic activity were selected for further characterization (Table 2.4). The selected phages were named according to the recommendations for phage nomenclature by Kropinski et al. (2009). Therefore, vB

(virus of bacteria), the first letter of the genus of the bacterial host (in capital letters), the first two letters of species of the bacterial host (in lower-case), the first letter of the phage family (in capitals), the first letters of the authors’ last names and isolation number comprise the name of the phage. The complete names of eleven selected phages, and the bacterial host that was used for their isolation and propagation are shown in Table 2.5.

Table 2.4. The host range of 11 selected Cronobacter phages against 21 Cronobacter strains based on the spot test (16 h incubation at 30ºC). The results were recorded as 3 (very clear zone of complete lysis on the bacterial lawn), 2 (clear/turbid zone of lysis), 1 (turbid lysis) and 0 (no lysis). C C. sakazakii C. sakazakii C. sakazakii C. sakazakii malonaticus C. C. sakazakii C. C. sakazakii C. malonaticus C. C. universalis C. C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii malonaticus C. . sakazakii

Bacteria muytjensii dublinensis muytjensii

Lytic Score

2855 2870 2871 2876 3253 3199 3290 130/3 236/04 324/04 974/03 1103/03

354/03 1084/04

51329 3270

3287 3169

3263 3267 1154/04

Phage Isolates GAP31 3 3 3 3 3 3 3 3 3 3 3 3 0 3 2 3 3 3 3 3 1 57 GAP32 3 2 0 3 0 3 2 2 3 1 3 3 0 3 2 3 1 3 3 3 0 43 GAP33 3 3 3 3 0 3 1 3 3 3 2 3 0 3 3 3 3 3 3 3 1 52 GAP52 3 2 1 3 0 3 1 3 2 3 3 3 1 3 0 0 3 3 3 3 0 43 GAP72 3 3 2 3 2 2 2 3 3 3 3 2 1 3 3 3 3 3 3 3 0 53 GAP136 3 3 2 2 0 3 0 3 3 3 2 2 0 1 3 3 3 3 3 1 1 44 GAP161 3 2 2 2 3 3 0 3 3 3 3 3 0 0 0 0 3 3 3 0 0 39 GAP184 0 3 3 3 0 3 0 3 3 3 3 3 1 3 1 1 1 2 2 3 1 42 GAP186 0 3 3 3 0 3 0 3 3 3 3 3 1 3 3 3 2 2 3 3 1 48 GAP188 0 3 3 3 0 3 3 3 3 3 3 3 0 3 1 1 3 3 3 3 1 48 GAP227 0 2 3 3 0 3 3 3 3 3 3 3 0 3 0 0 3 3 3 3 0 44

Table 2.5. Selected bacteriophages and the susceptible bacterial hosts used for propagation.

CRIFS culture collection Phage Bacterial Host number of the host vB_CsaM_GAP31 Cronobacter muytjensii 51329 vB_CsaM_GAP32 Cronobacter sakazakii 3290 vB_CsaM_GAP33 Cronobacter dublinensis 3169 vB_CsaP_GAP52 Cronobacter sakazakii 3199 vB_CsaM_GAP72 Cronobacter muytjensii 51329 vB_CsaM_GAP136 Cronobacter sakazakii 1084/04 vB_CsaM_GAP161 Cronobacter sakazakii 3253 vB_CsaP_GAP184 Cronobacter dublinensis 3169 vB_CsaP_GAP186 Cronobacter malonaticus 3267 vB_CsaP_GAP188 Cronobacter sakazakii 130/3 vB_CsaP_GAP227 Cronobacter malonaticus 3267

2.4.3. Characterization of the Selected Phages

2.4.3.1. Morphology

The electron micrographs of negatively-stained preparations of the eleven selected

phages revealed that they belong to Myoviridae and Podoviridae families. Figure 2.1 shows that phages GAP31, GAP32, GAP33, GAP72, GAP136, and GAP161 all have icosahedral heads and contractile tails, and therefore they are myoviruses (Ackermann,

2005). Isolated podoviruses include phages GAP52, GAP184, GAP186, GAP188, and

GAP227 which possess icosahedral heads and short tails (Figure 2.2). The heads of

GAP161, GAP52, and GAP188 are elongated. Dimensions, head shape, and related

phages for eleven selected bacteriophages are shown in Table 2.6. Among the selected

myoviruses, phages GAP31, GAP33, and GAP72 morphologically were similar to Felix

O1 a broad host range phage of Salmonella (Ackermann & DuBow, 1987; Ackermann,

1999). Phage species Felix O1 (also named O1) is able to infect 96-99.5% of Salmonella

serovars (Whichard, Sriranganathan, & Pierson, 2003), and it has been used as an agent

to identify Salmonella (Hirsh & Martin, 1983). Phages GAP32 and GAP136 possessed

the same morphotype, which is related to phage 121 of Proteus vulgaris (Nacesco,

Constantinesco, & Petrovici, 1969), and GAP161 is similar to T4 phage. Among the podoviruses, GAP52 and GAP188 phages are in the category of C3-morphotype phages that are extremely rare and were among the first viruses isolated and examined under the

electron microscope (Moazamie, Ackermann, & Murthy, 1979). These two phages are

morphologically related to Salmonella Newport phage 7-11 (Ackermann, Petrow, &

Kasatiya, 1974), and phages GAP184, GAP186, and GAP227 are morphologically T7-

like. The length of the heads ranged from 52 to 139 nm, and the tail dimensions were

from 10 to 129 nm long (Table 2.6).

Figure 2.1. Transmission electron micrographs of negatively stained (UA) phages GAP31, GAP32, GAP33, GAP72, GAP136, and GAP161 that belong to Myoviridae family.

Figure 2.2. Transmission electron micrographs of negatively stained (UA) of phages GAP52, GAP184, GAP186, GAP188, and GAP227 that belong to Podoviridae family.

Table 2.6. Dimensions, head shape, and related phages for the selected bacteriophages.

Head Tail Dimensions Phage Similar To Shape Length Width Length Width vB_CsaM_GAP31 Icosahedral 83 83 107 19 Felix O1 vB_CsaM_GAP32 Icosahedral 113 113 118 23 121 vB_CsaM_GAP33 Icosahedral 76 76 95 19 Felix O1 vB_CsaP_GAP52 Elongated 139 47 12 10 7-11 cylindrical vB_CsaM_GAP72 Icosahedral 78 78 129 24 Felix O1 vB_CsaM_GAP136 Icosahedral 119 119 109 24 121 vB_CsaM_GAP161 Prolate 110 74 113 17 T4 Icosahedral vB_CsaP_GAP184 Icosahedral 52 52 11 9 T7 vB_CsaP_GAP186 Icosahedral 55 55 11 9 T7 vB_CsaP_GAP188 Elongated 114 43 12 10 7-11 cylindrical vB_CsaP_GAP227 Icosahedral 60 60 10 9 T7

2.4.3.2. Cross infectivity

The infectivity of isolated phages was tested against 12 strains in the family

Enterobacteriaceae and two strains of Lactobacillus (Table 2.7). None of the phages was able to infect the Lactobacillus strains. GAP31 and GAP136 phages did not infect any of the Enterobacteriaceae, and GAP32, GAP33, and GAP184 phages infected only one of the strains incompletely. On the other hand, GAP186, GAP188, and GAP227 phages were able to infect at least 50% of bacterial strains tested although only in a few cases was the lysis complete. Enterobacter cloacae (13047) appeared to be the most susceptible bacterium with 7 out of the 11 selected phages being able to infect it.

Table 2.7. Cross infectivity of the selected phages to other bacterial species in Enterobacteriaceae and 2 strains of Lactobacillus. Enterobacter helveticus Enterobacter helveticus Enterobacter cloacae 13047 Enterobacter cloacae 35030 K21 coli Escherchia Escherchia coli 11229 C909 flexneri Shigella Klebsie Hafnia alvei C1194 C435 typhimurium Salmonella Serratia odorifera C1168 LJH408 enterocolitica Yersinia Lactobacillus paracasei C1268 Lactobacillus rhamnosusC1017 Bacteria

lla pneumoniae C1191

1129/04 1159/04

Phage

Isolate GAP31 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GAP32 0 0 0 0 0 0 0 0 0 0 1 0 0 0 GAP33 0 0 1 0 0 0 0 0 0 0 0 0 0 0 GAP52 0 0 2 0 0 0 0 0 0 3 0 0 0 0 GAP72 0 0 3 0 0 0 0 0 0 2 0 1 0 0 GAP136 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GAP161 1 2 2 0 0 0 0 0 0 0 1 2 0 0 GAP184 1 0 0 0 0 0 0 0 0 0 0 0 0 0 GAP186 0 0 1 2 3 1 0 2 0 2 1 1 0 0 GAP188 1 1 2 0 0 1 1 0 0 3 1 0 0 0 GAP227 1 1 1 1 0 0 1 0 1 0 1 0 0 0

2.5. DISCUSSION

Phages have emerged as a new biotechnological method to control bacterial contamination in food (Hagens & Loessner, 2007). To use phages as promising

biocontrol agents against food-borne pathogens, the first step is to isolate and

characterize the most promising phages. To be a suitable candidate for food safety

applications, a phage should possess strong lytic activity and a broad host range (Greer,

2005).

In the current investigation, two hundred and fifty two phages with varying plaque morphologies and sizes have been isolated from different environmental samples against

Cronobacter spp. The isolation of such a large number of phages supports the theory that phages are the most abundant and diverse microorganisms on earth, and they can be isolated from environments where their bacterial host can grow (Kutter & Sulakvelidze,

2005). The environmental sources of our Cronobacter phage isolates were similar to those previously investigated for the successful isolation of Cronobacter spp. (Gurtler,

Kornacki, & Beuchat, 2005; Iversen & Forsythe, 2003; Kandhai et al., 2004). All the plaques harvested from each individual host were purified and tested for host range by the spot test method. The isolated phages revealed different abilities to infect the tested bacterial strains, which were signified by different visual degrees of lysis in the spot test.

Although some of the isolated phages were highly specific and infected only one or a limited number of Cronobacter strains tested, many other phages were shown to have a broad host range. The susceptibility of tested bacterial strains to the various isolated phages differed, which may be due to lack of the specific receptor for the phages to attach or the development of resistance mechanisms [e.g., adsorption blocking (altering the

membrane receptors), abortive infection, modifying the restriction endonuclease system]

(Petty et al., 2007). Cronobacter universalis (3287) was the least susceptible bacterial

host, suggesting that this bacterium is less related to the other Cronobacter spp. tested.

This is in agreement with the results of a study that showed that the relatedness of C.

universalis to other species of the genus is from 45.9% (C. dublinensis) to 60.1% (C.

malonaticus) and it has 55.5% homology with C. sakazakii based on DNA-DNA

hybridization (Joseph et al., 2012a).

Because twenty-one bacterial strains were used in the enrichment step of the isolation process, it is probable that some of the phage isolates are identical, as they could possibly infect more than one of the Cronobacter strains used, and thus they could be isolated from more than one overlay. However, the eleven selected phages did not have similar host range patterns, and, therefore, they were presumed to be different from one another.

To select the most effective phages for biocontrol of Cronobacters, eleven phages were chosen for further study based on their lytic activity and broad host range.

Possessing a broad host range is one of the most critical characteristics of good lytic

phage candidates for biocontrol. A well-known example is phage Felix O1 that is able to lyse 96-99.5% of Salmonellae (Whichard, Sriranganathan, & Pierson, 2003). Because in some species of bacterial pathogens there are many sub-types, all of which must be controlled, choosing phages with a narrow host range is problematic for biocontrol purposes. However, applying phage cocktails could overcome this limitation of narrow host range (McIntyre et al., 2007).

The morphology of the selected phages, observed in electron micrographs, showed that among eleven selected phages six belong to the family of Myoviridae and five were in the family of Podoviridae. A survey in 2007 revealed that 96% of 5,568 phages examined since 1959 were tailed and belonged to one of three families of

Myoviridae, Siphoviridae, or Podoviridae (Ackermann & Prangishvili, 2012), and of these phages 24.8% and 14.2% were Myoviridae and Podoviridae, respectively.

Likewise, in our study more myoviruses were found among the selected phages than podoviruses. Morphologically, these selected phages were related to five phage species:

Felix O1, 121, T4, 7-11, and T7. Phages GAP31, GAP33, and GAP72 are morphologically similar to phage Felix O1. The genome of phage Felix O1 has not been completely sequenced (A. M. Kropinski, personal communication). Therefore, restriction endonuclease and PCR analysis of the DNA of phages GAP31, GAP33, and GAP72 could be conducted to confirm their relation to phage Felix O1.

Phages GAP32 and GAP136 are similar to phage 121; possessing a large head and relatively short tail, and the genome of no 121-like phage has been sequenced (H-W.

Ackermann, A. M. Kropinski, personal communication).

Moreover, phages GAP52 and GAP188 have elongated cylindrical heads and are similar to Salmonella Newport phage 7-11, which belongs to the C3 morphotype that is extremely rare and only constitutes 0.5% of phages examined by electron microscopy

(Ackermann, 2001). Enterobacter sakazakii phage C2, Erwinia herbicola phages E3 and

E16P (Grimont, Grimont, & du Pasquier, 1978; Grimont & Grimont, 1981a,b), and coliphage NJ01 (a new phiEco32-like phage) (Li et al., 2012) are some other C3 phages that have been previousely isolated and characterized.

Among the selected phages, five phages were considered as ambivalent as beside

Cronobacter (their original host bacteria), they were able to strongly infect bacteria from at least two other genera. For example, phages GAP186 and GAP188 could infect bacterial strains from 6 different genera, including Enterobacter cloacae, Escherichia coli, Salmonella Typhimurium, and Serratia odorifera. The wide host range of these two phages could be due to their ability to attach to possible receptor(s) that is common on cell surfaces [e.g., outer membrane proteins (OMP), lipopolysaccharide (LPS), and/or outer core oligosaccharide (OS)] of these suseptible bacteria (Goodridge, Gallaccio, &

Griffiths, 2003). These ambivalent phages with their broad host range would have the potential to be used for biocontrol of not only Cronobacters but also strains from another genus. Previously, ambivalent phages have been reported; phages AR1 and LG1 of E. coli could lyse Shigella dysenteriae, Proteus mirabilis, and two Salmonella strains

(Goodridge, Gallaccio, & Griffiths, 2003). A number of phages (including phages U2 and

LB related to T-even phages of E. coli) were also able to infect both E. coli K12 and some Salmonella strains (Krylov et al., 2006). However, applying ambivalent phages to infant formula used for feeding infants, whose gut microflora have not fully developed is not recommended. Instead, a cocktail of more host specific (within Cronobacter spp.) phages could be more suitable to control Cronobacter contamination in infant formulae.

Previous reports have demonstrated the efficacy of using phage cocktails to produce significant reductions in levels of food-borne pathogens, including Listeria monocytogenes, Salmonella and E. coli O157:H7 (Fiorentin, Vieira, & Barioni Junior,

2005; Leverentz et al., 2004; O'Flynn et al., 2004).

Interestingly, none of the selected phages were able to infect Lactobacillus

rhamnosus and Lactobacillus paracasei in the cross infectivity experiment. In a study on

the effects of the probiotics on Cronobacter, it was shown that specific probiotics,

especially L. rhamnosus and L. paracasei, reduced adhesion of Cronobacter to mucosal cells, and it was suggested that specific probiotics in infant formulae could reduce

Cronobacter contamination and its infection risk (Collado, Isolauri, & Salminen, 2008).

Therefore, application of our selected phages in infant formulae would not affect the probiotics that are potentially present in these food products.

In conclusion, many lytic phages have been isolated and eleven of them with the broadest host range and strongest lytic activity, which could potentially be considered as good candidates as biocontrol agents in food, have been characterized. Two of these phages are morphologically similar to phage 121 (the genome of which has not been sequenced), and the other two phages are from a very rare morphotype category. Based on the high intra-species specificity and interesting and rare morphology, five phages

(GAP31, GAP32, GAP52, GAP161, and GAP227) have been selected for complete genome sequence and proteomic analysis, which can assure the safety of using these phages in food. The genomic and proteomic analysis is described in the next two chapters. These selected Cronobacter phages have various host range profiles, therefore, using cocktails of these phages could enhance the scope of their application against a broader range of Cronobacters in infant formulae. Applying the phage cocktail to inhibit

Cronobacters in broth medium and infant formula is presented in chapter 6.

Chapter 3: SEQUENCING AND GENOME ANALYSIS OF

CRONOBACTER PHAGE GAP32

3.1. ABSTRACT

Cronobacter sakazakii is a pathogen found in milk-based infant formula that

causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage

vB_CsaM_GAP32 (GAP32), which we have found to possess the second largest phage

genome (358,663 bp) sequenced to date. A total of 571 genes including 545 open reading

frames and 26 tRNA were identified, more genes than the smallest bacterium

Mycoplasma genitalium G37. BLASTP and HHpred searches and proteomic analysis

reveal that only 20.9% of the putative proteins have defined functions. Some unique

proteins encoded by GAP32 include DNA polymerase III alpha and epsilon subunits,

toxic ion resistance protein, peptidyl-tRNA hydrolase and RNA polymerase. CoreGenes

analysis indicates GAP32 shows a peripheral relationship to the T4likevirus, but it lacked

13 proteins of the core proteome of that virus. A detailed proteomic analysis identified

49 structural proteins. These results suggest that the unusual and interesting phage

GAP32 belongs to a new genus of “Gap32likeviruses”.

3.2. INTRODUCTION

Cronobacter sakazakii is an opportunistic pathogen found in both the environment and a variety of foods (Gurtler, Kornacki, & Beuchat, 2005). Contaminated

milk-based powdered infant formulae have been the source of sporadic cases and

outbreaks of Cronobacter infections that cause sepsis, necrotizing enterocolitis, brain

abscess and meningitis in neonates and infants (Biering et al., 1989; Bowen & Braden,

2006; Centers for Disease Control and Prevention (CDC), 2002; Clark et al., 1990;

Gurtler, Kornacki, & Beuchat, 2005; Jarvis, 2005; Muytjens et al., 1983; Muytjens &

Kollee, 1990; Noriega et al., 1990). The mortality of the infections can reach 80%, and

antibiotics have no dramatic affect on clinical results (Drudy et al., 2006; Lai, 2001;

Norberg et al., 2012; Ray et al., 2007).

Bacteriophages (phages), viruses that attack bacteria, have been applied as antibacterial agents due to their high specificity and effectiveness in killing bacterial pathogens without affecting the host commensal microbiota. Phage biocontrol of many pathogens such as Salmonella, Campylobacter and Listeria in the food industry is recognized in the USA and Europe (Burrowes et al., 2011; Goodridge & Bisha, 2011;

Kropinski, 2006) and could be particularly relevant to the control of Cronobacter because of its intrinsic antibiotic resistance (Lai, 2001).

The objective of this study was to characterize a newly isolated phage for safe therapeutic application against C. sakazakii that could be used to reduce its prevalence

during processing of infant formula and in hospitals. The genomic sequencing helps

predict the likely reproductive behavior of the phage, and determine whether the phage

has lysogenic capability, an essential and important consideration for a phage intended for use in prophylaxis and therapy.

3.3. MATERIALS AND METHODS

3.3.1. Bacteria and Bacteriophage

Twenty-one Cronobacter strains (including 14 C. sakazakii strains) were used for

isolation, propagation, and determination of host range of phages, and all the strains were

grown in TSB and TSA as previously described in Chapter 2 section 3 (2.3).

Lytic bacteriophage vB_CsaM_GAP32 (GAP32) against Cronobacter spp. was

isolated from samples of untreated sewage from the Guelph Wastewater Treatment Plant

(Guelph, ON, Canada), using the method described by Kropinski et al. (2009) and

explained in Chapter 2 (2.3). After purification, and preparation of stocks, the host range

and cross infectivity were determined by spot test. Selected phages were examined by

electron microscopy of negatively stained preparations (2% uranyl acetate). All these

methods have been described in detail in Chapter 2 section 3.

3.3.2. Phage Purification, DNA Isolation and Sequencing

From the phages that were previously isolated and characterized (Chapter 2),

phage GAP32 was selected (based on host range, strong lytic activity, and electron

micrographs) for DNA sequencing and proteomic analysis. The phage was propagated to

high titre with the method previously described in Chapter 2, and it was highly purified

by a method described by Sambrook and Russell (Sambrook & Russell, 2001) with some

modifications. Briefly, the bacterial debris was removed from the crude phage lysate by

centrifugation at 14000 × g for 20 min at 4ºC (Beckman Coulter Inc., Mississauga, ON,

Canada). Pancreatic DNase 1 and RNase, were added to the supernatant to a final

concentration of 10 μg/ml each (Sigma-Aldrich Canada Ltd., Oakville, ON), and incubated at room temperature for 30 min to eliminate contaminating bacterial nucleic acids present in the supernatant. Sodium chloride was added to a final concentration of 1

M, and then the mixture was stirred on ice for 1 h, and the supernatant was collected after centrifugation at 14000 × g for 15 min at 4ºC. The phage particles were precipitated in the presence of 10% w/v (final concentration) PEG-8000 with stirring at 4ºC overnight.

The precipitated phage particles were recovered by centrifugation at 14000 × g for 20

min at 4ºC, and then resuspending the pellet in SM buffer. To purify the phage from the suspension, separation on a self-generating cesium chloride (CsCl) gradient (1.5 g/ml

CsCl, run at 21000 × g at 4ºC for 24 h) was used in a fixed angle, Beckman SW 90Ti rotor. After purification by a second passage through a CsCl gradient for another 24 hours, the phage was dialyzed against two changes of SM buffer (2 l each), using dialysis cassettes (3500 MWCO; Thermo Scientific, Fisher Scientific, Mississauga, ON). The highly purified, high-titer phage was stored at 4ºC prior to being used in the next step.

The DNA of phage GAP32 was extracted and purified by Midi Lambda DNA purification kit (Qiagen Midi25; Mississauga, ON, Canada), and the genomic sequence was determined using 454 pyrosequencing technology (McGill University and Génome

Québec Innovation Centre; Montreal, QC, Canada).

3.3.3. Bioinformatic Analysis

The genome was annotated with the help of Prof. Andrew M. Kropinski

(Laboratory for Foodborne Zoonoses, Guelph, ON) using a variety of online tools

(http://molbiol-tools.ca). The genome was annotated by Rapid Annotations using

Subsystems Technology (myRAST) with gene calls verified using Kodon (Applied

Maths, Austin, TX). Transfer RNAs were predicted using tRNAscan-SE

(http://lowelab.ucsc.edu/tRNAscan-SE/). For each protein the number of amino acids,

molecular weight and the isoelectric point were calculated by Batch MW and pI Finder

(http://greengene.uml.edu/programs/FindMW.html). Homologs were identified using

BatchBLAST (http://greengene.uml.edu/programs/NCBI_Blast.html). To predict protein

motifs the Transmembrane Hidden Markov Model (TMHMM) (http://www.cbs.dtu.dk

/services/TMHMM-2.0/), Phobius (http://phobius.sbc.su.se), and Pfam

(http://pfam.sanger.ac.uk/) were used. HHpred was used to detect remote protein

homology and predict its structure (Soding, Biegert, & Lupas, 2005) in collaboration with

Dr. John H.E. Nash (Laboratory for Foodborne Zoonoses, Guelph, ON). In addition, to

detect potential bacterial toxins the complete proteome of phage GAP32 was screened

against a proprietary database of 83 bacterial toxin proteins (including those from

Bacillus spp., Bordetella, Clostridium spp., Enterobacteriaceae, Listeria, Pseudomonas,

Staphylococcus, Streptococcus, and Vibrio) (Carter et al., 2012) using the BLASTP

feature of BioEdit (Tippmann, 2004).

3.3.4. Proteomic Analysis

Cesium chloride purified phage GAP32 was sent to the Laboratory of Gene

Technology (Katholieke Universiteit Leuven, Leuven, Belgium) for proteomic analysis.

Extraction of phage proteins from the high-titer GAP32 stock solution (>1011 PFU/ml)

was performed by methanol/chloroform extraction (1:1:0.75, v/v/v). The resulting protein

pellet was dissolved in loading buffer (1% SDS, 6% sucrose, 100 mM dithiothreitol, 10

mM Tris pH 6.8, 0.0625% bromophenol blue) (Moak and Molineux, 2004), loaded onto a

12% SDS-PAGE gel and subjected to gel electrophoresis. Subsequent staining with

Simply BlueTM Safe Stain (Invitrogen Ltd, Paisley, UK) revealed the GAP32 structural

proteome. Gel fragments were cut out, subjected to trypsinization (Shevchenko, 1996)

and analyzed using tandem electrospray ionization-mass spectrometry (ESI-MS/MS) on a

LCQ Classic (ThermoFinnigan) equipped with a nano-LC column switching system as

described by Lavigne et al. (2006).

3.3.5. Genome Sequence

The complete genome sequence of Cronobacter phage vB_CsaM_GAP32 is

available in GenBank under the accession number JN882285.

3.4. RESULTS

Phage GAP32 was able to lyse 85.7% of the Cronobacter sakazakii strains tested,

and it belongs to the Myoviridae family It possesses a very large icosahedral head (113

nm), and a comparatively short (118 × 23 nm) contractile tail as previously described

(Chapter 2). Phage GAP32 is related morphologically to phage 121 of Proteus vulgaris

(Nacesco, Constantinesco, & Petrovici, 1969). The genome of phage GAP32 consists of a

double-stranded DNA, and the complete DNA sequence analysis shows a giant genome

of 358,663 bp with a G+C content of 35.5%. The genome of GAP32 consists of 571

genes including 545 open reading frames (ORFs) and 26 tRNA genes. One hundred and

fourteen (20.9%) of the putative proteins of GAP32 were designated as possessing

defined function since they exhibited significant matches (E-value < l0-5) to known

proteins by BLASTP searches (88; 16.1%), HHpred (23; 4.2%), and proteomic analysis

(3; 0.6%). Furthermore, the proteome of GAP32 contains 43 (7.9%) proteins with

predicted transmembrane domains. Among the 81 (14.9%) conserved hypothetical

proteins, we have tentatively identified a cytidyltransferase, a co-chaperonin GroES

homolog, tRNAHis guanylyltransferase, acyl carrier protein, sigma 54 modulation

protein/ribosomal protein S30EA, DNA polymerase III alpha and epsilon subunits,

translation initiation factor IF-3, toxic ion resistance protein, peptidyl-tRNA hydrolase

and RpoD subfamily RNA polymerase sigma-70 subunit. Table 3.1 (in Appendix 2) shows the general features of ORFs in the DNA of phage GAP32 and homology to

proteins in the databases.

A detailed proteomic analysis identified 49 structural proteins, which are

processed in a similar way to Pseudomonas myovirus 201φ2-1 (Thomas et al., 2010).

From these 49 proteins only 12 have functions with significant matches, and only 3 of

them could not be identified by homology and HHpred. Table 3.2. (in Appendix 2) shows

the proteomic and HHpred analyses for phage GAP32. The screening of the proteome of

phage GAP32 against bacterial toxin proteins using the BLASTP feature of BioEdit

(Tippmann, 2004) recorded no hits (E value < 0.003).

CoreGenes (Mahadevan, King, & Seto, 2009) analysis indicates that GAP32 bears a peripheral relationship to the T4likevirus (56 homologs to coliphage T4 equivalent to

20.1% homology). However, this virus lacks 13 proteins that Petrov et al. (2010) conclude are part of the core proteome of T4-like phages. These include baseplate, neck,

RegA, gp33, gp45, and gp59 homologs.

Figure 3.1. The map of the linear genome of phage vB_CsaM_GAP32 in comparison to phage vB_KleM- RaK2 created with CGview using TBLASX (Grant & Stothard, 2008).

Phage GAP32 appears more closely related to Klebsiella phage vB_KleM-RaK2

(345,809 bp, 534 ORFs; GenBank accession number: JQ513383) (Simoliunas et al.,

2012), since CoreGenes (Mahadevan, King, & Seto, 2009) analysis indicates that these two phages share 238 homologs, which is equivalent to 44.6% homology. Figure 3.1 shows the map of the linear genome of phage GAP32 in comparison to phage RaK2

created with CGview using TBLASX (Grant & Stothard, 2008). However, phage GAP32

contains 44 genes with known function that are not present in RaK2, including colanic

acid-degrading protein, co-chaperonin GroES, tyrosyl-tRNA synthetase, tRNAHis

guanylyltransferase, N-4 cytosine-specific methyltransferase, acyl carrier protein, RNA

ligase, sigma 54 modulation protein/ribosomal protein S30EA, ribonuclease H,

nicotinamide-nucleotide adenylyltransferase, nicotinamide mononucleotide transporter

PnuC, and peptidyl-tRNA hydrolase.

3.5. DISCUSSION

Phage GAP32 possesses a large head and relatively short tail and is

morphologically similar to Proteus vulgaris phage 121 (Nacesco, Constantinesco, &

Petrovici, 1969). Since no genome of a 121-like phage had been sequenced (H-W.

Ackermann, personal communication), the sequencing of phage GAP32 makes it a first.

The giant genome (358,663 bp) of phage GAP32 makes it the second largest

sequenced phage genome after Bacillus megaterium phage G, which possesses a genome

of 497,513 bp (GenBank accession number JN638751). The genome of GAP32

comprises 571 genes, which is more than the smallest known bacterium Mycoplasma

genitalium G37, the genome of which encodes approximately 470 proteins (Fraser et al.,

1995; Glass et al., 2006).

The genomic analyses show that GAP32 and RaK2 share 44.6% homologous

proteins, and therefore, they are related and should be considered part of a single genus using the criteria laid out by Lavigne et al. (2008). However, presence of 44 genes with known function in phage GAP32 and their absence in Klebsiella phage RaK2 showes these two phages are not very closely related. For instance, existence of colanic acid-

degrading protein in phage GAP32, which is involved in the adsorption of phage, could suggest that GAP32 has a different mechanism for adsorption than phage RaK2. These

differences between GAP32 and RaK2 suggest that they do not belong to the same genus.

Therefore, a recommendation will be made to the International Committee on Taxonomy

of Viruses (ICTV) that GAP32 be placed in a new genus: the “Gap32likevirus”.

Alternatively, because, in theory, phage 121 was the first of its type the genus should be

called the “12unalikevirus.”

The strong lytic activity of GAP32, coupled with the absence of bacterial virulence genes and lysogenic markers, suggest that this phage can be used to control C. sakazakii after being tested in infant formula and in animal models.

Chapter 4. SEQUENCING AND GENOME ANALYSIS OF

CRONOBACTER PHAGES GAP31, GAP52, GAP161, AND GAP227

4.1. ABSTRACT

Cronobacter sakazakii is an opportunistic pathogen often associated with milk-

based infant formula that predominantly infects immunocompromised individuals and

causes meningitis in infants. Consideration of bacteriophages (phages) as alternative

control agents has been rekindled because of their high specificity and ability to kill

bacterial pathogens without affecting the host commensal microbiota. We have fully

sequenced the genome of four C. sakazakii phages. The genome of myovirus

vB_CsaM_GAP31 (GAP31) consists of 147,940 bp and has a G+C content of 46.3%. A

total of 295 genes including 269 open reading frames and 26 tRNA were identified. This

phage is related to Salmonella phage PVP-SE1 and coliphages vB_EcoM-FV3 and rV5.

Phages vB_CsaP_GAP52 (GAP52) and vB_CsaP_GAP227 (GAP227) are the first C. sakazakii podoviruses whose genomes have been sequenced. The DNA of GAP52

consists of 76,631 bp and has a G+C content of 44.2%. A total of 117 genes including

115 open reading frames and 2 tRNA were identified. This phage is related to Salmonella

phage 7-11 and coliphages KBNP135 and phiEco32.

The DNA of phage GAP227 consists of 41,796 bp and has a G+C content of

55.7%. Forty-nine open reading frames and no tRNA were identified. This phage is related to Yersinia phages φR8-01 and φ80-18, and Aeromonas phage phiAS7.

The genome of C. sakazakii myovirus vB_CsaM_GAP161 (GAP161) consists of

178,193 bp and has a G+C content of 44.5%. A total of 277 genes including 275 open

reading frames and two tRNA-encoding genes were identified. This phage is closely

related to coliphages RB16 and RB43 and Klebsiella phage KP15.

Observing no hits (E value < 0.003) against a database of 83 bacterial toxin proteins, suggests that these four phages could be applied to control Cronobacter sakazakii.

4.2. INTRODUCTION

Cronobacter sakazakii is a life-threatening opportunistic pathogen, which causes infections in immunocompromized individuals of all ages (Gurtler, Kornacki, & Beuchat,

2005). Cronobacter sakazakii infects infants as well as adults. Contaminated milk-based, powdered infant formulae have been the cause of rare but life-threatening cases of sepsis, necrotizing enterocolitis, brain abscess and meningitis in neonates and infants (Centers for Disease Control and Prevention (CDC), 2002; Gurtler, Kornacki, & Beuchat, 2005;

Lai, 2001). Bacteriophages (phages), have been used as alternative agents to control pathogens because of their high specificity and efficacy (Goodridge & Bisha, 2011;

Kropinski, 2006), and phage biocontrol of many pathogens such as Salmonella,

Campylobacter and Listeria in the food industry is approved for use in the USA and

Europe (Burrowes et al., 2011; Goodridge & Bisha, 2011; Kropinski, 2006). Therefore, due to the intrinsic antibiotic resistance of Cronobacter (Lai, 2001), phages could be particularly useful for the control of this food-borne pathogen. However, comprehensive knowledge of a phage as a potential therapeutic or control agent is required to ensure its safety before clinical application or addition to food. Presently, only ten Cronobacter phages have been reported including five myoviruses (GAP31, GAP161, ESSI-2, ES2,

and CR3) (Abbasifar et al., 2012a,b; Lee, Chang, & Park, 2011; Lee, Park, & Chang,

2011; Shin et al., 2012), three siphoviruses (ESP2949-1, phiES15, and ENT39118) (Lee

et al., 2012a,b; Lee & Park, 2012), one podovirus (GAP227) (Abbasifar et al., 2013), and

ENT47670 (unclassified; HQ201308). In this study, we have fully sequenced the genome of four C. sakazakii phages to ensure that they may be used safely for therapeutic and biocontrol applications to reduce the prevalence of this bacterium during processing of infant formula, especially in hospitals. Genome sequencing helps predict the likely reproductive behavior of the phage, and determine whether the phage has lysogenic capability, an essential and important consideration for a phage intended for use in therapy and biocontrol.

4.3. MATERIALS AND METHODS

4.3.1. Bacteria and Bacteriophages

Twenty-one Cronobacter strains (including 14 C. sakazakii strains) were used for isolation, propagation, and determination of host range of phages, and all the strains were grown in TSB and TSA as previously described in Chapter 2 section 3 (2.3).

Lytic bacteriophages vB_CsaM_GAP31 (GAP31), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161 (GAP161), and vB_CsaP_GAP227 (GAP227) against C. sakazakii were isolated from samples of untreated sewage from the Guelph Wastewater Treatment

Plant (Guelph, ON, Canada), using the method described by Kropinski et al. (2009) and explained in Chapter 2 (2.3). After purification, and preparation of stocks, the host range and cross infectivity were determined by spot test. Selected phages were examined by

electron microscopy of negatively stained preparations (2% uranyl acetate). Detailed

methods have been described in detail in Chapter 2 section 3.

4.3.2. Phage Purification, DNA Isolation and Sequencing

From the phages that were previously isolated and characterized (Chapter 2),

phages GAP31, GAP52, GAP161, and GAP227 were selected (based on host range,

strong lytic activity, and electron micrographs) for DNA sequencing and proteomic

analysis. Phages were propagated to high titre as described in Chapter 2, and they were

highly purified by a method described by Sambrook and Russell (Sambrook & Russell,

2001) with some modifications. Briefly, the bacterial debris was removed from the crude

phage lysate by centrifugation at 14000 × g for 20 min at 4ºC (Beckman Coulter Inc.,

Mississauga, ON, Canada). Pancreatic DNase 1 and RNase, were added to the

supernatant to a final concentration of 10 μg/ml each (Sigma-Aldrich Canada Ltd.,

Oakville, ON), and incubated at room temperature for 30 min to eliminate contaminating

bacterial nucleic acids present in the supernatant. Sodium chloride was added to a final

concentration of 1 M, and then the mixture was stirred on ice for 1 h, and the supernatant

was collected after centrifugation at 14000 × g for 15 min at 4ºC. The phage particles

were precipitated in the presence 10% w/v (final concentration) PEG-8000 with stirring at 4ºC overnight. The precipitated phage particles were recovered by centrifugation at

14000 × g for 20 min at 4ºC, followed by resuspension of the pellet in SM buffer. To purify the phage from the suspension, separation on a self-generating cesium chloride

(CsCl) gradient (1.5 g/ml CsCl, run at 21000 × g at 4ºC for 24 h) was used in a fixed angle, Beckman SW 90Ti rotor. After purification by a second passage through a CsCl

gradient for another 24 hours, the phage was dialyzed against two changes of SM buffer,

2 L each, using dialysis cassettes (3500 MWCO; Thermo Scientific, Fisher Scientific,

Mississauga, ON). The highly purified, high-titer phage was stored at 4ºC.

The DNA of four phages were extracted and purified using the Midi Lambda

DNA purification kit (Qiagen Midi25; Mississauga, ON, Canada), and their genomic sequence was determined using 454 pyrosequencing technology (McGill University and

Génome Québec Innovation Centre; Montreal, QC, Canada).

4.3.3. Bioinformatic Analysis

The genomes were annotated with the help of Prof. Andrew M. Kropinski

(Laboratory for Foodborne Zoonoses, Guelph, ON) using a variety of online tools

(http://molbiol-tools.ca). The genomes were annotated by Rapid Annotations using

Subsystems Technology (myRAST) with gene calls verified using Kodon (Applied

Maths, Austin, TX). Transfer RNAs were predicted using tRNAscan-SE

(http://lowelab.ucsc.edu/tRNAscan-SE/). For each protein the number of amino acids, molecular weight and the isoelectric point were calculated by Batch MW and pI Finder

(http://greengene.uml.edu/programs/FindMW.html). Homologs were identified using

BatchBLAST (http://greengene.uml.edu/programs/NCBI_Blast.html). To predict protein

motifs the Transmembrane Hidden Markov Model (TMHMM)

(http://www.cbs.dtu.dk/services/TMHMM-2.0/), Phobius (http://phobius.sbc.su.se), and

Pfam (http://pfam.sanger.ac.uk/) were used. HHpred was used to detect remote protein

homology and predict its structure (Soding, Biegert, & Lupas, 2005) in collaboration with

Dr. John H.E. Nash (Laboratory for Foodborne Zoonoses, Guelph, ON). In addition, to

detect potential bacterial toxins the complete proteome of phage GAP32 was screened

against a proprietary database of 83 bacterial toxin proteins (including those from

Bacillus spp., Bordetella, Clostridium spp., Enterobacteriaceae, Listeria, Pseudomonas,

Staphylococcus, Streptococcus, and Vibrio) (Carter et al., 2012) using the BLASTP feature of BioEdit (Tippmann, 2004).

4.3.4. Genome Sequences

The complete genome sequences of Cronobacter phages vB_CsaM_GAP31, vB_CsaP_GAP52, vB_CsaM_GAP161, and vB_CsaP_GAP227 are available in

GenBank under the accession numbers JN882284, JN882286, JN882287, and KC107834, respectively.

4.4. RESULTS

4.4.1. Features of the genome of phage GAP31

Phage GAP31 was able to lyse all 14 C. sakazakii strains tested, and this phage is a myovirus with an icosahedral head of 83 nm in diameter and a tail of 107 × 19 nm

(Chapter 2). The double-stranded DNA genome of phage GAP31 has 147,940 bp with a

G+C content of 46.3%. This genome encodes 295 genes including 269 open reading frames (ORFs) and 26 tRNA genes. Of these ORFs, 211 (78.4%) of the putative proteins of GAP31 were designated defined function with significant matches (E-value < l0-5) to

known proteins by BLASTP searches, while the rest were considered to be hypothetical

proteins unique to phage GAP31. Bacterial toxin and integrase homologs were absent in

the GAP31 genome. In addition, screening against the database of 83 bacterial toxin proteins using the BLASTP feature of BioEdit (Tippmann, 2004) showed no hits (E- value < 0.003). Table 4.1 shows the summary of the general features of the genome of phages GAP31, GAP52, GAP161, and GAP227. Table 4.2 (in Appendix 3) shows the details of the general features of ORFs in the DNA of phage GAP31 and homology to proteins in the databases.

Comparative proteomic analysis using CoreGenes (Zafar, Mazumder, & Seto,

2002) showed that GAP31 shares 202/244 (82.79%), 96/218 (44.04%), and 98/233

(42.06%) protein homology with Salmonella phage PVP-SE1 (Santos et al., 2011) and coliphages vB_EcoM-FV3 (Truncaite et al., 2012) and rV5 (DQ832317), respectively. In addition, this virus is peripherally related to Cronobacter phage CR3 with 94/265

(35.47%) homology (Shin et al., 2012). Figure 4.1 shows the map of the linear genome of phage GAP31 in comparison to Salmonella phage PVP-SE1 created by CGview using

TBLASX (Grant & Stothard, 2008).

Table 4.1. General features of the genome of phages GAP31, GAP52, GAP161, and GAP227 and their related phages based on proteomic analysis using CoreGenes (Zafar, Mazumder, & Seto, 2002).

DNA Length G+C No. of No. of Protein homology to:

(bp) content (%) ORFs tRNAs GAP31 147,940 46.3 269 26 Salmonella phage PVP-SE1 (83%), and coliphages vB_EcoM-FV3 (44%) and rV5 (42%) GAP52 76,631 44.2 115 2 Salmonella phage 7-11 (52%) GAP161 178,193 44.5 275 2 Coliphages RB16 (94%) and RB43 (87%), and phage KP15 (85%) of Klebsiella pneumoniae GAP227 41,796 55.7 49 0 Yersinia phages φR8-01 and φ80-18 (71%), and Aeromonas phage phiAS7 (63%)

Figure 4.1. The map of the linear genome of phage vB_CsaM_GAP31 in comparison to Salmonella phage PVP-SE1 created with CGview using TBLASX (Grant & Stothard, 2008).

4.4.2. Features of the genome of phage GAP52

Phage GAP52 lysed 11 of 14 (78.6%) of C. sakazakii strains tested, and GAP52 is

a podovirus (Ackermann, 2005) and morphologically similar to Salmonella Newport

phage 7-11, which belongs to the C3 morphotype. This morphotype is extremely rare and

only constitutes 0.5% of phages examined by electron microscopy (Ackermann, 2001),

and only 19 members are known (H. W. Ackermann, personal communication). GAP52

has an elongated cylindrical head of 139 × 47 nm and a tail of 12 × 10 nm (Chapter 2).

Enterobacter sakazakii phage C2, Erwinia herbicola phages E3 and E16P (Grimont,

Grimont, & du Pasquier, 1978; Grimont & Grimont, 1981a,b), coliphage phiEco32

(Savalia et al., 2008), Lactococcus phage KSY1 (Chopin et al., 2007), Vibrio phage 71A-

6 (Khan et al., 2001), and coliphage NJ01 (a new phiEco32-like phage) (Li et al., 2012)

are some other C3 phages that have been previousely isolated and characterized. The

genome of phage GAP52 is double-stranded DNA with 76,631 bp and a G+C content of

44.2%. Its genome encodes 117 genes including 115 ORFs and two tRNA genes. Ninety-

one (79.1%) of the putative proteins of GAP52 were designated a defined function with

significant matches (E-value < l0-5) to known proteins by BLASTP searches, and the rest were considered to be hypothetical proteins unique to phage GAP52. Bacterial toxin and integrase homologs were not found in the GAP52 genome. In addition, screening against

the database of 83 bacterial toxin proteins using the BLASTP feature of BioEdit

(Tippmann, 2004) showed no hits (E-value < 0.003). Table 4.3 (in Appendix 3) shows

the general features of ORFs in the DNA of phage GAP52 and homology to proteins in

the databases.

Comparative proteomic analysis using CoreGenes (Zafar, Mazumder, & Seto,

2002) reveals that the genome sequence of GAP52 shares 79/151 (52.32%) protein homology with Salmonella phage 7-11 (Kropinski, Lingohr, & Ackermann, 2011).

Additionally, GAP52 is peripherally related to coliphages KBNP135 (JX415536) and phiEco32 (Savalia et al., 2008) with 38/120 (31.67%), and 34/128 (26.56%) homology, respectively. Figure 4.2 shows the map of the linear genome of phage GAP52 in comparison to Salmonella phage 7-11 created with CGview using TBLASX (Grant &

Stothard, 2008).

Figure 4.2. The map of the linear genome of phage vB_CsaP_GAP52 in comparison to Salmonella phage 7-11 created with CGview using TBLASX (Grant & Stothard, 2008).

4.4.3. Features of the genome of phage GAP161

Phage GAP161 belongs to the Myoviridae family (Ackermann, 2005), with an

elongated cylindrical head 110 × 74 nm and a tail (113 × 17 nm), and has the characteristic morphology of a T4-like phage (Chapter 2). Phage GAP161 has a double- stranded DNA genome of 178,193 bp with a G+C content of 44.5%. This genome encodes 277 genes including 275 ORFs and two tRNA genes. Of these ORFs, 267

(97.1%) of the putative proteins of GAP161 were designated a defined function with significant matches (E-value < l0-5) to known proteins by BLASTP searches, while the

rest were considered to be hypothetical proteins unique to phage GAP161. Bacterial toxin

and integrase homologs were absent in the GAP161 genome. Also, no hit was found (E- value < 0.003) in the screening against the database of 83 bacterial toxin proteins using the BLASTP feature of BioEdit (Tippmann, 2004). Table 4.4 (in Appendix 3) shows the general features of ORFs in the DNA of phage GAP161 and homology to proteins in the databases.

Bioinformatic analysis using CoreGenes (Zafar, Mazumder, & Seto, 2002) shows

that GAP161 proteome shares 254/270 (94.07%), 225/258 (87.21%), and 249/292

(85.27%) homology with coliphages RB16 and RB43 (Petrov et al., 2006), and phage

KP15 of Klebsiella pneumoniae (Drulis-Kawa et al., 2011), respectively. Figure 4.3 shows the map of the linear genome of phage GAP161 in comparison to coliphage RB16

created with CGview using TBLASX (Grant & Stothard, 2008).

Figure 4.3. The map of the linear genome of phage vB_CsaM_GAP161 in comparison to coliphages RB16 created with CGview using TBLASX (Grant & Stothard, 2008).

4.4.4. Features of the genome of phage GAP227

The 41,796 bp dsDNA genome of phage GAP227 possesses a G+C content of

55.7%, and encodes 49 ORFs. Twenty-six (54.1%) of the putative proteins of GAP227 were designated a defined function with significant matches (E-value < l0-5) to known

proteins by BLASTP searches, and the rest were considered to be hypothetical proteins

unique to phage GAP227. Bacterial toxin and integrase homologs were not found in the

GAP227 genome. Also, no hits (E-value < 0.003) were found in the screening against the

database of 83 bacterial toxin proteins using the BLASTP feature of BioEdit (Tippmann,

2004). Table 4.5 (in Appendix 3) shows the general features of ORFs in the DNA of

phage GAP227 and homology to proteins in the databases.

Discontiguous megablast analysis (Zhang et al., 2000) revealed significant

sequence similarity to Yersinia phages φR8-01 (HE956707) and φ80-18 (HE956710), and Aeromonas phage phiAS7 (Kim et al., 2012). Comparative proteomic analyses using

CoreGenes (Zafar, Mazumder, & Seto, 2002) confirmed these results showing that

GAP227 shared 71.4% proteins with φR8-01 and φ80-18 and, 63.3% with phiAS7.

Figure 4.4 shows the map of the linear genome of phage GAP227 in comparison to

Yersinia phage φR8-01 created by CGview using TBLASX (Grant & Stothard, 2008).

Figure 4.4. The map of the linear genome of phage vB_CsaP_GAP227 in comparison to Yersinia phages φR8-01 created with CGview using TBLASX (Grant & Stothard, 2008).

4.5. DISCUSSION

These four selected phages have broad host ranges and strong lytic activity, therefore they were chosen for sequencing and genome analysis. Each one of these viruses has genes that are unique to that phage, which shows these phages are novel and

were sequenced for the first time. However, they have similarities to varying degrees

with other sequenced phages (Table 4.1).

In the case of phage GAP31, comparative genomic analysis using CoreGenes

(Zafar, Mazumder, & Seto, 2002) shows that its genome shares a high percentage of

protein homology with Salmonella phage PVP-SE1 (Santos et al., 2011) (82.79%), and low homology with coliphages vB_EcoM-FV3 (Truncaite et al., 2012) (44.04%) and rV5

(DQ832317) (42.06%). Therefore, GAP31 should not be considered part of a proposed

genus of “V5likeviruses” within the Myoviridae family (Lavigne et al., 2009), rather,

phages GAP31 and PVP-SE1 could belong to a new genus, the “Pvplikevirus”, within a

new subfamily, the “V5virinae”. In addition, phage GAP31 was described by Dr. H. W.

Ackermann morphologically similar to phage Felix O1. The genome of phage Felix O1

has not been completely sequenced (Whichard et al., 2010), revealing the problems of

relying on morphology alone to make decisions about the taxonomic position of phages.

As with phage GAP31, homologies of more than 85% of GAP161 with coliphages

RB16 and RB43 (Petrov et al., 2006), and phage KP15 of Klebsiella pneumoniae (Drulis-

Kawa et al., 2011) indicate that GAP161 is a member of the myoviral subfamily

Teequatrovirinae, genus T4likeviruses (Lavigne et al., 2009).

Due protein sequence homology that phage GAP52 has with Salmonella phage 7-

11 (Kropinski, Lingohr, & Ackermann, 2011) (52.32%) and coliphage phiEco32 (Savalia

et al., 2008) (26.56%) it could not be considered part of the genus of Phieco32likevirus.

Indeed that genus should be carefully examined with the aim of reclassifying many of the phages which have been grouped here.

For phage GAP227, discontiguous Megablast (Zhang et al., 2000) and CoreGenes

(Zafar, Mazumder, & Seto, 2002) analyses indicate significant sequence similarity to

Yersinia phages φR8-01 (HE956707) and φ80-18 (HE956710), and Aeromonas phage phiAS7 (Kim et al., 2012). Since the values of similarities (71.4% proteins with φR8-01 and φ80-18; and, 63.3% with phiAS7) are considerably higher than shared proteins with the type virus, φKMV (36.7%) (Lavigne et al., 2003), we propose that these four phages should be grouped in a new genus within the Autographivirinae. Since we have fully described GAP227 in a recent manuscript (Abbasifar et al., 2013) we propose that this genus should be called the “Gap227likevirus”.

Moreover, the absence of bacterial toxin proteins and integrase homologs along with their strong lytic activity, suggest that GAP31, GAP52, GAP161, and GAP227 meet many of the criteria necessary for consideration, either separately or as a cocktail, for the biocontrol of Cronobacters. Their efficacy in eliminating C. sakazakii in infant formula and in animal models will be outlined in chapters 5 and 6.

Chapter 5. EFFECTIVITY OF BACTERIOPHAGE THERAPY

AGAINST CRONOBACTER SAKAZAKII IN GALLERIA

MELLONELLA (GREATER WAX MOTH) LARVAE

5.1. ABSTRACT

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered

infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of

phages from various environments were purified, and tested in vitro against strains of

Cronobacter sakazakii. Based on host range and lytic activity, the T4-like phage

vB_CsaM_GAP161, which belongs to the Myoviridae family, was selected for evaluation

of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole

animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter

strains were evaluated for lethality in G. mellonella larvae. Different strains of

Cronobacter caused various rates of mortality; ranging from 0 to 98%. C. sakazakii HPB

5 3253 with a LD50 dose of ~2.0 × 10 CFU/larva (24 h, 37°C) was calculated for this study. Larvae infected with a dose of 5 × LD50, were treated with phage GAP161 at time

intervals (MOI of 8). The mortality rates were as high as 100% in the groups injected

only with bacteria, as compared to 16.6% in the group infected with bacteria and treated

with phage. Phage GAP161 showed the best protective activity against C. sakazakii in G.

mellonella in groups treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful

control agent against C. sakazakii. In addition, G. mellonella may be a useful whole

animal model for pre-screening phages for efficacy and safety, prior to clinical evaluation

in mammalian models.

5.2. INTRODUCTION

Cronobacter spp., known as yellow-pigmented Enterobacter cloacae prior to

1980, and Enterobacter sakazakii until 2007, are ubiquitous, opportunistic pathogens found in both the environment and a variety of foods (Farmer III et al., 1980; Iversen et

al., 2007a). Although the epidemiology and reservoir of this bacterium are unknown, it has been shown that contaminated milk-based powdered infant formulae were the source of sporadic cases and outbreaks of Cronobacter infections that cause sepsis, necrotizing enterocolitis, brain abscess and meningitis in neonates and infants (Biering et al., 1989;

Bowen & Braden, 2006; Centers for Disease Control and Prevention (CDC), 2002; Clark et al., 1990; Gurtler, Kornacki, & Beuchat, 2005; Jarvis, 2005; Muytjens et al., 1983;

Muytjens & Kollee, 1990; Noriega et al., 1990). Cronobacter spp. have been ranked by the International Commission on Microbiological Specifications for Foods as a “severe

hazard for restricted populations, life-threatening or substantial chronic sequelae or long

duration” (International Commission on Microbiological Specification for Foods, 2002).

The mortality rate for neonate/infant infections with this bacterium can approach

80%, with antibiotics not having a dramatic affect on clinical outcomes (Drudy et al.,

2006; Lai, 2001; Norberg et al., 2012; Ray et al., 2007). Almost all patients surviving

infections of the central nervous system (CNS) experience delays in mental and physical

development (Lai, 2001). In addition to its effects on neonates and infants, this

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nosocomial pathogen has been reported to produce rare instances of infections such as

urosepsis, bacteremia and pneumonia in adults, especially in the elderly (Emery &

Weymouth, 1997; Hawkins, Lissner, & Sanford, 1991; Jiménez & Giménez, 1982; Lai,

2001; Ongradi, 2002; Pribyl et al., 1985; See, Than, & Tang, 2007).

Consideration of bacteriophages (phages) as alternative control agents has been

rekindled because of their high specificity and effectiveness to kill bacterial pathogens

without affecting the host commensal microbiota. Since 1919, when Félix d’Hérelle

proved the efficacy of phages to treat bacillary dysentery and to reduce mortality due to

cholera, phages have been used safely in human and veterinary medicine (Sulakvelidze &

Barrow, 2005). Phage biocontrol of pathogens including Salmonella, Campylobacter and

Listeria in the food industry is recognized in the US and Europe (Burrowes et al., 2011;

Goodridge & Bisha, 2011; Kropinski, 2006), and may be particularly useful to control

Cronobacter because of its intrinsic antibiotic resistance (Lai, 2001).

Several studies have confirmed the efficacy of phage therapy in animal models for

the treatment of bacterial pathogens such as Pseudomonas aeruginosa, (Hagens et al.,

2004; McVay, Velasquez, & Fralick, 2007; Wang et al., 2006), Escherichia coli (Smith

& Huggins, 1982; Smith, Huggins, & Shaw, 1987), Staphylococcus aureus (Capparelli et

al., 2007; Fedhila et al., 2006), Klebsiella pneumoniae (Vinodkumar, Neelagund, &

Kalsurmath, 2005), and Campylobacter jejuni (Loc Carrillo et al., 2005). In general,

mammalian models, such as mice, have been favored to study human infectious diseases

due to their similarity to humans in anatomy, physiology, immune response, and

pathogen susceptibility. There are, however, disadvantages in the use of these models,

including high monetary costs, ethical issues, and difficulty in obtaining sufficient

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numbers of animals to achieve meaningful results (Glavis-Bloom, Muhammed, &

Mylonakis, 2012). As an alternative, invertebrates, including insects, have been used in pathogenesis studies (Scully & Bidochka, 2006). Some of the benefits of using insects as experimental models to study human pathogens include: i) large numbers of insects can be used, ii) short life cycle; iii) ease of manipulation (reducing time and cost of maintenance), iv) quicker infection process, leading to more rapid results, v) fewer ethical concerns related to the administration of pathogens (Scully & Bidochka, 2006), and vi) similarity between human and insect in infection and immune responses, such as phagocytosis and production of antimicrobial peptides (Salzet, 2011).

Among invertebrates used Galleria mellonella, the Greater Wax Moth, is of special interest because of its ability to survive at 37°C (important in studying temperature-sensitive virulence of pathogens), and relatively large larval size, which allows inoculation of precise quantities of pathogen by a syringe (Glavis-Bloom,

Muhammed, & Mylonakis, 2012). Moreover, a good correlation has been found when comparing G. mellonella and mammals (mice) as hosts for evaluating virulence and detecting virulence factors of some human pathogens, such as P. aeruginosa and Candida albicans (Brennan et al., 2002; Jander, Rahme, & Ausubel, 2000). Indeed, G. mellonella larvae have been successfully used to study human pathogens, including Bacillus cereus

(Fedhila et al., 2006), P. aeruginosa (Jander, Rahme, & Ausubel, 2000; Miyata et al.,

2003), Francisella tularensis (Aperis et al., 2007), and Burkholderia cepacia (Seed &

Dennis, 2009).

The objective of this study was to isolate specific phages against C. sakazakii and to evaluate their therapeutic efficacy and safety against this bacterium using the G.

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mellonella larva as a whole animal model. To date, a few Cronobacter (Enterobacter

sakazakii) phages have been isolated and characterized (Abbasifar et al., 2012a,b;

Abbasifar et al., 2013; Kim, Klumpp, & Loessner, 2007; Lee, Chang, & Park, 2011; Lee,

Park, & Chang, 2011; Lee et al., 2012b; Loessner et al., 1993). However, there is little published evidence showing the effectiveness of phage therapy for Cronobacter

infections in animal models; only one study is published on phage therapy of urinary tract

infection in mice that was induced by Cronobacter turicensis (Tothova et al., 2011). To

our knowledge, the current study is the first to explore the use of phage therapy against C.

sakazakii in any animal model.

5.3. MATERIALS AND METHODS

5.3.1. Bacterial Strains and Culture Condition

Twenty-one Cronobacter strains (including 14 C. sakazakii strains) used in this

study were obtained from the culture collection maintained by the Canadian Research

Institute for Food Safety (CRIFS), Franco Pagotto (Public Health Agency of Canada,

Ottawa, ON) and Roger Stephan (Institute for Food Safety and Hygiene, Zurich,

Switzerland). Cronobacter muytjensii 51329 was purchased from the ATCC (Manassas,

VA, USA) (Table 2.1). To grow Cronobacter strains and to isolate and propagate the

phages, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), and Tryptose Top Agarose

(TSB + 0.5% agarose) (Fisher Scientific, Ottawa, ON, Canada) were used as previously

described in Chapter 2. All the media were supplemented with CaCl2 to a final

concentration of 2 mM (Fisher Chemicals, Mississauga, ON, Canada). Pure cultures were

obtained from frozen stocks kept at -80°C, and maintained at 4°C on TSA. To maintain

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cell viability, cultures were re-streaked biweekly onto fresh medium. Cronobacter strains

were surface plated onto TSA and incubated at 37°C. Colonies from 18-hour cultures were collected aseptically and added to SM buffer (100 mM NaCl, 50 mM Tris-HCl, pH

7.5, 0.002% (w/v) gelatin, 8 mM MgSO4•7H2O) in order to prepare a bacterial inoculum

of ~1.0 × 1010 CFU/ml. The inoculum was then serially diluted in SM buffer. These suspensions were used for experimental infection.

5.3.2. Bacteriophage Isolation and Characterization

Lytic bacteriophages against C. sakazakii were isolated from 23 samples of

untreated sewage (from a local wastewater treatment plant, Guelph, ON, Canada), fresh

cattle manure and soil (Dairy Barn, University of Guelph, Guelph), and water (Fraser

River, False Creek, Burrard Inlet and Strait of Georgia; Vancouver, BC, Canada) using a

method previously described (Jamalludeen et al., 2007). Samples were centrifuged (5000

× g for 15 min) and the supernatant was filtered through a 0.45 µm filter (Corning Inc.,

NY, USA). Twenty milliliters of an 8 h-inoculum of Cronobacter isolates at 37ºC in

Tryptic Soy Broth (TSB; Fisher Scientific, Ottawa, ON) were added to 200 ml of filtered supernatant, plus 20 ml of bacteriophage broth and 20 ml of TSB containing 2 mM

CaCl2. This mixture was incubated at 37ºC overnight, centrifuged at 5000 × g for 15 min

at 4ºC (Beckman Avanti J-20 XPI, Beckman Coulter Inc., Mississauga, ON, Canada) and

the supernatant was treated by adding 1% v/v chloroform. Bacteriophage broth was

prepared as previously described (Jamalludeen et al., 2007) by adding 100 g peptone

(Difco Laboratories, Detroit, MI, USA), 30 g beef extract (Difco), 50 g yeast extract

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(Fisher), 25 g NaCl (Fisher), and 80 g potassium dihydrogen phosphate (BDH

Laboratory, Toronto, ON, Canada) to one liter of distilled water.

For isolation of the phages, the overlay method (Kropinski et al., 2009) was carried out using molten (47ºC) top agarose (5 g low-melt agarose/l TSB containing 2 mM CaCl2). The mixture was poured immediately onto TSA plates and allowed to solidify for 15 min on a level surface, before incubation for ~16 h at 30°C. After incubation, the plates were examined for the presence of plaques, and single plaques were picked by sterile plastic Pasteur pipettes, and each plaque was eluted in 1 ml of SM buffer containing 1 drop of chloroform. The tubes containing plaques and SM buffer were held at room temperature for 4 h to let the phages diffuse out of the top agarose, before being stored at 4ºC.

From the phages that were previously isolated and characterized (Chapter 2), phage vB_CsaM_GAP161 (GAP161) was selected and used in this study for phage therapy against C. sakazakii in G. mellonella larvae. Phages were propagated to high titre with the method previously described in Chapter 2, and they were highly purified by a method described by Sambrook and Russell (Sambrook & Russell, 2001) but with some modifications as detailed in Chapter 3 (section 3.3.2).

5.3.3. Efficiency of Bacteriophage Therapy Against C. sakazakii in G.

mellonella Larvae

Larvae were purchased from a commercial supplier (Recorp Inc. Georgetown,

ON, Canada), and those weighing between 200 to 300 mg were selected. They were stored in wood chips and paper towel at 12°C. In order to reduce the stress of temperature

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change, larvae were kept at 37°C for 24 h prior to the experiment. Bacteria or phage (4 µl

aliquots), when applicable, were injected into the hemolymph at the base of the second

set of thoracic legs using disposable 1-ml TB syringes, 32 gauge needles, and a

microinjector (Stephens, 1959). Phages were injected in the right side and bacteria in the

left where applicable. Following injection, G. mellonella larvae were immediately placed

in an incubator at 37°C in the dark. To study the virulence of Cronobacters in G.

mellonella, a fresh inoculum (~1.0 × 1010 CFU/ml) of 21 different Cronobacter strains was injected into the larvae and scored 24 h post-infection (p.i.) at 37°C. Larvae were scored as dead when they had no reaction to touch with thumb forceps. To determine the

7 50% lethal dose (LD50), a series of 10-fold serial dilutions from 4 × 10 to 0 CFU of C. sakazakii strain HPB 3253 in SM buffer were injected into larvae. A control group received 4 µl of only SM buffer to assess any potentially negative effects of the injection process. Three groups of ten larvae were injected for each dilution, and larvae were incubated at 37°C and monitored periodically and scored as alive or dead up to 48 h p.i.

Each experiment was repeated three times independently, and LD50 for G. mellonella was

calculated using the method of Miller and Tainter described by Randhawa (Randhawa,

2009).

In order to test the persistence of phage GAP161 in larval hemolymph over time,

the phage was injected into larvae (4 µl, 2.0 × 109 PFU/ml), and the titer of phage in

hemolymph was measured at 0, 24 and 48 h after injection. For the zero time point,

hemolymph was collected from the larvae 20 min after injection. At each time point, the

hemolymph was equally collected from 10 larvae, 20 µl each and combined into a

microcentrifuge tube, serially diluted, and phage was enumerated by the overlay method

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described by Kropinski et al. (2009). To quantify the phage titre, three groups of 10

larvae were used for each time point.

6 For the challenge trials, 4 µl of bacteria at the concentration of 5 × LD50 (~1 × 10

CFU/larva) were administered into the hemolymph. In the treatment groups, larvae were

injected with 2.0 × 109 PFU/mL of phage GAP161 (~8.0 × 106 PFU/larva) resulting in an

initial multiplicity of infection (MOI) of 8. This MOI was chosen based on the results of

preliminary trials (results not shown). Larvae infected by C. sakazakii 3253 were

immediately treated with phage GAP161 by separate injection (time 0). Furthermore, in

order to determine the prophylactic effect, phage was injected into larvae at different

times up to 1 h prior to the time of infection (time -1 h or -0.5 h). Also, to determine how

long treatment could be delayed, the phage was administered at various intervals up to 4

h after bacterial infection (time +1 h, +2 h, or +4 h). The untreated group received 4 µl of

SM buffer instead of phage (Infected group).

In addition, there were three groups of uninfected larvae, which received: i) no injection (not infected, not injected with phage nor buffer; Nil group), ii) one injection of

SM buffer following an injection of phage to determine any potential negative effect of the virus (Phage group); and iii) two injections of SM buffer to measure the effect of multiple injections on the larvae (Sham group). Following injection, G. mellonella larvae

were immediately incubated at 37°C for 48 h and monitored periodically, and larvae were

scored as alive or dead. In the third experiment, survival was measured at 23.5 h instead

of 24 h.

To verify if the effects of phage therapy were related to a nonspecific immune

activation response and not the phage itself, a control group was tested with heat-

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inactivated phage to determine the ability to rescue larvae infected by C. sakazakii. Phage

GAP161 (2.0 × 109 PFU/ml) was inactivated by heating at 85°C for 30 min. Following

this heat treatment, no viable phage was detected using the overlay method. Larvae were

6 injected with a 5 × LD50 dose of C. sakazakii 3253 (~1.0 × 10 CFU) and immediately

treated with heat-inactivated phage at a MOI of 8.

5.3.4. Statistical Analysis

In all experiments conducted in this study, each group had a total of 30 larvae that

consisted of 3 replicates, 10 larvae per replicate, and every experiment was repeated three

times independently. Survival data were analyzed using software from the SAS Institute

Inc., Cary, NC, USA, version 9.2. The General Linear Model (GLM) procedure with

Experiment, Time, Treatment and Treatment*Time in the model was used to evaluate

these effects (survival at 24 and 48 hours). Of the many comparisons of the various

treatments that could be made, single degree of freedom contrasts involving pairs or

groups of treatments were used to examine specific effects. In addition, survival curves

were plotted by the Kaplan-Meier method using GraphPad Prism (GraphPad Software,

Inc., La Jolla, CA, USA).

5.4. RESULTS

5.4.1. Mortality of C. sakazakii in G. mellonella Larvae

The mortality of different strains of Cronobacter varied between 0 to more than

98% in larvae injected with 4 × 107 CFU of the pathogen (Fig. 5.1). To study the effect of

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bacteriophage on C. sakazakii infection during 48 h incubation at 37ºC, C. sakazakii HPB

3253 strain was selected as the infectious agent because of its high virulence and its

sensitivity to bacteriophages.

Figure 5.1. Mortality rates of different Cronobacter strains compared with SM buffer or no injection in G. mellonella larvae 24 h p.i. at 37ºC (~4.0 × 107 CFU).

5.4.2. Bacteriophage and Its Persistence in G. mellonella Larvae

Based on host range, strong lytic activity, and preliminary results of phage therapy against C. sakazakii in G. mellonella larvae (results not shown), phage GAP161 was selected from a collection of 252 phage isolates (Chapter 2) as the therapeutic agent.

Phage GAP161 lysed 85.7% of the C. sakazakii strains tested. It is a T4-like phage belonging to the Myoviridae family (Fig. 5.2), with an elongated cylindrical head 110 nm long, 74 nm wide, and a tail 113 nm long by 17 nm wide. When persistence of the phage in the larvae was monitored, phage titres in hemolymph showed an approximate two-fold

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increase in titre 48 h after injection. However, statistical analysis (t-test) revealed that this difference was not significant.

Figure 5.2. Electron micrograph of phage vB_CsaM_GAP161 negatively stained with uranyl acetate.

5.4.3. Efficiency of Bacteriophage Therapy Against C. sakazakii in G.

mellonella Larvae

Cronobacter sakazakii HPB 3253 infection caused severe illness and death in G.

5 mellonella larvae injected with a LD50 dose of approximately 2 × 10 CFU (Fig. 5.3). At a dose of 5 × LD50, the mortality rates were between 96.6% and 100% in the control infected groups at 24 and 48 h p.i., respectively (Table 5.1; Fig. 5.3; Fig. 5.4).

Administration of phage GAP161 at a MOI of 8, in the opposite side of the larvae to the bacterial injection site, resulted in mortality as low as 16.6% after 24 and 48 h p.i. The survival of phage treated groups that received the phage before or immediately after infection was significantly higher than for the untreated groups (p <0.001).

In general, survival rates observed at 24 hours were not significantly different from those at 48 hours. When the heat-inactivated phage was administered, no significant

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difference was observed between infected, untreated larvae and those treated with the heat-inactivated GAP161; with both showing the same mortality rate of 83.3% at 48 h p.i.

Table 5.1. Least Square Means (LSM) of the survival of G. mellonella larvae following challenge with C. sakazakii HPB 3253 and/or phage GAP161 administration. These results were obtained by using General Linear Model (GLM) in SAS.

Experiment LSM* 1 12.95a 2 14.35b 3 16.75b Time 24 h 15.43 48h 13.93 Treatment Nil 29.17A Sham 27.00A Phage 27.33A Sakazaki 3.50D Sa Ph -1 17.50B Sa Ph -0.5 14.33B Sa Ph 0 16.50B Sa Ph +1 7.50D Sa Ph +2 3.00D Sa Ph +4 1.00D

* Standard error = 4.49. LSM with different lower case superscripts are different at P≤ 0.05; those with different upper case superscripts are different at P≤ 0.001.

Figure 5.3. G. mellonella larvae infected by C. sakazakii HPB 3253. A) Right to left: normal, sick, and dead. Images B and C show larvae 6 hours after infection: B) Treated with phage GAP161 immediately after infection, C) Received SM buffer immediately after infection.

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Figure 5.4. Survival of G. mellonella larvae infected with C. sakazakii and treated with phage GAP161 at different times prior to and after infection. Nil group: no injection; Phage group: one injection of SM buffer following an injection of phage; Sham group: two injections of SM buffer. Challenged groups infected with C. sakazakii HPB 3253 and treated with phage GAP161 at various times before infection (-1 h or -0.5 h) or after infection (0 h, +1 h, +2 h, or +4 h).

5.5 DISCUSSION

The variation in virulence of different Cronobacter strains observed in this study

as determined by larval mortality, agrees with a previous study on pathogenesis of this

bacterium (Pagotto et al., 2009), which showed significant differences in adhesion, invasion, and toxin production among strains. For instance, it was shown that strain 2855 has low and strain 3290 high ability to adhere to and invade human brain microvascular endothelial cells. Likewise, in our study the same strains produced no and 93% G. mellonella larval mortalities, respectively. This further indicates the relevance of this invertebrate model of pathogenesis.

An increase in the survival rates of infected larvae treated with phage GAP161 was observed, and the increase in survival rates within groups treated prior to or

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immediately after infection was significantly higher than the non-treated, infected control group. Phage GAP161 shows the best protective activity against C. sakazakii in G. mellonella in the groups treated with phage prior to or immediately after infection. In addition, when heat-inactivated phage was used, the survival rates were the same as for larvae inoculated with the pathogen alone, indicating that the survival of the larvae is not due to host immune stimulation but the effect of phage antibacterial activity. Moreover, the lack of significant differences in mortality among Nil, Sham, and Phage groups reveals that neither phage nor injection method used had an effect on the health of the larvae. Also, the analysis of the complete genome sequences of phage GAP161 and screening of its proteome against a database of 83 bacterial toxin proteins, have revealed the absence of toxin encoding genes in phage GAP161 (Abbasifar et al., 2012b).

There were small but significant differences among the results of the three repeated experiments. These differences could be due to variations between batches of larvae that were purchased due to the growing conditions such as temperature, moisture, and diet, as well as the age of the larvae. The effects of these differences were mitigated to some extent by only choosing healthy looking larvae and sorting them by weight for use in experiments.

In our study the phage was applied at various intervals prior to C. sakazakii infection in order to determine if this virus has a prophylactic effect. The results suggest that time of administration of phage plays a key role in therapy of C. sakazakii infection in larvae. When GAP161 was applied 1 h before infection the survival rates were the highest, however these rates were not significantly higher than the other groups that received the phage prior to or immediately after infection. On the other hand, the longer

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the time that phage administration occurs post infection the greater was the increase in the mortality rates. Although the survival rates in the groups that received phage 1 h p.i. or later were mostly higher than that of the infected, untreated group, the differences were not significant. These observations confirm that GAP161 is highly efficient in preventing infection in larvae. Likewise, Seed and Dennis (2009) showed that two of their three phages offered the best protection for larvae when applied immediately after infection with Burkholderia cepacia, however, treatments after 6 and 12 hours resulted in fewer survivors (Seed & Dennis, 2009). The higher mortality rates in groups treated with phage

GAP161 later than 1 h p.i. is likely due to the fact that by the time the phage has been administered, the bacteria have already caused irreversible damage to the larvae, that was visually manifested by a color change from yellow to gray or dark brown. In addition, larvae were more sensitive to the injection after 1 h p.i., as it caused bleeding in the hemolymph. The phage treatment of larvae infected with C. sakazakii helps them to survive not only in the short term (24 h p.i.) but also for longer time periods as there was no significant reduction in survival between 24 h p.i. and 48 h p.i. (Fig. 5.4).

Although the increase of phage titer in the persistency test was not statistically significant, the two-fold increase in phage titre can be explained by potential release of phage from an aggregated form, phage distribution from the tissues into the hemolymph or replication of phage in the bacterial microbiota of the gut of the larvae, which may contain strains susceptible to infection by the phage such as Enterobacter cloacae. The latter bacterium has been isolated previously from digestive tracts of G. mellonella larvae

(Lysenko & Weiser, 1974), and we have noted that phage GAP161 has a lytic effect on

E. cloacae strains (Chapter 2).

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This study confirms earlier work illustrating the usefulness of G. mellonella

larvae as an inexpensive animal model for evaluating the efficacy of phage therapy (Seed

& Dennis, 2009). Greater Wax Moth larvae have also been employed to evaluate other

antimicrobial compounds. For example, G. mellonella infected with Cryptococcus

neoformans, were treated by different antifungals (Mylonakis et al., 2005). The

combination of amphotericin B plus flucytosine, which is the recommended treatment in

humans for this fungal infection, also provides the best protection in G. mellonella. These studies along with our results suggest G. mellonella to be a valuable model to study antimicrobial agents including bacteriophages.

In conclusion, the newly isolated phage GAP161 is safe, and could potentially be used to control C. sakazakii during preparation of infant formula but would first have to be tested for its efficiency in this food matrix, and also clinically evaluated in mammalian models. More studies need to be conducted to validate the data from Greater Wax Moth model in a mammalian model. In addition, G. mellonella larvae proved to be a suitable

whole animal model for pre-screening of the phage for its killing efficacy against C.

sakazakii infection and safety.

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Chapter 6. USE OF COCKTAIL OF FIVE PHAGES TO CONTROL

CRONOBACTER SPP. IN BROTH MEDIA AND

IN INFANT FORMULA

6.1. ABSTRACT

Cronobacter spp. are opportunistic pathogens that can be found in milk-based,

powdered infant formulae and can cause rare, but life-threatening infections in newborns

and infants, with high fatality rates. Existing prevention and control processes are not

enough to guarantee production of infant formulae free of Cronobacter spp. In the current

study, the effectiveness of a cocktail of five lytic Cronobacter phages to control these

pathogens in infant formula was investigated. These five phages were selected from a

collection of 252 isolated and purified phages from various environmental sources. The

phage cocktail of these 5 phages (108 PFU/ml) was applied to prevent and control the

outgrowth of a mixture of three Cronobacter sakazakii strains in broth medium (TSB) and reconstituted infant formula (RIF) specially prepared for premature neonates and infants (at 101 to 104 CFU/ml; MOIs of 104 to 107) at 24°C. The phage cocktail effectively prevented the development of the tested Cronobacters at both high and low contamination doses in TSB and RIF. At these MOIs, the phage cocktail was able to prevent growth of Cronobacter strains in RIF. These results suggest that application of lytic Cronobacter phages is potentially a useful method to control against Cronobacter

spp. and to prevent the infection of infants through consumption of contaminated infant

formulae. In this study, we investigate the effectiveness of a cocktail of bacteriophages to

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biocontrol a cocktail of Cronobacter strains in RIF specially prepared for premature

neonates and infants.

6.2. INTRODUCTION

In 1860, Henri Nestlé developed the first infant formula because of high mortality

rates among infants with working-class mothers who had no time to breastfeed

(Estuningsih & Sani, 2008). That product was a mixture of cow’s milk and cereal and he

called it “Farine Lactee”. According to the Infant Formulae Directive, “infant formulae

are foodstuffs intended for particular nutritional use by infants during the first four to six

months of life and satisfying by themselves the nutritional requirements of this category

of persons” (Commission Directive 91/321/EEC).

Production of infant formula is a major industry with more than 461,000 tons

produced in 2005 worldwide. North America is the major producer of infant formula by

contributing more than 48% of the world production (Cordier, 2008). Infant formulae are produced in different forms: powder, concentrate and ready-to-feed. Powdered infant formula is a non-sterile product and the most popular one, because of its lower price and the ability to be stored for a longer period (O'Connor et al., 2008).

PIF is manufactured according to wet-mix process, dry-mix process, or combined process, and Cronobacter contamination may occur in all kinds of PIF manufacturing processes (Cordier, 2008). Because infant formula is used as the sole source of nutrition during the first months of infants’ life, it must be safe and meet the normal nutritional requirements of term infants.

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Cronobacters are ubiquitous and opportunistic pathogens found in a variety of

foods and the environment (Farmer III et al., 1980; Iversen et al., 2007a). The

epidemiology and reservoir of these bacteria is unknown; however, contaminated milk- based, powdered infant formulae were shown to be the source of sporadic cases and outbreaks of Cronobacter infections. These pathogens cause sepsis, necrotizing enterocolitis (NEC), brain abscess and meningitis in neonates and infants (Biering et al.,

1989; Bowen & Braden, 2006; Centers for Disease Control and Prevention (CDC), 2002;

Clark et al., 1990; Gurtler, Kornacki, & Beuchat, 2005; Jarvis, 2005; Muytjens et al.,

1983; Muytjens & Kollee, 1990; Noriega et al., 1990). The mortality rate for

Cronobacter spp. infection in neonates and infants is as high as 80%, with antibiotics not having a dramatic affect on clinical outcomes (Drudy et al., 2006; Lai, 2001; Norberg et al., 2012; Ray et al., 2007). Almost all patients surviving infections of the central nervous system (CNS) experience delays in mental and physical development (Lai, 2001). The

International Commission on Microbiological Specifications for Foods has ranked this pathogen as a “severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration” (International Commission on Microbiological

Specification for Foods, 2002).

Despite application of various procedures to ensure high hygiene and sanitation standards in food processing facilities, there are still reported outbreaks of food-borne diseases (Scallan et al., 2011), with substantial economic and health impact globally

(Mead et al., 1999; Scharff, 2012). For instance, effective control measures for

Salmonella are well established in PIF manufacturing plants (Iversen, Caubilla-Barron, &

Forsythe, 2004), however, Cronobacter cells have been isolated from samples that pass

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the quality control tests for both coliform and Salmonella. Another study on a milk powder processing line for the presence of Enterobacteriaceae and Cronobacter over an

11-month period, revealed that hygiene measures such as cleaning-in-place (CIP) process followed by heat treatment were insufficient to eliminate Cronobacter completely from all areas of the processing line (Hein et al., 2009).

Consideration of bacteriophages (phages) as alternative control agents has been revived because of their high specificity and effectiveness in killing bacterial pathogens without affecting the host commensal microbiota. Phages exist in all natural environments, including contaminated food, where bacteria can be found, and phages specific for bacterial pathogens are abundant in environments like soil and sewage

(Kutter & Sulakvelidze, 2005). Since 1919 when Félix d’Hérelle proved the effectiveness of phages to treat bacillary dysentery and to reduce mortality due to cholera, phages have been used safely in human and veterinary medicine (Sulakvelidze & Barrow, 2005). For food safety applications, phages with strong lytic activity are the best choice due to their ability to infect their bacterial hosts and lyse them, leading to the release of more phage particles that are then able to infect more bacterial cells (Greer, 2005). Phage biocontrol of pathogens including Salmonella, Campylobacter and Listeria in the food industry is recognized in the US and Europe (Burrowes et al., 2011; Goodridge & Bisha, 2011;

Kropinski, 2006) and may be particularly useful to control Cronobacter because of its intrinsic antibiotic resistance (Lai, 2001).

Using phages during the pre-harvest and post-harvest phases of food production to decrease bacterial pathogen loads is a promising method to achieve a safer food supply

(Strauch, Hammerl, & Hertwig, 2007). The application of lytic phages to disinfect

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surfaces in food plants (e.g. ready-to-eat products), decontaminate carcasses, or eliminate zoonotic pathogens from living animals, has been successful (Hagens & Loessner, 2010;

Strauch, Hammerl, & Hertwig, 2007). Although, phages are less active in food matrices than in liquid culture media, their application to control food-borne pathogens like

Escherichia coli O157:H7, Salmonella and Listeria monocytogenes has been beneficial

(Carlton et al., 2005; Greer, 2005; Hagens & Loessner, 2007; O'Flynn et al., 2004).

Indeed, the approval of the use of phage as food additives by the US FDA in 2006 has also increased the need for research on new applications of these natural killers of bacterial pathogens (FDA, 2006).

Two studies have evaluated phage biocontrol to prevent the growth of

Cronobacter in infant formula. In the first study, the efficacy of two Cronobacter phages

(ESP 1-3 and ESP 732-1) against this pathogen were investigated in reconstituted infant formula (RIF) as well as in ½ × Brain Heart Infusion (BHI) broth at 12, 24, and 37°C

(Kim, Klumpp, & Loessner, 2007). The bacterial concentration was 102 CFU/ml and phages were added at 107, 108, or 109 PFU/ml. The growth inhibition resulting from the addition of phages was dose-dependent, and phages present at the highest concentration exhibited the greatest effect on growth inhibition regardless of incubation temperature, with the maximum effect in infant formula obtained by phage ESP 732-1. Both phages were most effective at 24°C.

In the other publication, a cocktail of five phages against 40 Cronobacter strains in experimentally contaminated infant formula, and in BHI broth was investigated (Zuber et al., 2008). The RIF was inoculated with individual Cronobacter strains at 102 CFU/ml, and treated with the phage cocktail at 108 PFU/ml (MOI of 106). The outgrowth of 90%

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of the 40 tested Cronobacter strains was prevented at 30°C. On the other hand, the phage

cocktail was not able to prevent the bacterial outgrowth when it was applied at a MOI of

102 or 104. The findings of these studies suggested that bacteriophages could reduce the

number of Cronobacter cells through lysis. Thus, because of the high specificity of

bacteriophages, their application in infant formula may reduce the levels of the pathogen

in rehydrated PIF without affecting the normal flora in an infant’s gut when consumed.

It has been emphasized by Health Canada (2002) that neonates who are

immunosuppressed, born prematurely and have a low birth weight are at higher risk of

Cronobacter infection (Gurtler, Kornacki, & Beuchat, 2005; Pagotto et al., 2009).

Therefore, due to their nutritional needs, which are not met only by consuming breast

milk, these groups of neonates must be fed with infant formulae that are manufactured for

premature neonates/infants. However, in the only two studies that evaluated the effect of

phage on Cronobacter spp. in infant formula, the authors have not indicated if the infant formula used in their experiment was the formula for premature neonates/infant, since the nutrient composition of these infant formulae could affect phage efficiency. Also, the

enumeration of bacteria was conducted on non-selective and non-differential media (BHI

agar), and counting only based on the presence of yellow colonies. However, it has been

reported that not all Cronobacter strains produce yellow colonies (Druggan et al., 2004;

Farmer III et al., 1980; Iversen, Druggan, & Forsythe, 2004). The objective of the present study was to apply a cocktail of five newly isolated and characterized phages against a cocktail of three Cronobacter sakazakii strains in RIF taking into account the limitations

identified for the previous studies.

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6.3. MATERIALS AND METHODS

6.3.1. Bacteria and Bacteriophages

Three luminescent strains of Cronobacter (amp::lux) constructed previously by

Dr. Haifeng Wang, were obtained from the Canadian Research Institute for Food Safety

(CRIFS) Culture Collection at the University of Guelph, and used in this study (Table

6.1). To grow Cronobacter strains and to isolate and propagate the phages, Tryptic Soy

Broth (TSB), Tryptic Soy Agar (TSA), and Tryptic Top Agarose (TSB + 0.5% agarose)

(Fisher Scientific, Ottawa, ON) were used. All the media contained ampicillin (50 μg/ml) to select for the ampicillin-resistant Cronobacter (amp::lux) strains. These bacterial strains were used to artificially contaminate RIF.

Table 6.1. lux+ strains of Cronobacter (amp::lux) that were used for artificially contaminating of infant formula and TSB.

Genus Species/Serovar Identification number 1 Cronobacter sakazakii 2870-1 2 Cronobacter sakazakii 2876-3 3 Cronobacter sakazakii 3253-2

Five phages, previously isolated and characterized (Chapter 2), were used in this

study for phage biocontrol experiments in TSB and RIF. In order to propagate and titre

the phages, their susceptible bacterial host strains (obtained from CRIFS Culture

Collection; Table 6.2) were used, and all the media were supplemented with CaCl2 to a

final concentration of 2 mM (Fisher Chemicals, Mississauga, ON, Canada). Pure cultures

were obtained from frozen stocks kept at -80°C, and maintained at 4°C on TSA. To

maintain cell viability, cultures were re-streaked biweekly onto fresh medium.

The phages were propagated to high titre and purified using methods described in

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Chapters 2 and 3. The highly purified, high-titer phage was stored as stock at 4ºC until further application.

Table 6.2. Selected bacteriophages and the susceptible bacterial hosts used for propagation and titration.

CRIFS culture collection Phage Bacterial Host number of the host vB_CsaM_GAP31 Cronobacter muytjensii 51329 vB_CsaM_GAP32 Cronobacter sakazakii 3290 vB_CsaP_GAP52 Cronobacter sakazakii 3199 vB_CsaM_GAP161 Cronobacter sakazakii 3253 vB_CsaP_GAP227 Cronobacter malonaticus 3267

To prepare the phage cocktail, the five Cronobacter phages were mixed together.

The titration of each phage and the phage cocktail was performed by preparing serial 10- fold dilutions in SM buffer and tested using the previously described overlay method.

The phages and the phage cocktail were stored at 4°C.

6.3.2. Host Range Determination

The host range of the five selected phages against the three luminescent

Cronobacter (amp::lux) strains was determined by the spot test as described in Chapter 2.

A 6 h culture of each Cronobacter strain was inoculated on TSA (containing ampicillin,

50 μg/ml) plates by sterile cotton swabs and allowed to dry. Three drops of 20 µl of each phage sample were spotted onto the plate and allowed to dry. The plates were examined for zone of lysis, indicated by the presence of plaques, after 16 h incubation at 30ºC. The results were recorded as 3 (very clear zone of complete lysis on the bacterial lawn), 2

(clear/turbid zone of lysis), 1 (turbid lysis) and 0 (no lysis).

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6.3.3. Effect of the Phage Cocktail on C. sakazakii Strains in Broth and in

Reconstituted Infant Formula

To investigate the effect of the cocktail of five CsCl-purified phages on the three

C. sakazakii strains in TSB and RIF, each bacterial strain was grown in TSB broth overnight (18 h) at 30°C. The culture was diluted and added at different concentrations to

TSB or RIF in wells of sterile, 96-well, flat, clear-bottomed, polystyrene, black microplates (Corning Inc., Corning, NY, USA).

Shortly thereafter, the cocktail of phages was added to achieve a final concentration of 108 PFU/ml. The content of each well consisted of: bacteria, phages, and

medium (TSB or RIF). In the control groups, the bacteria, phages and RIF were replaced

by TSB, SM buffer and water, respectively. The final bacterial titer was 101, 102, 103, or

104 CFU/ml resulting in four multiplicities of infection (MOI - the ratio of the phage numbers to the number of bacterial cells, which was measured at the beginning of the experiment) of approximately 107 to 104. The final volume in each well was 210 μl, and

microplates were incubated for 24 h at 24°C (room temperature). For the experiment in

infant formula, a milk-based, powdered infant formula for premature neonates and infants

(4.1% fat; Similac Advance Neosure, Abbott, Ohio, USA) was purchased and

reconstituted using sterile water following the manufacturer’s recommendation on the

package. Table 6.3 shows the composition of the infant formula used in this assay.

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Table 6.3. Approximate analysis of Similac Advance Neosure infant formula used in the experiment as declared by the manufacturer.

Ingredient Per 100g of Powder Units Standard Dilution /100ml* Energy 2154 (515) kJ (Cal) 314 (75) Protein 14.4 g 2.1 Fat 28.3 g 4.1 Linoleic acid 3.86 g 0.564 Linolenic acid 0.515 g 0.075 Arachidonic acid 0.100 g 0.015 Docosahexaenoic acid 0.038 g 0.006 Carbohydrates 52.0 g 7.6 Ash 3.6 g 0.5 Moisture 2 g - Sodium 170 mg 25 Potasium 731 mg 107 Chloride 386 mg 56 Vitamin A 2368 IU 346 Vitamin D3 360 IU 53 Vitamin E 19 IU 2.7 Vitamin K1 0.057 mg 0.0083 Vitamin C 77 mg 11 Thiamine 1.13 mg 0.165 Riboflavin 0.772 mg 0.113 Niacin 10 mg 1.47 Vitamin B6 0.515 mg 0.075 Folic acid 0.129 mg 0.0188 Vitamin B12 0.00206 mg 0.00030 Pantothenic acid 4.12 mg 0.601 Biotin 0.0463 mg 0.0068 Calcium 541 mg 79 Phosphorus 319 mg 47 Magnesium 46 mg 7 Iron 9.3 mg 1.4 Zinc 6.2 mg 0.9 Copper 0.618 mg 0.0902 Manganese 0.0515 mg 0.0075 Iodine 0.0772 mg 0.0113 Selenium 0.0107 mg 0.00156 Choline 82 mg 12 Taurine 31 mg 4.5 Carnitine 28 mg 4.1 Inositol 180 mg 26 Nucleotides 49 mg 7.2 * Based on preparation by adding 1 unpacked level scoop of the powder formula to 60 ml of water.

In addition, to determine the possible contamination of the purchased infant formula with Cronobacter, the RIF was serially diluted in sterile 0.85% saline solution

(Sigma-Aldrich Canada Ltd.), and 100 µl of RIF and its dilutions were surface plated in

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duplicate onto Brilliance Enterobacter sakazakii Agar (DFI formulation) (Oxoid,

Fisher Scientific) a selective and differential medium, and incubated at 37°C.

The effect of various MOI values for the phage cocktail on the growth of the

cocktail of C. sakazakii strains in TSB and infant formula was determined by measuring

the luminescence produced by live bacteria. The amount of the luminescence produced in

each well was measured using a VICTOR3V 1420 Multilabel Plate Counter (Wallac Oy,

Turku, Finland) as counts per second (CPS) at 2 h intervals from 0 to 24 h. The experimental parameters used for all experiments were as follows: shaking for 5 s before measurements; 24°C incubation temperature; measurement time for 5 s; measuring mode by plate.

For the TSB experiment, the challenge wells contained TSB, phage, and bacteria

(initial concentrations of 101, 102, 103, or 104 CFU/ml). The control wells contained either phage and TSB, only TSB, or bacteria (at initial concentrations of 101, 102, 103, or

104 CFU/ml) and TSB, with the same total volume as test wells.

In the infant formula experiment, the challenge wells contained RIF, phage, and bacteria (initial concentrations of 101, 102, 103, or 104 CFU/ml). The wells of control

groups contained either phage and TSB and water, phage and TSB and RIF, RIF and TSB

and SM buffer, only TSB, bacteria at different initial concentrations (101, 102, 103, or 104

CFU/ml) and SM buffer and water, or bacteria (initial concentration of 101, 102, 103, or

104 CFU/ml) and SM buffer and RIF, with the same total volume as tested wells. All the

titers are final concentrations in the microtitre plate wells.

In addition, in order to determine the effect of phage and RIF diluents (SM buffer

and water, respectively) on the growth of Cronobacter strains, four control groups were

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considered containing different initial bacterial concentrations (101, 102, 103, or 104

CFU/ml) and SM buffer or water. These controls were called “medium controls”.

The correlation between CPS and CFU was determined in another experiment.

The wells contained i) RIF and phage and bacteria; ii) RIF and SM buffer and bacteria; iii) TSB and phage and bacteria; or iv) TSB and SM buffer and bacteria. The microplates were incubated at 24°C, and the initial concentrations of phages and bacteria were 108

PFU/ml and 105 CFU/ml, respectively. The CPS and CFU for each treatment were measured at 0, 4, 8, 12, and 24 h of incubation. To measure the plate count, samples were serially diluted in sterile 0.85% saline solution, and each dilution (100 µl) was surface plated in duplicate onto TSA containing ampicillin. After incubation at 37°C for 24 h, only colonies with bioluminescence were counted using the Night Owl Molecular Light

Imager (EG&G Berthold Technologies, Munich, Germany). The colonies were counted and the bacterial numbers in each broth or RIF sample were calculated (CFU/ml).

In each experiment all samples were tested in triplicate, and every experiment was repeated three times. The means of the triplicate CPS or plate count data were calculated and a growth curve was developed for each MOI value used in each experiment.

Appropriate negative controls (without addition of bacteria or bacteriophage) were tested along with inoculated samples. The data shown in the graphs are the averages of triplicate experiments, and the error bars indicate the standard deviation.

6.3.4. Statistical Analysis

The statistical analysis of the data was accomplished using Proc GLM and REG software, version 9.3 of the SAS (SAS Institute Inc., Cary, NC, USA). The Proc GLM

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was used for the analyses of variance (ANOVA). In all cases, the likelihood of differences between means occurring due to chance had to be less than 5%, P < 0.05, to be considered statistically significant.

6.4. RESULTS

6.4.1. Host Range

The lysates of five selected Cronobacter phages were tested against three bioluminescent strains of Cronobacter carrying an amp::lux fusion. The results were recorded from 0 to 3, based on the clearness of the plaque (0: no lysis; 3: very clear zone of complete lysis). Table 6.4 shows the host range of the selected phages. Phage GAP161 showed the broadest host range and strongest lytic activity. Phage GAP227 was also able to infect all three bacterial strains. On the other hand, phage GAP32 was only able to infect one Cronobacter (amp::lux) strain. C. sakazakii 2870-1 is susceptible to all five selected phages.

Table 6.4. The host range of five selected Cronobacter phages against luminescent strains of Cronobacter (amp::lux) based on the spot test (16 h incubation at 30ºC). The results were recorded as 3 (very clear zone of complete lysis on the bacterial lawn), 2 (clear/turbid zone of lysis), 1 (turbid lysis) and 0 (no lysis).

Phage Isolates

GAP31 GAP32 GAP52 GAP161 GAP227

Bacteria C. sakazakii 2870-1 3 2 3 3 2 C. sakazakii 2876-3 0 0 3 3 2 C. sakazakii 3253-2 2 0 0 3 2

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6.4.2. Effect of the Phage Cocktail on the Mixture of C. sakazakii Strains in

Broth

The VICTOR3V was used to determine the effect of the cocktail of five phages on the simultaneous growth of three C. sakazakii (amp::lux) strains (MOIs of approximately

104 to 107) in the liquid medium (TSB) by measuring the amount of luminescence produced during incubation for 24 hours at 24°C. For all tested MOI values, the addition of phage cocktail resulted in effective inhibition of the bacterial growth throughout the whole experiment. The CPS values of all samples treated with phage at 24 h were significantly lower than the CPS in the positive control groups (TSB and bacteria with initial concentration of 101-104 CFU/ml). Also, the CPS values of all these samples at 24 h p.i. (post inoculation) were not significantly higher than their CPS at 0 h p.i. (Fig. 6.1;

Table 6.5).

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Figure 6.1. Effect of the phage cocktail on the growth of different concentrations of the mixture of the three C. sakazakii strains in liquid medium (TSB); a) 101 CFU/ml (MOI of 107), b) 102 CFU/ml (MOI of 106), c) 103 CFU/ml (MOI of 105), and d) 104 CFU/ml (MOI of 104). The error bars indicate the standard deviation.

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When the Cronobacter spp. at initial concentrations of 101-103 CFU/ml were treated with phage, no significant changes in CPS values during 24 hours of incubation at

24°C were observed. The CPS values for initial population levels of 104 CFU/ml treated

with phage decreased from 453 + 9 at 0 h p.i. until it reached 209 + 2 after 10 h, which is

similar to the CPS values of negative controls (phage and TSB, or only TSB). However,

after 20 hours of incubation, this group showed bacterial re-growth and a slight increase

until 24 h when the CPS value reached 267 + 85 compared to 197 + 1 in the negative

control. These values are not significantly different (P > 0.05). In general, the CPS values of the phage treated groups with initial bacterial titre of 101-103 CFU/ml were not

significantly higher (P > 0.05) than the CPS values of negative controls throughout the

experiment. Likewise for phage treatment of bacterial populations of 104 CFU/ml, the

CPS values were not significantly higher than the negative controls after 8 h p.i. The CPS values for both negative controls were similar (phage and TSB, or only TSB). On the other hand, the CPS values in the positive control groups with initial bacterial concentrations of 101-104 CFU/ml showed increases of more than 2 log after 24 h

incubation (Fig. 6.1; Table 6.5). At the end of the experiment, the final CPS values

ranged from 196 + 1 to 267 + 85 for phage treated groups with MOIs of approximately

107 to 104, which are significantly lower than the CPS values of 39306 + 3852 to 58473 +

5068 observed in positive control groups with initial Cronobacter numbers of 101 to 104

CFU/ml, respectively.

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Table 6.5. Effect of phage cocktail on the growth of different concentrations of the mixure of three C. sakazakii strains in TSB. Values are the averages and standard deviations of luminescence (CPS) produced by live bacteria.

Bacteria Phage + Bacteria TSB 101 102 103 104 101 102 103 104 192 193 208 359 188 191 213 453 186 0 + 2 + 1 + 2 + 5 + 2 + 3 + 4 + 9 + 5 196 199 253 807 191 189 205 329 192 2 + 2 + 2 + 3 + 28 + 3 + 3 + 2 + 10 + 4 188 201 299 1255 196 198 213 374 196 4 + 3 + 2 + 11 + 28 + 2 + 3 + 5 + 8 + 1 199 223 620 6698 193 198 246 297 196 6 + 4 + 4 + 13 + 408 + 5 + 3 + 6 + 13 + 2 211 365 3271 10055 196 219 224 214 192 8 + 3 + 12 + 209 + 1037 + 2 + 7 + 9 + 2 + 5 306 2227 10189 13743 199 204 202 209 194

10 + 17 + 100 + 106 + 525 + 3 + 2 + 2 + 2 + 3 1249 8582 11129 23310 199 199 195 199 195 12 + 246 + 825 + 880 + 1777 + 10 + 9 + 5 + 2 + 2

Time (h) 6771 9362 19193 28314 198 196 198 202 194 14 + 1281 + 546 + 1726 + 2713 + 2 + 2 + 4 + 3 + 3 15692 16549 21712 32276 195 198 198 199 199 16 + 2258 + 529 + 2379 + 4594 + 5 + 0 + 5 + 5 + 2 19363 19978 28892 36896 195 196 201 206 197 18 + 5404 + 700 + 3011 + 5168 + 2 + 4 + 2 + 6 + 4 23914 36045 36107 39113 198 197 202 217 200 20 + 4398 + 4125 + 2927 + 4772 + 0 + 6 + 6 + 17 + 4 37492 47330 40056 39741 198 195 201 231 198 22 + 9481 + 1838 + 2326 + 4683 + 2 + 4 + 5 + 44 + 7 56488 58473 41108 39306 202 196 202 267 197 24 + 13034 + 5068 + 2255 + 3852 + 10 + 1 + 8 + 85 + 1

6.4.3. Effect of the Phage Cocktail on the Mixture of C. sakazakii Strains in

Reconstituted Infant Formula

To investigate the biocontrol of C. sakazakii by phage in RIF, the inhibitory effect of the phage cocktail (MOIs of approximately 104 to 107) on the growth of different concentrations of the C. sakazakii strain mixture in RIF was determined by measuring the amount of the luminescence as CPS for 24 hours at room temperature (24°C).

The cocktail of five phages in RIF was able to efficiently inhibit the growth of C. sakazakii at all four bacterial concentrations tested (initially 101-104 CFU/ml). Treatment

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of Cronobacter present at initial concentrations of 101-103 CFU/ml with phage resulted in

a constant luminescent output throughout the incubation period, and the CPS values

remained at a level similar to those of the negative controls (phage and RIF and TSB; SM

buffer and RIF and TSB; phage and SM buffer and water; or phage and TSB and SM

buffer) (Fig. 6.2; Table 6.6). The CPS values for all negative controls were similar. The

luminescence in the group with an initial bacterial concentration of 104 CFU/ml and

treated with phage decreased from 470 + 142 at time 0 to 199 + 3 at 2 h p.i., which was

similar to luminescence values observed for the negative control groups, and remained at

that level for the rest of the experiment. The changes in CPS in this group between 2 h and 6 h p.i. (209 + 12) were not significant. On the other hand, the bacteria-positive

control groups (containing only bacteria with an initial concentration of 101, 102, 103, or

104 CFU/ml) showed growth and their CPS values increased by 3 log cycles by the end of

the incubation period. In general, the CPS values of the phage treated groups with initial

bacterial titre of 101-103 CFU/ml were not significantly higher than the CPS values of

negative controls throughout the experiment. Likewise for phage treated Cronobacter at

an initial population 104 CFU/ml, the CPS values were not significantly higher than the negative controls after 6 h p.i. At the end of the experiment, the final CPS values were approximately 200 in all phage treated groups (MOIs of approximately 107 to 104), as

compared to values of about 300,000 and greater in bacteria-positive control groups with

initial concentrations of 101-104 CFU/ml (Fig. 6.2; Table 6.6).

134

135

Figure 6.2. Effect of the phage cocktail on the growth of different concentrations of the mixture of the three C. sakazakii strains in RIF; a) 101 CFU/ml (MOI of 107), b) 102 CFU/ml (MOI of 106), c) 103 CFU/ml (MOI of 105), and d) 104 CFU/ml (MOI of 104). The error bars indicate the standard deviation.

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Table 6.6. Effect of phage cocktail on the growth of different concentrations of three C. sakazakii in RIF. Values are the averages and standard deviations of luminescence (CPS) produced by live bacteria in phage treated, positive (Bacteria + SM buffer + RIF), negative (Phage+ TSB + RIF), and medium (Bacteria + SM buffer + Water) control groups.

Bacteria + SM buffer + RIF Phage + Bacteria Phage+ TSB + RIF Bacteria + SM buffer + Water

101 102 103 104 101 102 103 104 101 102 103 104 195 202 243 695 197 194 222 470 192 190 197 216 450 0 + 6 + 7 + 6 + 41 + 1 + 5 + 15 + 142 + 5 + 6 + 4 + 2 + 15 194 196 254 858 192 196 199 199 193 192 200 242 621 2 + 6 + 4 + 10 + 105 + 1 + 1 + 3 + 3 + 4 + 1 + 4 + 4 + 57 194 205 259 1407 191 194 199 220 188 192 201 291 1315 4 + 4 + 5 + 11 + 250 + 3 + 0 + 0 + 9 + 3 + 5 + 4 + 12 + 170 201 229 1288 33253 199 196 197 209 192 196 249 946 18229 6 + 7 + 8 + 227 + 8218 + 0 + 6 + 3 + 12 + 3 + 3 + 22 + 141 + 3269 247 1253 30607 163595 192 196 194 204 198 236 810 13706 22297 8 + 22 + 343 + 5554 + 25969 + 6 + 4 + 5 + 5 + 4 + 5 + 137 + 2811 + 2577 1370 28308 166136 254731 196 193 194 200 195 605 9867 25333 43955

10 + 510 + 8806 + 14109 + 37392 + 1 + 4 + 5 + 3 + 3 + 68 + 2449 + 1638 + 4291 31978 206542 278612 275962 193 197 196 201 201 8229 31088 40298 64570 12 + 11917 + 34432 + 19726 + 48132 + 5 + 2 + 2 + 2 + 3 + 491 + 4987 + 4887 + 8805

Time (h) 147320 369999 324203 362915 199 196 204 201 199 38130 45817 61766 78604 14 + 36636 + 33464 + 1845 + 40556 + 2 + 2 + 4 + 3 + 6 + 4041 + 6823 + 5594 + 8534 228897 405087 385609 340943 201 201 208 205 196 42580 62994 68195 69908 16 + 56808 + 47639 + 22612 + 54465 + 4 + 5 + 2 + 1 + 3 + 7545 + 14993 + 10973 + 11964 258429 546838 389937 354020 202 201 208 208 194 69962 84411 66101 58800 18 + 66086 + 54522 + 30821 + 55102 + 2 + 2 + 9 + 2 + 3 + 14007 + 17853 + 9399 + 9551 364305 567396 445744 371794 199 201 208 209 200 104090 94755 59814 53823 20 + 74406 + 41443 + 13875 + 46596 + 1 + 6 + 4 + 4 + 4 + 21634 + 14757 + 6573 + 5768 332497 540852 399211 318450 206 201 209 204 207 107267 80875 45875 40293 22 + 67058 + 47243 + 26844 + 52697 + 3 + 6 + 6 + 1 + 0 + 19336 + 16270 + 6484 + 6107 349994 511282 348408 282678 203 197 206 200 201 102881 72663 37751 32799 24 + 62464 + 41357 + 25544 + 50366 + 3 + 8 + 5 + 1 + 1 + 19586 + 14620 + 4667 + 4886

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The growth of the Cronobacter strains was slower in the “medium control”

groups than in RIF (Fig. 6.2; Table 6.6). At the end of the experiment, the final CPS

values of “medium control” groups with initial concentrations of 101-104 CFU/ml were

between approximately 33,000 and 100,000, which is significantly lower than observed

in control groups with the same initial concentrations of bacteria (approximately 280,000

to 510,000). However, these values were significantly higher than phage treated groups

and negative control groups.

The samples of RIF and its dilutions that were surface plated onto Brilliance

Enterobacter sakazakii Agar to determine possible contamination of the purchased infant

formula with Cronobacter, resulted in no blue-green colonies indicative of the presence

of the organism after 24 h incubation at 37°C.

6.4.4. The Correlation Between Luminescence and Plate Count

To determine the correlation between CPS and CFU/ml, the effect of phage mixture on three bioluminescent C. sakazakii strains in TSB and RIF was determined.

The initial concentrations of phage and bacteria were 108 PFU/ml and 105 CFU/ml,

respectively (MOI of approximately 103), and they were incubated at 24°C for 24 h. The

CPS values were measured using the VICTOR3V and the CFU/ml values were obtained by plating on TSA containing ampicillin and counting bioluminescent colonies using the

Night Owl. The correlation between the average of recorded CPS values and the average of the CFU/ml values was 0.905. The colonies were counted and the bacterial numbers were calculated (CFU/ml) in each sample in TSB or RIF.

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The phage cocktail was able to reduce the outgrowth of bacteria in both TSB and

RIF. In RIF, both luminescence and colony count showed that bacterial growth increased between 0 h to 24 h in the control group (bacteria and RIF), from 496.80 CPS and 1.19 ×

105 CFU/ml, to 874338 CPS and 5.83 × 108 CFU/ml, respectively (Fig. 6.3; Table 6.7).

On the other hand, in the phage treated group bacterial numbers increased from 0 h p.i.

(473 CPS; 1.06 × 104 CFU/ml) to 12 h p.i. (13191 CPS; 1.27 × 107 CFU/ml), but then declined at 24 h p.i. (1471 CPS; 7.97 × 106 CFU/ml). Throughout the 24 hours of

incubation at room temperature with initial concentrations of phage and bacteria of 108

PFU/ml and 105 CFU/ml, respectively, the phage cocktail could reduce the bacterial counts in RIF by 1-2 log CFU/ml at all sampling times compared to the control groups.

In broth (TSB), the Cronobacter count of the control group (bacteria and TSB) increased from 0 h p.i. (1.31 × 105 CFU/ml) to 12 h p.i. (2.24 × 108 CFU/ml), and then decreased slightly at 24 h p.i. (1.91 × 108 CFU/ml). However, this reduction is not significant. On the other hand, the CPS values for these groups increased constantly from

0 h p.i. (329) to the end of experiment (79573). In the groups treated by phage, the colony count increased by 1 log cycle between 0 h and 4 h, but it was reduced by 2 logs at 8 h, then increased again until 24 h p.i. The CPS values did not change significantly during storage from 0 h to 4 h, but then decreased until 12 h p.i., followed by an increase until the end of the incubation period. In general, the phage cocktail reduced the bacterial counts in TSB by 1-4 log CFU/ml compared to the control groups.

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Figure 6.3. Effect of phage cocktail on the growth of the mixture of three C. sakazakii strains (initial concentration of 105 CFU/ml) in RIF and TSB. Values are the averages and standard deviations of a) colony count (CFU/ml), and b) luminescence (CPS) produced by live bacteria. The error bars indicate the standard deviation.

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Table 6.7. Effect of phage cocktail on the growth of the mixture of three bioluminescent C. sakazakii strains (initial concentration of 105 CFU/ml) in RIF and TSB. Values are the averages and standard deviations of luminescence (CPS) produced by live bacteria, and also colony count (CFU/ml). Four combinations are: 1 (bacteria and RIF), 2 (bacteria, phage and RIF), 3 (bacteria and TSB), and 4 (bacteria, phage and TSB).

Luminescence (CPS) Colony Count (CFU/ml)

1 2 3 4 1 2 3 4 Combination 0 496 473 329 319 1.19 × 105 1.06 × 104 1.31 × 105 1.04 × 104 + 43 + 22 + 8 + 14 + 2.66 × 104 + 1.76 × 103 + 6.23 × 104 + 1.99 × 103 4 1191 281 1183 315 1.47 × 106 4.20 × 104 2.50 × 106 1.11 × 105 + 192 + 6 + 59 + 7 + 1.73 × 105 + 6.56 × 103 + 9.10 × 105 + 3.94 × 104 8 171830 1364 33222 257 6.63 × 107 5.97 × 105 1.79 × 107 1.17 × 103 + 19002 + 13 + 1734 + 6 + 1.50 × 107 + 2.08 × 104 + 5.57 × 105 + 1.44 × 103 8 7 8 4 Time (h) 12 331272 13191 51315 221 1.62 × 10 1.27 × 10 2.24 × 10 3.46 × 10 + 47193 + 790 + 2709 + 9 + 3.41 × 107 + 2.42 × 106 + 2.21 × 107 + 5.92 × 104 24 874338 1471 79573 1003 5.83 × 108 7.97 × 106 1.91 × 108 4.16 × 106 + 71804 + 520 + 25155 + 579 + 1.39 × 108 + 1.25 × 106 + 1.42 × 107 + 5.21 × 106

6.5. DISCUSSION

Consumption of contaminated reconstituted infant formula has been linked in several cases with Cronobacter infection in neonates and infants (Biering et al., 1989;

Bowen & Braden, 2006; Centers for Disease Control and Prevention (CDC), 2002; Clark et al., 1990; Gurtler, Kornacki, & Beuchat, 2005; Jarvis, 2005; Muytjens et al., 1983;

Muytjens & Kollee, 1990; Noriega et al., 1990). Cronobacter spp. possess physiological characteristics that enable RIF to serve as a vehicle for infection, such as: surviving desiccation and osmotic stress for long periods, longer than other species of

Enterobacteriaceae (Breeuwer et al., 2003); the ability to grow at a wide range of temperatures from 6 to 45°C (Iversen, Lane, & Forsythe, 2004); ability to form biofilms on the surface of different materials, including polycarbonate that is often used to make baby bottles (Iversen, Lane, & Forsythe, 2004; Lehner et al., 2005); and being more heat- resistant than other Enterobacteriaceae in RIF (Nazarowec-White & Farber, 1997b). Dry

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conditions, osmotic stress, and heat could be the forces that select for Cronobacter spp. in powdered infant formula and RIF over Campylobacter jejuni and Enterobacteriaceae, including Salmonella Typhimurium, E. coli, and Yersinia enterocolitica. Several methods have been proposed to control this pathogen and minimize transmission by RIF, including using hot water or microwaves, high pressure processing or gamma irradiation

(Edelson-Mammel & Buchanan, 2004; Gonzalez et al., 2006; Kindle et al., 1996; Lee et al., 2006). However, due to the disadvantages of these methods, such as potential loss of nutrients, risk of scalding of infants, cost efficiency and not being feasible, they are not well accepted.

One of the novel technologies applied to control bacterial pathogens in food is exploiting natural agents such as bacteriophages. Bacteriophages are non-destructive biocontrol agents that can be as effective as chemicals (Leverentz et al., 2001), and they have been used successfully against undesirable bacteria, and some of them have been approved for use in food (FDA, 2006).

In this study, we have applied a cocktail of five highly-purified phages in RIF for biocontrol of three C. sakazakii strains. These Cronobacter phages were not able to infect

E. coli strains (Chapter 2). E. coli is a common commensal of the human (including neonates) gut; therefore selected phages would not have lytic activity on these gut commensal bacteria. Also, selected phages were not able to infect Lactobacillus rhamnosus and Lactobacillus paracasei (Chapter 2), the probiotics that are used in infant formula to reduce the adhesion of Cronobacter to mucosal cells, and, therefore, its infection risk (Collado, Isolauri, & Salminen, 2008). Thus, the application of our selected phages in infant formulae would not affect probiotics with proven antibacterial effect on

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Cronobacters that are potentially present in these food products. However, the effect of

our phages on other probiotic bacteria used in infant formulae, such as Bifidobacteria,

needs to be investigated. In addition, the combination of these five phages can strongly

infect and lyse 100% of 17 Cronobacter sakazakii strains tested (14 strains in Chapter 2,

and 3 C. sakazakii (amp::lux) strains in this Chapter), and more than 91% of all 24

Cronobacter strain tested. Only two strains (C. universalis 3287 and C. malonaticus

1154/04) were weakly affected. C. malonaticus 1154/04 was also resistant to two

Cronobacter phages tested by Kim et al. (Kim, Klumpp, & Loessner, 2007). We have

noted that the lytic activity of the selected phages against the C. sakazakii lux+ mutant

strains (Table 6.4) is different from their lytic activity against the original wild (non- mutant) strains (Table 2.4). These differences could be due to the insertion of the lux

gene to the mutant strains, which affected their sensitivity to the phages. Phage resistance may be the result of lacking the specific receptor for the phages to attach or developing the resistance mechanisms [e.g., adsorption blocking (altering the membrane receptors), abortive infection, modifying the restriction endonuclease system] (Petty et al., 2007).

The use of highly purified phages imitates the real phage application in food products, because purification by density-gradient (CsCl) ultracentrifugation ensures the minimum possible level of contaminants (bacterial debris, medium components, and potential toxins or virulence factors, such as lipopolysaccharide from C. sakazakii) that

remain in the phage preparations even after regular centrifugation and filtration.

First, the effect of phage at four different MOIs (104 to 107) on C. sakazakii

strains and their growth in liquid medium (TSB) at room temperature (24°C) was

investigated. Based on observed luminescence produced by metabolically active bacteria,

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the addition of phage cocktail resulted in strong inhibition of bacterial growth for all

tested MOI values during the 24 hours of the experiment (Fig. 6.1; Table 6.5). The CPS

values of all samples treated with phage at 24 h were not only significantly lower than the

CPS in the positive control groups (only bacteria with initial concentration of 101-104

CFU/ml), but also the CPS values of all these samples at 24 h p.i. were not significantly

higher than the CPS observed upon inoculation with Cronobacter. The low CPS at time 0

in the groups with MOI of 107 and 106 could be due to the “lysis from without”

phenomenon (Delbruck, 1940). The CPS values in medium inoculated with 104 CFU/ml

and treated with phage decreased between 0 h and 10 hours until it reached levels similar

to those observed in negative controls, but at 20 h there was evidence of bacterial growth.

In a previous study, Kim et al. (2007) observed inhibition at 24°C using 109

PFU/ml of two phages separately against their single sensitive Cronobacter strain in

liquid medium (BHI), however, the inhibition started after 6 hours of incubation, and

although one of the phages (ESP 1-3) could reduce the turbidity of the medium, none of

the phage concentrations (107, 108, or 109 PFU/ml) could decrease the turbidity to the

initial level at the time of inoculation.

In another study on the biocontrol of Cronobacter in BHI broth by phage, Zuber and colleagues (2008) used two phages separately against single phage-sensitive

Cronobacter strains present at an initial bacterial concentration of 106 CFU/ml, and

phage titre of 108 PFU/ml at 30°C (Zuber et al., 2008). At an MOI of 102, after 3 h of outgrowth of the bacterium, the phage started to inhibit the growth that was observed until 6 h p.i., when the turbidity of the medium reached a similar level to that observed at the start of the experiment. However, after 12 h incubation bacterial growth was observed

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for one of the phages (F). These researchers also investigated the effect of phage F at

MOIs of 106 and 104 on 102 CFU/ml bacteria. At the MOI of 106, no bacterial growth was

observed until 9 h p.i. At a MOI of 104, bacteria grew for 6 h and at 7 h p.i., the cell count

decreased until 10 h p.i., however it was not reduced to the initial level. Therefore, our

phage cocktail is more effective against a cocktail of C. sakazakii strains in broth, than the phages in previous studies.

In general, our experiment on the efficiency of our phage cocktail against C. sakazakii strains in RIF was similar to that observed in TSB. The CPS values in RIF inoculated with 104 CFU/ml Cronobacter cells and treated with phage decreased after 2 h

incubation until it reached a level similar to that observed in uninoculated samples, and

remained at that level for the rest of the incubation period. Thus, the antibacterial effect

of the phage was observed 8 h earlier in RIF than in TSB.

Kim and colleagues (2007) obtained similar results in RIF with the greatest

degree of inhibition being achieved at the highest phage concentration (109 PFU/ml).

Both phages (ESP 1-3 and ESP 732-1) were most effective at 24°C, and phage concentrations of 108 and 109 PFU/ml were able to reduce the bacterial level to < 10

CFU/ml from 2 h until 14 h p.i., which was the length of the incubation period used in

their study. However, at the concentration of 108 PFU/ml of ESP 732-1 there was substantial growth of the Cronobacter strain tested. Also, none of the phages were able to inhibit the growth of the bacterium when present at phage populations of 107 PFU/ml.

Based on the contamination level that the authors mentioned (73,000 CFU/100 g) the bacterial level would be approximately 102 CFU/ml (based on the manufacturer’s

recommendation to prepare the RIF, as ~10g in 60 ml water), with a resulting MOI of

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105. Therefore, their phages were able to inhibit the single Cronobacter strain only at

MOIs of 106 and 107.

Zuber et al. (2008) have used a cocktail of five phages for the biocontrol of

Cronobacter spp. in RIF using 102 CFU/ml and 108 PFU/ml as initial bacterial and phage concentrations (MOI of 106), respectively. They reported that their phage cocktail could prevent the growth of 90% of the 40 tested Cronobacter strains tested over a 4-h incubation at 30°C. However, this incubation period was relatively short and does not give the chance to observe possible growth of bacteria due to emergence of phage- resistant mutants. In addition, the phage cocktail was not able to prevent the growth of the test strains at an MOI of 104. However, our phage cocktail was able to inhibit the growth of a cocktail of C. sakazakii strains (not just a single strain) even at an initial bacterial load of 104 CFU/ml and at MOIs as low as 104, suggesting that better biocontrol is achieved in RIF than previous studies.

There was a strong correlation (r = 0.905) between the CPS values and the

CFU/ml values for the luminescent strains of Cronobacter used in this study. This shows that using C. sakazakii (amp::lux) strains and measuring the CPS values by VICTOR3V is comparable with the traditional method of colony counting on plates and calculating the

CFU. In general, using CPS could be more cost efficient and less labor intensive than traditional methods. Although at a MOI of 103 the phage cocktail used in this study could reduce bacterial numbers by 2 to 3 log cycles (in TSB and RIF), the growth inhibition was not as strong as that obtained at MOIs of 104 to 107 in either TSB or RIF. However, it should be noted that the initial contamination level of 105 CFU/g may be unrealistic for natural levels of contamination of RIF by Cronobacter spp. Considering that the average

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generation time at room temperature (23°C) for Cronobacter is 40 minutes

(NazarowecWhite & Farber, 1997b) and the level of contamination is usually low (<1

CFU/100g) in powdered infant formula (FAO/WHO, 2004; FAO/WHO, 2006; Muytjens,

Roelofs-Willemse, & Jaspar, 1988; Nazarowec-White & Farber, 1997a), hypothetically,

it would take 16 h or more at room temperature for Cronobacter to reach levels of 105

CFU/ml in RIF.

Overall, at MOIs greater than 104 our phage cocktail more effectively inhibited

growth of the Cronobacter sakazakii strains studied when they were present in RIF rather

than in TSB. This is consistent with the results reported by Kim et al. (2007) and Zuber et

al. (2008). However, these previous studies focused on the effect of a single phage on a

single Cronobacter strain, although this scenario is unlikely to occur naturally. Using a phage cocktail is recommended to broaden the host range and overcome bacterial phage-

resistance (McIntyre et al., 2007; Hagens & Loessner, 2010). However, Kim et al. (2007)

did not employ a phage cocktail, and Zuber and colleagues (2008) applied their phage

cocktail for just a short period of time (4 h) at 30°C. Since usually the level of

Cronobacter contamination is low in PIF and growth occurs after rehydration and

preparation of formula when it is left at room temperature before use (FAO/WHO, 2004;

FAO/WHO, 2006; Muytjens, Roelofs-Willemse, & Jaspar, 1988; Nazarowec-White &

Farber, 1997a), incubation at 30°C does not seem realistic in most circumstances.

In this study, we investigated the effect of phage on Cronobacter spp. in infant formula specially prepared for premature neonates and infants. Premature neonates and infants are more susceptible to Cronobacter infection (Gurtler, Kornacki, & Beuchat,

2005; Pagotto et al., 2009), and they must be fed by infant formulae that meet their

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nutritional needs, which may not solely be provided by consuming breast milk. The

ingredients and their doses may differ between regular infant formulae and those

prepared for premature infants, and these factors may affect the efficiency of phage

biocontrol. For example, the fat content (including fatty acids) of the infant formula that

we used is 4.1% (Table 6.3), compared with a fat content of 3.5% in the formula used by

Kim et al. (2007) (Zuber and colleagues (2008) did not specify the fat content of their

formula). It has been shown that in the presence of exogenous unsaturated fatty acids, the

fatty acid composition of the membrane of the host bacterium (in the case of the study

cited it was S. Typhimurium) was altered, and phage genes showed differential

expression depending on the fat composition of the medium (Goyal & Chakravorty,

1989). Supplementation of culture with some fatty acids, such as linoleic and linolelaidic

acids, which are present in infant formula, but not oleic and palmitoleic acids, could

induce phage specific lysozyme and lead to bacterial count reduction. This could be a

reason for the better phage performance in RIF than in TSB. In another study on the

effect of fatty acids on phages, Reinhardt et al. (1978) have reported that palmitoleic and

oleic acids inhibited the entry process of lipid-containing phage PR4 into E. coli cells

(Reinhardt, Cadden, & Sands, 1978). Therefore, investigating phage biocontrol of

Cronobacter in premature infant formulae should be the priority, and the results could be

different for regular formulae. However, the other two studies (Kim, Klumpp, &

Loessner, 2007; Zuber et al., 2008) did not report whether the milk powders they used were formulated for premature babies.

In these previous studies on effect of phage on Cronobacter in infant formula

(Kim, Klumpp, & Loessner, 2007; Zuber et al., 2008), enumeration of bacteria was

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performed using non-selective and non-differential medium (BHI agar) and only yellow colonies, thought to be indicative of Cronobacter spp. were counted. However, it has been reported that not all Cronobacter strains produce yellow colonies (Druggan et al.,

2004; Farmer III et al., 1980; Iversen, Druggan, & Forsythe, 2004). Other members of

Enterobacteriaceae as well as some non-Enterobacteriaceae have been isolated from infant formulae, e.g., Acinetobacter sp., Escherichia hermanii, Cedaceae lepagii,

Leclercia acecarboxylata and Enterobacter aglomerans, which also produce yellow- pigmented colonies (Leuschner et al., 2004). Using ampicillin-resistant, luminescent

Cronobacter (amp::lux) strains and inoculating the samples onto an antibiotic-containing plate ensured that only cells of the inoculated Cronobacter strains were enumerated. The absence of Cronobacter spp. in the infant formula used in the experiments was confirmed using a selective differential agar (Brilliance Enterobacter sakazakii Agar). However, other studies did not report examination of the infant formula for possible contamination.

In Kim and colleagues’ study (2007), phage ESP 1-3 had the morphology of a B1

Siphovirus. This morphological group of phages, based on the systematic analysis of bacterial genomes, is the main source of integrated prophage genomes related to virulence genes (Canchaya et al., 2003). In addition, it is known that temperate phages from Enterobacteriaceae carry important bacterial virulence factors (Brussow, Canchaya,

& Hardt, 2004), so the safety of these phages is questionable and their use for food applications should be discouraged. Indeed, Zuber et al. (2008) commented that this

Lambdoid Siphovirus should be excluded from practical applications, and only phage

ESP 732-1 (a T4-like phage) out of six phages tested by Kim and colleagues could be safely used in food. Likewise, in the other study on phage biocontrol in infant formula

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(Zuber et al., 2008), only two phages out of five were assumed to be safe for food use as they were morphologically T4-like viruses. As these authors suggested, the other three phages need to be sequenced and checked for pathogenic traits.

In our study, all five phages used in the phage cocktail that was applied in the infant formula against Cronobacter strains, have been sequenced and screened and no undesirable gene was found (Chapters 3 and 4; Abbasifar et al., 2012a; Abbasifar et al.,

2012b; Abbasifar et al., 2013). In addition, in vivo administration of one of our phages

(GAP161) in the hemolymph of Galleria mellonella larvae showed no negative effect on the wellbeing of the larvae and could effectively prevent Cronobacter infection (Chapter

5). Therefore, our phage cocktail could be potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models.

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Chapter 7. CONCLUSIONS AND FUTURE WORK

7.1. THESIS SUMMARY AND GENERAL CONSLUSION

Contaminated reconstituted infant formula has been linked with Cronobacter

infection in neonates and infants in several cases due to physiological characteristics that

enable the organism to survive in this food matrix, such as surviving desiccation and

osmotic stress for long periods and the ability to form biofilms. Several methods have

been proposed to control this pathogen and minimize its transmission through

reconstituted infant formula (RIF) including using hot water or microwave heating, high

pressure processing or gamma irradiation. However, due to the disadvantages of these

methods, such as potential loss of nutrients, risk of scalding of infants, cost efficiency and

feasibility, they are not well accepted. One of the novel technologies applied to control

bacterial pathogens in food is through exploitation of natural antimicrobial agents such as

bacteriophages (phages). Phages are the largest group of viruses and there are an

estimated 1031 phage particles in the biosphere, making them the most abundant form of

life on the planet. The phage specificity to its host cell can be exploited to detect and

control pathogenic bacteria. Phage application has emerged as a new biotechnological

method to control bacterial contamination in food. Therefore, our objectives in this

research focused on the isolation of strong lytic phages, characterizing them, evaluating

their safety in vivo, and their application in RIF for effective biocontrol of Cronobacter

sakazakii.

To use phages as potential biocontrol agents against food-borne pathogens, the first step is to isolate and characterize potentially the most effective and biologicaly

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useful phages. To be a suitable candidate for food safety applications, a phage should have strong lytic activity against a broad host range. In this investigation, two hundred and fifty two phages with variant plaque morphologies and sizes that were active against

Cronobacter spp. have been isolated from different environmental samples. All the plaques harvested from each individual host were purified and tested for host range, and isolated phages revealed varying abilities to infect the tested twenty-one bacterial strains.

To select the best phages for biocontrol of Cronobacter, five phages were chosen for further study based on their lytic activity and the number of hosts they could infect.

As stated above, possession of a broad host range is one of the most critical characteristics of good lytic phage candidates for biocontrol applications.

The morphology of the selected phages showed that, of the eleven selected viruses, three belonged to the family of Myoviridae and two were in the family of

Podoviridae. Phages GAP32 and GAP136 are similar to phage 121 of Proteus vulgaris with a large head and relatively short tail. However, no 121-like phage genomes have been sequenced (H-W. Ackermann, personal communication). Moreover, phages GAP52 and GAP188 have elongated cylindrical heads and are similar to Salmonella phage 7-11, which belongs to C3 morphotype that is extremely rare and only constitutes 0.5% of phages examined by electron microscope.

Based on the high intra-species specificity, broad host range, strong lytic activity, and interesting and rare morphology, five phages (GAP31, GAP32, GAP52, GAP161, and GAP227) were selected for complete genome sequencing and proteomic analysis, which can provide information as to the safe use of these phages in food. These selected

Cronobacter phages have various host range patterns, therefore, using cocktails of these

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phages could enhance the scope of their application against a broader range of

Cronobacters in infant formulae.

These selected Cronobacter phages were not able to infect E. coli strains. Since E.

coli is a common commensal of the gastrointestinal tract of humans (including

newborns), the selected phages would not lyse these gut commensal bacteria. In addition,

selected phages were not able to infect probiotic Lactobacillus strains that are used in

infant formula and could reduce the adhesion of Cronobacter to mucosal cells, and limit

the risk of infection. Therefore, application of these selected phages in infant formulae

would not affect the probiotic bacteria that are potentially present in these food products.

Sequencing of phages to provide a comprehensive knowledge of their genome is

necessary for detection of lysogenic and/or pathogenic genes in phages intended to be

used for food applications. Phage GAP32 is the first 121-like morphotype phage to have

its DNA fully sequenced and analysed. Its giant genome (358,663 bp) makes it the

second largest sequenced phage genome after Bacillus megaterium phage G. The genome

of GAP32 consists of 571 genes, which is more than the smallest known bacterium,

Mycoplasma genitalium G37. The genomic analyses show that GAP32 and the Klebsiella

phage RaK2 are related, however, they do not belong to the same genus. Therefore, a

recommendation will be made to the International Committee on Taxonomy of Viruses

(ICTV) that GAP32 be placed in a new genus: the “Gap32likevirus”, alternatively, the

“12unalikevirus”.

The genome of GAP31 shares a high percentage of homologous proteins with

Salmonella phage PVP-SE1, as well as coliphages vB_EcoM-FV3, and rV5. Therefore,

phage GAP31 belongs to a proposed genus of “V5likeviruses” within the Myoviridae

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family. Likewise, homologies of more than 85% of GAP161 with coliphages RB16 and

RB43, and phage KP15 of Klebsiella pneumoniae, indicate that GAP161 is a member of the myoviral subfamily Teequatrovirinae, genus T4likevirus.

Phages GAP52 and GAP227 are the first Cronobacter podoviruses to have their genome fully sequenced and analysed. Phage GAP52 is a podovirus of the very rare C3 morphotype, of which only 19 members are known and only constitute 0.5% of phages examined by electron microscopy. Comparative genomic analysis reveals that the genome sequence of GAP52 shares more than 52% of homologous proteins with

Salmonella phage 7-11.

Bioinformatic analysis of DNA of phage GAP227 shows significant sequence similarity to Yersinia phages φR8-01 and φ80-18, and Aeromonas phage phiAS7. Since the values of similarities (71.4% proteins with φR8-01 and φ80-18; and, 63.3% with phiAS7) are considerably higher than shared proteins with the type virus, φKMV

(36.7%), we propose that these four phages should be grouped in a new genus within the

Autographivirinae.

Each one of these five phages has some genes that are unique to that phage, which suggests that they are novel and have been sequenced for the first time. However, they have similarities to varying degrees with some other sequenced phages. Moreover, observing no bacterial toxin protein and integrase homologs and absence of bacterial virulence genes and lysogenic markers, along with the strong lytic activity of phages

GAP31, GAP52, GAP161, and GAP227, indicates that these phages can be used to control C. sakazakii.

To evaluate the effectiveness of our phages against Cronobacter spp. in vivo, and

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the safety of their administration to live organisms, phage GAP161 was selected, based on host range and lytic activity, for evaluation of its efficacy against C. sakazakii HPB

3253 in Galleria mellonella larvae. The mortalities of larvae infected by different

Cronobacters were observed after administration of the bacterium in the hemolymph of the larvae. The variation in virulence of different Cronobacter strains observed in this study as determined by larval mortality, agrees with a previous study on pathogenesis of this bacterium, which showed significant differences in adhesion, invasion, and toxin production among the strains. An increase in the survival rates of infected larvae treated with phage GAP161 was observed, and the increase in survival rates within groups treated prior to or immediately after infection was significantly higher than for the non- phage treated, infected control group. Phage GAP161 showed the best protective activity against C. sakazakii in G. mellonella in the groups treated with phage prior to or immediately after infection. In addition, when heat-inactivated phage was used, the survival rates were the same as for larvae inoculated with the pathogen alone, indicating that the survival of larvae is not due to host immune stimulation but the effect of phage antibacterial activity. Moreover, lack of a significant difference among Nil, Sham, and

Phage groups reveals that neither phage nor injection method used had an effect on the health of the larvae. This reveals that the in vivo administration of the phage is safe. In addition, this study confirms earlier work illustrating the usefulness of G. mellonella larvae as an inexpensive animal model for evaluating the efficacy of phage therapy, and

G. mellonella larvae proved to be a suitable whole animal model for pre-screening of the phage for its killing efficacy and safety when applied to combat C. sakazakii infection. In conclusion, the newly isolated phage is safe, and could potentially be used to control C.

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sakazakii during preparation of infant formula but would first have to be tested for its

efficiency in infant formula.

The next step was to evaluate the efficacy of our selected phages against C.

sakazakii in infant formula. A cocktail containing five highly purified phages in RIF to

control three C. sakazakii strains were evaluated. The use of highly purified phages imitates the real phage application in food products, because purification by density- gradient (CsCl) ultracentrifugation ensures the minimum possible level of bacterial contaminants that remain in the phage preparations.

The cocktail of these five phages (108 PFU/ml) were applied to control and

prevent the outgrowth of a cocktail of three bioluminescent strains of Cronobacter

(amp::lux) in broth medium (TSB) and reconstituted infant formula (RIF) (101 to 104

CFU/ml; MOIs of 107 to 104) at room temperature (24°C) for 24 hours. The addition of the phage cocktail to artificially contaminated broth and RIF resulted in significant inhibition of Cronobacter growth at all tested MOI values during the 24 hours of the experiment. In the previous two studies of phage biocontrol of Cronobacter in infant formula, phages were able to inhibit a single Cronobacter strain when present at MOIs of greater than 105. Not only did our phage cocktail show a more effective performance

against the cocktail of C. sakazakii strains than the phages in two previous studies, but also we considered some aspects that were not addressed in the other studies. Our phage cocktail could efficiently inhibit the growth of a cocktail of C. sakazakii strains (not just a single strain as was the case in the other studies) even at bacterial loads of 104 CFU/ml

(MOI of 104), and showed a better biocontrol performance in RIF than previously

published work. Moreover, as there was a strong correlation (r = 0.905) between the

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luminescence of the C. sakazakii lux+ mutant and its cell count (CFU/ml), we demonstrated the suitability of using the C. sakazakii (amp::lux) strains to evaluate phage efficacy, which is more cost effective and less labor intensive than traditional methods.

Previous studies focused on the effect of a single phage on a single Cronobacter strain, although food contamination may be the result of multiple strains of the pathogen.

Using a phage cocktail is recommended to broaden the host range and to prevent emergence of bacterial phage-resistance. In a previous study using phage mixtures, Zuber and colleagues (2008) applied their phage cocktail for a short period (4 h) at 30°C. Since usually the level of Cronobacter contamination is low in PIF, the outgrowth occurs after rehydration and preparation of formula and leaving it at room temperature before use, incubation at 30°C may not represent the real-world situation.

Premature neonates and infants are more susceptible to Cronobacter infection, and they must be fed infant formulae that are prepared with their nutritional needs in mind. The ingredients and their portions may differ between regular infant formulae and those prepared for premature infants, and these factors may affect the efficiency of phage biocontrol. Therefore, investigating the phage biocontrol of Cronobacters in premature infant formulae should be the priority, and the results could be different for regular formulae. However, the other two studies did not report the nature of the formula used.

In conclusion, this thesis describes the successful isolation of efficacious lytic phages that are free of pathogenic and lysogenic genes, and are safe and efficient to use in vivo and in infant formula, and therefore can be considered as potential candidates for biocontrol applications for Cronobacter spp.

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7.2. FUTURE WORK

Our newly isolated phages were shown to be safe in vivo and efficient in infant formula, and could potentially be used to control C. sakazakii during preparation of infant formula. However, first these phages would have to be clinically evaluated in mammalian models. A previous study indicated that neonatal gerbils were the most promising non- primate models to study the pathogenesis of Cronobacter spp. (Pagotto & Farber, 2009).

Future in vitro studies could be performed using cell lines derived from intestinal cells of the gerbil to test the effectiveness and safety of phages against Cronobacters. This study could be followed by investigating the efficiency of our phage cocktail in controlling

Cronobacter infection in neonatal gerbils that are orally administered with artificially contaminated RIF. This would be a better simulation of real routes of infection that occur in infants consuming the contaminated RIF.

Moreover, the genome of the other giant phage (GAP136) in our phage collection that possessed a similar morphotype to phage GAP32 and phage 121 can be sequenced and analyzed. This could help to better understand these phages with very large genomes and their relation to one another. In addition, as phage GAP31 is morphologically similar to phage Felix O1, the genome of which has not been fully sequenced, using restriction endonuclease analysis and PCR of the DNA of phage GAP31 and Felix O1 could confirm their relationship. Also, isolation and in depth characterization of more phages would greatly help to promote this new emerging biocontrol tool and its application to improve food safety.

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APPENDIX

Appendix 1. Host range of isolated phages

Table 2.3. The host range of isolated Cronobacter phages against 21 Cronobacter strains based on the spot test (16 h incubation at 30ºC). The results were recorded as 3 (very clear zone of complete lysis on the bacterial lawn), 2 (clear/turbid zone of lysis), 1 (turbid lysis) and 0 (no lysis). C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii malonaticus C. C. sakazakii muytjensii C. C. sakazakii C. malonaticus C. muytjensii C. universalis C. C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii C. sakazakii malonaticus C.

Bacteria dublinensis

SUM 354/03 1084/04 2855 2870 2871 2876 3253 3199 3290 130/3 236/04 324/04 974/03 1103/03

51329 327 0

3287 3169

3263 3267 1154/04

Phage

Isolate

1 2 1 1 0 1 1 0 0 0 0 0 0 0 1 0 0 0 2 0 1 0 10

2 0 1 0 0 3 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 5

3 1 0 1 0 3 3 0 2 3 0 0 2 0 1 3 3 1 1 1 0 0 25

4 0 1 1 0 1 2 0 3 0 3 1 2 0 1 0 0 3 2 1 0 0 21

5 3 1 1 0 1 0 0 0 0 0 2 2 0 0 0 1 0 0 0 0 0 11

6 3 0 3 0 2 1 0 0 2 0 0 0 0 0 3 3 0 1 0 0 0 18

7 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 2 0 0 0 0 5

8 3 1 0 0 3 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 8

9 3 0 3 0 1 2 1 2 3 1 1 2 0 1 2 2 1 2 2 1 0 30

197

10 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

11 3 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 7

12 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

13 3 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 4

14 3 1 0 0 0 2 0 1 1 1 3 2 1 3 2 3 0 0 1 1 0 25

15 0 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 3

16 3 0 0 1 2 2 0 1 2 0 1 2 0 2 0 0 0 1 0 1 0 18

17 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

18 2 0 0 0 0 0 0 3 0 3 3 0 0 0 0 0 0 0 0 0 0 11

19 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2

20 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

21 1 0 0 0 0 2 0 2 3 1 1 2 0 0 2 3 1 2 3 0 0 23

22 2 3 0 0 0 3 1 3 2 2 2 3 0 3 3 3 1 1 1 2 0 35

23 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 5

24 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2

25 2 0 0 0 0 0 0 0 1 0 0 1 0 0 1 1 0 0 0 0 0 6

26 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 2

27 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

28 0 2 0 0 0 2 0 1 1 1 1 2 0 0 0 1 0 0 0 0 0 11

29 2 0 0 0 0 3 0 1 1 0 0 2 0 1 1 1 0 1 1 1 0 15

198

30 1 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 4

31 3 3 3 3 3 3 3 3 3 3 3 3 0 3 2 3 3 3 3 3 1 57

32 3 2 0 3 0 3 2 2 3 1 3 3 0 3 2 3 1 3 3 3 0 43

33 3 3 3 3 0 3 1 3 3 3 2 3 0 3 3 3 3 3 3 3 1 52

34 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0 3

35 1 0 0 0 0 1 0 0 1 0 2 2 0 0 1 2 0 0 1 0 0 11

36 0 0 0 0 0 0 0 0 0 0 1 3 0 0 0 0 0 0 0 0 0 4

37 2 1 1 0 0 1 1 3 2 1 1 2 0 2 0 0 0 3 1 3 0 24

38 2 0 0 0 0 0 0 0 0 1 0 2 0 1 3 2 0 0 1 1 0 13

39 1 0 0 0 0 0 0 0 0 1 0 2 0 0 3 2 0 0 0 0 0 9

40 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

41 1 0 1 0 0 0 0 0 1 1 0 2 0 1 0 0 0 0 0 1 0 8

42 1 0 1 0 0 0 0 0 1 0 0 2 0 0 0 0 0 0 0 1 0 6

43 2 0 1 0 0 0 0 2 1 1 0 1 0 2 0 0 0 2 1 3 0 16

44 2 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 1 1 1 0 7

45 1 0 1 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 1 1 0 7

46 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

47 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

48 0 1 1 0 0 0 0 3 3 2 3 3 0 3 2 2 0 3 0 3 0 29

49 0 1 1 0 0 0 0 3 1 3 3 3 0 3 0 0 0 0 0 3 0 21

199

50 0 0 0 2 0 0 2 0 1 0 2 3 0 3 0 0 2 0 0 3 0 18

51 0 0 0 0 2 0 1 0 0 0 2 2 1 2 2 2 0 0 1 1 0 16

52 3 2 1 3 0 3 1 3 2 3 3 3 1 3 0 0 3 3 3 3 0 43

53 0 2 3 3 0 2 0 3 1 3 3 3 0 3 0 0 0 1 1 3 0 31

54 1 0 0 3 0 1 2 2 2 1 2 2 1 2 1 1 3 0 1 2 0 27

55 0 2 3 3 0 3 0 3 2 3 3 3 1 3 0 0 1 2 1 3 0 36

56 0 0 0 0 0 0 0 0 0 0 2 3 1 2 1 1 0 0 1 1 0 12

57 0 1 0 0 0 0 0 3 1 3 3 3 1 3 1 1 0 0 1 3 0 24

58 0 0 0 0 0 1 0 0 1 0 3 3 1 3 1 2 0 0 2 3 0 20

59 0 0 0 1 0 0 1 1 1 0 2 2 0 3 0 0 2 0 0 2 0 15

60 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

61 0 0 0 0 0 1 0 0 1 0 2 2 1 1 1 2 0 0 1 1 0 13

62 0 1 1 0 0 0 0 3 1 3 3 3 0 3 0 0 0 2 0 3 0 23

63 0 0 0 0 0 0 0 3 0 3 3 2 0 1 0 0 0 0 1 1 1 15

64 0 1 1 0 0 0 0 3 1 3 3 2 0 3 0 0 0 2 0 3 0 22

65 0 1 0 0 3 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 5

66 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

67 1 0 1 0 3 0 0 0 1 0 0 0 0 0 1 2 0 0 0 0 0 9

68 2 0 1 2 0 1 1 1 1 0 0 0 1 3 0 0 1 2 1 2 0 19

69 0 0 0 0 2 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 5

200

70 1 0 0 0 1 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 1 7

71 3 0 1 1 0 1 0 1 2 0 1 1 1 3 2 2 1 3 1 3 1 28

72 3 3 2 3 2 2 2 3 3 3 3 2 1 3 3 3 3 3 3 3 0 53

73 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

74 3 2 1 1 0 3 0 2 3 2 2 3 0 3 2 2 1 1 1 2 0 34

75 2 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 5

76 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

77 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2

78 3 2 2 0 0 3 2 3 3 0 2 2 1 3 2 2 3 3 3 3 0 42

79 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

80 2 0 0 0 1 0 0 0 0 0 0 1 0 0 3 3 0 0 0 0 0 10

81 2 0 0 0 1 0 0 0 0 0 0 1 0 0 3 3 0 0 0 0 0 10

82 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 3

83 2 0 1 1 0 1 0 1 1 0 0 1 0 1 1 2 1 2 0 1 0 16

84 3 2 2 1 0 3 0 3 3 3 2 3 0 1 1 2 3 3 3 1 0 39

85 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 2 1 1 1 1 0 9

86 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 3

87 2 0 1 2 0 3 1 3 3 1 1 2 0 3 2 3 1 3 2 3 0 36

88 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

89 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 3

201

90 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

91 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

92 0 1 1 1 0 0 0 3 1 3 3 2 0 2 0 0 1 1 0 3 0 22

93 0 1 1 1 0 0 0 3 1 3 3 2 0 2 0 0 0 1 0 3 0 21

94 0 0 0 0 0 0 0 3 0 3 3 2 0 2 0 0 0 1 0 3 0 17

95 0 0 0 1 0 0 2 1 0 0 3 2 0 2 0 0 2 0 0 2 0 15

96 0 0 0 0 0 0 2 3 0 0 3 2 0 2 0 0 2 0 0 3 0 17

97 0 0 0 0 2 0 1 3 0 0 2 2 0 2 1 2 1 1 0 2 0 19

98 2 2 2 2 0 2 2 2 1 1 2 1 0 2 0 0 2 2 2 2 0 29

99 1 1 2 1 0 2 0 3 2 2 2 2 0 2 2 1 0 2 1 2 1 29

100 0 1 1 0 0 2 0 3 1 3 3 3 0 3 0 0 0 3 0 3 1 27

101 1 1 2 2 0 2 0 3 2 2 2 2 0 2 1 1 1 3 1 2 1 31

102 2 2 2 2 0 2 2 2 2 2 2 2 0 2 1 1 2 2 2 2 1 35

103 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 2

104 2 2 2 2 0 2 2 2 2 2 2 2 0 2 1 0 3 2 2 2 0 34

105 0 1 2 1 0 2 0 3 2 2 2 2 0 3 0 0 1 2 1 2 1 27

106 0 1 2 0 0 2 0 3 1 2 2 2 0 3 0 0 0 1 0 3 0 22

107 0 1 1 0 0 0 0 3 1 2 2 2 0 3 0 0 0 1 0 3 1 20

108 0 0 0 2 0 0 1 3 1 2 3 1 0 2 0 0 3 1 1 3 1 24

109 0 0 0 1 0 0 2 1 0 0 2 1 0 2 0 0 2 0 1 3 2 17

202

110 0 1 1 0 0 0 0 3 1 3 3 1 0 3 0 0 0 2 0 3 1 22

111 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 1 4

112 0 0 0 1 0 0 2 1 0 0 2 1 0 2 0 0 2 0 1 2 0 14

113 2 3 3 3 0 2 2 3 2 3 3 2 0 2 0 1 2 2 3 2 1 41

114 0 1 2 2 0 3 0 3 2 3 3 2 0 3 0 0 1 2 1 3 1 32

115 0 1 2 2 0 2 0 3 2 3 3 3 0 3 0 0 0 2 1 3 0 30

116 0 0 0 0 0 0 0 3 0 0 3 3 0 3 0 0 0 1 0 3 0 16

117 0 1 3 1 0 2 0 3 2 3 3 3 0 3 0 0 0 2 1 3 0 30

118 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 1 7

119 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

120 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

121 0 2 0 0 0 3 0 1 0 1 1 1 0 0 0 0 0 0 0 0 0 9

122 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

123 0 0 1 0 0 0 0 2 1 0 0 0 0 0 0 0 0 0 0 0 0 4

124 2 2 2 2 0 2 1 2 3 2 2 2 0 2 3 2 3 3 3 2 0 40

125 2 0 0 0 0 2 0 1 2 0 0 1 0 1 2 2 1 1 1 1 0 17

126 2 0 0 0 0 1 0 3 0 1 0 0 0 0 2 2 0 0 0 0 0 11

127 2 2 2 2 0 2 0 2 2 2 3 2 0 0 2 2 3 3 2 0 0 33

128 3 0 0 0 0 1 0 1 0 1 0 2 0 0 0 0 0 0 0 0 0 8

129 3 0 1 0 0 2 0 2 2 0 0 2 0 2 2 2 1 3 1 2 0 25

203

130 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 2

131 3 0 1 0 1 1 0 1 1 0 0 1 0 0 3 3 0 1 1 0 0 17

132 3 0 1 0 2 1 0 1 1 0 0 1 0 0 3 3 1 1 0 0 0 18

133 2 0 1 0 1 0 0 0 0 0 0 1 0 0 2 2 0 0 0 0 0 9

134 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

135 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

136 3 3 2 2 0 3 0 3 3 3 2 2 0 1 3 3 3 3 3 1 1 44

137 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1

138 3 1 0 0 0 3 0 2 3 0 0 2 0 2 3 3 2 3 2 3 1 33

139 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

140 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

141 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

142 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 1 6

143 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 1 6

144 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

145 0 0 0 1 3 0 0 0 0 0 0 2 0 0 1 1 0 0 0 0 0 8

146 0 0 3 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 6

147 3 0 1 0 0 1 0 3 0 0 0 3 0 0 0 0 0 1 1 1 0 14

148 0 0 1 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 0 0 5

149 0 0 0 0 0 0 0 0 0 0 1 3 1 0 1 1 0 0 0 0 0 7

204

150 2 0 0 0 0 0 0 0 0 0 1 3 1 0 3 3 1 0 0 0 0 14

151 1 0 0 0 0 0 0 0 0 0 1 3 0 0 3 3 0 0 0 0 0 11

152 3 0 1 0 0 0 0 1 0 0 0 2 0 0 0 1 1 1 0 0 0 10

153 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 1 2 1 0 0 0 7

154 3 0 1 0 0 0 0 0 0 0 0 2 0 0 1 1 0 1 1 1 0 11

155 0 0 1 0 0 0 0 0 0 0 0 1 0 0 1 1 0 1 1 1 0 7

156 3 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 6

157 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1

158 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

159 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 2

160 3 0 0 0 0 2 0 1 1 0 0 2 1 0 0 0 2 0 0 0 0 12

161 3 2 2 2 3 3 0 3 3 3 3 3 0 0 0 0 3 3 3 0 0 39

162 1 0 0 0 0 2 0 2 1 2 0 3 0 0 0 0 2 2 2 0 0 17

163 1 2 0 0 0 3 0 2 2 2 2 3 0 0 0 0 3 3 3 0 0 26

164 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

165 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 4

166 3 0 0 0 0 1 0 1 1 2 0 3 0 0 0 0 1 1 2 0 0 15

167 0 0 0 0 0 1 0 1 1 1 0 3 0 0 0 0 1 1 2 0 0 11

168 2 1 0 0 0 2 1 2 1 0 1 3 1 2 2 1 0 1 1 1 0 22

169 1 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0 7

205

170 2 0 0 0 0 0 0 0 0 0 0 1 0 0 3 3 0 0 0 0 0 9

171 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1

172 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1

173 0 0 0 0 0 1 0 1 1 1 0 3 0 0 0 0 2 2 2 0 0 13

174 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

175 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 4

176 3 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4

177 0 1 0 0 0 0 0 3 1 2 3 3 0 3 1 1 0 0 0 3 0 21

178 0 1 0 0 0 0 0 3 1 1 3 3 0 3 1 1 0 0 0 3 0 20

179 0 1 0 2 0 1 2 2 0 1 3 3 0 3 1 1 2 0 1 3 0 26

180 0 1 0 0 0 3 0 3 1 1 3 3 1 3 2 2 0 0 0 3 0 26

181 1 1 0 2 0 2 1 2 0 2 3 2 0 3 0 0 2 1 1 3 0 26

182 0 3 3 0 0 3 0 3 3 3 3 2 0 3 1 1 1 2 0 3 0 34

183 0 3 3 3 0 3 0 3 3 3 3 2 0 3 1 1 1 2 2 3 1 40

184 0 3 3 3 0 3 0 3 3 3 3 3 1 3 1 1 1 2 2 3 1 42

185 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 3

186 0 3 3 3 0 3 0 3 3 3 3 3 1 3 3 3 2 2 3 3 1 48

187 0 0 0 0 0 0 0 0 0 0 2 2 0 0 1 1 0 0 0 2 0 8

188 0 3 3 3 0 3 3 3 3 3 3 3 0 3 1 1 3 3 3 3 1 48

189 1 0 0 0 0 0 0 1 0 0 1 2 0 0 2 2 0 0 0 0 0 9

206

190 0 0 0 0 0 0 0 0 0 0 1 2 0 0 1 1 0 0 0 0 0 5

191 0 0 1 0 0 0 0 1 0 0 3 2 0 3 0 1 0 0 0 2 0 13

192 0 0 0 0 0 0 0 1 0 0 1 2 0 0 0 0 1 0 0 1 0 6

193 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

194 0 0 0 0 0 1 0 0 0 0 3 3 1 0 0 0 0 0 0 0 0 8

195 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

196 0 1 3 3 0 1 0 3 1 3 3 3 0 3 0 0 2 1 1 3 0 31

197 0 1 3 3 0 1 0 3 0 3 3 3 0 3 0 0 1 1 0 3 0 28

198 0 0 0 0 0 0 0 1 0 0 0 0 0 0 2 2 0 0 0 0 0 5

199 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 2

200 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

201 0 0 0 0 3 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 6

202 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 2

203 2 1 1 1 0 3 0 3 2 3 1 3 0 1 0 0 2 2 3 2 0 30

204 2 2 0 3 0 3 2 2 2 2 3 3 2 3 2 2 2 3 3 3 0 44

205 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 2

206 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1

207 3 1 2 1 1 3 0 3 2 2 2 3 0 0 2 3 2 3 3 0 0 36

208 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1

209 1 0 0 0 1 0 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0 8

207

210 1 0 0 0 1 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 6

211 1 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 5

212 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

213 2 1 2 1 0 2 0 2 2 2 1 2 0 0 2 2 2 2 2 0 0 27

214 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 4

215 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

216 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

217 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

218 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 2

219 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2

220 2 0 0 0 0 1 0 1 1 0 0 0 0 0 1 1 0 0 0 0 0 7

221 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

222 1 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 1 0 0 0 6

223 0 0 1 0 0 0 0 2 0 3 2 0 0 0 0 0 1 0 0 0 0 9

224 1 0 0 0 0 0 0 1 0 0 1 0 0 0 1 1 1 0 0 0 0 6

225 0 2 3 0 0 0 0 3 1 3 3 2 0 3 0 0 0 1 0 3 0 24

226 0 0 0 0 0 2 0 0 0 0 2 2 1 0 0 1 0 0 0 0 0 8

227 0 2 3 3 0 3 3 3 3 3 3 3 0 3 0 0 3 3 3 3 0 44

228 0 1 0 1 0 1 2 2 2 0 3 3 0 3 0 0 3 1 2 3 0 27

229 0 1 3 2 0 2 0 3 1 3 3 3 1 3 0 0 2 2 2 3 1 35

208

230 0 1 3 2 0 2 0 3 1 3 3 3 1 3 0 0 2 2 1 3 1 34

231 0 1 3 3 0 2 0 3 1 3 3 3 1 3 0 0 2 2 2 3 1 36

232 3 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 5

233 0 0 0 0 3 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 6

234 2 0 0 0 1 0 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0 9

235 2 0 0 0 1 0 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0 9

236 0 1 3 2 0 0 0 3 1 3 3 3 0 3 0 0 0 2 2 3 0 29

237 0 1 3 2 0 1 0 3 1 3 3 3 0 3 0 0 1 2 2 3 0 31

238 0 1 3 3 0 2 0 3 1 3 3 3 0 3 0 0 2 2 2 3 1 35

239 0 2 3 2 0 2 0 3 1 3 3 3 0 3 0 0 1 3 2 3 1 35

240 0 0 0 0 0 2 0 0 0 0 3 3 1 0 0 1 0 0 0 0 0 10

241 0 1 3 2 0 1 0 3 1 3 3 3 0 3 0 0 0 2 1 3 1 30

242 0 1 3 3 0 1 0 3 1 3 3 3 0 3 0 0 1 2 1 3 1 32

243 0 1 3 3 0 1 0 3 1 3 3 3 0 3 0 0 1 2 1 3 1 32

244 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 6

245 0 0 0 0 0 0 0 3 0 2 0 0 0 0 0 0 0 0 0 0 0 5

246 0 0 0 0 0 2 0 0 0 0 3 3 1 0 0 1 0 0 0 0 0 10

247 0 0 0 0 0 2 0 0 0 2 3 3 1 0 0 0 0 0 0 0 0 11

248 0 0 0 0 0 2 0 0 0 1 2 2 1 0 0 0 0 0 0 0 0 8

249 3 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 3 0 0 0 9

209

250 0 1 3 3 0 2 0 3 1 3 3 3 1 3 1 1 1 1 1 3 1 35

251 0 1 2 0 0 1 0 3 1 3 3 3 0 3 0 0 0 0 0 3 0 23

252 0 0 0 0 0 2 0 0 0 1 3 3 1 0 1 1 0 0 0 0 0 12

Sensitivity 238 126 166 129 76 196 55 286 177 215 289 361 35 248 165 181 154 208 150 252 38 Score

210

Appendix 2. Genome of phage GAP32

Table 3.1. General features of ORFs in the DNA of phage GAP32 and homology to proteins in the databases.

DNA Protein Sequence Similarity to: Gene Product Coordinates Strand Length Mass pI Residues (Homologs & motifs)

orf001 301..1458 - 1158 41450 6.9 385 conserved hypothetical C-terminus YP_004412035.1 regulator protein of chromosome condensation RCC1 [Spirochaeta coccoides DSM 17374] orf002 1804..2436 - 633 22802 6.3 210 hypothetical protein orf003 3147..3284 + 138 5162 4.1 45 hypothetical protein orf004 4330..4503 + 174 6582 10.3 57 hypothetical protein

orf005 4583..5257 + 675 24723 5.1 224 hypothetical protein orf006 5405..5674 + 270 10111 89 hypothetical protein orf007 5797..6189 + 393 15107 4.7 130 hypothetical protein orf008 6259..6543 + 285 10453 9.8 94 hypothetical protein orf009 6964..7236 + 273 10090 90 conserved hypothetical AEL79639.1 hypothetical protein protein [Escherichia phage vB_EcoP_G7C] orf010 7301..7747 + 447 16859 7.7 148 conserved hypothetical ZP_06243690.1 hypothetical protein protein Vvad_PD2293 [Victivallis vadensis ATCC BAA-548]; PB005443 Pfam- B_5443 orf011 7824..8279 + 456 17165 6.6 151 hypothetical protein orf012 8357..8779 + 423 16323 9.2 140 hypothetical protein orf013 9319..9675 + 357 13923 4.3 118 hypothetical protein orf014 9749..10129 + 381 14458 8.9 126 conserved hypothetical C-terminus YP_002003681.1 protein hypothetical protein rv5_gp179 [Escherichia phage rv5] orf015 10176..10430 + 255 10030 84 hypothetical protein orf016 10435..10644 + 210 8072 4.2 69 hypothetical protein

211

orf017 10934..11167 + 234 8561 5.1 77 hypothetical protein orf018 11325..11558 + 234 8904 4.3 77 hypothetical protein orf018A 11630..11740 + 111 4212 36 hypothetical protein orf019 11753..11956 + 204 7557 9.9 67 hypothetical protein orf020 11956..12132 + 177 6789 4.9 58 hypothetical protein orf021 12209..12412 + 204 7560 6.2 67 hypothetical protein orf022 12491..12793 + 303 11348 8.8 100 hypothetical protein orf023 13030..13275 + 246 9014 4.4 81 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf024 13313..13513 + 201 7547 9.0 66 hypothetical protein orf025 13523..14380 + 858 30839 5.4 285 conserved hypothetical NP_758633.1 putative protease protein [Pseudomonas resinovorans]; Pfam PF01145.19 Band_7 orf026 14513..14707 + 195 7941 64 hypothetical protein orf027 14674..15021 + 348 12972 5.1 115 hypothetical protein orf028 15034..15321 + 288 10888 9.8 95 hypothetical protein orf029 15384..15575 + 192 7206 10.1 63 hypothetical protein orf030 15637..16002 + 366 13572 6.6 121 hypothetical protein orf031 16051..16293 + 243 9555 10.7 80 hypothetical protein orf032 16348..16869 + 522 19041 9.4 173 hypothetical protein orf033 16934..17278 + 345 12912 5.4 114 hypothetical protein orf034 17610..17837 + 228 8619 4.6 75 hypothetical protein orf035 18056..18631 + 576 22788 4.7 191 conserved hypothetical YP_002003682.1 hypothetical protein protein rv5_gp180 [Escherichia phage rv5]; PB003131 Pfam-B_3131 orf036 18934..19278 + 345 13546 114 hypothetical protein orf037 19566..19808 + 243 9265 7.8 80 hypothetical protein orf039 19900..20787 + 888 33696 295 conserved hypothetical CAZ15859.1 conserved hypothetical protein protein [Xanthomonas albilineans]; PF06067.5 DUF932 orf040 20864..21202 + 339 13443 5.7 112 hypothetical protein orf041 21466..21687 + 222 8423 5.1 73 hypothetical protein orf042 21787..22134 + 348 13345 4.7 115 hypothetical protein orf043 22337..22531 + 195 7278 4.6 64 hypothetical protein

212

orf044 22592..22768 + 177 6496 4.5 58 hypothetical protein

orf045 23183..23533 + 351 12985 5.0 116 hypothetical protein orf046 23773..24042 + 270 10388 4.5 89 hypothetical protein orf047 24113..24406 + 294 11078 4.4 97 hypothetical protein orf047A 24488..24688 + 201 7142 9.7 66 hypothetical protein orf048 24917..25498 + 582 23449 4.8 193 hypothetical protein orf049 25499..26122 + 624 23834 5.2 207 hypothetical protein orf050 26100..26672 + 573 22231 5.7 190 hypothetical protein orf051 26674..27291 + 618 24452 4.6 205 hypothetical protein orf052 27413..28009 + 597 22833 5.7 198 hypothetical protein orf053 28006..28509 + 504 20023 9.2 167 hypothetical protein orf054 28521..29072 + 552 21740 5.2 183 hypothetical protein orf055 29069..29497 + 429 16525 5.5 142 hypothetical protein orf056 29475..30011 + 537 21106 178 hypothetical protein orf057 30004..30447 + 444 17080 5.0 147 hypothetical protein orf058 30527..31003 + 477 18315 4.8 158 hypothetical protein orf059 31007..31177 + 171 6382 56 hypothetical protein orf060 31181..32068 + 888 35485 8.4 295 hypothetical protein orf061 32052..32372 + 321 12758 5.3 106 hypothetical protein orf062 32416..32811 + 396 14706 5.1 131 hypothetical protein orf063 32834..33184 + 351 13439 4.6 116 hypothetical protein orf064 33227..33553 + 327 12956 8.6 108 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf065 33550..34308 + 759 29418 252 hypothetical protein orf066 34308..35090 + 783 30513 4.8 260 hypothetical protein orf067 35805..36329 + 525 21026 5.5 174 hypothetical protein orf068 36342..36884 + 543 20685 5.5 180 DNA N-6-adenine- YP_001254197.1 DNA N-6-adenine- methyltransferase methyltransferase of bacteriophage [Clostridium botulinum A str. ATCC 3502]; PF05869.5 Dam orf069 36888..37445 + 558 21173 5.6 185 cytitidyltransferase C-terminus YP_214426.1 cytitidyltransferase [Prochlorococcus phage P-SSM2] orf070 37429..37710 + 282 10718 4.6 93 hypothetical protein orf071 37755..38330 + 576 22368 4.1 191 hypothetical protein

213

orf072 38314..38463 + 150 5796 4.7 49 hypothetical protein orf073 38444..38593 + 150 5772 9.8 49 hypothetical protein orf074 38593..39237 + 645 24903 5.6 214 conserved hypothetical EGH81543.1 hypothetical protein protein PLA107_00320 [Pseudomonas syringae pv. lachrymans str. M301315] orf075 39239..39457 + 219 7992 7.8 72 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf076 39438..40535 + 1098 41930 4.8 365 conserved hypothetical YP_003973383.1 hypothetical protein protein BATR1942_07565 [Bacillus atrophaeus 1942] orf077 40577..41308 + 732 28155 4.9 243 hypothetical protein orf078 41351..41644 + 294 11023 4.8 97 hypothetical protein orf079 41641..41937 + 297 11509 5.7 98 hypothetical protein orf080 42001..43152 + 1152 44144 5.5 383 RnlA RNA ligase 1 and YP_002922568.1 RnlA RNA ligase 1 tail fiber attachment and tail fiber attachment catalyst catalyst [Enterobacteria phage JS10]; PF09511.4 RNA_lig_T4_1 orf081 43182..43418 + 237 9238 10.4 78 hypothetical protein orf082 43411..43635 + 225 8591 5.1 74 hypothetical protein orf083 43648..43872 + 225 8544 8.8 74 hypothetical protein orf084 43860..44783 + 924 35456 6.3 307 PseT polynucleotide 5'- YP_003969353.1 PseT polynucleotide kinase and 3'- 5'-kinase and 3'-phosphatase [Aeromonas phosphatase phage phiAS5]; PF06414.6 Zeta_toxin orf085 44840..45049 + 210 7881 4.8 69 hypothetical protein orf086 45042..45245 + 204 8073 4.5 67 hypothetical protein orf087 45249..45416 + 168 6381 8.0 55 conserved hypothetical ZP_05845037.1 hypothetical protein membrane protein Rsw2DRAFT_3024 [Rhodobacter sp. SW2]; 1-2 transmembrane domains shown using TMHMM 2.0 and Phobius orf088 45406..45687 + 282 11243 3.9 93 hypothetical protein orf089 45684..46022 + 339 13003 3.9 112 hypothetical protein orf090 46024..46251 + 228 8337 75 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius

214

orf091 46232..46480 + 249 9599 4.3 82 hypothetical protein orf092 46477..46833 + 357 14195 5.3 118 conserved hypothetical YP_004301266.1 hypothetical protein protein 65p430 [Aeromonas phage 65] orf093 46833..47078 + 246 9812 3.9 81 conserved hypothetical YP_003734262.1 hypothetical protein protein [Enterobacteria phage IME08] orf094 47123..47764 + 642 24796 4.6 213 conserved hypothetical YP_003579994.1 hypothetical protein protein KP-KP15_gp133 [Klebsiella phage KP15] orf095 47764..48543 + 780 29950 5.0 259 putative serine/threonine YP_004306516.1 putative protein phosphatase serine/threonine protein phosphatase [Enterobacteria phage SPC35]; PF00149.22 Metallophos orf096 48543..48848 + 306 11699 4.9 101 hypothetical protein orf097 48848..49306 + 459 17263 5.4 152 conserved hypothetical YP_003969337.1 hypothetical protein protein phiAS5_ORF0048 [Aeromonas phage phiAS5] orf098 49317..50537 + 1221 47342 6.3 406 conserved hypothetical ADF58145.1 hypothetical protein protein PJG4_143 [Pseudomonas phage JG004] orf099 50563..51978 + 1416 53040 5.1 471 DNA ligase CBZ42332.1 hypothetical protein [Campylobacter phage CP81] & YP_004421617.1 gp30 DNA ligase [Campylobacter phage NCTC12673]; PF01068.1 DNA_ligase_A_M orf100 51975..52397 + 423 16657 5.2 140 hypothetical protein orf101 52375..53166 + 792 30701 5.8 263 metallophosphoesterase YP_911130.1 metallophosphoesterase [Chlorobium phaeobacteroides DSM 266]; PF12850.1 Metallophos_2 orf102 53168..53608 + 441 14893 4.5 146 conserved hypothetical NP_899386.1 hypothetical protein protein KVP40.0139 [Vibrio phage KVP40] orf103 53610..54302 + 693 25559 230 ATP-dependent Clp YP_536059.1 ATP-dependent Clp protease proteolytic protease proteolytic subunit subunit [Lactobacillus salivarius UCC118]; PF00574.17 CLP_protease orf104 54319..54606 + 288 10457 4.5 95 hypothetical protein

215

orf105 54611..55069 + 459 17825 8.7 152 hypothetical protein orf106 55114..55677 + 564 22739 5.1 187 hypothetical protein orf107 55662..56189 + 528 20768 5.6 175 hypothetical protein orf108 56179..56673 + 495 19833 5.9 164 hypothetical protein orf109 56630..57103 + 474 17590 6.0 157 CMP/dCMP deaminase YP_003893084.1 CMP/dCMP deaminase protein [Sulfurimonas autotrophica DSM 16294] & YP_004010351.1 Cd dCMP deaminase [Acinetobacter phage Acj9]; PF00383.1 dCMP_cyt_deam_1 orf110 57107..57373 + 267 10220 9.7 88 hypothetical protein orf111 57416..58327 - 912 30181 9.1 303 Ig domain-containing YP_003191420.1 Ig domain-containing protein protein group 2 domain-containing protein [Desulfotomaculum acetoxidans DSM 771]; PF02368.12 Big_2 orf112 58569..59666 + 1098 42296 6.2 365 bifunctional NP_442622.1 bifunctional nicotinamide nicotinamide mononucleotide mononucleotide adenylyltransferase/ADP-ribose adenylyltransferase/ADP pyrophosphatase[Synechocystis sp. PCC -ribose pyrophosphatase 6803]; PF01467.1 CTP_transf_2 & PF00293.1 NUDIX orf113 59713..60228 + 516 19816 4.4 171 hypothetical protein orf114 60288..61775 + 1488 55580 5.9 495 nicotinamide YP_438032.1 putative nicotinate phosphoribosyltransferas phosphoribosyltransferase [Hahella e chejuensis KCTC 2396]; PB002198 Pfam-B_2198 & PF04095.10 NAPRTase orf115 61818..62030 + 213 7945 7.8 70 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf116 62017..62955 + 939 36914 4.6 312 conserved hypothetical NP_944069.1 hypothetical protein protein Aeh1p191 [Aeromonas phage Aeh1] orf117 62958..63158 + 201 7584 9.5 66 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius

216

orf118 63166..63612 + 447 16654 5.5 148 conserved hypothetical YP_418159.1 hypothetical protein protein PPEV_gp126 [Pseudomonas phage EL] orf118A 63625..63861 + 237 9030 8.6 78 conserved hypothetical NP_891661.1 hypothetical protein protein RB49p090 [Enterobacteria phage RB49] orf119 63858..64655 + 798 29889 4.8 265 putative Sir2-like protein YP_006930.1 putative Sir2-like protein [Enterobacteria phage T5]; PF02146.1 SIR2 orf120 64655..65560 + 906 34711 301 conserved hypothetical YP_001194957.1 hypothetical protein protein Fjoh_2611 [Flavobacterium johnsoniae UW101]; PF00293.1 NUDIX & PB001179 Pfam-B_1179 orf121 65560..65862 + 303 11115 6.0 100 hypothetical protein orf122 65933..67375 + 1443 53726 4.9 480 PhoH family protein YP_003555102.1 PhoH family protein [Shewanella violacea DSS12]; PF02562.10 PhoH orf123 67423..67605 + 183 6983 6.1 60 hypothetical protein orf124 67776..68474 + 699 27438 5.9 232 hypothetical protein orf124A 68471..68677 + 207 7450 6.5 68 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf125 68684..68833 + 150 5628 5.0 49 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf126 68908..69450 + 543 22174 9.4 180 hypothetical protein orf127 69476..70288 + 813 31073 4.5 270 conserved hypothetical NP_899283.1 hypothetical protein protein KVP40.0035 [Vibrio phage KVP40] orf128 70351..72372 + 2022 74299 5.8 673 colanic acid-degrading C-terminus ADI49571.1 colanic acid- protein degrading protein [Phage NST1]; PB001187 Pfam-B_1187 orf129 72427..73041 + 615 22579 4.9 204 conserved hypothetical AEJ59754.1 hypothetical protein protein UMNF18_5326 [Escherichia coli UMNF18]; PB000661 Pfam-B_661 orf130 73041..73322 + 282 10250 93 co-chaperonin GroES YP_002152263.1 10 Kda chaperonin [Proteus mirabilis HI4320]; PF00166.15 Cpn10

217

orf131 73332..74594 + 1263 47439 5.1 420 tyrosyl-tRNA synthetase YP_002349927.1 tyrosyl-tRNA synthetase [Listeria monocytogenes HCC23]; PF00579.19 tRNA-synt_1b & PF01479.19 S4 orf132 74596..74802 + 207 7812 68 hypothetical protein orf133 74950..75591 + 642 25454 4.5 213 hypothetical protein orf134 75587..75946 + 360 13520 9.6 119 Cd dCMP deaminase NP_803826.1 ORF260 [Pseudomonas phage phiKZ]; PF00383.1 dCMP_cyt_deam_1 orf135 75999..76193 + 195 7559 8.0 64 hypothetical protein orf136 76193..76744 + 552 21268 5.0 183 conserved hypothetical CBA33504.1 hypothetical protein protein Csp_B19390 [Curvibacter putative symbiont of Hydra magnipapillata]; PB002350 Pfam-B_2350 & PB000221 Pfam-B_221 orf137 76754..77044 + 291 11205 5.3 96 hypothetical protein orf138 77166..78968 + 1803 68063 5.7 600 NrdD anaerobic NTP YP_002003610.1 anaerobic reductase large subunit ribonucleoside-triphosphate reductase [Escherichia phage rv5]; PF01228.15 Gly_radical orf139 78978..79421 + 444 17749 4.7 147 hypothetical protein orf140 79440..80183 + 744 27972 4.8 247 conserved hypothetical YP_003579901.1 hypothetical protein protein KP-KP15_gp027 [Klebsiella phage KP15] orf141 80180..80653 + 474 17829 5.1 157 anaerobic ZP_02085286.1 hypothetical protein ribonucleoside- CLOBOL_02822 [Clostridium bolteae triphosphate reductase ATCC BAA-613] activating protein orf142 80646..80822 + 177 6651 3.8 58 hypothetical protein orf143 80900..83608 + 2709 98358 6.0 902 conserved hypothetical C-terminus YP_418148.1 hypothetical protein protein PPEV_gp115 [Pseudomonas phage EL] orf144 83618..86356 + 2739 99595 5.5 912 conserved hypothetical C-terminus YP_418148.1 hypothetical membrane protein protein PPEV_gp115 [Pseudomonas phage EL]; 1 transmembrane domains

218

shown using TMHMM 2.0 and Phobius orf145 86368..89118 + 2751 99719 5.7 916 conserved hypothetical YP_418148.1 hypothetical protein protein PPEV_gp115 [Pseudomonas phage EL] orf146 89248..89820 + 573 22318 7.1 190 hypothetical protein orf147 89821..90600 + 780 29856 7.6 259 putative tRNAHis YP_001957040.1 putative Thg1 guanylyltransferase [Pseudomonas phage 201phi2-1]; COG4021, Uncharacterized conserved protein & Thg1[pfam04446], tRNAHis guanylyltransferase; PF04446.6 Thg1 orf148 90566..91075 + 510 19919 169 hypothetical protein orf149 91075..91377 + 303 11277 5.2 100 NrdA.1 conserved YP_003969388.1 NrdA.1 conserved hypothetical protein hypothetical protein [Aeromonas phage phiAS5]; PB002144 Pfam-B_2144 orf150 91384..91863 + 480 18514 9.2 159 hypothetical protein orf151 91860..92480 + 621 24504 8.6 206 hypothetical protein orf152 92489..93349 + 861 33159 5.3 286 putative N-4 cytosine- CBX30922.1 hypothetical protein specific N47_E44340 [uncultured methyltransferase Desulfobacterium sp.]; PF01555.12 N6_N4_Mtase orf153 93346..93627 + 282 10698 5.1 93 hypothetical protein orf154 93636..94877 + 1242 48691 5.6 413 conserved hypothetical YP_001869348.1 hypothetical protein protein Npun_F6116 [Nostoc punctiforme PCC 73102]; PF10127.3 Nuc-transf orf155 94879..95292 + 414 15972 4.2 137 hypothetical protein orf156 95296..95802 + 507 19299 4.1 168 hypothetical protein orf157 95804..96280 + 477 18476 4.3 158 hypothetical protein orf158 96264..96839 + 576 21669 5.3 191 hypothetical protein orf159 96973..97227 + 255 138 acyl carrier protein YP_003564666.1 acyl carrier protein [Bacillus megaterium QM B1551]; PF00550.19 PP-binding orf160 97303..97680 + 378 14447 9.3 125 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius

219

orf161 97682..97978 + 297 11137 5.5 98 hypothetical protein orf162 98053..98442 + 390 15417 5.4 129 hypothetical protein orf163 98453..98710 + 258 10299 4.3 85 hypothetical protein orf164 99365..99736 + 372 14752 5.9 123 hypothetical protein orf165 99770..100210 + 441 17239 4.5 146 MobD.6 conserved ADQ52884.1 MobD.6 conserved hypothetical protein hypothetical protein [Aeromonas phage PX29]; PB014259 Pfam-B_14259 orf166 100255..100785 + 531 20633 4.0 176 hypothetical protein orf167 100823..101257 + 435 17250 3.9 144 conserved hypothetical YP_004508620.1 hypothetical protein protein SCRM01_187 [Synechococcus phage S- CRM01] orf168 101241..101528 + 288 11103 4.4 95 hypothetical protein orf169 101534..101833 + 300 11527 9.8 99 hypothetical protein orf170 101847..102416 + 570 21423 5.1 189 thymidine kinase NP_873886.1 thymidine kinase [Haemophilus ducreyi 35000HP]; PF00265.12 TK orf171 102473..102979 + 507 19695 9.1 168 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf172 102976..103557 + 582 22149 7.2 193 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf173 103554..103913 + 360 13685 6.3 119 conserved hypothetical YP_004301202.1 hypothetical protein protein 65p365 [Aeromonas phage 65] orf174 103916..104491 + 576 22422 4.9 191 hypothetical protein orf175 104503..105768 + 1266 47841 4.9 421 RNA ligase YP_238935.1 hypothetical protein PHG31p206 [Aeromonas phage 31]; PHA02142, putative RNA ligase & RNA_ligase[pfam09414], RNA ligase orf176 105937..107451 + 1515 55693 9.1 504 conserved hypothetical NP_899547.1 hypothetical protein protein KVP40.0300 [Vibrio phage KVP40]; PF11443.2 DUF2828 orf177 107502..107642 + 141 5021 46 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius

220

orf178 107696..108118 + 423 17245 8.4 140 conserved hypothetical ZP_02927560.1 hypothetical protein protein VspiD_12960 [Verrucomicrobium spinosum DSM 4136] orf179 108111..108686 + 576 21849 5.2 191 hypothetical protein orf180 108801..109733 + 933 35328 9.3 310 lysozyme EGB43418.1 phage lysozyme [Escherichia coli H120]; bacteriophage_T4- like_lysozyme[cd00735]; 1 transmembrane domains shown using TMHMM 2.0 and Phobius orf181 109751..110122 + 372 14828 9.7 123 conserved hypothetical ADA82448.1 hypothetical protein protein [Escherichia phage K1ind3] orf182 110119..110364 + 246 9354 7.8 81 hypothetical protein orf183 110382..110717 + 336 12806 5.1 111 sigma 54 modulation YP_004547118.1 ribosomal subunit protein/ribosomal protein interface protein [Desulfotomaculum S30EA ruminis DSM 2154]; PF02482.13 Ribosomal_S30AE orf184 110801..111196 + 396 15606 5.2 131 hypothetical protein orf185 111183..111392 + 210 8287 69 hypothetical protein orf186 111395..112510 + 1116 42839 371 multifunctional tRNA EGT81514.1 putative tRNA nucleotidyl nucleotidyltransferase-like protein transferase/2'3'-cyclic [Haemophilus haemolyticus M21639]; phosphodiesterase/2'nucl PF01743.14 PolyA_pol & PF12627.1 eotidase/phosphatase PolyA_pol_RNAbd orf187 112578..113006 + 429 16587 4.7 142 NudE nudix hydrolase YP_003580023.1 NudE nudix hydrolase [Klebsiella phage KP15]; PF00293.1 NUDIX orf188 113032..113268 + 237 8714 4.5 78 glutaredoxin YP_003007685.1 glutaredoxin, GrxA family [Aggregatibacter aphrophilus NJ8700]; PF00462.18 Glutaredoxin orf189 113270..113683 + 414 15194 4.9 137 conserved hypothetical EGH82440.1 hypothetical protein protein PLA107_04839 [Pseudomonas syringae pv. lachrymans str. M301315]; PF09424.4 YqeY orf190 113685..114029 + 345 13358 5.5 114 hypothetical protein

221

orf191 114077..114319 + 243 9311 80 hypothetical protein orf192 114364..114861 + 498 18500 5.4 165 conserved hypothetical YP_003579992.1 Tk.4 [Klebsiella protein phage KP15]; PF01661.15 Macro orf193 114888..115214 + 327 11876 4.5 108 hypothetical protein orf194 115218..115580 + 363 13561 120 hypothetical protein orf195 115629..116414 + 786 29634 5.3 261 neck protein YP_004324705.1 neck protein [Synechococcus phage S-SSM5]; PB009499 Pfam-B_9499 orf196 116453..116704 + 252 9639 3.8 83 hypothetical protein orf197 116743..117429 + 687 26437 5.0 228 putative deoxynucleoside YP_003969712.1 putative monophosphate kinase deoxynucleoside monophosphate kinase [Cafeteria roenbergensis virus BV-PW1] orf198 117516..120167 + 2652 95643 4.7 883 tail sheath monomer C-terminus of YP_214361.1 tail sheath monomer [Prochlorococcus phage P- SSM2]; PF04984.8 Phage_sheath_1 orf199 120275..120964 + 690 25392 4.7 229 hypothetical protein orf200 121045..121821 + 777 29095 5.2 258 hypothetical protein orf201 121832..122926 + 1095 39841 5.0 364 hypothetical protein orf202 122926..123384 + 459 17733 9.5 152 head completion protein YP_195238.1 head completion protein [Synechococcus phage S-PM2]; PB000562 Pfam-B_562 & PF08722.5 Tn7_Tnp_TnsA_N orf203 123386..123955 + 570 21751 4.7 189 hypothetical protein orf204 123983..124528 + 546 20976 4.7 181 hypothetical protein orf205 124531..125106 + 576 20760 5.0 191 hypothetical protein orf206 125119..126540 + 1422 52727 5.2 473 hypothetical protein orf207 126558..127514 + 957 34322 3.8 318 hypothetical protein orf208 127514..127918 + 405 14719 3.7 134 hypothetical protein orf209 127922..128296 + 375 14303 4.9 124 hypothetical protein orf210 128437..129780 + 1344 50484 5.2 447 ATPase YP_003891484.1 ATPase [Sulfurimonas autotrophica DSM 16294]; PB003814 Pfam-B_3814 orf211 129823..131178 + 1356 51071 5.1 451 conserved hypothetical YP_003610479.1 hypothetical protein protein BC1002_7228 [Burkholderia sp. CCGE1002]; PF09967.3 DUF2201 &

222

PB015028 Pfam-B_15028 orf212 131286..131522 + 237 8607 78 hypothetical protein orf213 131524..132210 + 687 25914 5.2 228 hypothetical protein orf214 132210..132518 + 309 11588 4.9 102 hypothetical protein orf215 132505..133092 + 588 22242 5.8 195 conserved hypothetical XP_383559.1 hypothetical protein protein FG03383.1 [Gibberella zeae PH-1]; PB014196 Pfam-B_14196 orf216 133099..133452 + 354 13652 4.5 117 hypothetical protein orf217 133415..134344 + 930 35302 5.2 309 thymidylate synthase YP_003656853.1 thymidylate synthase [Arcobacter nitrofigilis DSM 7299]; PF00303.13 Thymidylat_synt orf218 134375..136312 - 1938 68385 4.9 645 conserved hypothetical YP_001970885.1 putative glycine-rich protein autotransporter protein [Stenotrophomonas maltophilia K279a] orf219 136401..137039 - 639 24157 4.6 212 conserved hypothetical CBJ94334.1 hypothetical phage protein protein [Campylobacter phage CPt10]; PB014973 Pfam-B_14973 orf220 137053..141726 - 4674 154065 4.2 1557 conserved hypothetical EGG24419.1 hypothetical protein protein; putative DFA_06569 [Dictyostelium coliphage T4 gp34 long fasciculatum] tail fiber, proximal subunit orf221 141861..142724 - 864 30144 4.6 287 hypothetical protein orf222 142803..143942 - 1140 41425 4.2 379 hypothetical protein orf223 144104..144544 + 441 16657 146 hypothetical protein orf224 144555..145163 + 609 21770 5.3 202 hypothetical protein orf225 145174..146283 + 1110 40647 4.9 369 hypothetical protein orf226 146285..146752 - 468 17735 4.7 155 MutT/NUDIX hydrolase YP_001949983.1 MutT/NUDIX family protein hydrolase family protein [Ralstonia phage RSL1]; PF00293.1 NUDIX orf227 146763..148100 - 1338 51312 4.8 445 tail sheath stabilizer and YP_003969298.1 tail sheath stabilizer completion protein (gp15 and completion protein [Aeromonas homolog) phage phiAS5]; PB006635 Pfam-

223

B_6635 orf228 148097..149593 - 1497 57329 4.6 498 hypothetical protein orf229 149699..151993 + 2295 84483 4.9 764 ATP-dependent Clp YP_004474389.1 ATP-dependent Clp protease, ATP-binding protease, ATP-binding subunit clpA subunit clpA [Pseudomonas fulva 12-X]; PB001281 Pfam-B_1281 & PF00004.23 AAA & PF07724.8 AAA_2 & PF10431.3 ClpB_D2-small orf230 152032..162057 - 10026 381192 4.7 3341 hypothetical protein orf231 162139..165603 - 3465 131411 4.8 1154 baseplate wedge YP_004322992.1 baseplate wedge [Synechococcus phage S-SM1] orf232 165679..166068 - 390 14846 4.9 129 base plate protein; phage NP_861891.1 baseplate wedge subunit T4 gp25 homolog [Enterobacteria phage RB69]; PB001307 Pfam-B_1307 & PF04965.8 GPW_gp25 orf233 166079..166567 - 489 18106 9.1 162 baseplate hub subunit YP_003969622.1 baseplate hub subunit and tail lysozyme; phage and tail lysozyme [Aeromonas phage T4 gp5 homolog phiAS5]; PF00959.13 Phage_lysozyme orf234 166603..169287 - 2685 96994 5.6 894 baseplate hub subunit YP_003969622.1 baseplate hub subunit and tail lysozyme; phage and tail lysozyme [Aeromonas phage T4 gp5 homolog phiAS5]; PF00959.13 Phage_lysozyme orf235 169287..171674 - 2388 88940 4.8 795 hypothetical protein orf236 171686..172000 - 315 12183 104 hypothetical protein orf237 172000..172737 - 738 27686 6.1 245 hypothetical protein orf238 172855..173715 + 861 33534 286 sigma factor for late YP_004322799.1 sigma factor for late transcription; T4 gp55 transcription [Synechococcus phage S- homolog ShM2]; 55[PHA02547], RNA polymerase sigma factor orf239 173702..174733 + 1032 39961 5.2 343 recombination YP_004324210.1 recombination endonuclease subunit; endonuclease subunit [Synechococcus phage T4 gp47 homolog phage S-SSM7]; PF12850.1 Metallophos_2

224

orf240 174746..176887 + 2142 81951 4.8 713 recombination YP_003734204.1 gp46 recombination endonuclease subunit; endonuclease subunit [Enterobacteria phage T4 gp46 homolog phage IME08]; PB010468 Pfam- B_10468 orf241 176902..177396 - 495 18831 9.5 164 EndoVII packaging and YP_002003595.1 EndoVII packaging recombination and recombination endonuclease endonuclease; phage T4 [Escherichia phage rv5]; 49[PHA02565], gp49 homolog recombination endonuclease VII; orf242 178201..178377 - 177 6852 6.2 58 hypothetical protein orf243 178398..179255 - 858 32653 4.5 285 hypothetical protein orf244 179319..181481 + 2163 78421 5.2 720 conserved hypothetical YP_003663488.1 phage infection protein protein [Bacillus thuringiensis BMB171] orf245 181500..183242 + 1743 65245 4.8 580 portal vertex protein of NP_899604.1 portal vertex protein of head; phage T4 gp20 head [Vibrio phage KVP40]; PF07230.5 homolog Phage_T4_Gp20 orf246 183299..183529 + 231 8657 76 hypothetical protein orf247 183637..184479 + 843 31015 4.1 280 hypothetical protein orf248 184502..185134 + 633 23378 5.8 210 prohead core scaffold YP_004322543.1 prohead core scaffold and protease; phage T4 and protease [Prochlorococcus phage P- gp21 homolog HM1]; PF03420.7 Peptidase_U9 orf249 185204..186331 + 1128 42708 5.8 375 hypothetical protein orf250 186409..187584 + 1176 42103 5.8 391 precursor of major head YP_004323268.1 precursor of major subunit; phage T4 gp23 head subunit [Prochlorococcus phage P- homolog RSM4]; PF07068.5 Gp23 orf251 187674..187907 + 234 8568 4.5 77 conserved hypothetical C-terminus of YP_002922206.1 protein hypothetical protein EpJSE_00134 [Enterobacteria phage JSE] orf252 187968..188384 + 417 15980 4.8 138 hypothetical protein orf253 188413..188724 + 312 12019 5.4 103 conserved hypothetical YP_001836995.1 hypothetical protein protein AGC_0072 [Enterobacteria phage EPS7]; PF10544.3 T5orf172 orf254 188769..190388 + 1620 56894 6.2 539 putative tail fiber protein YP_002003535.1 putative tail fiber protein [Escherichia phage rv5]

225

orf255 190381..190794 + 414 16163 4.8 137 caudovirales tail fibre YP_002923765.1 phage tail assembly assembly protein chaperone [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] orf256 190881..191183 + 303 11335 4.9 100 hypothetical protein orf257 191245..194277 + 3033 115975 5.0 1010 DNA polymerase; phage ADQ52734.1 gp43 DNA polymerase T4 gp43 homolog [Aeromonas phage PX29]; PF03104.13 DNA_pol_B_exo1 & PF00136.15 DNA_pol_B orf258 194293..194802 + 510 18983 169 hypothetical protein orf259 194866..195597 + 732 26735 5.3 243 hypothetical protein orf260 195646..196251 + 606 23490 6.1 201 putative phosphoesterase YP_004300651.1 putative or phosphohydrolase phosphoesterase or phosphohydrolase [Acinetobacter phage 133]; PB001300 Pfam-B_1300 orf261 196271..196480 + 210 8036 6.0 69 hypothetical protein Protein motif: PF09723.4 CxxC_CxxC_SSSS orf262 196502..196789 + 288 10282 4.7 95 co-chaperonin GroES YP_003848396.1 Chaperonin Cpn10 [Gallionella capsiferriformans ES-2]; PF00166.15 Cpn10 orf263 196782..197501 + 720 28686 4.7 239 hypothetical protein orf264 197546..198202 - 657 22445 5.1 218 hypothetical protein orf265 198218..198778 - 561 19138 4.6 186 hypothetical protein orf266 198974..199960 + 987 38114 328 RNaseH ribonuclease CBZ42347.1 hypothetical protein [Campylobacter phage CP81] & YP_004421603.1 putative RNaseH [Campylobacter phage NCTC12673]; PF02739.10 5_3_exonuc_N orf267 199960..200460 + 501 18983 4.4 166 hypothetical protein orf268 200504..201211 + 708 27395 6.5 235 terminase DNA YP_004323947.1 terminase DNA packaging enzyme large packaging enzyme large subunit subunit; phage T4 gp17 [Synechococcus phage Syn19] homolog

226

orf269 201198..202847 + 1650 62998 5.5 549 DNA terminase YP_003097347.1 DNA terminase packaging enzyme large packaging enzyme large subunit subunit; phage T4 gp17 [Synechococcus phage S-RSM4]; homolog PF03237.9 Terminase_6 orf270 202884..204173 + 1290 48636 6.2 429 hypothetical protein orf271 204399..205442 + 1044 38586 5.3 347 ssDNA binding protein; YP_004324057.1 ssDNA binding phage T4 gp32 homolog protein [Synechococcus phage S-SSM7] orf272 205501..206631 + 1131 41473 4.9 376 UvsX RecA-like protein YP_214708.1 UvsX RecA-like protein [Prochlorococcus phage P-SSM4]; PF00154.15 RecA orf273 206633..207097 + 465 17920 5.4 154 hypothetical protein orf274 207155..208063 + 909 34589 8.6 302 conserved hypothetical YP_001295409.1 hypothetical protein protein FP0485 [Flavobacterium psychrophilum JIP02/86] orf275 208050..208895 + 846 32628 4.5 281 putative DNA YP_003183945.1 DNA polymerase III, polymerase III, epsilon epsilon subunit [Alicyclobacillus subunit acidocaldarius subsp. acidocaldarius DSM 446]; PF00929.18 RNase_T & cd06127, DEDDh 3'-5' exonuclease domain family orf276 208919..210403 + 1485 56077 6.2 494 RNA-DNA + DNA- YP_214374.1 RNA-DNA + DNA-DNA DNA helicase helicase [Prochlorococcus phage P- SSM2]; PF04851.9 ResIII orf277 210457..210897 + 441 17196 3.6 146 hypothetical protein orf278 210907..211410 + 504 19199 4.4 167 hypothetical protein orf279 211410..212255 + 846 32336 5.1 281 hypothetical protein orf280 212242..212961 + 720 28741 8.2 239 hypothetical protein orf281 212958..213308 + 351 13451 6.5 116 hypothetical protein orf282 213274..213924 + 651 25050 5.7 216 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf283 213908..214261 - 354 14194 8.8 117 hypothetical protein orf284 214319..215290 + 972 36566 5.3 323 hypothetical protein orf285 215353..216399 + 1047 40474 5.8 348 putative DNA primase NP_853172.2 DNA primase [Mycoplasma gallisepticum str. R(low)]

227

orf286 216407..217909 + 1503 55919 5.6 500 DNA primase-helicase YP_214418.1 DNA primase-helicase [Prochlorococcus phage P-SSM2] & coliphage gp41 DNA primase-helicase; PF03796.9 DnaB_C orf287 217912..218181 + 270 10382 6.1 89 hypothetical protein orf288 218183..218341 + 159 5660 10.1 52 hypothetical protein orf289 218345..218743 + 399 14731 5.3 132 hypothetical protein orf290 218789..219736 + 948 34670 4.4 315 hypothetical protein orf291 219736..219954 + 219 8396 72 hypothetical protein orf292 219951..220754 + 804 30275 4.7 267 hypothetical protein orf293 220787..221434 + 648 23770 215 hypothetical protein orf294 221446..221781 + 336 12144 4.2 111 hypothetical protein orf295 221821..222522 + 702 26568 5.1 233 conserved hypothetical YP_265907.1 hypothetical protein protein SAR11_0482 [Candidatus Pelagibacter ubique HTCC1062]& YP_003097237.1 putatitive exonulcease [Synechococcus phage S-RSM4]; PF12705.1 PDDEXK_1 orf296 222526..223077 + 552 20284 4.7 183 hypothetical protein orf297 223106..223684 + 579 20927 5.0 192 hypothetical protein orf298 223756..226089 + 2334 87807 5.7 777 ribonucleotide- YP_003614008.1 ribonucleotide- diphosphate reductase diphosphate reductase alpha subunit alpha subunit [Enterobacter cloacae subsp. cloacae ATCC 13047]; PF03477.10 ATP-cone & PF00317.15 Ribonuc_red_lgN & PF02867.1 Ribonuc_red_lgC orf299 226147..226884 + 738 28846 9.1 245 conserved hypothetical YP_131801.1 hypothetical protein protein PBPRB0128 [Photobacterium profundum SS9] orf300 226897..228021 + 1125 43176 4.8 374 ribonucleotide- YP_004427291.1 ribonucleotide- diphosphate reductase diphosphate reductase subunit beta subunit beta [Alteromonas macleodii str. 'Deep ecotype']; PF00268.15 Ribonuc_red_sm orf301 228061..228267 + 207 7879 4.3 68 hypothetical protein orf302 228310..228543 + 234 9088 9.8 77 hypothetical protein

228

orf303 228555..228935 + 381 14838 4.6 126 hypothetical protein orf304 228935..229651 + 717 26508 4.9 238 conserved hypothetical YP_265439.1 hypothetical protein protein SAR11_0011 [Candidatus Pelagibacter ubique HTCC1062]; PF01503.11 PRA- PH orf305 229680..230324 - 645 24072 7.6 214 hypothetical protein orf306 230336..231046 - 711 26388 7.6 236 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf307 231179..231553 + 375 14214 9.5 124 hypothetical membrane 2-3 transmembrane domains shown protein using TMHMM 2.0 and Phobius orf308 231550..232068 + 519 19567 5.9 172 dihydrofolate reductase YP_002720345.1 dihydrofolate reductase [Brachyspira hyodysenteriae WA1]; PF00186.13 DHFR_1 orf309 232069..232503 + 435 16192 6.1 144 ribonuclease H YP_004305109.1 RNase H [Polymorphum gilvum SL003B-26A1]; PF00075.18 RNase_H orf310 232500..233840 + 1341 51391 5.7 446 DNA helicase Dda YP_003579895.1 Dda DNA helicase [Klebsiella phage KP15]; PF05970.8 PIF1 & PB010441 Pfam-B_10441 orf311 233911..234456 + 546 19858 4.7 181 hypothetical protein orf312 234477..234671 + 195 7286 5.0 64 hypothetical protein orf313 234744..235250 + 507 18857 9.4 168 translation initiation YP_003432420.1 translation initiation factor IF-3 factor 3 [Hydrogenobacter thermophilus TK-6]; PF05198.10 IF3_N & PF00707.16 IF3_C orf314 235317..235850 + 534 20167 177 ATP-dependent Clp YP_001355850.1 ATP-dependent Clp protease protease, adaptor protein ClpS [Nitratiruptor sp. SB155-2]; PF02617.11 Clp orf315 235913..236446 + 534 20694 9.4 177 conserved hypothetical YP_003591270.1 hypothetical protein protein Cseg_0125 [Caulobacter segnis ATCC 21756] orf316 236436..238043 + 1608 61777 5.4 535 conserved hypothetical ZP_08695409.1 hypothetical protein protein FVAG_02046 [Fusobacterium varium ATCC 27725] orf317 238043..240577 + 2535 95930 5.1 844 hypothetical protein

229

orf318 240587..240952 + 366 13946 7.1 121 conserved hypothetical YP_004301135.1 hypothetical protein membrane protein 65p298 [Aeromonas phage 65]; 1 transmembrane domains shown using TMHMM 2.0 and Phobius; PF11750.2 DUF3307 orf319 241048..243015 + 1968 74055 6.2 655 DNA gyrase, subunit B YP_002334711.1 DNA gyrase B subunit [Thermosipho africanus TCF52B]; PF02518.20 HATPase_c & PF00204.19 DNA_gyraseB orf320 243067..244473 + 1407 53217 5.0 468 DNA topoisomerase II XP_765033.1 DNA topoisomerase II [Theileria parva strain Muguga]; PF00521.1 DNA_topoisoIV orf321 244559..246931 + 2373 88301 4.8 790 hypothetical protein orf322 246941..247246 + 306 11696 5.1 101 hypothetical protein orf323 247206..247724 + 519 20520 4.6 172 DNA polymerase III, YP_003306784.1 DNA polymerase III, alpha subunit alpha subunit [Streptobacillus moniliformis DSM 12112]; PF07733.6 DNA_pol3_alpha orf324 247771..248145 + 375 13939 5.7 124 hypothetical protein orf325 248156..248476 + 321 11640 5.6 106 hypothetical protein orf326 248521..248811 + 291 11219 4.9 96 hypothetical protein orf327 248814..249770 + 957 36222 4.8 318 sliding clamp loader YP_004421559.1 gp44 sliding clamp subunit; phage T4 gp44 loader subunit [Campylobacter phage homolog NCTC12673]; PF00004.23 AAA orf328 249779..250399 + 621 24045 4.7 206 conserved hypothetical NP_049815.1 gp30.2 conserved protein; phage T4 gp30.2 hypothetical protein [Enterobacteria homolog phage T4] orf329 250457..251029 + 573 21826 4.2 190 hypothetical protein orf330 251029..251712 + 684 25838 4.3 227 hypothetical protein orf331 251727..252506 + 780 30120 7.6 259 hypothetical protein orf332 252645..253196 + 552 19812 5.4 183 hypothetical protein orf333 253233..253433 + 201 7023 66 hypothetical protein orf334 253494..254684 + 1191 45651 5.0 396 hypothetical protein orf335 254773..255921 + 1149 42784 5.4 382 toxic ion resistance YP_416739.1 toxic ion resistance protein protein [Staphylococcus aureus RF122]; PF05816.5 TelA

230

orf336 255989..256321 + 333 12997 4.2 110 hypothetical protein orf337 256321..257001 + 681 25805 4.7 226 putative metallopeptidase YP_004306560.1 putative metallopeptidase [Enterobacteria phage SPC35] orf338 257057..258154 + 1098 42114 7.6 365 conserved hypothetical YP_003969376.1 hypothetical protein membrane protein phiAS5_ORF0087 [Aeromonas phage phiAS5]; 2-3 transmembrane domains shown using TMHMM 2.0 and Phobius orf339 258151..258705 + 555 20860 184 conserved hypothetical NP_944184.1 hypothetical protein protein Aeh1p306 [Aeromonas phage Aeh1] orf340 258809..258991 + 183 6586 60 hypothetical protein orf341 259002..259349 + 348 13082 8.4 115 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf342 259390..259875 + 486 18905 5.1 161 conserved hypothetical YP_003358497.1 hypothetical protein protein [Shigella phage phiSboM-AG3] orf343 259933..261381 + 1449 54847 4.4 482 hypothetical protein orf344 262291..262701 + 411 15955 4.2 136 hypothetical protein orf345 262704..262907 + 204 7963 67 hypothetical protein orf346 262907..263254 + 348 13607 4.7 115 hypothetical protein orf347 263256..263558 + 303 11937 4.4 100 hypothetical protein orf348 263573..264292 + 720 27715 4.5 239 conserved hypothetical YP_209413.1 YdfA [Salmonella protein enterica subsp. enterica serovar Choleraesuis str. SC-B67]; PB005443 Pfam-B_5443 & PB007575 Pfam- B_7575 orf349 264282..264530 + 249 9852 5.8 82 hypothetical protein orf350 264535..264729 + 195 7642 4.3 64 hypothetical protein orf351 264742..265764 + 1023 40018 4.6 340 conserved hypothetical YP_001875870.1 hypothetical protein protein Emin_0980 [Elusimicrobium minutum Pei191] orf352 265774..266145 + 372 14152 4.7 123 conserved hypothetical YP_003580022.1 hypothetical protein protein KP-KP15_gp165 [Klebsiella phage KP15]

231

orf353 266164..267210 + 1047 40203 5.9 348 nicotinamide-nucleotide YP_001837009.1 nicotinamide- adenylyltransferase nucleotide adenylyltransferase [Enterobacteria phage EPS7]; PF01467.1 CTP_transf_2 orf354 267207..268010 + 804 30245 9.0 267 nicotinamide YP_003309907.1 nicotinamide mononucleotide mononucleotide transporter PnuC transporter PnuC [Sebaldella termitidis ATCC 33386]; 7-9 transmembrane domains shown using TMHMM 2.0 and Phobius; PF04973.6 NMN_transporter orf355 268000..268626 + 627 24672 8.4 208 conserved hypothetical YP_224229.1 hypothetical protein protein LPPPVgp01 [Listonella phage phiHSIC] orf356 268613..268855 + 243 9155 4.5 80 conserved hypothetical NP_930177.1 hypothetical protein protein plu2943 [Photorhabdus luminescens subsp. laumondii TTO1] orf357 268904..269326 + 423 16964 5.5 140 hypothetical protein orf358 269323..269634 + 312 11692 5.3 103 hypothetical protein orf359 269643..269978 + 336 12455 4.6 111 hypothetical protein orf360 269962..270321 + 360 14007 4.5 119 hypothetical protein orf361 270332..270517 + 186 7285 61 hypothetical protein orf362 270634..271083 + 450 17242 4.5 149 hypothetical protein orf363 271209..271508 + 300 11382 3.8 99 hypothetical protein orf364 271501..271920 + 420 16097 4.5 139 hypothetical protein orf365 271917..272741 + 825 31150 4.9 274 conserved hypothetical YP_001516926.1 hypothetical protein protein AM1_2605 [Acaryochloris marina MBIC11017]; PB002278 Pfam-B_2278 orf366 272741..274003 + 1263 49216 4.9 420 conserved hypothetical NP_899514.1 hypothetical protein protein KVP40.0268 [Vibrio phage KVP40] orf367 274092..275309 + 1218 46251 4.8 405 conserved hypothetical AEK82040.1 putative ATPase protein [Salmonella phage 7-11] orf367A 275385..275516 + 132 4615 4.4 43 hypothetical protein orf368 275687..276160 + 474 18209 6.0 157 hypothetical protein orf369 276184..276516 + 333 12997 6.1 110 hypothetical protein

232

orf370 276520..277107 + 588 21972 6.0 195 putative 5'3'- YP_003145252.1 5 nucleotidase deoxy deoxyribonucleotidase cytosolic type C [Kangiella koreensis DSM 16069]; PF06941.6 NT5C orf371 277101..277661 + 561 21543 4.4 186 hypothetical protein orf372 277780..278358 + 579 22167 5.8 192 hypothetical protein orf373 278826..279515 + 690 26494 9.2 229 HNH endonuclease YP_955954.1 HNH endonuclease [Mycobacterium vanbaalenii PYR-1]; PB000761 Pfam-B_761 orf374 279512..279838 + 327 12971 4.4 108 conserved hypothetical AEH03461.1 hypothetical protein protein [Pseudomonas phage PhiPA3] orf374A 280174..280323 + 150 5336 9.1 49 hypothetical protein orf375 280323..280577 + 255 9838 4.3 84 hypothetical protein orf376 280628..281365 + 738 30017 5.0 262 conserved hypothetical YP_632368.1 hypothetical protein protein MXAN_4193 [Myxococcus xanthus DK 1622]; PB002278 Pfam-B_2278 orf377 281358..281717 + 360 14299 5.6 119 hypothetical protein orf378 281689..282153 + 465 17914 4.8 154 hypothetical protein orf379 282150..282419 + 270 10483 4.0 89 hypothetical protein orf380 282416..282973 + 558 22441 185 hypothetical protein orf381 283251..283379 + 129 4820 42 hypothetical protein orf382 283354..283782 + 429 16811 6.9 142 peptidyl-tRNA hydrolase NP_143399.1 peptidyl-tRNA hydrolase [Pyrococcus horikoshii OT3]; PF01981.10 PTH2 orf383 283782..284135 + 354 13523 5.1 117 hypothetical protein orf384 284388..284747 + 360 13779 5.6 119 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf385 284737..284970 + 234 8731 9.0 77 hypothetical protein orf386 284951..285220 + 270 10753 9.0 89 hypothetical protein orf387 285373..285627 + 255 10046 6.1 84 hypothetical protein orf388 285637..286008 + 372 14015 4.4 123 hypothetical protein orf389 285992..286456 + 465 18301 5.3 154 hypothetical protein orf390 286477..287343 + 867 33037 5.2 288 DNA-cytosine ADX89637.1 putative DNA- methyltransferase methyltransferase, type II restriction- modification system (Enterobacteria phage RB16) [Vibrio phage ICP1_2004_A]; PF00145.11

233

DNA_methylase

orf391 287330..287899 + 570 21612 4.7 189 hypothetical protein orf392 287902..288243 + 342 13277 8.6 113 hypothetical protein orf393 288240..288941 + 702 27282 4.6 233 hypothetical protein orf394 288952..289332 + 381 15049 5.6 126 hypothetical protein orf395 289304..289501 + 198 7892 65 hypothetical protein orf396 289795..290124 + 330 12655 109 hypothetical protein orf397 290231..290464 + 234 8800 6.7 77 hypothetical protein orf398 290461..290601 + 141 5334 4.7 46 hypothetical protein orf398A 290609..290803 + 195 7954 10.1 64 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf399 290716..291027 + 312 11881 6.1 103 conserved hypothetical EGH82531.1 hypothetical protein protein PLA107_05316 [Pseudomonas syringae pv. lachrymans str. M301315] orf400 291075..291518 + 444 17674 147 hypothetical protein orf401 291515..291703 + 189 7382 3.7 62 hypothetical protein orf402 291705..292055 + 351 13300 116 hypothetical protein orf403 292636..292926 + 291 10923 9.6 96 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf404 292929..293180 + 252 9457 9.5 83 conserved hypothetical YP_004327451.1 putative protein uncharacterised protein [Salmonella phage Vi01] orf405 293461..293670 + 210 8021 6.6 69 hypothetical protein orf406 293678..293833 + 156 5525 51 conserved hypothetical AEJ81265.1 gp006 [Erwinia phage protein vB_EamM-M7] orf407 293896..294699 - 804 31268 9.5 267 hypothetical protein orf408 294959..295111 + 153 5795 6.5 50 hypothetical protein orf409 295120..295320 + 201 7976 66 hypothetical protein orf410 295320..295526 + 207 7487 9.3 68 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf411 295568..295723 + 156 5697 7.8 51 hypothetical protein

234

orf412 295723..296166 + 444 16983 4.6 147 hypothetical protein orf413 296279..296473 + 195 7209 6.2 64 conserved hypothetical AEK82041.1 hypothetical protein protein [Salmonella phage 7-11] orf414 296613..297065 + 453 17772 4.6 150 hypothetical protein orf415 297140..297949 + 810 31348 4.7 269 hypothetical protein orf416 297942..298349 + 408 15978 5.1 135 conserved hypothetical YP_004009942.1 hypothetical protein protein CC31p084 [Enterobacteria phage CC31] orf417 298520..298813 + 294 11671 5.6 97 hypothetical protein orf418 298891..299169 + 279 9906 5.4 92 hypothetical protein orf419 299172..299369 + 198 7826 4.5 65 hypothetical protein orf420 299353..299718 + 366 14420 4.6 121 hypothetical protein Protein motif: PB001179 Pfam-B_1179 orf421 299718..300074 + 357 13890 6.2 118 hypothetical protein Protein motif: PB001179 Pfam-B_1179 orf422 300226..300459 + 234 8608 9.0 77 hypothetical protein orf423 300691..301053 + 363 13629 4.9 120 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf424 301305..303038 + 1734 62024 4.8 577 adhesin AEJ81565.1 adhesin [Erwinia phage vB_EamP-S6]; PF03895.9 YadA orf425 303094..303603 + 510 19615 4.7 169 hypothetical protein orf426 303753..303968 + 216 8292 9.2 71 hypothetical protein orf427 304033..304275 + 243 9435 80 hypothetical protein orf428 304288..304758 + 471 17474 9.2 156 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf429 304793..305212 + 420 16021 4.3 139 conserved hypothetical YP_001836948.1 hypothetical protein protein AGC_0025 [Enterobacteria phage EPS7]

orf430 305370..305945 + 576 22839 191 conserved hypothetical YP_238979.1 hypothetical protein protein RB43ORF003c [Enterobacteria phage RB43]; PF10263.3 SprT-like orf431 306115..306261 + 147 5345 4.4 48 hypothetical protein orf431A 306886..307005 + 120 4635 9.8 39 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf432 307086..307487 + 402 15882 4.7 133 hypothetical protein orf433 307507..307716 + 210 8020 5.3 69 hypothetical protein orf434 307811..308035 + 225 8346 74 conserved hypothetical YP_004009376.1 hypothetical protein protein Ac42p014 [Acinetobacter phage Ac42]

235

orf435 308035..308430 + 396 16009 4.8 131 hypothetical protein orf436 309130..309729 + 600 23064 5.7 199 hypothetical protein orf437 309762..310115 + 354 13687 9.5 117 conserved hypothetical YP_004620440.1 hypothetical protein protein Rta_33090 [Ramlibacter tataouinensis TTB310]; PF07087.5 DUF1353 orf438 310269..310403 + 135 4792 44 hypothetical protein orf439 310615..310848 + 234 9115 4.4 77 hypothetical protein orf439A 311282..311440 + 159 5809 52 hypothetical protein orf440 311538..311864 + 327 12746 6.5 108 hypothetical protein orf441 311953..312189 + 237 9313 4.4 78 hypothetical protein orf442 312182..312667 + 486 19115 6.6 161 hypothetical protein orf443 312669..312872 + 204 8200 4.4 67 hypothetical protein orf444 312982..313200 + 219 8495 5.5 72 hypothetical protein orf445 313197..313457 + 261 9890 9.7 86 hypothetical protein orf446 313634..313855 + 222 8746 6.3 73 conserved hypothetical YP_002425630.1 hypothetical protein protein AFE_1176 [Acidithiobacillus ferrooxidans ATCC 23270] orf447 313852..314169 + 318 12551 4.5 105 hypothetical protein orf448 314200..314595 + 396 15564 4.7 131 hypothetical protein orf449 314599..315009 + 411 16109 5.2 136 hypothetical protein orf449A 314978..315397 + 420 16678 4.7 139 hypothetical protein orf450 315512..315712 + 201 7981 4.9 66 hypothetical protein orf451 315702..316016 + 315 12192 10.0 104 conserved hypothetical YP_003969537.1 hypothetical protein protein phiAS5_ORF0248 [Aeromonas phage phiAS5] orf452 316013..316453 + 441 16846 4.6 146 hypothetical protein orf453 316465..316917 + 453 17485 4.7 150 hypothetical protein orf453A 316892..317140 + 249 9746 82 hypothetical protein Protein motif: PB000525 Pfam-B_525 orf454 317302..317748 + 447 16765 148 hypothetical protein orf455 317790..317963 + 174 6631 57 hypothetical protein orf456 317950..318198 + 249 9197 9.6 82 hypothetical protein orf457 318301..318483 + 183 7012 4.2 60 hypothetical protein orf458 318458..318586 + 129 5108 42 hypothetical protein orf459 318586..318900 + 315 12040 9.5 104 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius

236

orf460 318900..319253 + 354 13058 7.6 117 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf461 319250..319564 + 315 11698 7.6 104 hypothetical protein orf462 319576..319872 + 297 11036 8.9 98 hypothetical protein orf462A 319865..320059 + 195 7179 9.8 64 hypothetical protein orf463 320056..320268 + 213 8123 4.6 70 hypothetical protein orf464 320258..320740 + 483 18191 4.4 160 conserved hypothetical YP_656352.1 hypothetical protein protein PHG25ORF117c [Aeromonas phage 25] orf465 320779..321165 + 387 14027 128 hypothetical protein orf466 321212..321346 + 135 4927 44 hypothetical protein orf467 321386..321889 + 504 19777 5.1 167 hypothetical protein orf468 321882..322511 + 630 24441 209 hypothetical protein orf469 322595..322954 + 360 14067 4.4 119 hypothetical protein orf470 323070..323258 + 189 7349 9.5 62 hypothetical protein orf471 323266..323469 + 204 7617 6.7 67 hypothetical protein orf472 323551..324291 - 741 28576 8.9 246 hypothetical protein orf473 324781..325458 + 678 24471 4.5 225 hypothetical protein orf474 325514..326056 + 543 21236 5.4 180 conserved hypothetical XP_001445153.1 hypothetical protein protein [Paramecium tetraurelia strain d4-2] orf475 326071..327204 - 1134 41213 4.4 377 hypothetical protein orf476 327382..327960 + 579 21894 6.7 192 hypothetical protein orf477 327971..328273 + 303 11490 8.5 100 hypothetical protein orf478 328277..328759 + 483 18320 9.5 160 hypothetical protein orf479 328820..329347 + 528 19864 8.7 175 hypothetical protein orf480 329395..329853 + 459 18250 7.7 152 hypothetical membrane 2-3 transmembrane domains shown protein using TMHMM 2.0 and Phobius orf481 329932..330177 + 246 9283 5.4 81 hypothetical protein orf482 330170..330898 + 729 28493 5.1 242 hypothetical protein orf483 330891..331010 + 120 3926 8.0 39 hypothetical protein orf484 331012..331458 + 447 16858 9.4 148 conserved hypothetical YP_003969072.1 hypothetical protein membrane protein phiAS4_ORF0054 [Aeromonas phage phiAS4]; 2-3 transmembrane domains shown using TMHMM 2.0 and Phobius orf485 331473..332009 + 537 20761 5.8 178 hypothetical protein orf486 332015..332320 + 306 11336 7.8 101 hypothetical protein

237

orf487 332320..332703 + 384 14677 5.6 127 hypothetical protein orf487A 333386..333553 + 168 6074 5.0 55 hypothetical protein orf488 333974..334204 + 231 8718 6.2 76 transcriptional regulator EGS73983.1 helix-turn-helix family protein [Vibrio cholerae BJG-01]; PF12844.1 HTH_19 orf489 334201..334350 + 150 5596 49 hypothetical protein orf490 334445..335440 + 996 37916 4.8 331 RpoD subfamily RNA YP_002462259.1 RpoD subfamily RNA polymerase sigma-70 polymerase sigma-70 subunit subunit [Chloroflexus aggregans DSM 9485]; PF04539.10 Sigma70_r3 orf491 335453..335962 + 510 19777 9.6 169 HNH endonuclease EFY74812.1 HNH endonuclease [Salmonella enterica subsp. enterica serovar Montevideo str. 366867]; PF00847.14 AP2 orf492 335965..336672 + 708 26770 4.5 235 conserved hypothetical AEJ81576.1 gp057 [Erwinia phage protein vB_EamP-S6] orf493 336681..336872 + 192 7150 9.9 63 hypothetical protein orf494 336928..337902 + 975 37106 9.2 324 conserved hypothetical YP_001469475.1 hypothetical protein protein phi1p130.1 [Enterobacteria phage Phi1] orf495 337899..338129 + 231 8573 4.2 76 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf496 338131..338304 + 174 6447 9.0 57 hypothetical protein orf497 338375..338614 + 240 9260 4.8 79 conserved hypothetical YP_001595358.1 hypothetical protein protein EpJS98_gp229 [Enterobacteria phage JS98] orf498 338619..339059 + 441 17301 7.8 146 DenV endonuclease V YP_004063810.1 DenV endonuclease V N-glycosylase UV repair N-glycosylase UV repair enzyme enzyme [Enterobacteria phage vB_EcoM-VR7]; PF03013.8 Pyr_excise orf499 339116..339301 + 186 7249 61 hypothetical protein orf500 339368..339778 + 411 16393 5.6 136 hypothetical protein orf501 339775..339972 + 198 7223 9.4 65 hypothetical membrane 2 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf502 339969..340202 + 234 8969 5.1 77 hypothetical protein orf503 340204..340665 + 462 17599 4.4 153 hypothetical protein orf503A 341071..341184 + 114 4193 8.9 37 hypothetical protein

238

orf504 341317..342117 + 801 31187 4.7 266 hypothetical protein orf505 342177..343175 + 999 39095 332 hypothetical protein orf506 343241..343765 + 525 19460 5.1 174 hypothetical protein orf507 343775..344578 + 804 30931 4.5 267 hypothetical protein orf508 344646..345143 + 498 19203 8.4 165 HNH endonuclease YP_004216392.1 HNH endonuclease [Acidobacterium sp. MP5ACTX9]; PF01844.17 HNH orf509 345146..345463 + 318 12345 9.1 105 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf510 345463..346674 + 1212 45597 5.1 403 conserved hypothetical NP_803774.1 ORF208 [Pseudomonas protein phage phiKZ]; PF00004.23 AAA orf511 346727..346912 + 186 7126 4.9 61 hypothetical protein orf512 346912..347178 + 267 10620 9.6 88 hypothetical protein orf513 347171..347347 + 177 6767 58 hypothetical protein orf514 347325..347492 + 168 6296 55 hypothetical protein orf515 347526..347798 + 273 10343 9.8 90 hypothetical protein orf516 347844..348623 + 780 30706 9.2 259 HNH endonuclease NP_944871.1 Phage conserved protein; putative endonuclease VII [Enterobacteria phage Felix 01] orf517 348764..349222 + 459 16857 8.4 152 hypothetical protein orf518 349219..350403 + 1185 45572 6.2 394 conserved hypothetical YP_001781643.1 hypothetical protein protein CLD_2545 [Clostridium botulinum B1 str. Okra]; PF03235.8 DUF262 orf519 350624..351457 + 834 32297 277 putative DNA N6- YP_002003598.1 putative DNA N6- adenine adenine methyltransferase [Escherichia methyltransferase phage rv5] orf520 351544..352458 + 915 35487 9.3 304 hypothetical protein orf521 352731..353513 + 783 29256 6.0 260 nrdC.10 hypothetical YP_002854431.1 nrdC.10 hypothetical protein protein [Enterobacteria phage RB14] orf522 353515..353838 + 324 12470 9.0 107 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf523 353850..354209 + 360 13427 9.4 119 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf524 354221..354985 + 765 29018 8.8 254 hypothetical protein

239

orf525 355031..355384 + 354 13554 8.2 117 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf526 355381..355719 + 339 12904 4.6 112 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf526A 355716..356063 + 348 13945 8.7 115 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf527 356060..356404 + 345 12586 9.1 114 hypothetical membrane 3 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf528 356474..356752 + 279 10695 4.6 92 hypothetical membrane 1 transmembrane domains shown using protein TMHMM 2.0 and Phobius orf529 356806..357429 + 624 22518 9.3 207 conserved hypothetical YP_001480496.1 hypothetical protein protein Spro_4274 [Serratia proteamaculans 568]; PF06693.5 DUF1190 orf530 357458..358111 - 654 23409 8.9 217 hypothetical protein orf531 358121..358636 - 516 19184 9.3 171 hypothetical protein

240

Table 3.2. Proteomic and HHpred analysis of phage GAP32.

HHpred Peptide Report

PDB Gene Function Probability E-value HHpred Phage E-value Accession #

orf001 3mvd Regulator of chromosome condensation {Drosophila 98.7 8.7E-13 Regulator of 1.7e-66 melanogaster} chromosome condensation (RCC1)

orf025 3bk6 PH stomatin {Pyrococcus horikoshii} 99.12 3.00E-15 FtsH protease regulator 3.6e-51 HflC

orf069 1o6b Phosphopantetheine adenylyltransferase {Bacillus subtilis} 99.00 2e-14

orf074 1vj7 Bifunctional RELA/SPOT {Streptococcus dysgalactiae subsp} 98.25 1.3e-10

orf080 2c5u RNA ligase, T4 RNA ligase 1 {Bacteriophage T4} 100 1.80E-46

orf084 1ltq Polynucleotide kinase {Enterobacteria phage T4} 99.88 1.40E-28

orf095 1g5b Serine/threonine protein phosphatase {Enterobacteria phage 99.81 1.2e-25 lambda}

orf099 1a0i DNA ligase {Enterobacteria phage T7} 99.78 2.2e-24

orf101 3rl5 Metallophosphoesterase mpped2 {Rattus norvegicus} 99.29 1.00E-16

orf103 3p2l ATP-dependent CLP protease proteolytic subunit {Francisella 99.86 1.20E-27 tularensis subsp}

orf109 1vq2 DCMP deaminase, deoxycytidylate deaminase {Enterobacteria 99.92 1.80E-31 phage T4}

241

orf111 2l04 Major tail protein V {Enterobacteria phage lambda} 99.35 3.2e-17 Major tail protein V Lambda 2.5e-16 orf112 1ej2 Nicotinamide mononucleotide adenylyltransferase 98.32 6.20E-11 {Methanothermobacter thermautotrophicusorganism_taxid} orf114 1vlp Naprtase, nicotinate phosphoribosyltransferase {Saccharomyces 99.95 6.70E-34 cerevisiae} orf118 2'-5' RNA ligase / 4.7e-28 orf119 1s5p NAD-dependent deacetylase {Escherichia coli} 99.83 2.20E-26 orf120 3kvh Protein syndesmos {Homo sapiens} 98.08 6.7e-10 orf122 3b85 Phosphate starvation-inducible protein {Corynebacterium 98.82 2.9e-13 glutamicum atcc 13032} orf124 1wot Putative minimal nucleotidyltransferase {Thermus 91.73 3.50E-03 thermophilus} orf128 1rmg Rgase A, rhamnogalacturonase A {Aspergillus aculeatus} 92.63 2.10E-03 orf129 1t1j Hypothetical protein; structural genomics {Pseudomonas 96.33 8.3e-06 aeruginosa} orf130 1g31 chaperone, CO-chaperonin {Enterobacteria phage T4} 98.55 8.1e-12 orf131 2yxn Tyrosyl-tRNA synthetase {Escherichia coli str} 100 6.30E-60 orf134 1vq2 DCMP deaminase, deoxycytidylate deaminase {Enterobacteria 92.92 1.80E-03 phage T4} orf136 2jaq Deoxyguanosine kinase {Mycoplasma mycoides subsp} 97.25 1.1e-07 orf138 1h7b Anaerobic ribonucleotide-triphosphate reductase large chain 100 3.90E- {Bacteriophage T4} 121

242

orf140 2ih2 Modification methylase TAQI; DNA, DNA methyltransferase 98.36 4.20E-11 {Thermus aquaticus} orf141 3c8f Pyruvate formate-lyase 1-activating enzyme {Escherichia coli} 98 8.4e-10 orf147 3otd tRNA(His) guanylyltransferase {Homo sapiens} 100 7.90E-39 orf152 1boo Protein (N-4 cytosine-specific methyltransferase PVU II) 99.92 2.5e-31 {Proteus vulgaris} orf159 2cgq Acyl carrier protein ACPA {Mycobacterium tuberculosis} 97.46 4.20E-08 orf165 2xgw Peroxide resistance protein; metal binding protein 94.14 6.00E-04 {Streptococcus pyogenes} orf170 1w4r Thymidine kinase; type II {Homo sapiens} 100 1e-46 orf175 1s68 RNA ligase 2 {Enterobacteria phage T4} 99.08 5.80E-15 orf176 1yvr RO autoantigen, 60-kDa SS-A/RO ribonucleoprotein, 60 kDa 99.77 4.1e-24 {Xenopus laevis} orf180 1swy Lysozyme; RB+ binding sites {Enterobacteria phage T4} 99.10 4.3e-15 orf183 2rql Probable sigma-54 modulation protein {Escherichia coli} 98.37 3.8e-11 orf186 3h38 tRNA nucleotidyl transferase-related protein {Thermotoga 100 5.2E-37 maritima} orf187 2kdv RNA pyrophosphohydrolase; nudix family {Escherichia coli} 98.91 7e-14 orf188 1ego Glutaredoxin; electron transport; NMR {Escherichia coli} 98.97 3.1e-14 orf189 1zq1 Glutamyl-tRNA(Gln) amidotransferase subunit E {Pyrococcus 89.88 9.40E-03 abyssi}

243

orf192 3gpg NSP3, non-structural protein 3 {Chikungunya virus} 97.91 2.00E-09 orf197 1dek Deoxynucleoside monophosphate kinase; phosphotransferase; 99.83 2.40E-26 {Enterobacteria phage T4} orf198 Phage tail sheath T4 1.00E- protein gp18 37 orf210 2xsz RUVB-like 2; hydrolase {Homo sapiens} 96.59 2.6e-06 orf211 1ijb VON willebrand factor; dinucleotide-binding fold, blood 96.72 1.50E-06 clotting; {Homo sapiens} orf217 1tis Thymidylate synthase; transferase(methyltransferase); 100 5.50E-82 {Enterobacteria phage T4} orf226 2yvp NDX2, MUTT/nudix family protein; nudix protein, ADP-ribose 98.2 1.80E-10 {Thermus thermophilus} orf227 tail sheath stabilizer P-SSM2 4.3e-70 and completion protein gp15 orf229 1jbk CLPB protein {Escherichia coli} 99.79 4.80E-25 orf231 baseplate wedge P1 5.8e-64 subunit orf232 2ia7 Tail lysozyme, putative {Geobacter sulfurreducens} 97.88 2.50E-09 Gene 25-like lysozyme T4 5.3e-21 base plate protein orf233 2o4w Lysozyme {Enterobacteria phage T4} 99.66 1.50E-21 orf234 1swy Lysozyme {Enterobacteria phage T4} 99.55 8.40E-20 bacteriophage_T4- T4 2.6e-30 like_lysozyme base plate protein

244

orf239 3tho Exonuclease, putative; adenosine triphosphate, DNA breaks, 99.21 5.50E-16 DNA repair enzymes {Thermotoga maritima} orf240 3qks DNA double-strand break repair RAD50 ATPase; RECA-like 98.86 1.50E-13 fold, ATPase, {Pyrococcus furiosus} orf241 1e7l GP49, recombination endonuclease VII {Bacteriophage T4} 98.24 1.70E-10 orf245 Bacteriophage T4-like T4 2.4e- capsid assembly protein 139 (Gp20) orf248 Prohead core protein T4 9.6e-84 protease U9 (gp21) orf250 1yue Head vertex protein GP24 {Enterobacteria phage T4} 100 8.10E-41 major capsid protein T4 1.4e- gp23 138 orf257 3qex DNA polymerase, GP43 {Enterobacteria phage RB69} 100.00 8.10E-60 orf260 2a22 Vacuolar protein sorting 29; alpha-beta-BETA-alpha sandwich 96.54 3.20E-06 {Cryptosporidium parvum} orf261 1qyp RNA polymerase II; transcription, RPB9 {Thermococcus celer} 95.59 5.80E-05 orf262 1g31 GP31; chaperone, CO-chaperonin, groes {Enterobacteria phage 98.07 6.60E-10 T4} orf266 3h7i Ribonuclease H, RNAse H; BPT4 RNAse H, 5'-3' exonuclease 99.29 1.10E-16 {Enterobacteria phage T4} orf268 3cpe Terminase, DNA packaging protein GP17 {Bacteriophage T4} 98.99 2.50E-14

245

orf269 3cpe Terminase, DNA packaging protein GP17 {Bacteriophage T4} 100 2.50E-44 orf271 2a1k GP32 single stranded DNA binding protein {Enterobacteria 97.53 3.40E-08 phage RB69} orf272 3io5 Recombination and repair protein; RECA like core domain 99.9 1.00E-29 {Enterobacteria phage T4} orf275 2p1j POLIII, DNA polymerase III POLC-type {Thermotoga maritima 99.45 1.80E-18 MSB8} orf276 2pl3 Probable ATP-dependent RNA helicase DDX10 {Homo 99.04 1.00E-14 sapiens} orf285 2au3 DNA primase {Aquifex aeolicus} 98.39 3.50E-11 orf286 3bh0 DNAB-like replicative helicase {Bacillus phage SPP1} 99.51 3.50E-19 orf295 3h4r Exodeoxyribonuclease 8; exonuclease {Escherichia coli} 95.72 4.30E-05 orf298 2xap Ribonucleoside-diphosphate reductase 1 subunit alpha 100 6.60E- {Escherichia coli} 134 orf300 3n37 Ribonucleoside-diphosphate reductase 2 subunit BE 100 1.50E-45 {Escherichia coli} orf304 2yf4 MAZG-like nucleoside triphosphate pyrophosphohydr 99.49 8.50E-19 {Deinococcus radiodurans} orf308 3dau Dihydrofolate reductase {Escherichia coli} 99.89 2.10E-29 orf309 3h08 RNH (ribonuclease H); RNAse H {Chlorobaculum tepidum} 99.88 2.40E-28

246

orf310 1w36 RECD, exodeoxyribonuclease V alpha chain; recombination, 99.92 6.50E-31 helicase {Escherichia coli} orf313 1tif IF3-N, translation initiation factor 3 {Geobacillus 98.84 2.40E-13 stearothermophilus} orf314 3o1f ATP-dependent CLP protease adapter protein CLPS 97.7 9.80E-09 {Escherichia coli} orf319 3m4i DNA gyrase subunit B {Mycobacterium tuberculosis} 99.94 5.40E-33 orf320 3ilw DNA gyrase subunit A {Mycobacterium tuberculosis} 100.00 2.60E-51 orf323 2hnh DNA polymerase III alpha subunit {Escherichia coli} 97.06 3.70E-07 orf327 2gno DNA polymerase III, gamma subunit-related protein 99.65 1.90E-21 {Thermotoga maritima} orf328 3m9l Hydrolase, haloacid dehalogenase-like family {Pseudomonas 97.41 4.30E-08 fluorescens} orf342 2b3w Hypothetical protein YBIA; structure, NESG {Escherichia coli} 97.85 3.30E-09 orf353 1lw7 Transcriptional regulator NADR; NMN, NMN adenylyl 100.00 1.20E-39 transferase, ribosylnicotinamide KINA transferase {Haemophilus influenzae} orf367 3bos Putative DNA replication factor {Shewanella amazonensis} 98.47 1.30E-11 orf370 1q92 5(3)-deoxyribonucleotidase; alpha-beta rossman fold, hydrolase 97.24 1.20E-07 {Homo sapiens} orf382 1xty PTH, peptidyl-tRNA hydrolase {Pyrococcus abyssi} 98.53 8.80E-12

247

orf390 2qrv DNA (cytosine-5)-methyltransferase 3A; DNA 99.94 4.90E-33 methyltransferase 3A (DNMT3A) and ITS regulatory factor {Homo sapiens} orf412 2i79 Acetyltransferase, GNAT family; acetyl coenzyme *A 96.29 7.10E-06 {Streptococcus pneumoniae} orf424 2gr8 Trimeric autotransporter, protein secretion, {Haemophilus 99.55 1.20E-19 influenzae} orf429 1ly1 Polynucleotide kinase; PNK, phosphatase, transferase 96.83 9.10E-07 {Enterobacteria phage T4} orf436 3smj Poly [ADP-ribose] polymerase 14; transferase, NAD+, ADP- 95.9 2.60E-05 ribosylat transferase-transferase inhibitor complex {Homo sapiens} orf474 3pfg N-methyltransferase; N,N-dimethyltransferase {Streptomyces 95.35 8.20E-05 fradiae} orf488 3g5g Regulatory protein; transcriptional regulator, restriction- 97.85 3.00E-09 modification {Enterobacter SP} orf490 2a6h RNA polymerase sigma factor RPOD {Thermus thermophilus} 99.5 5.30E-19 orf491 1u3e HNH homing endonuclease {Bacillus phage SPO1} 98.99 2.60E-14 orf498 2end Endonuclease V {Enterobacteria phage T4} 100 9.90E-51 orf508 2qgp HNH endonuclease {Geobacter metallireducens gs-15} 97.59 1.90E-08 orf510 2x8a Nuclear valosin-containing protein-like {Homo sapiens} 98.59 3.90E-12 orf519 2ih2 Modification methylase TAQI; DNA, DNA methyltransferase 98.27 9.60E-11 {Thermus aquaticus}

248

orf521 3co5 Putative two-component system transcriptional RES regulator 95.29 9.30E-05 {Neisseria gonorrhoeae} orf530 4dqa Uncharacterized protein; two domains structure, DUF 1735, 95.54 6.20E-05 laminin_G_3 concanavalin A- lectin/glucanases superfamily domain {Bacteroides ovatus}

249

Appendix 3. Genome of phages GAP31, GAP52, GAP161, and GAP227

Table 4.2. General features of ORFs in the DNA of phage GAP31 and homology to proteins in the databases.

Gene Name Position Gene Length Function Homology

orf001 1..1995 1995 rIIA protein ADP02397.1| rIIA protein [Salmonella phage PVP-SE1] orf002 2007..2180 174 hypothetical protein orf003 2185..2727 543 cell wall hydrolase SleB ADP02639.1 cell wall hydrolase SleB [Salmonella phage PVP-SE1]

orf004 2758..3168 411 conserved hypothetical protein ADP02638.1| conserved hypothetical protein [Salmonella phage PVP-SE1] orf005 3146..3373 228 hypothetical protein orf006 3348..3527 180 conserved hypothetical protein ADP02636.1| hypothetical protein [Salmonella phage PVP-SE1] orf007 3527..4573 1047 RnlB RNA ligase 2 ADP02635.1| RnlB RNA ligase 2 [Salmonella phage PVP-SE1] orf008 4566..4931 366 conserved hypothetical protein ADP02633.1| hypothetical protein [Salmonella phage PVP-SE1] orf009 4918..5190 273 conserved hypothetical protein ADP02632.1| hypothetical protein [Salmonella phage PVP-SE1] orf010 5180..5422 243 conserved hypothetical protein ADP02631.1| hypothetical protein [Salmonella phage PVP-SE1] orf011 5422..5622 201 conserved membrane protein ADP02629.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf012 5619..5801 183 hypothetical protein orf013 5801..6295 495 putative phosphatase ADP02628.1| putative phosphatase [Salmonella phage PVP-SE1] orf014 6292..6804 513 conserved hypothetical protein YP_004009937.1| hypothetical protein CC31p079 [Enterobacteria phage CC31] orf015 6805..7017 213 hypothetical protein orf016 7014..7190 177 conserved hypothetical protein ADP02625.1| hypothetical protein [Salmonella phage PVP-SE1] orf017 7183..7458 276 hypothetical protein orf018 7455..7652 198 conserved hypothetical protein ADP02624.1| hypothetical protein [Salmonella phage PVP-SE1] orf019 7646..7939 294 conserved hypothetical protein ADP02623.1| hypothetical protein [Salmonella phage PVP-SE1] orf020 7936..8088 153 hypothetical protein orf021 8085..8276 192 conserved hypothetical protein ADP02622.1| conserved hypothetical protein [Salmonella phage PVP-SE1] orf022 8269..8610 342 hypothetical protein

250

orf023 8669..9358 690 hypothetical protein orf024 9368..10576 1209 MoxR ATPase ADP02621.1| MoxR ATPase [Salmonella phage PVP-SE1] orf025 10563..10805 243 conserved hypothetical protein ADP02620.1 hypothetical protein [Salmonella phage PVP-SE1] orf026 10790..11161 372 conserved hypothetical protein ADP02619.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf027 11154..11651 498 conserved hypothetical protein ADP02618.1 hypothetical protein [Salmonella phage PVP-SE1] orf028 11653..11862 210 conserved hypothetical protein ADE35053.1 hypothetical protein [Klebsiella phage KP15] orf029A 11727..12083 357 conserved hypothetical protein Sequence similarity to:ADP02617.1 hypothetical protein [Salmonella phage PVP-SE1] orf029 12083..12601 519 conserved hypothetical protein ADP02616.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

orf030 12565..13677 1113 conserved hypothetical protein ADP02615.1 hypothetical protein [Salmonella phage PVP-SE1] orf031 13727..14659 933 anti-sigma factor ADP02614.1 anti-sigma factor [Salmonella phage PVP-SE1] orf032 14659..14919 261 conserved hypothetical protein ADP02613.1 hypothetical protein [Salmonella phage PVP-SE1] orf033 14977..16413 1437 conserved hypothetical protein Sequence similarity to:ADP02612.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf034 16397..16783 387 conserved hypothetical protein ADP02611.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf035 16780..17076 297 conserved hypothetical protein ADP02609.1 hypothetical protein [Salmonella phage PVP-SE1] orf036 17353..17556 204 hypothetical protein orf036A 17618..17794 177 hypothetical protein orf037 17763..18407 645 conserved membrane protein ADP02606.1 hypothetical membrane protein [Salmonella phage PVP-SE1]

orf038 18404..18742 339 conserved hypothetical protein ADP02605.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

orf039 18739..18942 204 conserved hypothetical protein ZP_02468167.1 hypothetical protein Bpse38_32715 [Burkholderia thailandensis MSMB43] orf040 18939..19277 339 conserved hypothetical protein ADP02604.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf041 19279..19428 150 conserved hypothetical protein Sequence similarity to:ADP02602.1 hypothetical protein [Salmonella phage PVP-SE1] orf042 19553..20377 825 conserved hypothetical protein ADP02601.1 hypothetical protein [Salmonella phage PVP-SE1] orf043 20434..20565 132 conserved hypothetical protein ADP02599.1 hypothetical protein [Salmonella phage PVP-SE1] orf043A 20552..20833 282 conserved hypothetical protein Sequence similarity to:ADP02598.1 hypothetical protein [Salmonella phage PVP- SE1]. Poor ribosome-binding site

251

orf044 21224..21415 192 conserved hypothetical protein ADP02597.1 hypothetical protein [Salmonella phage PVP-SE1] orf045 21598..22128 531 conserved hypothetical protein ADP02596.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf046 22202..22498 297 hypothetical protein orf047 22589..22918 330 conserved hypothetical protein ADP02594.1 hypothetical protein [Salmonella phage PVP-SE1] orf048 22980..23177 198 hypothetical protein orf049 23258..23551 294 hypothetical protein orf050 23631..24029 399 conserved hypothetical protein ADP02592.1 hypothetical protein [Salmonella phage PVP-SE1] orf051 24031..24174 144 hypothetical protein orf052 24187..24369 183 conserved hypothetical protein ADP02591.1 hypothetical protein [Salmonella phage PVP-SE1] orf053 24369..24596 228 conserved hypothetical protein ACY75978.1 predicted protein [Prochlorococcus phage P-SSM2] orf054 24695..25261 567 conserved hypothetical protein ADP02590.1 hypothetical protein [Salmonella phage PVP-SE1] orf055 25265..25612 348 conserved hypothetical protein ADP02589.1 hypothetical protein [Salmonella phage PVP-SE1] orf056 25627..25830 204 conserved hypothetical protein ADP02588.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf057 26159..26449 291 conserved hypothetical protein ADP02587.1 hypothetical protein [Salmonella phage PVP-SE1] orf058 26629..26847 219 conserved hypothetical protein ADP02585.1 hypothetical protein [Salmonella phage PVP-SE1] orf059 26858..27154 297 conserved hypothetical protein ADP02584.1 hypothetical protein [Salmonella phage PVP-SE1] orf060 27234..27350 117 conserved membrane protein ADP02583.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf061 27366..27707 342 conserved hypothetical protein ADP02582.1 hypothetical protein [Salmonella phage PVP-SE1] orf062 27761..27994 234 hypothetical protein orf063 27997..28329 333 conserved hypothetical protein ADP02581.1 hypothetical protein [Salmonella phage PVP-SE1] orf064 28416..28805 390 conserved hypothetical protein ADP02580.1 hypothetical protein [Salmonella phage PVP-SE1] orf065 28856..29080 225 conserved hypothetical protein ADP02579.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf066 29140..29304 165 conserved hypothetical protein ADP02578.1 hypothetical protein [Salmonella phage PVP-SE1] orf067 29447..29923 477 conserved hypothetical protein ADP02577.1 hypothetical protein [Salmonella phage PVP-SE1] orf068 29934..30251 318 hypothetical protein orf069 30319..30441 123 hypothetical protein orf070 30707..31072 366 hypothetical protein orf071 31150..31473 324 hypothetical protein orf072 31523..31789 267 hypothetical protein orf073 32659..32805 147 hypothetical protein orf074 33055..33213 159 hypothetical protein orf075 33588..33941 354 hypothetical protein orf076 33951..34190 240 hypothetical protein orf077 34201..34383 183 hypothetical protein

252

orf078 34386..34499 114 hypothetical protein orf079 34623..35051 429 conserved hypothetical protein AEG18624.1 hypothetical protein MSWAN_1613 [Methanobacterium sp. SWAN-1]

orf081 35052..35336 285 hypothetical protein orf082 35339..35530 192 hypothetical protein orf083 35518..35943 426 hypothetical protein orf084 35953..36342 390 hypothetical protein orf085 36326..36565 240 hypothetical protein orf086 36562..36789 228 hypothetical protein orf088 36870..37154 285 hypothetical protein orf089 37164..37343 180 hypothetical protein orf090 37451..37591 141 hypothetical protein orf091 37591..37758 168 hypothetical protein orf092 37831..38076 246 hypothetical protein orf093 38174..38368 195 hypothetical protein orf094A 38365..38481 117 hypothetical protein orf094 38561..39214 654 conserved hypothetical protein ADP02406.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf095 39255..39404 150 conserved hypothetical protein Sequence similarity to:ADP02574.1 hypothetical protein [Salmonella phage PVP-SE1] orf096 39394..39567 174 hypothetical protein orf097 39564..39758 195 hypothetical protein orf098 39755..40003 249 hypothetical protein orf099A 40000..40257 258 conserved hypothetical protein ADP02572.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

orf099 40251..40409 159 conserved hypothetical protein ADP02571.1 hypothetical protein [Salmonella phage PVP-SE1] orf100 40406..40615 210 conserved hypothetical protein ADP02570.1 hypothetical protein [Salmonella phage PVP-SE1] orf101 40605..41753 1149 conserved hypothetical protein EFE12627.1 conserved hypothetical protein [Clostridium sp. M62/1]

orf102 41873..42121 249 hypothetical protein orf103 42121..42435 315 conserved hypothetical protein ADP02569.1 hypothetical protein [Salmonella phage PVP-SE1] orf104 42458..42952 495 conserved hypothetical protein ADP02568.1 hypothetical protein [Salmonella phage PVP-SE1] orf105 42937..43332 396 conserved hypothetical protein ADP02567.1 hypothetical protein [Salmonella phage PVP-SE1] orf106 43332..43943 612 hypothetical protein ADO99013.1 structural protein [Prochlorococcus phage P-SSM7] orf107 43943..44425 483 conserved membrane protein ADP02566.1 hypothetical membrane protein [Salmonella phage PVP-SE1]. N.B. could begin 5 amino acids uipstream. orf108 44499..45149 651 hypothetical protein ABG84639.1 putative capK protein [Clostridium perfringens ATCC 13124]

253

orf109A 45157..45387 231 conserved hypothetical protein ADP02565.1 hypothetical protein [Salmonella phage PVP-SE1] orf109 45384..45539 156 hypothetical protein orf110 45536..45712 177 conserved hypothetical protein ADP02564.1 hypothetical protein [Salmonella phage PVP-SE1] orf111 45699..45869 171 conserved hypothetical protein ADP02563.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf112 45904..46977 1074 conserved hypothetical protein ADP02562.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf113 46986..47315 330 conserved hypothetical protein ADP02561.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf114 47312..47488 177 hypothetical protein orf115 47490..47825 336 conserved hypothetical protein ADP02560.1 hypothetical membrane protein [Salmonella phage PVP-SE1]

orf116 47828..48316 489 conserved hypothetical protein ADP02558.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf117 48313..48540 228 hypothetical protein orf118 48544..48858 315 hypothetical protein orf119 48868..49074 207 conserved hypothetical protein ADP02556.1 hypothetical protein [Salmonella phage PVP-SE1]; AEK81976.1 hypothetical protein [Salmonella phage 7-11] orf120 49079..50332 1254 tRNA nucleotidyltransferase ADP02555.1 tRNA nucleotidyl transferase [Salmonella phage PVP-SE1]; YP_001436496.1 multifunctional tRNA nucleotidyl transferase/2'3'-cyclic phosphodiesterase/2'nucleotidase/phosphatase [Cronobacter sakazakii ATCC BAA-894] orf121 50342..50608 267 hypothetical protein orf122 50605..50916 312 conserved hypothetical protein ADP02553.1 hypothetical protein [Salmonella phage PVP-SE1] orf123 50906..51157 252 hypothetical protein EEH95690.1 predicted protein [Citrobacter sp. 30_2] orf124 51154..51306 153 hypothetical protein orf125 51296..51685 390 conserved hypothetical protein ADP02552.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf126 51682..51900 219 conserved hypothetical protein ADP02551.1 hypothetical protein [Salmonella phage PVP-SE1] orf127 51900..52172 273 conserved hypothetical protein ADP02550.1 hypothetical protein [Salmonella phage PVP-SE1] orf128 52260..52409 150 conserved hypothetical protein ADP02549.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

orf129 52372..53052 681 conserved hypothetical protein ADP02548.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf130 53052..53984 933 ClpP ATP-dependent ADP02547.1 ClpP ATP-dependent protease subunit [Salmonella phage PVP-SE1] protease subunit orf131 53986..54468 483 conserved hypothetical protein ADP02546.1 hypothetical protein [Salmonella phage PVP-SE1] orf132 54476..54784 309 conserved hypothetical protein ADP02545.1 hypothetical protein [Salmonella phage PVP-SE1] orf133 54771..55403 633 conserved hypothetical protein ADP02544.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

254

orf134 55414..56214 801 PhoH-like protein ADP02543.1 PhoH-like protein [Salmonella phage PVP-SE1]; ABU77596.1 hypothetical protein ESA_02349 [Cronobacter sakazakii ATCC BAA-894] orf135 56226..56780 555 lysozyme ADP02542.1 lysozyme [Salmonella phage PVP-SE1]; ABU76288.1 hypothetical protein ESA_01019 [Cronobacter sakazakii ATCC BAA-894] orf136 56793..57224 432 conserved hypothetical protein ADP02540.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf137 57233..57373 141 conserved hypothetical protein Sequence similarity to:ADP02539.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf138 57451..57717 267 glutaredoxin 1 ADP02536.1 glutaredoxin 1 [Salmonella phage PVP-SE1] orf139 57719..58075 357 conserved hypothetical protein ADP02535.1 hypothetical protein [Salmonella phage PVP-SE1] orf140 58085..59173 1089 ribonucleoside diphosphate Sequence similirarity to: ADP02534.1 ribonucleoside diphosphate reductase beta reductase beta chain chain [Salmonella phage PVP-SE1] orf141 59212..61527 2316 ribonucleoside triphosphate ADP02533.1 ribonucleoside triphosphate reductase alpha chain [Salmonella phage reductase alpha chain PVP-SE1]; gb|EGL72037.1 ribonucleotide-diphosphate reductase subunit alpha [Cronobacter sakazakii E899] orf142 61537..61767 231 conserved hypothetical protein ADP02532.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf143 61767..62243 477 conserved hypothetical protein ADP02531.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf144 62325..63323 999 thymidylate synthase ADP02529.1 thymidylate synthase [Salmonella phage PVP-SE1] orf145 63323..63646 324 conserved hypothetical protein ADP02528.1 hypothetical protein [Salmonella phage PVP-SE1] orf146 63643..63837 195 conserved hypothetical protein ADP02527.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf147 63837..64439 603 conserved hypothetical protein ADP02526.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf148 64432..65037 606 conserved hypothetical protein ADP02525.1 conserved hypothetical protein [Salmonella phage PVP-SE1]; AEM24788.1 ATP-binding protein [Cronobacter phage ESP2949-1] orf149 65069..66043 975 conserved hypothetical protein ADP02523.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf150 66043..66255 213 conserved hypothetical protein ADP02522.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf151 66245..66793 549 EndoVII packaging and ADP02521.1 EndoVII packaging and recombination endonuclease [Salmonella recombination endonuclease phage PVP-SE1] orf152 66775..67410 636 conserved hypothetical protein ADP02520.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf153 67403..67708 306 conserved hypothetical protein ADP02519.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf154 67705..68826 1122 putative exodeoxyribonuclease ADP02518.1 exonuclease [Salmonella phage PVP-SE1]; ABI79160.1 putative exodeoxyribonuclease [Escherichia phage rv5]

255

orf155 68835..69272 438 putative HNH endonuclease ADP02517.1 putative HNH endonuclease [Salmonella phage PVP-SE1] orf157 69274..69780 507 conserved hypothetical protein ADP02516.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf158 69792..69986 195 hypothetical membrane protein ADP02514.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf159 70050..70940 891 conserved hypothetical protein ADP02513.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf160 70933..71097 165 conserved hypothetical protein ADP02512.1 hypothetical protein [Salmonella phage PVP-SE1] orf161 71081..71284 204 hypothetical membrane protein ADP02511.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf162 71284..71643 360 conserved hypothetical protein ADP02510.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf163 71712..71933 222 conserved hypothetical protein ADP02509.1 hypothetical protein [Salmonella phage PVP-SE1] orf164 71987..72676 690 conserved hypothetical protein ADP02508.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf165 72686..73057 372 hypothetical membrane protein ADP02507.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf166 73057..73503 447 conserved hypothetical protein ADP02506.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf167 73503..74807 1305 DNA ligase ADP02505.1 DNA ligase [Salmonella phage PVP-SE1] orf168 74807..75031 225 hypothetical protein orf169 75038..75445 408 conserved hypothetical protein ADP02503.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf170 75455..75799 345 conserved hypothetical protein ADP02502.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf171 75796..76032 237 hypothetical protein orf172 76029..76991 963 RNA ligase 1 and tail ADP02501.1 RNA ligase 1 and tail attachment protein [Salmonella phage PVP-SE1] attachment protein orf173 77141..77497 357 conserved hypothetical protein ADP02499.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf174 77499..78092 594 putative phosphoesterase ADP02498.1 putative phosphoesterase [Salmonella phage PVP-SE1] orf175 78089..78328 240 hypothetical protein orf176 78325..78609 285 transposase-like protein ADP02497.1 transposase-like protein [Salmonella phage PVP-SE1] orf177 78606..78881 276 conserved hypothetical protein ADP02496.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf178 78993..79337 345 conserved hypothetical protein ADP02495.1 hypothetical protein [Salmonella phage PVP-SE1] orf179 79616..79933 318 hypothetical protein orf180 79935..80747 813 phosphoribosylpyrophosphate ADP02492.1 phosphoribosylpyrophosphate synthetase [Salmonella phage PVP- synthetase SE1]; EGL72884.1 hypothetical protein CSE899_09382 [Cronobacter sakazakii E899] orf181 80747..81280 534 conserved hypothetical protein, AEO93591.1 gp332 [Bacillus phage G] Bacillus phage G gp332 homolog

256

orf182 81334..83037 1704 nicotinamide phosphoribosyl ADP02491.1 nicotinamide phosphoribosyl transferase [Salmonella phage PVP-SE1]; transferase EGL72885.1 putative nicotinate phosphoribosyltransferase [Cronobacter sakazakii E899] orf183 83086..83397 312 conserved hypothetical protein ADP02490.1 hypothetical protein [Salmonella phage PVP-SE1] orf184 83390..83599 210 conserved hypothetical protein ADP02488.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf185 83679..83924 246 conserved hypothetical protein ADP02487.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf186 84049..84237 189 conserved hypothetical protein ADP02485.1 hypothetical protein [Salmonella phage PVP-SE1] orf187 84352..84519 168 conserved hypothetical protein ADP02483.1 hypothetical protein [Salmonella phage PVP-SE1] orf188 84589..84918 330 conserved hypothetical protein ADP02482.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf189 85382..85993 612 conserved hypothetical protein ADP02479.1 hypothetical protein [Salmonella phage PVP-SE1] orf190 86071..86718 648 conserved hypothetical protein ADP02478.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf191 87885..88250 366 conserved hypothetical protein ADP02477.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf192 89185..89340 156 hypothetical protein orf193 90847..92271 1425 conserved hypothetical protein ADP02474.1 hypothetical protein [Salmonella phage PVP-SE1]; ADP02475.1 hypothetical protein [Salmonella phage PVP-SE1] orf194 92268..92672 405 conserved hypothetical ADP02473.1 conserved hypothetical membrane protein [Salmonella phage PVP- membrane protein SE1] orf195 92669..92959 291 conserved hypothetical protein ADP02472.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf196 93058..94557 1500 terminase large subunit ADP02471.1 terminase large subunit [Salmonella phage PVP-SE1] orf197 94573..96123 1551 conserved hypothetical protein ADP02470.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf198 96193..96819 627 conserved hypothetical protein ADP02469.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf199 96816..97817 1002 conserved hypothetical protein ADP02468.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf200 97838..98257 420 head stabilization/decoration ADP02467.1 head stabilization/decoration protein [Salmonella phage PVP-SE1] protein orf201 98279..99292 1014 putative major head protein ADP02466.1 putative major head protein [Salmonella phage PVP-SE1] orf202 99427..99951 525 conserved hypothetical protein ADP02465.1 hypothetical protein [Salmonella phage PVP-SE1] orf203 99961..101718 1758 Phage tail fiber protein ADP02464.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

257

orf204 101754..102398 645 conserved hypothetical protein ADP02463.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf205 102412..102801 390 conserved hypothetical protein ADP02462.1 hypothetical protein [Salmonella phage PVP-SE1] orf206 102824..103228 405 hypothetical membrane protein ADP02461.1 hypothetical membrane protein [Salmonella phage PVP-SE1] orf207 103363..103881 519 conserved hypothetical protein ADP02460.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf208 103947..104423 477 conserved hypothetical protein ADP02459.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf209 104423..104857 435 conserved hypothetical protein ADP02458.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf210 104857..105387 531 conserved hypothetical protein ADP02457.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf211 105518..106930 1413 structural protein ADP02456.1 structural protein [Salmonella phage PVP-SE1]; CBA33461.1 hypothetical protein CTU_34230 [Cronobacter turicensis z3032] orf212 106934..107407 474 structural protein ADP02455.1 structural protein [Salmonella phage PVP-SE1] orf213 107483..107956 474 conserved hypothetical protein ADP02454.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf214 107968..108234 267 conserved hypothetical protein ADP02453.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf215 108288..110681 2394 conserved hypothetical ADP02452.1 conserved hypothetical membrane protein [Salmonella phage PVP- membrane protein SE1] orf216 110763..111635 873 conserved hypothetical protein ADP02451.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf217 111635..111985 351 conserved hypothetical protein ADP02450.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf218 111988..112977 990 conserved hypothetical protein ADP02449.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf219 112977..113678 702 conserved baseplate assembly ADP02448.1 putative baseplate assembly protein [Salmonella phage PVP-SE1] protein orf220 113688..114305 618 conserved hypothetical protein ADP02447.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

258

orf221 114316..114987 672 conserved hypothetical protein ADP02446.1 conserved hypothetical protein [Salmonella phage PVP-SE1]; ABU77566.1 hypothetical protein ESA_02317 [Cronobacter sakazakii ATCC BAA- 894]; ADZ13622.1 hypothetical protein [Cronobacter phage ENT47670]

orf222 114987..117776 2790 conserved hypothetical protein ADP02445.1 conserved hypothetical protein [Salmonella phage PVP-SE1]; ABU77565.1 hypothetical protein ESA_02316 [Cronobacter sakazakii ATCC BAA-894] orf223 117786..119924 2139 putative fusion protein ADP02444.1 putative fusion protein [Salmonella phage PVP-SE1]; ABU76442.1 hypothetical protein ESA_01175 [Cronobacter sakazakii ATCC BAA-894] orf224 120033..121526 1494 conserved hypothetical protein ADP02443.1 conserved hypothetical protein [Salmonella phage PVP-SE1]; YP_003211776.1 hypothetical protein CTU_34130 [Cronobacter turicensis z3032]

orf225 121537..122169 633 conserved hypothetical protein ADP02442.1 conserved hypothetical protein [Salmonella phage PVP-SE1]

orf226 122181..123293 1113 conserved tail fiber protein ADP02441.1 putative tail fiber protein [Salmonella phage PVP-SE1]

orf227 123305..123820 516 tail fiber assembly protein EFE23075.1 putative tail fiber assembly protein [Edwardsiella tarda ATCC 23685]; ADX32387.1 tail fiber assembly protein [Cronobacter phage ESSI-2]

orf228 123830..124144 315 conserved hypothetical ADP02439.1 conserved hypothetical membrane protein membrane protein [Salmonella phage PVP-SE1] orf229 124141..124614 474 conserved hypothetical ADP02438.1 hypothetical membrane protein [Salmonella phage PVP-SE1] membrane protein orf230 124630..124899 270 conserved hypothetical Sequence similarity to:ADP02437.1 conserved hypothetical membrane protein membrane protein [Salmonella phage PVP-SE1] orf231 124910..126718 1809 putative tail fiber protein ADP02436.1 putative tail fiber protein [Salmonella phage PVP-SE1] orf232 126763..128466 1704 conserved hypothetical protein ADP02435.1 hypothetical protein [Salmonella phage PVP-SE1] orf233 128499..129680 1182 conserved hypothetical protein ADP02434.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf234 129694..130041 348 conserved hypothetical protein ADP02433.1 hypothetical protein [Salmonella phage PVP-SE1] orf235 130104..130310 207 conserved hypothetical ADP02432.1 hypothetical membrane protein [Salmonella phage PVP-SE1] membrane protein orf237A 130326..130547 222 conserved hypothetical protein ADP02431.1 hypothetical protein [Salmonella phage PVP-SE1] orf237 130544..131116 573 conserved hypothetical protein ADP02430.1 hypothetical protein [Salmonella phage PVP-SE1] orf238 131116..131412 297 hypothetical protein orf239 131405..131692 288 conserved hypothetical protein ADP02429.1 hypothetical protein [Salmonella phage PVP-SE1]

259

orf240 131683..132054 372 conserved hypothetical ADP02428.1 hypothetical membrane protein [Salmonella phage PVP-SE1] membrane protein orf241 132058..132627 570 conseved hypothetical protein CAX61391.1 Conserved uncharacterized protein [Erwinia billingiae Eb661]; EGL73719.1 hypothetical protein CSE899_04203 [Cronobacter sakazakii E899] orf242 132642..132821 180 hypothetical protein orf243 132818..133525 708 nicotinamide mononucleotide ADP02427.1 nicotinamide mononucleotide transport [Salmonella phage PVP-SE1]; transport YP_001438676 hypothetical protein ESA_02595 [Cronobacter sakazakii ATCC BAA-894] orf244 133546..133776 231 hypothetical protein orf245 133769..134056 288 conserved hypothetical protein BAG75040.1 hypothetical protein [Pseudomonas phage PAJU2] orf246 134049..134387 339 conserved hypothetical protein ADP02424.1 hypothetical protein [Salmonella phage PVP-SE1] orf247 134380..134811 432 conserved hypothetical protein ADP02423.1 hypothetical protein [Salmonella phage PVP-SE1] orf248 134813..135886 1074 nicotinamide-nucleotide ADP02421.1 nicotinamide-nucleotide adenylyltransferase [Salmonella phage PVP- adenylyltransferase SE1]; YP_001439419.1 nicotinamide-nucleotide adenylyltransferase [Cronobacter sakazakii ATCC BAA-894] orf249 135883..136155 273 conserved hypothetical protein ADP02420.1 hypothetical protein [Salmonella phage PVP-SE1] orf250 136158..136475 318 conserved hypothetical protein ADP02419.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf251 136509..136715 207 conserved hypothetical ADP02418.1 hypothetical membrane protein [Salmonella phage PVP-SE1] membrane protein orf252 136726..136944 219 hypothetical protein orf253 136987..137385 399 conserved hypothetical protein ADP02416.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf254 137382..137582 201 conserved hypothetical protein EER47714.1 hypothetical protein AM305_06361 [Actinobacillus minor NM305]; YP_003209497.1 hypothetical protein CTU_11340 [Cronobacter turicensis z3032] orf255 137617..140331 2715 DNA polymerase ADP02411.1 DNA polymerase [Salmonella phage PVP-SE1] orf256 140387..140884 498 baseplate wedge protein gp25 AEI91225.1 gp25 [Escherichia phage phiEB49] orf257 140874..142931 2058 DNA replicative ADP02409.1 primase/helicase [Salmonella phage PVP-SE1] helicase/primase orf258 142941..143087 147 conserved hypothetical protein ADP02407.1 hypothetical protein [Salmonella phage PVP-SE1] orf259 143096..143788 693 conserved hypothetical protein EFO45124.1 Dcm methylase [Vibrio parahaemolyticus AQ4037]; AEM24785.1 site- specific DNA methylase [Cronobacter phage ESP2949-1]; ADP02405.1 conserved hypothetical protein [Salmonella phage PVP-SE1] orf260 143799..144080 282 conserved hypothetical protein ADP02404.1 hypothetical protein [Salmonella phage PVP-SE1] orf261 144080..144361 282 conserved hypothetical protein ADP02403.1 hypothetical protein [Salmonella phage PVP-SE1] orf262 144354..144677 324 conserved hypothetical protein ADP02402.1 hypothetical protein [Salmonella phage PVP-SE1]

260

orf263 144658..144837 180 conserved hypothetical protein ADP02401.1 hypothetical protein [Salmonella phage PVP-SE1] orf264 144839..145348 510 conserved hypothetical protein ADP02400.1 hypothetical protein [Salmonella phage PVP-SE1] orf265 145360..146667 1308 putative helicase ADP02399.1 putative helicase [Salmonella phage PVP-SE1] orf266 146667..147887 1221 rIIB protein ADP02398.1 rIIB protein [Salmonella phage PVP-SE1]

261

Table 4.3. General features of ORFs in the DNA of phage GAP52 and homology to proteins in the databases.

Gene Position Gene Mass pI AA Function Homology E-value Motifs Pfam Motifs Pfam Length (TMHMM ;Phobius) E-value

orf001 1712..2065 353 13004 7.9 117 Hypothetical AEK81916.1 hypothetical protein 2.00E-61 0 protein [Salmonella phage 7-11]

orf002 2173..2245 72 5742 10.6 54 Hypothetical ABV74305.1 hypothetical protein 5.00E-08 0 protein BA3_0020 [Thalassomonas phage BA3_0020 BA3]

orf003 2553..3155 602 23218 4.9 200 Hypothetical 0 protein

orf004 3209..3376 167 6423 8.1 55 Hypothetical AEK81918.1 hypothetical protein 4.00E-18 0 protein [Salmonella phage 7-11]

orf005 3376..4860 1484 56155 5.3 494 Terminase AEK81919.1 terminase large subunit 0.0 0 PF03237.9 3.10E-42 large subunit [Salmonella phage 7-11] Terminase_6

orf006 4871..6448 1577 58826 4.6 525 Portal protein AEK81920.1 portal protein 0.0 0 [Salmonella phage 7-11]

orf007 6411..7151 740 27995 5.9 246 Portal protein AEK81920.1 portal protein 7.00E-82 0 [Salmonella phage 7-11]

orf008 7152..7391 239 9065 4.7 79 Hypothetical AEK81921.1 hypothetical protein 5.00E-19 0 protein [Salmonella phage 7-11]

orf009 7391..8407 1016 38335 4.2 338 Scaffolding AEK81922.1 scaffolding protein 3.00E-81 0 protein [Salmonella phage 7-11]

orf010 8547..9503 956 34847 6.5 318 Major head AEK81923.1 major head protein E-169 0 protein [Salmonella phage 7-11]

orf011 9584..10684 1100 39647 4.8 366 Major head AEK81924.1 major head protein E-151 0 protein [Salmonella phage 7-11]

262

orf012 10687..11127 440 14693 5.4 146 Hypothetical BAH15186.1 hypothetical protein 8.00E-08 0 protein [Serratia phage KSP100] orf013 11202..11810 608 22563 9.4 202 Hypothetical AEK81925.1 hypothetical protein 9.00E-32 0 protein [Salmonella phage 7-11] orf014 11820..12002 182 7062 8.0 60 Conserved AEK81926.1 hypothetical protein 1.00E-20 0 hypothetical [Salmonella phage 7-11] protein orf015 12015..12704 689 26666 4.6 229 Conserved AEK81927.1 hypothetical protein E-120 0 hypothetical [Salmonella phage 7-11] protein orf016 12719..13498 779 28803 4.7 259 Conserved AEK81928.1 hypothetical protein 9.00E-84 0 hypothetical [Salmonella phage 7-11] protein orf017 13491..16394 2903 10944 5.1 967 Conserved AEF43943.1 phage tail protein I 0.0 0 2 hypothetical [Serratia sp. AS9] protein orf018 16394..16852 458 16833 4.3 152 Hypothetical AEK81930.1 hypothetical protein 5.00E-32 0 protein [Salmonella phage 7-11] orf018 16920..17171 251 9108 6.6 83 Hypothetical AEK81931.1 hypothetical protein 3.00E-37 0 A protein [Salmonella phage 7-11] orf019 17168..18046 878 31646 6.4 292 Putative AEK81932.1 putative structural E-125 0 structural protein [Salmonella phage 7-11] protein orf020 18057..19796 1739 66283 4.9 579 Conserved AEK81933.1 hypothetical protein 0.0 0 hypothetical [Salmonella phage 7-11] protein orf021 19796..20065 269 10328 6.1 89 Hypothetical AEK81934.1 hypothetical protein 3.00E-21 0 protein [Salmonella phage 7-11]

263

orf022 20077..21309 1232 42732 8.9 410 Putative DNA AEK81935.1 putative DNA injection E-138 0 injection protein [Salmonella phage 7-11] protein orf023 21343..22185 842 27233 9.7 280 Hypothetical AEK81936.1 hypothetical protein 4.00E-13 0 protein [Salmonella phage 7-11] orf024 22196..22729 533 19950 4.9 177 Hypothetical AEK81937.1 hypothetical protein 1.00E-49 0 protein [Salmonella phage 7-11] orf025 22729..24375 1646 59529 5.5 548 Hypothetical AEK81938.1 hypothetical protein 1.00E-68 0 protein [Salmonella phage 7-11] orf026 24422..28336 3914 14117 5.2 130 Hypothetical AEK81939.1 hypothetical protein 7.00E-24 0 1 4 protein [Salmonella phage 7-11] orf027 28363..28632 269 10370 9.2 89 Hypothetical 0 protein orf028 28439..28882 443 15847 5.4 147 Hypothetical AEK81945.1 hypothetical protein 9.00E-06 0 PF05367.5 1.10E-09 A protein [Salmonella phage 7-11] Phage_endo_I orf028 28879..29316 437 17431 9.5 145 Putative AEK81944.1 putative endonuclease 7.00E-71 0 endonuclease [Salmonella phage 7-11] orf029 29363..29491 128 5223 10.1 42 Hypothetical 0 protein orf030 29540..30163 623 23125 9.2 207 Putative ADM73658.1 putative nicotinamide 3.00E-07 7 PF04973.6 1.60E-25 nicotinamide mononucleotide transporter NMN_transport mononucleotid er e transporter [Lactococcus phage 949] orf031 30241..30549 308 11456 9.1 102 Endonucelase AAP92668.1 endonucelase VII 3.00E-19 0 PF02945.9 3.10E-23 VII [Synechococcus phage S-PM2] Endonuclease_7 orf032 30585..30716 131 5045 5.3 43 Hypothetical 0 protein

264

orf033 30679..30942 263 9977 6.0 87 Conserved AEK81948.1 hypothetical protein 2.00E-31 0 PF11753.2 4.10E-14 hypothetical [Salmonella phage 7-11] DUF3310 protein orf034 30939..31346 407 15897 6.8 135 Hypothetical AEK81955.1 hypothetical protein 1.00E-37 0 protein [Salmonella phage 7-11] orf035 31336..31893 557 21066 4.7 185 Hypothetical AAX92355.1 ORF060 3.00E-19 0 protein [Staphylococcus phage Twort] orf036 31880..32092 212 8305 5.8 70 Hypothetical 0 protein orf037 32092..32421 329 11689 6.7 109 Hypothetical 0 protein orf038 32414..33502 1088 41316 8.2 362 ATP- AEK81958.1 ATP-dependent DNA E-143 0 PF01068.1 4.60E-19 dependent ligase [Salmonella phage 7-11] DNA_ligase_A DNA ligase _M orf039 33557..33925 368 13543 4.0 122 Hypothetical AEK81959.1 hypothetical protein 1.00E-12 0 protein [Salmonella phage 7-11] orf040 33897..34793 896 34259 4.8 298 5'-3' AEK81960.1 5'-3' exonuclease E-162 0 exonuclease [Salmonella phage 7-11] orf041 34793..34987 194 7358 5.0 64 Hypothetical AEK81961.1 hypothetical protein 2.00E-19 0 protein [Salmonella phage 7-11] orf042 35000..35557 557 22209 5.6 185 RNA AEK81962.1 RNA polymerase ECF E-100 0 PF08281.6 7.00E-07 polymerase sigma factor [Salmonella phage 7-11] Sigma70_r4_2 ECF sigma factor orf043 35632..36399 767 28256 5.1 255 Hypothetical AEK81963.1 hypothetical protein E-161 0 protein [Salmonella phage 7-11]

265

orf044 36396..36761 365 13873 8.6 121 Prophage AAN00738.1AE014276_19 prophage 6.00E-21 0 PF07463.5 2.50E-12 LambdaSa2, LambdaSa2, HNH endonuclease NUMOD4 HNH endonuclease family protein family protein [Streptococcus agalactiae 2603V/R] orf045 36837..37292 455 16967 6.3 151 Hypothetical AEK81964.1 hypothetical protein 7.00E-39 0 protein [Salmonella phage 7-11] orf046 37320..37712 392 14810 9.2 130 Putative AEK81965.1 putative endolysin 2.00E-58 0 endolysin [Salmonella phage 7-11] orf047 37693..37905 212 7765 3.9 70 Bcep22gp31 AAQ54964.2 Bcep22gp31 5.00E-04 0

[Burkholderia phage Bcep22] orf048 37902..38120 218 8223 4.0 72 Hypothetical AEK81968.1 hypothetical protein 7.00E-14 0 protein [Salmonella phage 7-11] orf049 38120..38269 149 5644 9.1 49 Hypothetical 0 protein orf050 38271..38513 242 8985 10.1 80 Hypothetical 0 protein orf051 38525..40360 1835 69038 9.1 611 DNA AEK81978.1 DNA polymerase 0.0 0 PF00476.14 1.20E-26 polymerase [Salmonella phage 7-11] DNA_pol_A orf052 40375..40683 308 11398 4.5 102 Hypothetical AEK81979.1 hypothetical protein 2.00E-35 0 protein [Salmonella phage 7-11] orf053 40683..40958 275 10392 8.0 91 Hypothetical AEK81980.1 hypothetical protein 1.00E-05 0 protein [Salmonella phage 7-11] orf054 41129..41467 338 12036 11.6 112 Hypothetical AEK81983.1 hypothetical protein 2.00E-41 0 protein [Salmonella phage 7-11]

266

orf055 41469..41903 434 16566 9.7 144 Conserved ACJ71863.1 hypothetical protein 6.00E-15 0 A hypothetical [Enterobacteria phage WV8] protein orf055 41827..41976 149 5597 4.5 49 Hypothetical AEK81984.1 hypothetical protein 1.00E-05 0 protein [Salmonella phage 7-11] orf056 42155..42655 500 18953 6.1 166 NrdG ADJ55554.1 NrdG anaerobic 6.00E-46 0 PF04055.15 3.00E-06 anaerobic nucleotide reductase subunit Radical_SAM nucleotide [Enterobacteria phage RB16] reductase subunit orf057 42665..42907 242 8695 5.3 80 Hypothetical AEK81956.1 hypothetical protein 2.00E-04 0 protein [Salmonella phage 7-11] orf058 42910..44037 1127 41351 6.4 375 Conserved YP_001949958.1 hypothetical 7.00E-58 0 PF01139.11 6.30E-98 hypothetical protein RSL1_gp083 UPF0027 protein [Ralstonia phage RSL1] orf059 44037..44525 488 18099 7.7 162 Conserved AEK81986.1 hypothetical protein 8.00E-31 0 hypothetical [Salmonella phage 7-11] protein orf060 44564..45052 488 18901 9.8 162 Putative ADJ55360.1 putative homing 6.00E-27 0 PF00847.14 2.40E-09 homing endonuclease RB16 2 [Enterobacteria AP2 endonuclease phage RB16] orf061 45027..47159 2132 79630 5.2 710 NrdD ADJ55548.1 NrdD anaerobic 0.0 0 PF03477.10 9.10E-16 anaerobic ribonucleotide reductase subunit ATP-cone ribonucleotide [Enterobacteria phage RB16] reductase subunit orf062 47211..47663 448 16889 4.6 150 DNA-binding AEK81988.1 DNA-binding protein 1.00E-51 0 PF00210.18 3.60E-09 protein [Salmonella phage 7-11] Ferritin

267

orf063 47660..47929 269 10120 10.1 89 Conserved AEK81993.1 hypothetical protein 8.00E-33 0 hypothetical [Salmonella phage 7-11] protein orf064 47929..48192 263 9540 5.1 87 Thiol- ABY52866.1 thiol-disulphide 6.00E-13 0 PF00085.14 1.40E-14 disulphide isomerase and thioredoxin Thioredoxin isomerase and [Enterobacteria phage phiEco32] thioredoxin orf065 48185..48739 554 20198 5.0 184 dCTP AEK81996.1 dCTP deaminase E-112 0 deaminase [Salmonella phage 7-11] orf066 48739..48852 113 4457 3.3 37 Hypothetical 0 protein orf067 48883..49377 494 18957 4.8 164 DNA AEK81997.1 DNA polymerase 2.00E-79 0 PF01612.14 4.60E-07 polymerase [Salmonella phage 7-11] DNA_pol_A_e xo1 orf068 49374..51173 1799 66662 5.7 599 Primase/ AEK81999.1 primase/helicase 0.0 0 PF03796.9 8.00E-07 [Salmonella phage 7-11] DnaB_C Helicase orf069 51223..51732 509 18401 6.6 169 Hypothetical AEK82000.1 hypothetical protein 5.00E-18 0 protein [Salmonella phage 7-11] orf070 51754..52188 434 16557 4.8 144 Conserved AEK82001.1 hypothetical protein 2.00E-54 0 PF06094.1 8.40E-12 hypothetical [Salmonella phage 7-11] AIG2 protein orf071 52254..52847 693 22174 5.1 197 Hypothetical AEK82009.1 hypothetical protein 3.00E-10 0 protein [Salmonella phage 7-11] orf072 52877..53842 965 36312 5.4 321 RnlB RNA ADR32568.1 RnlB RNA ligase 2 1.00E-11 0 PF09414.4 2.10E-21 ligase 2 [Enterobacteria phage vB_EcoM- RNA_ligase VR7] orf073 53848..54360 512 18801 9.7 170 Hypothetical AEK82010.1 hypothetical protein 6.00E-28 0 protein [Salmonella phage 7-11]

268

orf074 54360..54545 185 6886 6.7 61 Hypothetical 0 protein orf075 54550..54777 227 9098 9.1 75 Conserved AEK82012.1 hypothetical protein 9.00E-39 0 hypothetical [Salmonella phage 7-11] protein orf076 54777..55310 533 19391 5.2 177 Hypothetical 0 PF00293.1 3.90E-06 protein NUDIX orf077 55319..55564 245 9473 5.1 81 Hypothetical AEK82013.1 hypothetical protein 4.00E-12 0 protein [Salmonella phage 7-11] orf078 55549..56733 1184 42961 5.7 394 ATP-grasp AEK82014.1 ATP-grasp enzyme E-119 0 enzyme [Salmonella phage 7-11] orf079 56801..57019 218 8793 4.7 72 Hypothetical 0 protein orf080 57012..57245 233 8840 9.1 77 Hypothetical AEK82016.1 hypothetical protein 1.00E-33 0 protein [Salmonella phage 7-11] orf081 57250..59223 1973 73999 5.1 657 Glutamine-- AEK82017.1 glutamine E-160 0 PF00310.1 8.00E-06 fructose-6- amidotransferase [Salmonella phage GATase_2 phosphate 7-11] transaminase orf082 59278..60819 1541 58186 5.5 513 Conserved AEK82018.1 hypothetical protein E-155 0 hypothetical [Salmonella phage 7-11] protein orf083 60789..61367 578 21787 5.1 192 Hypothetical AEK82019.1 hypothetical protein 2.00E-48 0 protein [Salmonella phage 7-11] orf084 61367..62164 797 29879 5.4 265 Conserved AEK82020.1 hypothetical protein E-131 0 hypothetical [Salmonella phage 7-11] protein

269

orf085 62176..63120 944 36799 7.7 314 Hypothetical AEK82021.1 hypothetical protein 2.00E-63 0 protein [Salmonella phage 7-11] orf086 63132..64004 872 33389 4.4 290 Hypothetical AEK82022.1 hypothetical protein 6.00E-39 0 protein [Salmonella phage 7-11] orf087 64017..64205 188 7097 6.5 62 Hypothetical 0 protein orf088 64252..64623 371 14093 8.8 123 Hypothetical AEK82023.1 hypothetical protein 2.00E-09 0 protein [Salmonella phage 7-11] orf089 64636..65184 548 19924 9.7 182 Hypothetical 0 protein orf090 65184..65396 212 7543 5.1 70 Hypothetical AEK82024.1 hypothetical protein 2.00E-05 0 protein [Salmonella phage 7-11] orf091 65406..65831 425 16438 10.0 141 Putative AEK82026.1 putative endolysin 5.00E-60 0 PF07486.6 4.00E-18 endolysin [Salmonella phage 7-11] Hydrolase_2 orf092 65891..66550 659 24022 5.7 219 Stress protein, YP_004607621.1 stress protein, 1.00E-21 0 PF02342.12 6.10E-06 tellurium tellurium resistance protein TerZ TerD resistance [Helicobacter bizzozeronii CIII-1] protein TerZ orf093 66660..66896 236 8593 6.3 78 Hypothetical 0 protein orf094 66926..67501 575 20259 4.4 191 Tellurium EEL52267.1 tellurium resistance 1.00E-12 0 PF02342.12 2.50E-06 resistance protein, TerD [Bacillus cereus TerD protein, TerD Rock3-44] orf095 67501..67707 206 8068 6.1 68 Hypothetical 0 protein

270

orf096 67761..68681 920 33647 5.7 306 Putative phage AAG55319.1AE005273_12 putative 2.00E-75 9 PF03741.10 1.10E-46 inhibition, phage inhibition, colicin resistance TerC colicin and tellurite resistance protein resistance and [Escherichia coli O157:H7 str. tellurite EDL933] resistance protein orf097 68682..68876 194 7457 9.9 64 Hypothetical 1 protein orf098 68876..70540 1664 61842 5.3 554 Nicotinamide AEK82034.1 nicotinamide 0.0 0 PF04095.10 1.20E-46 phosphoribosyl phosphoribosyl transferase NAPRTase transferase [Salmonella phage 7-11] orf099 70537..71394 857 31253 5.7 285 Putative ribose- AEK82035.1 putative ribose- 4.00E-70 0 PF00156.21 2.50E-10 phosphate phosphate pyrophosphokinase Pribosyltran pyrophospho- [Salmonella phage 7-11] kinase orf100 71399..71653 254 9569 6.2 84 Hypothetical CBW54780.1 hypothetical protein 5.00E-10 0 protein [Pantoea phage LIMElight] orf101 71647..72138 491 18617 8.9 163 Putative anti- AEK82044.1 putative anti-sigma 1.00E-20 0 sigma factor factor Srd [Salmonella phage 7-11] Srd orf102 72125..72436 311 11798 8.8 103 Hypothetical AEK82045.1 hypothetical protein 1.00E-12 0 protein [Salmonella phage 7-11] orf103 72579..73013 434 17080 8.8 144 Hypothetical AEK82057.1 hypothetical protein 8.00E-35 0 protein [Salmonella phage 7-11] orf104 73033..73440 407 15122 5.3 135 Hypothetical 0 protein orf105 73437..73652 215 8470 8.8 71 Hypothetical 0 protein

271

orf106 73658..73918 260 9466 6.5 86 Phage/conjugal EGF61212.1 phage/conjugal plasmid 2.00E-26 0 PF01258.11 zf- 1.40E-10 plasmid C-4 C-4 type zinc finger protein, TraR dskA_traR type zinc finger family [Klebsiella sp. MS 92-3] protein, TraR family orf107 74100..74222 122 4164 9.3 40 Hypothetical 1 protein orf108 74222..74371 149 5522 5.0 49 Hypothetical 0 protein orf109 74365..74547 182 6956 9.9 60 Hypothetical 0 protein orf110 74544..74921 377 14572 10.0 125 Hypothetical 0 protein orf111 74989..75576 587 21329 9.2 195 Hypothetical YP_001671875.1 hypothetical 1.00E-06 0 protein protein [Pseudomonas phage LUZ24] orf112 75608..75949 341 13081 10.8 113 Hypothetical 0 protein

272

Table 4.4. General features of ORFs in the DNA of phage GAP161 and homology to proteins in the databases.

Motifs (TMHMM; Gene Pfam E- Gene Position Lenth Mass pI Function Homology E-value Phobius) Pfam Motifs value orf001 1..2250 2249 87289 8.1 RIIA protector from gb|ADJ55306.1| rIIA protector from 0.0 0 prophage-induced early prophage-induced early lysis lysis [Enterobacteria phage RB16] orf002 2261.. 236 9042 8.7 Conserved hypothetical gb|ADJ55307.1| conserved 8.00E-32 1 phage protein hypothetical phage protein 2497 [Enterobacteria phage RB16] orf003 2494.. 539 20707 9.6 Conserved hypothetical gb|ADJ55308.1| conserved 2.00E-88 0 PF10263.3 2.50E-12 protein hypothetical protein [Enterobacteria SprT-like 3033 phage RB16] orf004 3107.. 1286 48881 6.9 gp52 DNA topoisomerase gb|ADJ55309.1| gp52 DNA 0.0 0 PF00521.1 1.30E-92 subunit topoisomerase subunit DNA_topoisoI 4393 [Enterobacteria phage RB16] V orf005 4393.. 1895 70610 5.9 gp39plus60 DNA gb|ADJ55310.1| gp39plus60 DNA 0.0 0 PF02518.20 0.00000000 topoisomerase II large topoisomerase II large subunit HATPase_c ; 000014 6288 subunit [Enterobacteria phage PF00204.19 RB16] DNA_gyraseB orf006 6356.. 164 6185 8.4 gp39.2 hypothetical gb|ADJ55312.1| gp39.2 hypothetical 6.00E-30 0 PF09723.4 3.80E-12 protein protein [Enterobacteria phage RB16] CxxC_CxxC_S 6520 SSS orf007 6520.. 431 16688 5.9 motB.2 hypothetical gb|AAX78530.1| motB.2 2.00E-87 0 protein [Enterobacteria phage RB43] 6951 orf008 6972.. 383 14101 5.5 Conserved hypothetical gb|AAX78531.1| hypothetical protein 1.00E-61 0 protein RB43ORF009c [Enterobacteria phage 7355 RB43]

273

orf009 7367.. 203 7848 9.2 Conserved hypothetical gb|ADJ55314.1| conserved 1.00E-19 0 phage protein hypothetical phage protein 7570 [Enterobacteria phage RB16] orf010 7557.. 299 11223 6.4 Conserved hypothetical gb|ADJ55315.1| conserved 9.00E-45 0 phage protein hypothetical phage protein 7856 [Enterobacteria phage RB16] orf011 7853.. 590 22111 6.3 Conserved hypothetical gb|ADJ55316.1| conserved E-112 1 phage protein hypothetical phage protein 8443 [Enterobacteria phage RB16] orf012 8476.. 332 12540 9.3 Conserved hypothetical gb|ADJ55317.1| conserved 1.00E-54 0 phage protein hypothetical phage protein 8808 [Enterobacteria phage RB16] orf013 8894.. 203 7682 9.7 Conserved hypothetical gb|ADJ55319.1| conserved 2.00E-31 0 phage protein hypothetical phage protein 9097 [Enterobacteria phage RB16] orf014 9085.. 161 5810 8.9 Conserved hypothetical gb|AAX78537.1| hypothetical protein 8.00E-28 0 phage protein RB43ORF015c [Enterobacteria phage 9246 RB43] orf015 9335.. 305 11514 8.1 Conserved hypothetical gb|ADE34846.1| hypothetical protein 9.00E-23 1 phage protein [Klebsiella phage KP15] 9640 orf016 9640.. 209 8043 10.6 Conserved hypothetical gb|ADJ55322.1| conserved 1.00E-16 0 phage protein hypothetical phage protein 9849 [Enterobacteria phage RB16] orf017 9859.. 143 5131 4.6 Conserved hypothetical gb|ADJ55323.1| conserved 1.00E-13 1 phage protein hypothetical phage protein 10002 [Enterobacteria phage RB16] orf018 10071.. 281 10500 8.7 Conserved hypothetical gb|AAX78542.1| hypothetical protein 1.00E-43 0 PF01476.14 3.70E-09 protein RB43ORF020c [Enterobacteria phage LysM 10352 RB43]

274

orf019 10349.. 671 25901 5.3 DexA exonuclease A gb|ADJ55324.1| DexA exonuclease A E-154 0 [Enterobacteria phage RB16] 11020 orf020 11001.. 311 11976 5.1 Conserved hypothetical gb|ADJ55325.1| conserved 1.00E-68 0 phage protein hypothetical phage protein 11312 [Enterobacteria phage RB16] orf021 11309.. 311 12051 7.8 Conserved hypothetical gb|ADJ55326.1| conserved 3.00E-70 0 phage protein hypothetical phage protein 11620 [Enterobacteria phage RB16] orf022 11647.. 1331 50677 6.6 Dda DNA helicase gb|AAX78546.1| Dda DNA helicase 0.0 0 PF05970.8 3.30E-09 [Enterobacteria phage RB43]; PIF1 12978 gb|ADJ55327.1| Dda DNA helicase [Enterobacteria phage RB16] orf023 12975.. 287 11346 8.6 Conserved hypothetical gb|ADJ55328.1| conserved 4.00E-49 0 phage protein hypothetical phage protein 13262 [Enterobacteria phage RB16] orf024 13441.. 533 20436 5.0 gp56 dCTPase gb|ADJ55329.1| gp56 dCTPase E-124 0 PF08761.5 3.70E-17 [Enterobacteria phage RB16] dUTPase_2 13974 orf025 13971.. 497 18687 9.7 Hypothetical protein gb|AAX78549.1| hypothetical protein E-105 0 PF00847.14 2.70E-06 RB43ORF027c RB43ORF027c AP2 14468 [Enterobacteria phage RB43] orf026 14441.. 1028 39774 8.9 gp61 DNA primase gb|AAX78550.1| gp61 DNA primase 0.0 0 PF08275.5 8.30E-06 subunit subunit [Enterobacteria phage RB43] Toprim_N 15469 orf027 15478.. 221 8812 9.5 Hypothetical protein gb|ADJ55331.1| hypothetical protein 3.00E-36 0 RB16p027 RB16p027 15699 [Enterobacteria phage RB16]

275

orf028 15696.. 491 18742 9.6 gp61.1 conserved gb|ADJ55332.1| gp61.1 conserved 5.00E-96 0 PF07068.5 3.90E-06 hypothetical protein hypothetical protein [Enterobacteria Gp23 16187 phage RB16] orf029 16187.. 548 20696 9.9 Putative homing gb|ADJ55445.1| putative homing 9.00E-41 0 endonuclease RB16 6 endonuclease RB16 6 [Enterobacteria 16735 phage RB16] orf030 16741.. 740 27782 4.7 Putative gb|ADJ55333.1| putative E-177 0 methyltransferase methyltransferase [Enterobacteria 17481 phage RB16] orf031 17462.. 386 14541 3.7 Conserved hypothetical gb|ADJ55334.1| conserved 3.00E-88 0 phage protein hypothetical phage protein 17848 [Enterobacteria phage RB16] orf032 17845.. 230 8100 9.7 Conserved hypothetical gb|ADJ55335.1| conserved 1.00E-47 1 phage protein hypothetical phage protein 18075 [Enterobacteria phage RB16] orf033 18171.. 254 9476 4.4 Conserved hypothetical gb|ADJ55336.1| conserved 2.00E-47 0 phage protein hypothetical phage protein 18425 [Enterobacteria phage RB16] orf034 18505.. 209 7699 10.7 Hypothetical protein gb|AAX78556.1| hypothetical protein 3.00E-16 1 RB43ORF034c RB43ORF034c 18714 [Enterobacteria phage RB43] orf035 18748.. 1439 53761 5.6 gp41 replication and gb|ADJ55338.1| gp41 replication and 0.0 0 PF03796.9 1.90E-14 recombination DNA recombination DNA helicase DnaB_C 20187 helicase [Enterobacteria phage RB16] orf036 20197.. 305 11889 4.9 gp40 membrane- gb|ADJ55339.1| gp40 membrane- 4.00E-65 0 PF11113.2 4.50E-23 associated initiation of associated initiation of head vertex Phage_head_ch 20502 head vertex [Enterobacteria phage RB16] ap orf037 20542.. 1148 42595 4.9 UvsX RecA-like gb|ADJ55340.1| UvsX RecA-like 0.0 0 PF00154.15 1.60E-12 recombination protein recombination protein [Enterobacteria RecA 21690 phage RB16]

276

orf038 21770.. 2705 104678 6.2 gp43 DNA polymerase gb|ADJ55341.1| gp43 DNA 0.0 0 PF03104.13 8.7E-24 polymerase DNA_pol_B_ex 24475 o1 [Enterobacteria phage RB16] orf039 24468.. 317 12468 7.9 Conserved hypothetical gb|ADJ55342.1| conserved 4.00E-33 0 phage protein hypothetical phage protein 24785 [Enterobacteria phage RB16] orf040 24854.. 362 14367 9.3 RegA early gene gb|ADJ55343.1| RegA early gene 4.00E-82 0 PF01818.11 9.90E-60 translational repressor translational repressor [Enterobacteria Translat_reg 25216 phage RB16] orf041 25226.. 572 22413 5.8 gp62 clamp-loader gb|ADJ55344.1| gp62 clamp-loader E-115 0 subunit subunit [Enterobacteria phage RB16] 25798 orf042 25801.. 998 37021 6.5 gp44 clamp-loader gb|ADJ55345.1| gp44 clamp-loader 0.0 0 PF00004.23 1.90E-15 subunit subunit [Enterobacteria phage RB16] AAA 26799 orf043 26864.. 665 24641 5.0 gp45 sliding clamp DNA gb|ADJ55346.1| gp45 sliding clamp E-140 0 PF02916.9 3.9E-25 polymerase DNA polymerase [Enterobacteria DNA_PPF 27529 phage RB16] orf044 27550.. 284 10682 6.4 RpbA RNA polymerase gb|AAX78566.1| RpbA RNA 3.00E-47 0 PF10789.3 6.60E-10 binding protein polymerase binding protein Phage_RpbA 27834 [Enterobacteria phage RB43] orf045 27880.. 980 35560 4.9 gp32 ssDNA binding gb|ADJ55348.1| gp32 ssDNA binding 0.0 0 PF08804.4 3.60E-41 protein protein [Enterobacteria phage RB16] gp32 28860 orf046 28864.. 665 26350 9.3 gp59 loader of gene 41 gb|AAX78568.1| gp59 loader of gene E-151 0 PF08993.4 T4- 7.5e-42 DNA helicase 41 DNA helicase [Enterobacteria helicase_N 29529 phage RB43 orf047 29526.. 251 9270 4.4 gp33 late promoters gb|ADJ55350.1| gp33 late promoters 1.00E-50 0 transcription transcription [Enterobacteria phage 29777 RB16]

277

orf048 29779.. 263 9979 5.4 DsbA dsDNA binding gb|ADE34876.1| DsbA dsDNA 7.00E-49 0 PF11126.2 4.00E-31 binding [Klebsiella phage KP15] Phage_DsbA 30042 orf049 30052.. 935 36284 6.4 RnaseH ribonuclease gb|ADJ55352.1| RnaseH ribonuclease 0.0 0 PF02739.10 3.4e-08 [Enterobacteria phage RB16] 5_3_exonuc_N 30987 orf050 31311. 515 20260 5.1 gp55 Sigma factor for late gb|ADJ55353.1| gp55 Sigma factor E-112 0 transcription for late transcription [Enterobacteria .31826 phage RB16] orf051 31823.. 227 8626 4.1 a-gt.5 conserved gb|ADJ55354.1| a-gt.5 conserved 1.00E-40 0 hypothetical protein hypothetical protein [Enterobacteria 32050 phage RB16] orf052 32034.. 344 13698 7.9 a-gt.4 conserved gb|AAX78574.1| a-gt.4 conserved 2.00E-68 0 PF10849.2 2.90E-34 hypothetical protein hypothetical protein [Enterobacteria DUF2654 32378 phage RB43] orf053 32405.. 1019 38992 4.9 gp47 recombination gb|AAX78575.1| gp47 recombination 0.0 0 PF12850.1 1.60E-08 endonuclease subunit endonuclease subunit [Enterobacteria Metallophos_2 33424 phage RB43] orf054 33417.. 281 10667 5.1 Conserved hypothetical gb|ADJ55357.1| conserved 7.00E-45 0 phage protein hypothetical phage protein 33698 [Enterobacteria phage RB16] orf055 33682.. 1691 63742 8.0 gp46 recombination gb|AAX78577.1| gp46 recombination 0.0 0 PF02463.13 6.10E-08 endonuclease subunit endonuclease subunit [Enterobacteria SMC_N 35373 phage RB43] orf056 35360.. 710 27086 9.5 Putative site-specific gb|ABI94878.1| putative site-specific 8.00E-59 0 intron-like DNA intron-like DNA endonuclease 36070 endonuclease [Enterobacteria phage RB32] orf057 36125.. 191 7645 4.9 Hypothetical protein gb|ADO51678.1| hypothetical protein 3.00E-35 0 [Klebsiella phage KP15] 36316

278

orf058 36388.. 1700 63479 9.4 Conserved hypothetical gb|ADJ55362.1| conserved 0.0 0 PF00226.25 9.20E-08 phage protein hypothetical phage protein DnaJ 38088 [Enterobacteria phage RB16] orf059 38146.. 227 8552 4.0 Conserved hypothetical gb|ADJ55363.1| conserved 2.00E-49 0 phage protein hypothetical phage protein 38373 [Enterobacteria phage RB16] orf060 38352.. 275 10576 5.9 Conserved hypothetical gb|ADJ55364.1| conserved 6.00E-60 0 phage protein hypothetical phage protein 38627 [Enterobacteria phage RB16] orf061 38624.. 581 21234 6.7 Frd dihydrofolate gb|ADJ55365.1| Frd dihydrofolate E-140 0 PF00186.13 4.70E-20 reductase reductase [Enterobacteria phage DHFR_1 39205 RB16] orf062 39207.. 854 32236 5.7 Td thymidylate synthetase gb|ADJ55366.1| Td thymidylate 0.0 0 PF00303.13 5.70E-95 synthetase [Enterobacteria phage Thymidylat_syn 40061 RB16] t orf063 40102.. 2249 84074 5.6 NrdA ribonucleotide gb|ADJ55367.1| NrdA ribonucleotide 0.0 0 PF03477.10 3.6e-11 reductase A subunit reductase A subunit [Enterobacteria ATP-cone 42351 phage RB16] orf064 42381.. 488 18837 9.5 Putative homing gb|ADJ55368.1| putative homing E-120 0 PF00847.14 1.10E-11 endonuclease RB16 3 endonuclease RB16 3 [Enterobacteria AP2 42869 phage RB16] orf065 42871.. 1175 45960 5.5 NrdB ribonucleotide gb|ADJ55369.1| NrdB ribonucleotide 0.0 0 PF00268.15 2.2e-08 reductase B subunit reductase B subunit [Enterobacteria Ribonuc_red_s 44046 phage RB16] m orf066 44113.. 482 18464 9.1 Putative homing gb|ADJ55445.1| putative homing 3.00E-40 0 PF00847.14 1.30E-09 endonuclease RB16 6 endonuclease RB16 6 [Enterobacteria AP2 44595 phage RB16] orf067 44567.. 416 16480 9.2 DenA endonuclease II gb|ADJ55371.1| DenA endonuclease 6.00E-89 0 PF01541.18 2.40E-11 II [Enterobacteria phage RB16] GIY-YIG 44983

279

orf068 44970.. 1112 42351 4.8 RnlA RNA ligase gb|ADJ55372.1| RnlA RNA ligase 0.0 0 PF09511.4 3.10E-42 [Enterobacteria phage RB16] RNA_lig_T4_1 46082 orf069 46079.. 326 12317 8.7 PseT.3 conserved gb|ADJ55373.1| PseT.3 conserved 2.00E-66 1 hypothetical protein hypothetical protein [Enterobacteria 46405 phage RB16] orf070 46402.. 317 11793 9.2 PseT.2 conserved gb|ADJ55374.1| PseT.2 conserved 6.00E-45 0 hypothetical protein hypothetical protein [Enterobacteria 46719 phage RB16] orf071 46704.. 173 7017 5.3 Hypothetical protein 0

46877 orf072 46874.. 887 33512 6.4 PseT 3'phosphatase, gb|ADJ55375.1| PseT 3'phosphatase, 0.0 0 PF06414.6 2.80E-07 5'polynucleotide kinase 5'polynucleotide kinase Zeta_toxin 47761 [Enterobacteria phage RB16] orf073 47822.. 266 10038 9.3 Conserved hypothetical gb|ADJ55376.1| conserved 8.00E-48 0 phage protein hypothetical phage protein 48088 [Enterobacteria phage RB16] orf074 48085.. 254 9637 5.2 Conserved hypothetical gb|ADJ55377.1| conserved 1.00E-36 0 phage protein hypothetical phage protein 48339 [Enterobacteria phage RB16] orf075 48339.. 530 19934 6.5 Cd dCMP deaminase gb|AAX78597.1| Cd dCMP 5.00E-90 0 PF00383.1 1.20E-14 deaminase [Enterobacteria phage dCMP_cyt_dea 48869 RB43] m_1 orf076 48862.. 356 12835 5.6 gp31 co-chaperonin for gb|ADJ55379.1| gp31 co-chaperonin 4.00E-62 0 PF00166.15 9.10E-11 GroEL for GroEL [Enterobacteria phage Cpn10 49218 RB16] orf077 49221.. 251 9435 7.8 Conserved hypothetical gb|ADJ55380.1| conserved 4.00E-51 0 phage protein hypothetical phage protein 49472 [Enterobacteria phage RB16]

280

orf078 49787.. 419 16248 5.7 Conserved hypothetical gb|ADJ55381.1| conserved 3.00E-68 0 phage protein hypothetical phage protein 50206 [Enterobacteria phage RB16] orf079 50249.. 569 22030 9.3 Conserved hypothetical gb|ADJ55382.1| conserved E-144 1 phage protein hypothetical phage protein 50818 [Enterobacteria phage RB16] orf080 50823.. 362 13613 6.3 Vs.6 conserved gb|ADJ55383.1| Vs.6 conserved 1.00E-79 0 PF01228.15 3.10E-23 hypothetical protein hypothetical protein [Enterobacteria Gly_radical 51185 phage RB16] orf081 51185.. 281 10788 4.8 Vs.4 conserved gb|ADJ55384.1| Vs.4 conserved 7.00E-60 0 hypothetical protein hypothetical protein [Enterobacteria 51466 phage RB16] orf082 51582.. 293 10318 10.5 Conserved hypothetical gb|ADJ55385.1| conserved 2.00E-44 0 phage protein hypothetical phage protein 51875 [Enterobacteria phage RB16] orf083 51966.. 440 16563 9.3 Conserved hypothetical gb|ADJ55386.1| gp30.3 conserved 2.00E-93 0 PF08010.5 1.30E-78 phage protein hypothetical protein [Enterobacteria Phage_30_3 52406 phage RB16] orf083 52403.. 206 7890 8.8 Hypothetical protein gb|ADB81584.1| hypothetical protein 2.00E-04 1 A CC31p088 CC31p088 [Enterobacteria phage 52609 CC31] orf084 52618.. 269 9925 5.7 Conserved hypothetical gb|ADJ55387.1| conserved 6.00E-58 1; 0 phage protein hypothetical phage protein 52887 [Enterobacteria phage RB16] orf085 52890.. 221 8762 4.4 Conserved hypothetical gb|ADJ55388.1| conserved 1.00E-48 0 phage protein hypothetical phage protein 53111 [Enterobacteria phage RB16] orf086 53108.. 617 23423 5.7 gp30.2 gb|AAX78616.1| gp30.2 E-135 0 [Enterobacteria phage RB43] 53725

281

orf087 53703.. 1523 57272 5.3 gp30 DNA ligase gb|ADJ55391.1| gp30 DNA ligase 0.0 0 PF01068.1 2.60E-17 [Enterobacteria phage RB16] DNA_ligase_A 55226 _M orf088 55318.. 272 10123 4.2 Conserved hypothetical gb|ADJ55392.1| conserved 2.00E-59 0 phage protein hypothetical phage protein 55590 [Enterobacteria phage RB16] orf089 55652.. 179 6387 5.3 Conserved hypothetical gb|ADJ55393.1| conserved 1.00E-35 0 phage protein hypothetical phage protein 55831 [Enterobacteria phage RB16] orf090 55828.. 191 7333 5.5 Conserved hypothetical gb|ADJ55394.1| conserved 5.00E-44 0 phage protein hypothetical phage protein 56019 [Enterobacteria phage RB16] orf091 56016.. 287 11134 8.2 Conserved hypothetical gb|ADJ55395.1| conserved 9.00E-60 0 phage protein hypothetical phage protein 56303 [Enterobacteria phage RB16] orf092 56300.. 374 14833 5.0 Conserved hypothetical gb|ADJ55396.1| conserved 2.00E-85 0 phage protein hypothetical phage protein 56674 [Enterobacteria phage RB16] orf093 56671.. 1103 41813 5.0 gp27 baseplate hub gb|ADJ55397.1| gp27 baseplate hub 0.0 0 PF09097.4 3.50E-39 subunit subunit [Enterobacteria phage RB16] Phage-tail_1 57774 orf094 57837.. 578 22450 6.3 gp28 baseplate distal hub gb|ADJ55398.1| gp28 baseplate distal E-127 0 PF11110.2 4.80E-10 subunit hub subunit [Enterobacteria phage Phage_hub_GP 58415 RB16] 28 orf095 58418.. 1748 65304 5.3 Conserved hypothetical gb|ADJ55399.1| conserved 0.0 0 phage protein hypothetical phage protein 60166 [Enterobacteria phage RB16] orf096 60178.. 1094 39447 6.2 gp48 baseplate tail tube gb|ADJ55400.1| gp48 baseplate tail 0.0 0 PF11091.2 4.80E-120 cap tube cap [Enterobacteria phage RB16] T4_tail_cap 61272

282

orf097 61282.. 863 31852 5.1 gp54 baseplate-tail tube gb|ADJ55401.1| gp54 baseplate-tail 0.0 0 PF06841.6 8.90E-11 initiator tube initiator [Enterobacteria phage Phage_T4_gp19 62145 RB16] orf098 62173.. 743 28654 5.1 gp51 baseplate hub gb|ADJ55402.1| gp51 baseplate hub E-151 0 PF12322.2 9.30E-44 assembly catalyst assembly catalyst [Enterobacteria T4_baseplate 62916 phage RB16] orf099 62909.. 557 21365 5.2 gp26 baseplate hub gb|ADJ55403.1| gp26 baseplate hub E-117 0 PF12322.2 6.10E-40 subunit subunit [Enterobacteria phage RB16] T4_baseplate 63466 orf100 63468.. 392 14489 4.4 gp25 baseplate wedge gb|ADJ55404.1| gp25 baseplate 1.00E-82 0 PF04965.8 8.30E-18 subunit wedge subunit [Enterobacteria phage GPW_gp25 63860 RB16] orf101 63888.. 497 18863 5.3 Conserved hypothetical gb|ADJ55405.1| conserved E-114 0 phage protein hypothetical phage protein 64385 [Enterobacteria phage RB16] orf102 64396.. 230 8584 5.2 Conserved hypothetical gb|ADJ55406.1| conserved 7.00E-48 0 phage protein hypothetical phage protein 64626 [Enterobacteria phage RB16] orf103 64623.. 215 7969 5.3 Conserved hypothetical gb|ADJ55407.1| conserved 2.00E-33 0 phage protein hypothetical phage protein 64838 [Enterobacteria phage RB16] orf104 64918.. 653 24940 6.9 Conserved hypothetical gb|ADJ55408.1| conserved 3.00E-22 0 phage protein hypothetical phage protein 65571 [Enterobacteria phage RB16] orf105 65590.. 290 11105 4.3 Conserved hypothetical gb|ADJ55409.1| conserved 2.00E-50 0 phage protein hypothetical phage protein 65880 [Enterobacteria phage RB16] orf106 65884.. 161 6086 9.6 Hypothetical protein gb|ADB81610.1| hypothetical protein 6.00E-20 0 A CC31p114 CC31p114 [Enterobacteria phage 66045 CC31]

283

orf106 66014.. 467 17731 4.3 Hypothetical protein; gb|ADE34929.1| hypothetical protein 1.00E-78 0 Conserved hypothetical [Klebsiella phage KP15] 66481 phage protein orf107 66478.. 191 7172 8.1 Conserved hypothetical gb|ADJ55411.1| conserved 3.00E-26 1 phage protein hypothetical phage protein 66669 [Enterobacteria phage RB16] orf108 66666.. 626 23846 5.1 Conserved hypothetical gb|ADJ55412.1| conserved 4.00E-89 0 phage protein hypothetical phage protein 67292 [Enterobacteria phage RB16] orf109 67334.. 2246 83814 5.2 Conserved hypothetical gb|ADJ55413.1| conserved 0.0 0 phage protein hypothetical phage protein 69580 [Enterobacteria phage RB16] orf110 69581.. 335 12997 5.2 Hypothetical protein gb|ADE34933.1| hypothetical protein 4.00E-44 0 [Klebsiella phage KP15] 69916 orf111 69916.. 512 19807 4.7 Conserved hypothetical gb|ADJ55415.1| conserved E-115 0 phage protein hypothetical phage protein 70428 [Enterobacteria phage RB16] orf112 70425.. 233 8703 4.5 Hypothetical protein 0

70658 orf113 70655.. 242 9328 7.7 Hypothetical protein gb|ADJ55417.1| hypothetical protein 2.00E-46 0 RB16p113 RB16p113 [Enterobacteria phage 70897 RB16] orf114 70894.. 272 9815 4.2 Conserved hypothetical gb|ADJ55418.1| conserved 7.00E-13 0 phage protein hypothetical phage protein 71166 [Enterobacteria phage RB16] orf115 71209.. 278 10604 5.7 Conserved hypothetical gb|ADJ55420.1| conserved 2.00E-23 0 phage protein hypothetical phage protein 71487 [Enterobacteria phage RB16]

284

orf116 71535.. 440 17373 9.5 Conserved hypothetical gb|ADJ55421.1| conserved 1.00E-77 0 phage protein hypothetical phage protein 71975 [Enterobacteria phage RB16] orf117 71972.. 911 34076 8.4 Putative DNA- gb|ADJ55423.1| putative DNA- 0.00E+00 0 PF00145.11 2.00E-89 methyltransferase, type II methyltransferase, type II restriction- DNA_methylas 72883 restriction-modification modification system [Enterobacteria e system phage RB16] orf118 72880.. 260 9775 4.1 Conserved hypothetical gb|ADJ55424.1| conserved 1.00E-36 0 phage protein hypothetical phage protein 73140 [Enterobacteria phage RB16] orf119 73140.. 242 9164 8.9 Hypothetical protein gb|ADO51694.1| hypothetical protein 5.00E-34 0 [Klebsiella phage KP15] 73382 orf120 73379.. 1001 38134 6.3 NrdC.11 gb|AAX78653.1| NrdC.11 0.0 0 PF10127.3 1.20E-15 [Enterobacteria phage RB43 Nuc-transf 74380 orf121 74377.. 395 14845 9.2 Hypothetical protein gb|AAX78654.1| hypothetical protein 4.00E-32 0 RB43ORF132c RB43ORF132c [Enterobacteria phage 74772 RB43] orf122 74769.. 227 8560 6.1 Hypothetical protein gb|AAX78655.1| hypothetical protein 2.00E-40 0 RB43ORF133c RB43ORF133c [Enterobacteria phage 74996 RB43] orf123 75017.. 557 20355 8.9 Hypothetical protein gb|AAX78656.1| hypothetical protein 1.00E-35 0 RB43ORF134c RB43ORF134c [Enterobacteria phage 75574 RB43] orf124 75571.. 272 10401 5.3 Hypothetical protein gb|AAX78658.1| hypothetical protein 2.00E-55 0 RB43ORF136c RB43ORF136c [Enterobacteria phage 75843 RB43] orf125 75840.. 194 7007 5.2 Hypothetical protein gb|ADJ55432.1| hypothetical protein 1.00E-36 0 RB16p128 [Enterobacteria phage 76034 RB16]

285

orf126 76119.. 1055 40695 7.2 Putative bifunctional gb|ADJ55433.1| putative bifunctional 0.0 0 PF01467.1 3.7e-07 protein: NMN protein: NMN CTP_transf_2 77174 adenylyltransferase/NUDI adenylyltransferase/NUDIX X hydrolase hydrolase [Enterobacteria phage RB16] orf127 77352.. 914 34035 6.7 Putative SPFH domain- gb|ADJ55434.1| putative SPFH E-176 0 PF01145.19 4.00E-17 containing protein/band 7 domain-containing protein/band 7 Band_7 78266 family protein family protein [Enterobacteria phage RB16] orf128 78340.. 383 14467 5.1 rI.-1 conserved gb|ADJ55435.1| rI.-1 conserved 1.00E-82 0 hypothetical protein hypothetical protein [Enterobacteria 78723 phage RB16] orf129 78733.. 284 10860 9.0 rI lysis inhibition gb|ADJ55436.1| rI lysis inhibition 7.00E-58 0 regulator regulator [Enterobacteria phage 79017 RB16] orf130 79125.. 392 15412 9.7 Conserved hypothetical gb|ADJ55437.1| conserved 6.00E-85 0 phage protein hypothetical phage protein 79517 [Enterobacteria phage RB16] orf131 79548.. 584 22285 6.9 Tk thymidine kinase gb|ADJ55438.1| Tk thymidine kinase E-130 0 PF00265.12 5.10E-49 [Enterobacteria phage RB16] TK 80132 orf132 80125.. 197 7810 6.9 Hypothetical protein gb|AAQ15346.1| hypothetical protein 4.00E-14 0 A RB49ORF089c RB49ORF089c [Enterobacteria phage 80322 RB49] orf132 80315.. 215 8016 9.0 Hypothetical protein 0

80530 orf133 80523.. 212 8095 4.3 Conserved hypothetical gb|ADJ55439.1| conserved 2.00E-09 0 phage protein hypothetical phage protein 80735 [Enterobacteria phage RB16] orf134 80732.. 497 18189 7.6 Tk.4 gb|ADE34948.1| Tk.4 [Klebsiella 4E-71 0 PF01661.15 4.90E-09 phage KP15] Macro

286

81229 orf135 81308.. 134 5351 4.4 Conserved hypothetical gb|ADJ55440.1| conserved 3.00E-15 0 phage protein hypothetical phage protein 81442 [Enterobacteria phage RB16] orf136 81454.. 320 12236 6.1 Conserved hypothetical gb|ADJ55441.1| conserved 3.00E-53 0 phage protein hypothetical phage protein 81774 [Enterobacteria phage RB16] orf137 81784.. 611 24044 6.0 Hypothetical protein gb|AAX78673.1| hypothetical protein 1.00E-51 0 RB43ORF151c RB43ORF151c [Enterobacteria phage 82395 RB43] orf138 82397.. 404 15312 6.3 Conserved hypothetical gb|ADJ55442.1| conserved 8.00E-46 0 phage protein hypothetical phage protein 82801 [Enterobacteria phage RB16] orf139 82869.. 647 24216 9.6 Vs.1; Vs.1 conserved gb|AAX78675.1| Vs.1 E-131 0 PF10715.3 1.80E-15 hypothetical protein [Enterobacteria phage RB43] REGB_T4 83516 orf140 83568.. 569 22379 6.0 Hypothetical protein gb|ADJ55446.1| hypothetical protein 3.00E-74 0 RB16p142 RB16p142 [Enterobacteria phage 84137 RB16] orf141 84134.. 170 6153 8.4 Conserved hypothetical gb|ADJ55447.1| conserved 1.00E-16 0 phage protein hypothetical phage protein 84304 [Enterobacteria phage RB16] orf142 84312.. 194 7258 8.7 Hypothetical protein; gb|ADO51700.1| hypothetical protein 5.00E-30 0 Conserved hypothetical [Klebsiella phage KP15] 84506 phage protein orf143 84543.. 1346 49396 5.0 Hypothetical protein gb|AAX78679.1| hypothetical protein 0.0 0 PF01850.15 9.8e-07 RB43ORF157c RB43ORF157c [Enterobacteria phage PIN 85889 RB43] orf144 85899.. 524 19672 9.4 Putative homing gb|ADE34956.1| RB16 HNH(AP2) 1 E-120 0 PF00847.14 3.10E-10 endonuclease RB16 5 [Klebsiella phage KP15] AP2 86423

287

orf145 86423.. 470 17508 8.7 Conserved hypothetical gb|ADJ55450.1| conserved 2.00E-66 0 phage protein hypothetical phage protein 86893 [Enterobacteria phage RB16] orf146 86903.. 395 14717 9.4 Putative endolysin ADJ55451.1| putative endolysin 9.00E-79 0 [Enterobacteria phage RB16] 87298 orf147 87366.. 398 15306 5.8 Conserved hypothetical gb|ADE34959.1| hypothetical protein 4.00E-79 0 phage protein [Klebsiella phage KP15] 87764 orf148 87820.. 320 12240 4.5 Hypothetical protein gb|ADE34961.1| hypothetical protein 8.00E-70 0 [Klebsiella phage KP15] 88140 orf149 88177.. 263 9996 4.5 Hypothetical protein gb|ADE34962.2| hypothetical protein 2.00E-62 0 [Klebsiella phage KP15] 88440 orf150 88513.. 263 9784 7.6 gp39.1 conserved gb|ADJ55455.1| gp39.1 conserved 8.00E-48 0 hypothetical protein hypothetical protein [Enterobacteria 88776 phage RB16] orf151 88780.. 428 16269 4.7 Hypothetical protein gb|ADE34963.1| hypothetical protein 8.00E-69 0 [Klebsiella phage KP15] 89208 orf152 89205.. 353 13467 5.4 Conserved hypothetical gb|ADJ55457.1| conserved 6.00E-54 0 phage protein hypothetical protein [Enterobacteria 89558 phage RB16] orf153 89533.. 380 14788 5.6 Conserved hypothetical gb|ADJ55458.1| conserved 1.00E-38 0 phage protein hypothetical phage protein 89913 [Enterobacteria phage RB16] orf154 89913.. 293 11268 5.5 Conserved hypothetical gb|ADJ55459.1| conserved 4.00E-13 0 phage protein hypothetical phage protein 90206 [Enterobacteria phage RB16] orf155 90206.. 1169 43619 5.7 Putative radical SAM gb|ADJ55460.1| putative radical 0.0 0 protein SAM protein [Enterobacteria phage

288

91375 RB16] orf156 91386.. 203 7912 9.2 Conserved hypothetical gb|ADJ55461.1| conserved 5.00E-37 0 phage protein hypothetical phage protein 91589 [Enterobacteria phage RB16] orf157 91589.. 260 10372 9.9 Conserved hypothetical gb|ADJ55462.1| conserved 6.00E-54 0 phage protein hypothetical phage protein 91849 [Enterobacteria phage RB16] orf158 91916.. 200 7724 4.3 Hypothetical protein gb|ADJ55463.1| hypothetical protein 5.00E-41 0 RB16p159 RB16p159 [Enterobacteria phage 92116 RB16] orf159 92113.. 146 5612 4.9 Conserved hypothetical gb|ADJ55464.1| conserved 1.00E-27 0 phage protein hypothetical phage protein 92259 [Enterobacteria phage RB16] orf160 92256.. 251 9757 5.4 Hypothetical protein gb|ADJ55465.1| hypothetical protein 8.00E-55 0 RB16p161 RB16p161 [Enterobacteria phage 92507 RB16] orf161 92579.. 278 10485 4.9 Frd.1 conserved gb|ADJ55467.1| Frd.1 conserved 6.00E-65 0 hypothetical protein hypothetical protein [Enterobacteria 92857 phage RB16] orf162 92951.. 590 23008 5.8 Conserved hypothetical gb|ADJ55468.1| conserved 1.00E-131 0 phage protein hypothetical phage protein 93541 [Enterobacteria phage RB16] orf163 93538.. 404 15910 8.6 Conserved hypothetical gb|ADJ55469.1| conserved 5.00E-95 0 phage protein hypothetical phage protein 93942 [Enterobacteria phage RB16] orf164 93953.. 116 4169 4.8 Hypothetical protein 0

94069

289

orf165 94121.. 281 10699 8.8 Conserved hypothetical gb|ADJ55470.1| conserved 1.00E-42 0 phage protein hypothetical phage protein 94402 [Enterobacteria phage RB16]* orf166 94404.. 308 11613 4.9 Conserved hypothetical gb|ADJ55471.1| conserved 4.00E-61 0 phage protein hypothetical phage protein 94712 [Enterobacteria phage RB16] orf167 94709.. 533 20681 6.1 Conserved hypothetical gb|ADJ55472.1| conserved 1.00E-118 0 PF03235.8 1.30E-16 phage protein hypothetical protein [Enterobacteria DUF262 95242 phage RB16] orf168 95235.. 329 12015 6.4 Conserved hypothetical gb|ADJ55473.1| conserved 1.00E-54 1 phage protein hypothetical phage protein 95564 [Enterobacteria phage RB16] orf169 95572.. 260 9601 4.9 Conserved hypothetical gb|ADJ55474.1| conserved 3.00E-42 0 phage protein hypothetical protein [Enterobacteria 95832 phage RB16] orf170 95829.. 380 14208 4.7 Hypothetical protein gb|AAX78707.1| hypothetical protein 3.00E-45 0 RB43ORF185c RB43ORF185c [Enterobacteria phage 96209 RB43] orf171 96206.. 413 15481 5.9 NudE nudix hydrolase gb|ADJ55476.1| NudE nudix 2.00E-81 0 PF00293.1 4.90E-15 hydrolase [Enterobacteria phage NUDIX 96619 RB16] orf172 96619.. 239 8819 6.0 Conserved hypothetical gb|ADJ55477.1| conserved 1.00E-33 0 phage protein hypothetical phage protein 96858 [Enterobacteria phage RB16] orf173 96855.. 266 10021 6.1 Conserved hypothetical gb|ADJ55478.1| conserved 7.00E-54 0 phage protein hypothetical phage protein 97121 [Enterobacteria phage RB16] orf174 97105.. 302 11528 4.3 Conserved hypothetical gb|ADJ55479.1| conserved 9.00E-71 0 phage protein hypothetical phage protein 97407 [Enterobacteria phage RB16]

290

orf175 97404.. 338 12607 8.4 Conserved hypothetical gb|ADJ55480.1| conserved 2.00E-55 0 phage protein hypothetical phage protein 97742 [Enterobacteria phage RB16] orf176 97753.. 140 5233 9.0 Conserved hypothetical gb|ADJ55481.1| conserved 3.00E-27 1 phage protein hypothetical phage protein 97893 [Enterobacteria phage RB16] orf177 97983.. 140 5283 9.0 Conserved hypothetical gb|ADJ55483.1| conserved 2.00E-26 0 phage protein hypothetical phage protein 98123 [Enterobacteria phage RB16] orf178 98190.. 539 20846 9.4 Hypothetical protein gb|AAX78717.1| hypothetical protein 8.00E-83 0 RB43ORF195c RB43ORF195c [Enterobacteria phage 98729 RB43] orf179 98751.. 572 21600 5.1 gp57B conserved gb|AAX78718.1| gp57B E-130 0 hypothetical protein [Enterobacteria phage RB43] 99323 orf180 99433.. 266 9437 4.3 gp57A chaperone for tail gb|ADJ55486.1| gp57A chaperone for 2.00E-38 0 fiber formation tail fiber formation [Enterobacteria 99699 phage RB16] orf181 99703.. 668 25271 8.7 gp1 dNMP kinase gb|ADJ55487.1| gp1 dNMP kinase 1.00E-102 0 [Enterobacteria phage RB16] 100371 orf182 100368.. 551 20732 4.6 gp3 head-proximal tip of gb|ADJ55488.1| gp3 head-proximal E-122 0 PF06841.6 3.70E-10 tail tube tip of tail tube [Enterobacteria phage Phage_T4_gp19 100919 RB16] orf183 101176.. 791 29105 8.2 Conserved hypothetical gb|ADJ55489.1| conserved 1.00E-158 0 101967 phage protein hypothetical phage protein [Enterobacteria phage RB16] orf184 102004.. 854 33121 10.1 gp2 DNA end protector gb|ADJ55490.1| gp2 DNA end E-178 0 protein protector protein [Enterobacteria 102858 phage RB16]

291

orf185 102855.. 689 26190 9.7 putative Seg-like homing gb|ADJ55491.1| putative Seg-like E-167 0 endonuclease, GIY-YIG homing endonuclease, GIY-YIG 103544 family family [Enterobacteria phage RB16] orf186 103541.. 365 14468 9.5 gp4 head completion gb|ADJ55492.1| gp4 head completion 7.00E-79 0 protein protein [Enterobacteria phage RB16] 103906 orf187 104058.. 554 21931 5.4 gp53 baseplate wedge gb|ADJ55493.1| gp53 baseplate E-133 0 PF11246.2 2.00E-77 subunit wedge subunit [Enterobacteria phage Phage_gp53 104612 RB16] orf188 104609.. 1766 64529 5.1 gp5 baseplate hub subunit gb|ADJ55494.1| gp5 baseplate hub 0.0 0 PF06714.5 1e-61 and tail lysozyme subunit and tail lysozyme Gp5_OB 106375 [Enterobacteria phage RB16] orf189 106378.. 2087 76187 5.3 Conserved hypothetical gb|ADJ55495.1| conserved 0.0 0 phage protein hypothetical phage protein 108465 [Enterobacteria phage RB16] orf190 108489.. 1913 71079 4.7 gp6 baseplate wedge gb|ADJ55497.1| gp6 baseplate wedge 0.0 0 subunit subunit [Enterobacteria phage RB16] 110402 orf191 110483.. 3080 118259 5.4 gp7 baseplate wedge gb|ADJ55498.1| gp7 baseplate wedge 0.0 1 subunit subunit [Enterobacteria phage RB16] 113563 orf192 113563.. 992 38146 4.7 gp8 baseplate wedge gb|ADJ55499.1| gp8 baseplate wedge 0.0 0 PF09215.4 8.30E-157 subunit subunit [Enterobacteria phage RB16] Phage-Gp8 114555 orf193 114567.. 863 31121 5.5 gp9 baseplate wedge tail gb|ADJ55500.1| gp9 baseplate wedge 0.0 0 PF07880.5 4.80E-97 fiber connector tail fiber connector [Enterobacteria T4_gp9_10 115430 phage RB16] orf194 115427.. 1817 66761 4.7 gp10 baseplate wedge gb|ADJ55501.1| gp10 baseplate 0.0 0 PF07880.5 1.10E-90 subunit and tail pin wedge subunit and tail pin T4_gp9_10 117244 [Enterobacteria phage RB16]

292

orf195 117245.. 662 24177 4.8 gp11 baseplate wedge gb|ADJ55502.1| gp11 baseplate 1.00E-144 0 PF08677.4 3.10E-82 subunit and tail pin wedge subunit and tail pin GP11 117907 [Enterobacteria phage RB16] orf196 117917.. 1388 49545 6.5 gp12 short tail fibers gb|ADJ55503.1| gp12 short tail fibers 0.0 0 PF07484.6 1.20E-12 [Enterobacteria phage RB16]* Collar 119305 orf197 119318.. 2039 72520 4.6 Wac whisker protein gb|ADJ55504.1| Wac whisker protein 0.0 0 PF07921.6 1.20E-20 [Enterobacteria phage RB16] Fibritin_C 121357 orf198 121394.. 923 33623 4.7 gp13 neck protein gb|ADJ55505.1| gp13 neck protein 0.0 0 [Enterobacteria phage RB16] 122317 orf199 122326.. 746 28816 4.4 gp14 neck protein gb|ADJ55506.1| gp14 neck protein E-169 0 PF11649.2 1.00E-95 [Enterobacteria phage RB16] T4_neck- 123072 protein orf200 123131.. 821 31930 5.5 gp15 tail sheath stabilizer gb|ADJ55508.1| gp15 tail sheath 0.0 0 and completion protein stabilizer and completion protein 123952 [Enterobacteria phage RB16] orf201 123955.. 539 19797 4.6 gp16 terminase DNA gb|ADJ55509.1| gp16 terminase DNA E-106 0 PF11053.2 3.40E-53 packaging enzyme, small packaging enzyme, small subunit DNA_Packagin 124494 subunit [Enterobacteria phage RB16] g orf202 124463.. 1829 69619 5.7 gp17 terminase subunit, gb|ADJ55510.1| gp17 terminase 0.0 0 PF03237.9 1.10E-69 nuclease and ATPase subunit, nuclease and ATPase Terminase_6 126292 [Enterobacteria phage RB16] orf203 126311.. 1991 72371 5.1 gp18 tail sheath protein gb|ADJ55511.1| gp18 tail sheath 0.0 0 PF04984.8 1.10E-143 protein [Enterobacteria phage RB16] Phage_sheath_1 128302 orf204 128348.. 485 18283 4.9 gp19 tail tube protein gb|ADJ55512.1| gp19 tail tube protein E-116 0 PF06841.6 3.60E-14 [Enterobacteria phage RB16] Phage_T4_gp19 128833

293

orf205 128887.. 1574 60723 5.1 gp20 portal vertex protein gb|ADJ55513.1| gp20 portal vertex 0.0 0 PF07230.5 1.40E-227 protein [Enterobacteria phage RB16] Phage_T4_Gp2 130461 0 orf206 130461.. 254 9646 4.2 gp67 prohead core protein gb|ADJ55514.1| gp67 prohead core 9.00E-25 0 protein [Enterobacteria phage RB16] 130715 orf207 130728.. 407 15319 10.1 gp68 prohead core protein gb|ADJ55515.1| gp68 prohead core 5.00E-69 0 protein [Enterobacteria phage RB16] 131135 orf208 131138.. 659 24181 5.4 gp21 prohead core and gb|ADJ55516.1| gp21 prohead core E-145 0 PF03420.7 1.90E-86 protease and protease [Enterobacteria phage Peptidase_U9 131797 RB16] orf209 131824.. 794 29558 4.4 gp22 prohead core protein gb|ADJ55517.1| gp22 prohead core E-159 0 protein [Enterobacteria phage RB16] 132618 orf210 132638.. 1574 56077 5.1 gp23 major capsid protein gb|ADJ55518.1| gp23 major capsid 0.00E+00 0 PF07068.5 5.40E-262 protein [Enterobacteria phage RB16] Gp23 134212 orf211 134299.. 635 23845 9.5 Putative Seg-like homing gb|ADJ55519.1| putative Seg-like E-111 0 endonuclease, GIY-YIG homing endonuclease, GIY-YIG 134934 family family [Enterobacteria phage RB16] orf212 134974.. 1298 47722 4.8 gp24 capsid vertex gb|ADJ55520.1| gp24 capsid vertex 0.0 0 PF07068.5 1.20E-16 protein protein [Enterobacteria phage RB16] Gp23 136272 orf213 136538.. 422 16072 6.8 UvsY recombination, gb|ADJ55521.1| UvsY 1.00E-77 0 PF11056.2 9.90E-48 repair and ssDNA binding recombination, repair and ssDNA UvsY 136960 protein binding protein [Enterobacteria phage RB16] orf214 136962.. 182 6740 4.2 UvsY.-2 conserved gb|ADJ55522.1| UvsY.-2 conserved 1.00E-21 0 PF10886.2 3.60E-16 hypothetical protein hypothetical protein [Enterobacteria DUF2685 137144 phage RB16]

294

orf215 137153.. 137 5307 4.7 Hypothetical protein 0

137290 orf216 137636.. 230 8757 4.8 UvsW.1 conserved gb|ADJ55523.1| UvsW.1 conserved 2.00E-34 0 PF11637.2 6.60E-28 hypothetical protein hypothetical protein [Enterobacteria UvsW 137866 phage RB16] orf217 137876.. 1499 57415 9.1 UvsW RNA- and DNA- gb|ADJ55524.1| UvsW RNA- and 0.0 0 PF04851.9 2.40E-10 helicase DNA-helicase [Enterobacteria phage ResIII 139375 RB16] orf218 139446.. 659 24724 4.3 Inh minor capsid protein gb|ADJ55525.1| Inh minor capsid E-124 0 inhibitor of 21 protease protein inhibitor of 21 protease 140105 [Enterobacteria phage RB16] orf219 140175.. 347 13351 6.1 Hypothetical protein gb|ADE35025.1| hypothetical protein 8.00E-65 0 [Klebsiella phage KP15] 140522 orf220 140816.. 320 11926 4.7 Conserved hypothetical gb|ADJ55527.1| conserved 2.00E-58 0 phage protein hypothetical phage protein 141136 [Enterobacteria phage RB16] orf221 141164.. 503 18188 4.4 Hoc large outer capsid gb|ADJ55528.1| Hoc large outer 1.00E-99 0 protein capsid protein [Enterobacteria phage 141667 RB16] orf222 141688.. 500 19396 5.7 Conserved hypothetical gb|ADJ55529.1| conserved E-111 0 phage protein hypothetical phage protein 142188 [Enterobacteria phage RB16] orf223 142223.. 467 17445 8.5 Conserved hypothetical gb|ADJ55530.1| conserved 9.00E-91 0 phage protein hypothetical phage protein 142690 [Enterobacteria phage RB16] orf224 142740.. 833 30293 9.3 Conserved hypothetical gb|ADJ55531.1| conserved 0.0 0 PF06414.6 4.20E-07 phage protein hypothetical phage protein Zeta_toxin 143573 [Enterobacteria phage RB16]

295

orf225 143688.. 869 33867 7.2 Hypothetical protein gb|AAX78764.1| hypothetical protein 0.0 0 RB43ORF242w RB43ORF242w [Enterobacteria 144557 phage RB43] orf226 144594.. 317 11593 5.9 Conserved hypothetical gb|ADJ55533.1| conserved 3.00E-67 0 phage protein hypothetical phage protein 144911 [Enterobacteria phage RB16] orf227 144908.. 173 6317 4.4 Hypothetical protein gb|ADE35035.1| hypothetical protein 1.00E-23 1 [Klebsiella phage KP15] 145081 orf228 145074.. 365 14246 4.8 Hypothetical protein 0

145439 orf229 145436.. 326 11935 5.0 Conserved hypothetical gb|ADJ55535.1| conserved 4.00E-44 0 phage protein hypothetical phage protein 145762 [Enterobacteria phage RB16] orf230 145765.. 344 13419 10.2 Conserved hypothetical gb|ADJ55536.1| conserved 2.00E-60 0 phage protein hypothetical phage protein 146109 [Enterobacteria phage RB16] orf231 146119.. 542 20203 9.7 Srd postulated decoy of gb|ADJ55537.1| Srd postulated decoy 3.00E-76 0 host sigma70 or sigmaS of host sigma70 or sigmaS 146661 [Enterobacteria phage RB16] orf232 146665.. 1046 38788 5.3 RnlB RNA ligase 2 gb|ADJ55538.1| RnlB RNA ligase 2 0.0 0 PF09414.4 1.40E-47 [Enterobacteria phage RB16] RNA_ligase 147711 orf233 147701.. 581 21942 6.2 Conserved hypothetical gb|ADJ55539.1| conserved E-123 0 phage protein hypothetical phage protein 148282 [Enterobacteria phage RB16] orf234 148352.. 443 16855 6.4 Conserved hypothetical gb|ADJ55540.1| conserved 8.00E-52 0 phage protein hypothetical phage protein 148795 [Enterobacteria phage RB16]

296

orf235 148883.. 251 9107 4.5 Conserved hypothetical gb|ADJ55541.1| conserved 6.00E-47 0 phage protein hypothetical phage protein 149134 [Enterobacteria phage RB16] orf236 149144.. 464 17623 8.7 Conserved hypothetical gb|ADJ55542.1| conserved 5.00E-57 0 phage protein hypothetical phage protein 149608 [Enterobacteria phage RB16] orf237 149632.. 1433 53387 5.4 Putative nicotinate gb|ADJ55543.1| putative nicotinate 0.0 0 PF04095.10 4.70E-50 phosphoribosyltransferase phosphoribosyltransferase NAPRTase 151065 [Enterobacteria phage RB16] orf238 151046.. 308 11661 8.5 Conserved hypothetical gb|ADJ55544.1| conserved 3.00E-65 0 151354 phage protein hypothetical phage protein [Enterobacteria phage RB16] orf239 151351.. 851 33318 6.3 Dam DNA adenine gb|AAX78779.1| Dam DNA adenine E-178 0 PF02086.9 2.60E-34 methylase methylase [Enterobacteria phage MethyltransfD1 152202 RB43] 2 orf240 152209.. 269 10150 6.3 NrdC thioredoxin gb|ADJ55546.1| NrdC thioredoxin 2.00E-53 0 PF00462.18 4.50E-09 [Enterobacteria phage RB16] Glutaredoxin 152478 orf241 152511.. 482 18662 9.4 gp49 recombination gb|AAX78781.1| gp49 recombination E-111 0 PF02945.9 4.6e-19 endonuclease VII endonuclease VII [Enterobacteria Endonuclease_7 152993 phage RB43] orf242 152990.. 2111 79382 6.7 NrdD anaerobic gb|ADJ55548.1| NrdD anaerobic 0.0 0 PF03477.10 6e-17 ribonucleotide reductase ribonucleotide reductase subunit ATP-cone 155101 subunit [Enterobacteria phage RB16] orf243 155155.. 851 32393 9.2 SegH gb|ABI48949.1| SegH [Enterobacteria 2.00E-74 0 PF07460.5 1.10E-08 phage RB3] NUMOD3 156006 orf244 156056.. 596 21871 6.3 e.6 conserved gb|AAX78788.1| e.6 [Enterobacteria 3.00E-48 0 hypothetical protein phage RB43] 156652

297

orf245 156682.. 947 36586 5.8 Hypothetical protein gb|AAX78790.1| hypothetical protein 7.00E-44 0 RB43ORF268w RB43ORF268w [Enterobacteria 157629 phage RB43] orf246 157623.. 284 10580 5.0 Conserved hypothetical gb|ADJ55551.1| conserved 1.00E-58 0 phage protein hypothetical phage protein 157907 [Enterobacteria phage RB16] orf247 157953.. 194 7573 8.6 Conserved hypothetical gb|ADJ55552.1| conserved 1.00E-41 0 phage protein hypothetical phage protein 158147 [Enterobacteria phage RB16] orf248 158144.. 530 20132 6.1 Hypothetical protein gb|AAX78793.1| hypothetical protein 4.00E-86 0 PF00888.16 9.10E-06 RB43ORF271w RB43ORF271w [Enterobacteria Cullin 158674 phage RB43] orf249 158674.. 491 18843 7.7 NrdG anaerobic gb|ADJ55554.1| NrdG anaerobic 1.00E-117 0 PF04055.15 8.90E-09 nucleotide reductase nucleotide reductase subunit Radical_SAM 159165 subunit [Enterobacteria phage RB16] orf250 159174.. 209 8036 9.4 Hypothetical protein 0

159383 orf151 159388.. 953 34495 9.6 Conserved hypothetical gb|ADJ55555.1| conserved 1.00E-167 0 phage protein hypothetical phage protein 160341 [Enterobacteria phage RB16] orf252 160389.. 734 27670 5.2 Conserved hypothetical gb|ADJ55556.1| conserved E-154 0 phage protein hypothetical phage protein 161123 [Enterobacteria phage RB16] orf253 161083.. 278 10787 8.5 NrdH glutaredoxin gb|ADJ55557.1| NrdH glutaredoxin 2.00E-62 0 PF00462.18 8.30E-07 [Enterobacteria phage RB16] Glutaredoxin 161361 orf254 161362.. 338 13196 9.4 gp55.2 conserved gb|ADJ55558.1| gp55.2 conserved 1.00E-64 0 hypothetical protein hypothetical protein [Enterobacteria 161700 phage RB16]

298

orf255 161697.. 1427 55142 8.6 Conserved hypothetical gb|ADJ55559.1| conserved 0.0 0 phage protein hypothetical phage protein 163124 [Enterobacteria phage RB16] orf256 163181.. 3659 131342 6.1 gp34 long tail fiber gb|ADJ55560.1| gp34 long tail fiber 0.0 0 proximal subunit proximal subunit [Enterobacteria 166840 phage RB16] orf257 166846.. 1592 58426 5.0 gp35 hinge connector of gb|ADJ55561.1| gp35 hinge 0.0 0 long tail fiber proximal connector of long tail fiber proximal 168438 connector connector [Enterobacteria phage RB16] orf258 168490.. 2147 76327 5.7 gp36 small distal tail fiber gb|ADJ55562.1| gp36 small distal tail 0.0 0 PF03903.7 2.30E-22 subunit fiber subunit [Enterobacteria phage Phage_T4_gp36 170637 RB16] orf259 170700.. 2210 78741 6.1 gp37 large distal tail fiber gb|AAX78803.1| gp37 large distal tail 0.0 0 PF03903.7 2.00E-16 subunit fiber subunit [Enterobacteria phage Phage_T4_gp36 172910 RB43] orf260 172937.. 518 18773 9.5 gp38 distal long tail fiber gb|ADJ55564.1| gp38 distal long tail 2E-75 0 PF05268.5 2.30E-18 assembly catalyst fiber assembly catalyst GP38 173455 [Enterobacteria phage RB16] orf261 173509.. 644 24754 7.7 T holin lysis mediator gb|ADJ55565.1| T holin lysis E-145 1 PF11031.2 1.20E-68 mediator [Enterobacteria phage Phage_holin_T 174153 RB16] orf262 174136.. 275 10333 9.5 Conserved hypothetical gb|ADJ55566.1| conserved 5.00E-48 0 phage protein hypothetical phage protein 174411 [Enterobacteria phage RB16] orf263 174408.. 302 11672 4.8 Conserved hypothetical gb|ADJ55567.1| conserved 2.00E-45 0 phage protein hypothetical phage protein 174710 [Enterobacteria phage RB16] orf264 174707.. 440 16570 8.9 Conserved hypothetical gb|ADJ55568.1| conserved 2.00E-58 0 phage protein hypothetical phage protein 175147 [Enterobacteria phage RB16]

299

orf265 175224.. 227 8833 8.8 Hypothetical protein 0

175451 orf266 175448.. 329 12270 4.3 Conserved hypothetical gb|ADJ55569.1| conserved 1.00E-72 0 phage protein hypothetical phage protein 175777 [Enterobacteria phage RB16] orf267 175752.. 137 5438 10.5 Conserved hypothetical gb|ADJ55570.1| conserved 2.00E-22 0 phage protein hypothetical phage protein 175889 [Enterobacteria phage RB16] orf268 175886.. 230 8774 9.3 Conserved hypothetical gb|ADJ55571.1| conserved 1.00E-47 0 phage protein hypothetical phage protein 176116 [Enterobacteria phage RB16] orf269 176161.. 254 9476 6.3 Hypothetical protein gb|ADO51716.1| hypothetical protein 1.00E-46 0 [Klebsiella phage KP15] 176415 orf270 176424.. 380 14615 8.9 Ndd nuclear disruption gb|ADJ55573.1| Ndd nuclear 5.00E-87 0 protein disruption protein [Enterobacteria 176804 phage RB16] orf271 176836.. 413 15392 9.5 Conserved hypothetical gb|ADJ55574.1| conserved 1.00E-57 2 phage protein hypothetical phage protein 177249 [Enterobacteria phage RB16] orf272 177295.. 746 28158 7.1 rIIB protector from gb|ADJ55305.1| rIIB protector from E-159 0 prophage-induced early prophage-induced early lysis 178041 lysis [Enterobacteria phage RB16]

300

Table 4.5. General features of ORFs in the DNA of phage GAP227 and homology to proteins in the databases.

Gene Position Gene Mass pI Function Homology E-value Motifs Pfam Pfam Name Length (TMHMM; Motifs Phobius) E-value

orf01 656..958 302 11494 10.6 Hypothetical 0 protein

orf02 1506..2045 539 19824 9.0 Conserved |YP_001294652.1| hypothetical protein 9.00E-13 0 hypothetical ORF059 [Pseudomonas phage PA11] protein

orf03 2119..2235 116 4420 4.8 Hypothetical 0 protein

orf04 2306..2710 404 14980 4.5 Hypothetical |CBW54764.1| hypothetical protein [Pantoea 5.00E-11 0 protein phage LIMElight]

orf05 2773..3267 494 18135 5.5 Hypothetical |CBW54774.1| hypothetical protein [Pantoea 9.00E-11 0 protein phage LIMElight]

orf06 3260..3502 242 9261 10.2 Hypothetical 0 protein

orf07 3581..3808 227 8882 9.5 Hypothetical 0 protein

orf08 3819..4346 527 20252 7.8 Hypothetical 0 protein

orf09 4339..4722 383 14640 7.1 Hypothetical 0 protein

orf10 4719..5015 296 10602 9.2 Hypothetical 1 protein

orf11 5012..5440 428 15355 5.5 Hypothetical 0 protein

301

orf12 5442..5768 326 11751 4.8 Hypothetical 0 protein orf13 5774..6037 263 9330 6.3 Hypothetical 0 protein orf14 6037..6261 224 8698 10.3 Hypothetical 0 protein orf15 6274..6990 716 27034 9.1 Putative DNA |YP_001522861.1| putative DNA primase 1.00E-03 0 primase [Enterobacteria phage LKA1] orf16 6993..8237 1244 46570 5.9 DNA_B |YP_002727836.1| DNA_B helicase 3.00E-69 0 helicase [Pseudomonas phage phikF77] orf17 8270..8407 137 5282 9.2 Hypothetical 0 protein orf18 8400..8591 191 6872 4.9 Hypothetical 0 protein orf19 8588..9493 905 35019 6.5 ATP- |CBW54778.1| putative ATP-dependent DNA 2.00E-74 0 PF01068.1 1.2E-11 dependent ligase [Pantoea phage LIMElight] DNA_ligase_ DNA ligase A_M orf20 9559..12027 2468 92967 5.5 DNA directed |YP_003345482.1| predicted phage DNA E-149 0 PF00476.14 1.9E-26 DNA Polymerase [Pseudomonas phage phi-2] DNA_pol_A polymerase orf21 12095..12910 815 30406 5.1 Conserved YP_001522872.1| hypothetical protein 3.00E-17 0 hypothetical PPLKA1_gp31 [Enterobacteria phage LKA1] protein orf22 12910..13872 962 36229 8.8 DNA |YP_001522873.1| putative DNA exonuclease 4.00E-33 0 Exonuclease [Enterobacteria phage LKA1] orf23 13877..14290 413 15525 4.6 Hypothetical 0 protein

302

orf24 14283..14747 464 17195 10.1 DNA |ADQ12729.1| putative DNA endonuclease 2.00E-17 0 PF02945.9 1.6E-09 Endonuclease VII [Acinetobacter phage phiAB1] Endonuclease VII _7

orf25 14747..15790 1043 38403 6.3 Conserved |YP_003347674.1| hypothetical protein KP- E-130 0 hypothetical KP34p23 [Klebsiella phage KP34] protein orf25A 15777..16118 341 12487 4.9 Conserved |YP_004327088.1| hypothetical protein 9.00E-08 0 hypothetical ErPhphiEa104_gp113 [Erwinia phage protein phiEa104]

orf26 16015..16344 329 12241 5.3 Conserved |ABF88598.1| hypothetical protein 2.00E-26 0 hypothetical MXAN_1823 [Myxococcus xanthus DK protein 1622]

orf27 16344..16655 311 11269 6.3 Conserved |CBW54793.1| hypothetical protein [Pantoea 8.00E-31 0 hypothetical phage LIMElight] protein

orf28 16639..16758 119 4475 6.0 Hypothetical 0 protein

orf29 16758..19208 2450 92506 6.5 DNA- |ADD21661.1| DNA-dependent RNA E-137 0 PF00940.13R 4.8E-92 dependent polymerase [Caulobacter phage Cd1] NA_pol RNA polymerase

orf30 19343..19522 179 6257 8.1 Hypothetical 0 protein

orf31 19515..19943 428 16676 9.2 Hypothetical 0 protein

orf32 19943..20362 419 14817 9.6 Hypothetical 0 protein

orf33 20378..21898 1520 56889 5.1 Head-to-tail |ADD21666.1| head-to-tail joining protein E-114 0 PF12236.2 3.7E-75 joining [Caulobacter phage Cd1] Head-tail_con protein

303

orf34 21910..22746 836 29124 4.6 Scaffolding |ADD21667.1| scaffold-like protein 7.00E-10 0 protein [Caulobacter phage Cd1]

orf35 22834..23853 1019 36738 5.1 Capsid protein |ADD21668.1| putative major capsid protein 4.00E-61 0 [Caulobacter phage Cd1]

orf36 23928..24533 605 22606 6.1 Tail tuber |YP_002213722.1| tail tuber protein A 6.00E-29 0 protein A [Ralstonia phage RSB1]

orf37 24536..27169 2633 97575 6.6 Tail tubular |ADD21670.1| tail tubular protein B 0.0 0 protein B [Caulobacter phage Cd1]

orf38 27173..27967 794 28383 8.6 Hypothetical 0 protein

orf39 27977..30229 2252 82366 5.4 Conserved |ZP_02468143.1| hypothetical protein 2.00E-14 0 hypothetical Bpse38_32595 [Burkholderia thailandensis] protein

orf40 30232..34029 3797 138309 5.8 Lytic |YP_002213726.1| putative transglycosylase 9.00E-49 0 PF01464.14 9.3E-12 transglycosyla [Ralstonia phage RSB1] SLT se, catalytic

orf41 34101..36677 2576 92087 5.6 Putative tail |YP_002213727.1| putative phage tail fiber 4.00E-22 0 PF03906.8 4.1E-13 fiber protein protein [Ralstonia phage RSB1] Phage_T7_tai l orf41A 36688..36876 188 6822 6.0 Hypothetical 1 PF10746.3 1.6E-05 protein Phage_holin_ 6

orf42 36860..37195 335 12597 4.7 Hypothetical 0 protein

orf43 37195..39120 1925 72278 5.8 DNA |ADD21678.1| DNA maturase B [Caulobacter E-146 0 maturase B phage Cd1]

304

orf44 39164..40075 911 32283 4.8 Wac whisker |YP_239189.1| Wac whiskers [Enterobacteria 3.00E-19 0 PF10746.3 2.6E-06 protein phage RB43] Phage_holin_ 6 orf45 40112..40660 548 20034 9.0 Lysozyme |EGB64298.1| phage lysozyme [Escherichia 8.00E-26 0 PF00959.13 3.6E-14 coli TA007] Phage_lysoz yme orf46 40657..41022 365 13230 6.7 Hypothetical 0 protein orf47 41006..41188 182 6645 5.0 Hypothetical 0 protein

______

305