Download Molecular Biology, Genetics and Applications of Yersiniophages

TURUN YLIOPISTON JULKAISUJA
ANNALES UNIVERSITATIS TURKUENSIS
SARJA – SER. D OSA TOM.
MEDICA – ODONTOLOGICA
Molecular Biology, Genetics and Applications of
Yersiniophages
by
Saija Kiljunen
TURUN YLIOPISTO
Turku 2006
From the Department of Medical Biochemistry and Molecular Biology, Institute of
Biomedicine, University of Turku, Turku, Finland and the Department of
Bacteriology and Immunology, Haartman Institute, University of Helsinki
Supervised by
Professor Mikael Skurnik
Department of Bacteriology and Immunology
Haartman Institute
University of Helsinki
Helsinki, Finland
Reviewed by
Saija Kiljunen. Molecular biology, genetics and applications of yersiniophages.
Department of Medical Biochemistry and Molecular Biology, Institute of
Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.
Annales Universitatis Turkuensis, Medica-Odontologica Series D, Turku, Finland,
2006.
ABSTRACT
The genus Yersinia in the family Enterobacteriaceae consists of 12 species, three of
which are human pathogens. Yersinia enterocolitica and Yersinia pseudotuberculosis
cause primarily gastrointestinal infections, whereas Yersinia pestis is the causative
agent of plague.
Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant
and versatile group of organisms on Earth and have a significant impact on microbial
ecosystems. Several phages infecting Yersinia are known, but only a few have been
characterized in detail. The aim of this thesis was to obtain more information about
yersiniophages, with a special interest in understanding factors that determine their
host specificity.
The phages studied in this thesis were φYeO3-12 and φR1-37, infecting Y.
enterocolitica serotype O:3 (YeO3), and Y. pestis phage φA1122. φYeO3-12 and
φA1122 are T7 – related members of Podoviridae, whereas φR1-37 belongs to the
viral family Myoviridae.
These phages utilize different parts of lipopolysaccharide as their receptor.
For φYeO3-12 the receptor is the YeO3 O-antigen. In this work, the YeO3 outer core
was identified as the receptor for φR1-37 and the core of Y. pestis and Y.
pseudotuberculosis as the receptor for φA1122.
The basic biological and genetic features of phages φYeO3-12 and φR1-37 were
elucidated in this work. φR1-37 was found to be exceptional in having its genome
composed of DNA where thymidine is replaced by deoxyuridine. According to the Nterminal amino acid sequences of structural proteins and the partial genomic
sequence, no close relatives of φR1-37 have been described. For φYeO3-12, the nonessential regions in the genome were identified and the genes coding for DNA ligase
and lysozyme were shown to be evolutionary factors important in adaptation of
φYeO3-12 to grow on Yersinia.
KEYWORDS: Yersinia, bacteriophage, receptor, lipopolysaccharide, evolution
Saija Kiljunen. Yersiniofagien molekyylibiologia, genetiikka ja sovellutukset.
Lääketieteellinen Biokemia ja Molekyylibiologia, Biolääketieteen laitos, Turun
yliopisto, Kiinamyllynkatu 10, 20520 Turku.
Annales Universitatis Turkuensis, Medica-Odontologica Series D, Turku, 2006.
TIIVISTELMÄ
Enterobacteriaceae – heimoon kuuluva Yersinia – suku sisältää 12 lajia, joista kolme
on ihmiselle patogeenisiä. Yersinia enterocolitica ja Yersinia pseudotuberculosis
aiheuttavat suolistoalueen infektioita, kun taas Yersinia pestis on pelätty ruttobakteeri.
Bakteriofagit (faagit) ovat bakteereja infektoivia viruksia. Ne ovat maapallon
runsaslukuisin ja monimuotoisin eliöryhmä ja ne vaikuttavat huomattavasti
mikrobiologisiin ekosysteemeihin. Kirjallisuudessa on kuvattu useita Yersinia –
suvun bakteereja infektoivia faageja, mutta näistä vain muutama tunnetaan tarkasti.
Tämän väitöskirjatyön tavoitteena oli saada lisää tietoa yersinioiden bakteriofageista,
erityisesti tekijöistä jotka vaikuttavat niiden isäntäspesifisyyteen.
Tässä työssä tutkitut bakteriofagit olivat Y. enterocolitica serotyyppi O:3:a (YeO3)
infektoivat φYeO3-12 ja φR1-37 sekä Y. pestis -spesifinen φA1122. Näistä φYeO3-12
ja φA1122 ovat T7 -sukuisia Podoviridae –heimon jäseniä, kun taas φR1-37 kuuluu
virusten heimoon Myoviridae.
Nämä faagit käyttävät reseptoreinaan eri kohtia bakteerin lipopolysakkaridista.
φYeO3-12:n reseptori on YeO3:n O-antigeeni. Tässä työssä osoitettiin, että φR1-37
käyttää reseptorinaan YeO3:n ulompaa ydinsokeria ja φA1122:n reseptori on Y.
pestiksen ja Y. pseurotuberculosiksen ydinsokeri.
Väitöskirjatyössä karakterisoitiin faagien φYeO3-12 ja φR1-37 biologisia ja
geneettisiä ominaisuuksia. Työssä osoitettiin että φR1-37 on poikkeuksellinen
bakteriofagi, jonka genomisessa DNA:ssa tymidiini on korvautunut deoksiuridiinilla.
Faagin rakenneproteiinien aminoterminaalisten aminohapposekvenssien ja osittaisen
genomisen DNA-sekvenssin perusteella sille ei ole kuvattu läheisiä sukulaisia.
Lisäksi työssä identifioitiin φYeO3-12:n genomin alueita jotka eivät ole
välttämättömiä faagin elinkyvylle ja osoitettiin, että faagin DNA ligaasia ja
lysotsyymiä koodaavat geenit ovat faagin isäntäspesifisyyteen vaikuttavia
evoluutiotekijöitä.
AVAINSANAT: Yersinia, bakteriofagi, reseptori, lipopolysakkaridi, evoluutio
CONTENTS
ABBREVIATIONS
LIST OF ORIGINAL PUBLICATIONS
1. INTRODUCTION
2. REVIEW OF LITERATURE
2.1. The genus Yersinia
2.1.1. Yersinia enterocolitica
2.1.2. Yersinia pseudotuberculosis
2.1.3. Yersinia pestis
2.1.4. Lipopolysaccharides of Yersinia
2.2. Bacteriophages
2.2.1. Phage taxonomy
2.2.2. Phage genomes
2.2.3. Phage evolution
2.2.4. The impact of phages on bacterial evolution
2.2.5. Phage receptors
2.3. Applications of phages
2.3.1. Bacteriophage therapy
2.3.2. Phages in bacterial diagnostics
2.3.3. Other applications
2.4. Bacteriophages of Yersinia
2.4.1. φYeO3-12
2.4.2. φA1122
2.4.3. PY54
2.4.4. Applications of yersiniophages
3. AIMS OF THE PRESENT STUDY
4. MATERIALS AND METHODS
4.1. Bacterial strains, phages and plasmids
4.2. Biological characterization of bacteriophages
4.2.1. Host specificity, efficiency of plating, growth curve and fitnessanalysis
4.2.2. Antisera (I)
4.2.3. Receptor characterization and adsorption analysis
4.2.4. LPS isolation and analysis
4.3. Structural characterization of bacteriophages
4.3.1. Electron microscopy
4.3.2. SDS-PAGE, Western-analysis and N-terminal sequencing of
structural proteins
4.4. Molecular biology techniques
4.4.1. General DNA techniques
4.4.2. In vitro transposon mutagenesis
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24
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30
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37
37
37
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4.4.3. mRNA isolation and analysis
4.4.4. Analysis of modified nucleotides
4.5. Luminescence measurements and the construction of the
reporter phage
4.5.1. Luminescence measurements
4.5.2. Construction of the reporter phage
5. RESULTS AND DISCUSSION
5.1. Characterization of φYeO3-12
5.1.1. φYeO3-12 is related to T3 and T7
5.1.2. Transposon insertions in the early genomic region of φYeO3-12
cause growth defects on Y. enterocolitica
5.1.3. φYeO3-12 DNA ligase and lysozyme are needed for growth on
Y. enterocolitica
5.2. Characterization of φR1-37
5.2.1. Biological and structural features of φR1-37
5.2.2. The φR1-37 receptor
5.2.3. The size and composition of the of φR1-37 genome
5.2.4. Sequencing of the φR1-37 DNA
5.3. Identification of the φA1122 receptor
5.3.1. The φA1122 receptor is a LPS core structure present in Y. pestis
and Y. pseudotuberculosis but not in Y. enterocolitica
5.3.2. The φA1122 receptor is blocked by the heterologous expression
of Y. enterocolitica O:3 outer core
5. 4. Reporter phages
5.4.1. Construction of the reporter phage by homologous recombination
5.4.2. Phages φ::lucFF1.2 and φ::lucFF5.3
5.4.3. Phage φ::lucFF1
6. SUMMARY
ACKNOWLEDGEMENTS
REFERENCES
ORIGINAL PUBLICATIONS
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37
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38
38
39
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42
42
43
43
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ABBREVIATIONS
aa
bp
BER
CDC
CFU
dA
dC
dG
DIG
DNA
DOC
ds
dU
dUTP
EM
EMBOSS
EOP
GFP
ICTV
IM
kb
kDa
Kdo
LC-MS/MS
LPS
mRNA
NCBI
OM
OMP
PAGE
PCR
PFGE
PFU
RLU
RNA
RNAP
RT
RT-PCR
SDS
ss
T
YeO3
YeO8
amino acid(s)
base pair(s)
base-excision repair pathway
Centers for Disease Control and Prevention
colony forming unit(s)
deoxyadenosine
deoxycytidine
deoxyguanosine
digoxigenin
deoxyribonucleic acid
deoxycholate
double stranded
deoxyuridine
deoxyuridine triphosphate
electron microscopy
The European Molecular Biology Open Software Suit
efficiency of plating
green fluorescent protein
The International Committee for Taxonomy of Viruses
inner membrane
kilo base pair(s)
kilodalton(s)
3-deoxy-D-manno-octulosonic acid
Liquid Chromatography Mass Spectrometry/Mass Spectrometry
lipopolysaccharide
messenger RNA
The National Center for Biotechnology Information
outer membrane
outer membrane protein
polyacrylamide gel electrophoresis
polymerase chain reaction
pulse-field gel electrophoresis
plaque forming unit(s)
relative light unit(s)
ribonucleic acid
RNA polymerase
room temperature (20oC to 25oC)
reverse transcriptase-PCR
sodium dodecyl sulphate
single stranded
thymidine
Yersinia enterocolitica serotype O:3
Yersinia enterocolitica serotype O:8
8
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications and some unpublished
results. The publications reproduced with the kind permission of the copyright
holders will be referred to in the text by the Roman numerals (I-IV).
I.
Pajunen, M., Kiljunen, S., and Skurnik, M. (2000) Bacteriophage φYeO312 specific for Yersinia enterocolitica serotype O:3 is related to
coliphages T3 and T7. J. Bacteriol 182: 5114-5120.
II.
Kiljunen, S., Vilen, H., Savilahti, H., and Skurnik, M. (2005) Nonessential genes of the phage φYeO3-12 include genes involved in
adaptation to growth on Yersinia enterocolitica serotype O:3. J. Bacteriol
187: 1405-1414.
III. Kiljunen, S., Hakala, K., Pinta, E., Huttunen, S., Pluta, P:, Gador, A.,
Lönnberg, H., and Skurnik, M. (2005) Yersiniophage φR1-37 is a tailed
bacteriophage having a 270 kb DNA genome with thymidine replaced by
deoxyuridine. Microbiology 151: 4093-4102.
IV.
Kiljunen, S., Bengoechea, J. A., Holst, O. and Skurnik, M. Identification
of LPS core of Yersinia pestis and Yersinia pseudotuberculosis as the
receptor for bacteriophage φA1122. Manuscript.
9
1. INTRODUCTION
The bacterial genus Yersinia belongs to the family Enterobacteriaceae. Yersiniae
are Gram-negative, rod-shaped bacteria that inhabit a wide variety of ecological
environments. The genus includes 12 species, of which Yersinia enterocolitica,
Yersinia pseudotuberculosis and Yersinia pestis are pathogenic to humans. The
others are considered nonpathogenic or environmental (Bottone, 1997, Carniel et
al., 2005, Sprague & Neubauer, 2005, Wauters et al., 1991, Wren, 2003).
Bacteriophages or phages are viruses that infect bacteria. They were independently
discovered by Frederick Twort in 1915 and Felix d’Herelle in 1917, even though at
the time of their discovery, their biological nature was not quite understood (Stone,
2002, Summers, 1999, Summers, 2001). Phages inhabit every possible
environment and generally outnumber their bacterial hosts by an order of
magnitude. The phage abundance in sea water may be as high as 107 PFU/ml and
the global phage population has been estimated to approach 1031, which makes
phages the most numerous organisms on Earth (Breitbart & Rohwer, 2005, Pedulla
et al., 2003, Weinbauer, 2004).
The history of yersiniophages roots back to d’Herelle, who isolated “an antiplague
phage” and used it to cure plague patients in 1925 (Stone, 2002, Summers, 1999,
Summers, 2001). After that, several Yersinia –specific phages have been isolated,
mainly to develop phage typing schemes for Yersinia diagnostics (Baker & Farmer
III, 1982, Calvo et al., 1981, Garcia et al., 2003, Kawaoka et al., 1987, Nilehn,
1969, Nunes & Suassuna, 1978). However, not many of these phages have been
characterized in detail.
In the Skurnik research group, bacteriophages specific for Yersinia lipopolysaccharide (LPS) have been isolated and used to study the LPS structure and biosynthesis
(Al-Hendy et al., 1991, Skurnik et al., 1995, Skurnik & Zhang, 1996, Zhang &
Skurnik, 1994). The present PhD thesis work focused on the characterization of
two of these phages, φYeO3-12 and φR1-37, which both infect Y. enterocolitica
serotype O:3. This characterization included the study of the basic biological and
genetic properties of these phages. For φYeO3-12, a detailed analysis of the
function of the phage genome was carried out and the possibility to utilize the
phage in bacterial diagnostics was evaluated. In addition, the cell surface receptor
for the Y. pestis –specific phage φA1122 was identified.
10
2. REVIEW OF LITERATURE
2.1. The genus Yersinia
The yersiniae in the family Enterobacteriaceae are Gram-negative coccobacilli.
The genus consists of 12 bacterial species: Yersinia aldovae, Yersinia aleksiciae
(introduced in 2005), Yersinia bercovieri, Yersinia enterocolitica, Yersinia
frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii,
Yersinia pestis, Yersinia pseudotuberculosis, Yersinia rohdei and Yersinia rückeri.
Out of these, Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis are pathogenic
to humans. Y. rückeri is a fish pathogen and the others are considered nonpathogenic or environmental. For Y. aleksiciae, though, the apathogenicity still
needs to be confirmed. (Bottone, 1997, Carniel et al., 2005, Sprague & Neubauer,
2005, Wauters et al., 1991, Wren, 2003).
2.1.1. Yersinia enterocolitica
Y. enterocolitica causes primarily gastrointestinal infections, which normally ensue
after the ingestion of contaminated food or water (Bottone, 1997). In human
yersiniosis, the most frequent clinical manifestations are diarrhea, gastro-enteritis,
and mesenteric lymphadenitis. These may be followed by sequelae such as reactive
arthritis, erythema nodosum, and septicemia (Bottone, 1997, Saken et al., 1994). In
Finland there are approx. 500 – 600 laboratory-confirmed Y. enterocolitica cases a
year, which makes it the third most common enterobacterial pathogen (Holmström
et al., 2004). Y. enterocolitica is also a well recognized cause of transfusionassociated bacteremia. Even though a rare event, the posttransfusional sepsis
caused by Y. enterocolitica may have mortality rate as high as 64 % (Bottone,
1997, Leclercq et al., 2005).
Y. enterocolitica is a heterogenous species and is separated into six biotypes (1A,
1B, 2, 3, 4, and 5) based on biochemical behavior (Bottone, 1997, Bottone, 1999).
Out of these, biotype 1A lacks the Yersinia virulence plasmid pYV (Cornelis et al.,
1998, Skurnik, 1985), and has often been considered nonpathogenic. Some biotype
1A strains have, however, been isolated from clinical samples (Tennant et al.,
2003), and some of them were recently found to have virulence-associated genes
that are related to the insecticidal toxin complex genes of other bacterial species
(Tennant et al., 2005). Pathogenic biotypes 1B to 5 rely on pYV and several
chromosomally encoded virulence factors for their virulence (Revell & Miller,
2001). Biotype 1B is considered as a high pathogenicity type, since strains
belonging to it are lethal in a mouse model and can cause systemic infection in
humans. Biotypes 2 to 5 are regarded as low pathogenicity types and can kill mice
or cause systemic infection in humans only on condition of iron overload (Bottone,
1997, Bottone, 1999, Wren, 2003).
Y. enterocolitica is divided into over 30 serotypes based on differences in Oantigen structures (Wauters et al., 1991). The distribution of Y. enterocolitica
11
biotypes and serotypes is shown in Table 1. Clinically, the most significant Y.
enterocolitica serotypes are O:3, O:5,27, O:8 and O:9 (Bottone, 1997, FredrikssonAhomaa & Korkeala, 2003).
Table 1. The distribution of the most commonly encountered Y. enterocolitica
biotypes and serotypes.
Biotype
1A
Serotype(s)
O:5, O:6,30, O:6,31, O:7,8, O:7,13, O:10, O:14, O:16, O:19,8, O:22, O:25, O:37,
O:41,42, O:41,43, O:46, O:47, O:57, nontypeable
1B
O:4, O:4,32, O:8, O:13a, O:13b, O:13,7, O:13,8 O:18, O:20, O:21, O:25, O:41,42
2
O:5,27, O:9, O:27
3
O:1,2,3, O:5,27
4
O:3
5
O:2,3
Modified from (Bottone, 1997, Bottone, 1999, Tennant et al., 2003, Wauters et al., 1991).
2.1.2. Yersinia pseudotuberculosis
Y. pseudotuberculosis, too, causes gastrointestinal infections, but the disease is
slightly different to Y. enterocolitica (Wren, 2003). A typical manifestation is
mesenteric lymphadenitis, followed by fever and abdominal pain. Septicemia is a
rare outcome of Y. pseudotuberculosis infection, and usually requires an underlying
disorder like diabetes, liver disease, or iron overload (Carniel et al., 2005,
Ljungberg et al., 1995). Y. pseudotuberculosis infections occur mostly in the
northern hemisphere, with about 50 – 200 cases a year in Finland (Holmström et
al., 2004, Jalava et al., 2004).
Y. pseudotuberculosis is separated into 21 serotypes: O:1 to O:15, of which O:1
and O:2 are divided into subtypes a, b, and c, and O:4 and O:5 into a and b
(Bogdanovich et al., 2003, Carniel et al., 2005, Skurnik, 2004). Of these, serotypes
O:1a and O:1b are predominantly isolated in Western countries, whereas serotypes
O:4b and O:5 are more common in the Far East (Carniel et al., 2005).
2.1.3. Yersinia pestis
Y. pestis is the causative agent of bubonic, septicemic, and pneumonic plague
(Gage & Kosoy, 2005, Perry & Fetherston, 1997, Titball et al., 2003). The
bacterium has a complex zoonotic life cycle, where various mammalian species
(primarily rodents) may function as reservoir, and fleas serve as vectors (Perry &
Fetherston, 1997, Wren, 2003). Humans usually encounter the disease by being
bitten by an infected flea (Jarrett et al., 2004). From the flea bite, the infection
rapidly spreads in regional lymph nodes, which enlarge to form a “bubo”.
Septicemia usually follows after 2 – 6 days. Primary septicemic plague may occur
if the infection spreads in the bloodstream without the formation of the bubo.
Occasionally, the infection spreads in lungs to produce a secondary pneumonic
plague. This form of plague is extremely contagious and may spread from human
12
to human by the airborne route to cause primary pneumonic plague (Perry &
Fetherston, 1997, Sebbane et al., 2005, Titball et al., 2003, Zhou et al., 2005).
For untreated bubonic plague, the mortality is ca. 60 %, and for untreated
septicemic and pneumonic forms it is close to 100 %. Effective antibiotic therapy
decreases the mortality to 5 % and 33 % for bubonic and septicemic forms,
respectively. For primary pneumonic plague the treatment may decrease the
mortality to 10 %, but the medication needs to be started within 24 hours from
infection to be efficient (Zhou et al., 2005). According to the World Health
Organization, there are annually ca. two thousand plague cases in the world. This
number may, however, be largely underestimated due to inadequate diagnostics and
reporting in some endemic countries (World Health Organization, 2004).
Y. pestis is evolutionary a very new organism and it is believed to have evolved
from Y. pseudotuberculosis only 1500 – 20000 years ago (Achtman et al., 1999).
The species is classically divided into three biovars, Antiqua, Mediaevalis and
Orientalis, according to the ability to ferment glycerol and reduce nitrate (Achtman
et al., 2004, Wren, 2003, Zhou et al., 2005). The biovar assignment does not seem
to correlate with the virulence. A fourth biovar, Microtus, was recently proposed
(Zhou et al., 2004). Strains belonging to this biovar are virulent to small rodents
like mice, but avirulent to larger mammals like guinea pigs, rabbits and humans. A
more detailed classification of Y. pestis into subspecies is being used in the
countries of the Former Soviet Union and Mongolia (Anisimov et al., 2004).
2.1.4. Lipopolysaccharides of Yersinia
The outer surface of Gram-negative bacteria is composed of an inner membrane
(IM), a periplasmic space and an outer membrane (OM). IM is a bilayer composed
of phospholipids, integral proteins and lipoproteins. The periplasm constitutes ca.
10% of the cell volume and contains a peptidoglycan layer and soluble proteins, eg.
protein folding and trafficking factors. OM is an asymmetric structure, having
phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet.
In the OM there are integral OM proteins and lipoproteins that participate in solute
and protein translocation, pathogenesis and signal transduction (Bond & Sansom,
2004, Bos & Tommassen, 2004, Koebnik et al., 2000, Ruiz et al., 2006).
LPS is an amphipathic molecule that is attached to OM by the hydrophobic anchor
lipid A (Raetz & Whitfield, 2002, Skurnik & Zhang, 1996). Lipid A is fairly
conserved among Gram-negative bacteria, the basic molecule being a disaccharide
of two phosphorylated glucosamine residues acylated with five to seven fatty acid
chains (Holst, 2003, Raetz & Whitfield, 2002, Skurnik & Toivanen, 1993, Skurnik
& Zhang, 1996). The schematic representation of lipid A is shown in Figure 1. The
structures of Y. enterocolitica and Y. pestis lipid A have been determined, and
shown to vary according to the growth temperature of the bacteria (Holst, 2003,
Knirel et al., 2005a, Knirel et al., 2005b). Lipid A, also called endotoxin, is
recognized by the innate immune system and is responsible for the endotoxic shock
during Gram-negative sepsis (Raetz & Whitfield, 2002).
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The core oligosaccharide is attached to the lipid A via 3-deoxy-D-mannooctulosonic acid (Kdo) (Holst, 2003, Skurnik & Toivanen, 1993). The LPS core
structures of Gram-negative bacteria generally contain heptoses, glucose and
galactose, and only limited diversity is seen between different species (Bruneteau
& Minka, 2003, Raetz & Whitfield, 2002). This illustrates the importance of core
oligosaccharide to outer membrane stability and function (Raetz & Whitfield, 2002,
Skurnik et al., 1999). The core structures of Y. enterocolitica serotypes O:3, O:8,
and O:9 have been determined (Muller-Loennies et al., 1999, Oertelt et al., 2001,
Radziejewska-Lebrecht et al., 1994), as well as that of Y. pestis (Vinogradov et al.,
2002). For Y. pseudotuberculosis only a partial structure is known (Bruneteau &
Minka, 2003). The structure of Y. enterocolitica O:3 LPS core differs from other
Yersinia in having inner and outer cores (Figure 1).
The outermost structure of smooth LPS is O-polysaccharide or O-antigen. Oantigen structures are hypervariable and they determine the serological specificity
within a species (Bruneteau & Minka, 2003, Raetz & Whitfield, 2002). Y. pestis
LPS differs from other Yersinia in being rough, i.e. having no O-antigen. This is
due to multiple mutations in genes coding for enzymes in O-antigen synthetic
pathway (Skurnik et al., 2000). For Y. enterocolitica and Y. pseudotuberculosis Oantigen expression is temperature-regulated, being expressed only at temperatures
below 30ºC. Other signals, like bacterial growth phase, iron concentration, pH, and
ionic strength, may, however, also affect this regulation. Thus, the O-antigen may
be expressed at some stages of mammalian infection (Skurnik & Bengoechea,
2003). Structurally O-antigens may be either hetero- or homopolysaccharides.
Heteropolysaccharides are composed of repeating units made of different sugars.
Such O-antigens are more common, and all studied Y. pseudotuberculosis and most
Y. enterocolitica serotypes fall into this category. Y. enterocolitica serotypes O:3
and O:9 have homopolymeric O-antigens, composed of various number of 6deoxy-L-altrose and 4-deoxy-4-formamido-D-rhamnose residues, respectively
(Figure 1) (Bruneteau & Minka, 2003, Holst, 2003, Raetz & Whitfield, 2002).
The outer membrane and LPS as its major constituent has a critical role in the
interaction of a Gram-negative bacterium with its environment. The lipid A – core
region is important for the membrane integrity. Bacteria with defective core are
hypersensitive to compounds such as antimicrobial peptides, detergents, and
hydrophobic antibiotics. Such cells are also leaky, and release periplasmic enzymes
into the growth medium (Raetz & Whitfield, 2002, Skurnik et al., 1999). The Oantigen part is often involved in bacterial virulence, which is shown e.g. for Y.
enterocolitica (Skurnik & Bengoechea, 2003) and Y. pseudotuberculosis (Mecsas
et al., 2001). LPS containing at least lipid A and Kdo is essential for Escherichia
coli and Salmonella enterica serovar Typhimurium (Galloway & Raetz, 1990, Rick
& Osborn, 1977), whereas Y. pestis was recently shown to be viable with LPS
containing only lipid A (Tan & Darby, 2005). The only known Gram-negative
bacterium that can survive without lipid A is Neisseria meningitidis (Steeghs et al.,
1998).
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A.
B.
Glc
Gal
Nac
Outer
Core
Gal
n
O-Antigen
Gal
Nac
6dAlt
Fuc
Nac
Glc
Hep
Hep
Glc
Gal
Nac
Hep
Hep
Glc
Kdo
P
GlcN
GlcN
Hep
Hep
Hep
Glc
Kdo
Sug1
GlcN
GlcN
Sug2
Core
Inner
Core
P
AraN
P
P
AraN
Lipid A
Lipid A
Figure 1. Model of Y. enterocolitica serotype O:3 (A) and Y. pestis (B) LPS. The attachment site of
Y. enterocolitica O:3 O-antigen has not been unequivocally determined. GlcN; glucosamine, Kdo;
3-deoxy-D-manno-octulosonic acid, Hep; heptose, Glc; glucose, FucNac; 2-acetamido-2,6dideoxygalactose, GalNac; 2-acetamido-2-deoxygalactose, Gal; galactose, AraN; 4-amino-4deoxyarabinose, Sug1; Kdo or D-glycero-D-talo-oct-2-ulosonic acid, Sug2 galactose or D-glyceroD-manno-heptose. Modified from (Frirdich & Whitfield, 2005, Holst, 2003, Knirel et al., 2005b,
Skurnik, 2004, Skurnik & Bengoechea, 2003, Skurnik et al., 1995, Skurnik & Zhang, 1996,
Vinogradov et al., 2002).
2.2. Bacteriophages
Bacteriophages, i.e. the viruses of bacteria, are the most abundant and the most
versatile group of organisms on Earth. In environmental samples phages usually
outnumber their bacterial hosts three to ten times, and the global phage population
has been estimated to approach 1031 (Breitbart & Rohwer, 2005, Hendrix, 2002,
Pedulla et al., 2003, Wommack & Colwell, 2000). Phages are active components of
their ecosystems and have important roles in food web processes, biogeochemical
cycles, gene transfer and prokaryotic diversity (Weinbauer, 2004, Weinbauer &
Rassoulzadegan, 2004, Wommack & Colwell, 2000). As an example, phages were
recently found to significantly influence the seasonal occurrence of Vibrio cholerae
and cholera epidemics in Bangladesh and India (Faruque et al., 2005b, Faruque et
al., 2005c).
Phages can be divided into lytic and temperate based on their life cycles. The
infection by a lytic, or virulent, phage results in the lysis of the bacterial cell and
release of a new phage progeny (Ackermann & DuBow, 1987a). The first step of a
15
lytic cycle is the adsorption of the phage on the bacterial surface (see Chapter
2.2.5.). The infection then proceeds by the entry of the phage nucleic acid into the
bacterial cytoplasm and consequent transcription and translation of phage proteins
as well as synthesis of multiple copies of the phage genome takes place. New
phage particles are assembled and finally released upon the lysis of the host
bacterium. The time until the new phage progeny is beginning to be assembled is
called an eclipse period, and the time until the host cell lysis is a latent period. The
number of new phage particles released from an infected cell is called a burst size
(Ackermann & DuBow, 1987a). The most studied examples of lytic phages are T4
and T7, both infecting E. coli (Mathews et al., 1983, Molineux, 1999).
Temperate phages can choose between lytic and lysogenic life cycles (Ackermann
& DuBow, 1987a, Little, 2005). In lysogenic, or prophage, state, the phage genome
is most often integrated into the bacterial chromosome and replicated as part of it.
The expression of lytic genes is repressed, but other genes in the phage genome
may be expressed in a state called lysogenic conversion. The expression of these
extra genes may benefit the prophage by enhancing the fitness of the lysogenic
bacterium, thus indirectly improving the replication of the phage genome (
Brüssow et al., 2004). Examples of lysogenic conversion are toxin genes carried by
Pseudomonosa aeruginosa phage φCTX (Nakayama et al., 1999), E. coli O:157:H7
phage 933W (Plunkett et al., 1999), Clostridium botulinum phage c-st (Sakaguchi
et al., 2005) and V. cholerae phage CTXφ (Miller, 2003, Waldor & Mekalanos,
1996). Lysogenic conversion and its effects on phage and bacterial evolution will
be discussed in more detail in Chapter 2.2.4.
Even though most temperate phages acquire the prophage state by integrating their
genome into the bacterial chromosome, also other strategies exist. E. coli phage
N15 and Y. enterocolitica phage PY54 establish the lysogenic state by residing as a
linear plasmid (see Chapter 2.4.3) (Hertwig et al., 2003a, Ravin et al., 2000), and E.
coli phage P1 and C. botulinum phage c-st lysogenize their hosts as circular
plasmids (Lobocka et al., 2004, Sakaguchi et al., 2005). The choice between lytic
and lysogenic cycles, so called lysis-lysogeny decision, is a complex process and
understood well for few phages only (Little, 2005), the best-characterized example
being E. coli phage λ (Hendrix et al., 1983, Little, 2005 #1129).
2.2.1. Phage taxonomy
The International Committee for Taxonomy of Viruses (ICTV) describes viruses as
“Elementary biosystems that posses some of the properties of living systems such
as having a genome and being able to adapt to a changing environment” (van
Regenmortel & Mahy, 2004). Viruses are classified into taxonomic categories
based on characteristics like particle morphology, genome type and organization
and the strategy of replication. The hierarchy ranks are species, subfamily, family,
genus and order. A virus species is defined as “a polyethic class of viruses that
constitute a replicating lineage and occupy a particular ecological niche” (BüchenOsmond, 2003, van Regenmortel & Mahy, 2004). Members of a particular species
16
share a number of properties but do not necessary have any single property in
common. A viral family, instead, is “a universal class sharing a number of
properties that are both necessary and sufficient for class membership” (van
Regenmortel & Mahy, 2004).
Bacteriophages are divided into one order and thirteen families (Table 2)
(Ackermann, 2003, Ackermann & DuBow, 1987a, Ackermann & DuBow, 1987b).
The order Caudovirales (tailed phages) consists of three families, Myoviridae,
Siphoviridae, and Podoviridae, and comprises ca. 96 % of known phages. There
are six genera in the family Myoviridae, six in the Siphoviridae and three in the
Podoviridae (Ackermann, 2003, Ackermann, 2001, Maniloff & Ackermann, 1998).
Polyhedral, filamentous and pleomorphic phages are classified into ten families
altogether (Table 2), each having one to four genera.
The present, morphology-based, taxonomy has faced a major criticism during the
past few years (Lawrence et al., 2002, Nelson, 2004). The ICTV taxonomy is
dependent on electron microscopic (EM) images and does not take into account
the rapidly increasing genomic and proteomic data. However, there are several
phages for which there is no EM image available, but whose genomes have been
completely sequenced. This specially concerns lysogenic prophages and phages of
nonculturable bacteria. It has been estimated that at the moment, half of the phages
whose genome has been deposited in databanks are not classified properly by the
current taxonomic system (Nelson, 2004). Also, looking only at the phage
morphology largely underestimates the diversity of phage genomes and may result
in mistakes in classification. A clear example of such a case is Salmonella phage
P22, which has been classified as a member of Podoviridae based on its short tail,
even though it biologically resembles lambdoid phages in the family Siphoviridae
(Vander Byl & Kropinski, 2000). Phage genomes are highly mosaic (Campbell,
2003, Pedulla et al., 2003), and it is now becoming more and more clear that a
strictly hierarchical taxonomy can not represent the complex relationships between
viral species. There is thus an increasing consensus that in the future, phage
classification should be based on genomic data (Lawrence et al., 2002, Nelson,
2004, Proux et al., 2002).
New systems for phage taxonomy have been proposed by a number of authors:
Rohwer and Edwards (2002) presented a “phage proteome tree”. The authors stated
that no single gene, or even a DNA sequence motif, is conserved enough to be used
as a base for a taxonomical system. Instead, they used the overall similarity of
phage genomes (Rohwer & Edwards, 2002). Lawrence et al. (2002) proposed a
system where the highest taxonomic levels, “domains” and “divisions”, would be
founded on the nature of genetic material and the possibility for genetic exchange,
respectively. The lower levels, “modi”, would be based on phenotypic characters
(i.e. common genetic modules) in such a way that most phages would belong to
more than one group (Lawrence et al., 2002). Proux et al. (2002) and ChibaniChennoufi et al. (2004) suggested a classification scheme based on the comparative
genomics of structural genes (Chibani-Chennoufi et al., 2004, Proux et al., 2002).
17
A recent proposal came from Chen and Schneider (2005), who used the sequence
information of phage promoters and RNA polymerases to build a phylogeny model
for T7-like phages (Chen & Schneider, 2005). Most these sequence-based models
result in a classification scheme that is fairly well compatible with the current
ICTV system. A somewhat different approach is presented by Bamford et al.
(2002) who introduced a term “viral self” to describe the fundamental structural
principles of the virion, like particle assembly and genome packaging. The authors
propose that these conserved structural features could be used to develop a viral
phylogeny, which would be based on the ancient viral lineages (Bamford, 2003,
Bamford et al., 2002, Benson et al., 1999, Benson et al., 2004, Saren et al., 2005).
However, more molecular data is needed before a universal system that would be
informative and generally accepted by virologists can be created (ChibaniChennoufi et al., 2004, Nelson, 2004).
2.2.2. Phage genomes
Bacteriophage genomes may be composed of DNA or RNA, which both can be
either double-stranded (ds) or single-stranded (ss) (Table 2). Tailed phages, which
constitute ca. 96% of known phages, all have dsDNA genomes (Ackermann, 2003,
Ackermann & DuBow, 1987a). The sizes of phage dsDNA genomes in databanks
range from 2,4 kb of Oenococcus oeni (formerly Leuconostoc oenos) phage L5 that
only contains eight genes (accession no. NC003695) to 280 kb of P. aeruginosa
phage φKZ (Mesyanzhinov et al., 2002).
A number of phages have dsDNA genomes where one of the normal nucleotides is
completely substituted by a modified base (Gommers-Ampt & Borst, 1995,
Warren, 1980). The most thoroughly studied example is T4 DNA, where cytosine
is replaced by 5-hydroxymethylcytosine (Wyatt & Cohen, 1953), which is further
glucosylated by α- (70%) or β- (30%) bonds (Lehman & Pratt, 1960). Other
examples are Bacillus subtilis phages φe, H1, SPO1, SP8, SP82G, 2C, and φ25,
where thymine is substituted by 5-hydroxymethyluracil (Hemphill & Whiteley,
1975) and PBS1 and PBS2, where thymine is replaced by deoxyuridine (Takahashi
& Marmur, 1963). In some phages, only a portion of certain base residues is
chemically modified (Gommers-Ampt & Borst, 1995, Warren, 1980). These
include E. coli phage Mu, where ca. 15% of adenine residues are substituted by N6carboxymethyladenines (Swinton et al., 1983) and B. subtilis phage SP15, in which
62% of thymine residues are modified to phosphoglucuronated 5dihydroxypentyluracil (Ehrlich & Ehrlich, 1981). The role of modified nucleotides
in phage DNA is in many cases to confer resistance to host or phage encoded
nucleases (Gommers-Ampt & Borst, 1995, Warren, 1980), and many restriction
endonucleases have been shown to digest modified DNA slowly if at all (Huang et
al., 1982). Modified bases may also serve as signals for transcription by phageencoded RNA polymerases and phage DNA packaging.
18
19
Filamentous
Pleomorphic
Plasmaviridae
Polyhedral
Tectiviridae
Rudiviridae
Polyhedral
Polyhedral
Microviridae
Corticoviridae
Polyhedral
Polyhedral
Filamentous
Filamentous
Tailed
Podoviridae
Leviviridae
Cystoviridae
Inoviridae
Lipothrixviridae
Shape
Tailed
Tailed
Family
Myoviridae
Siphoviridae
Siphoviridae
Lipothrixviridae
Plasmaviridae
Rudiviridae
Tectiviridae
Podoviridae
Fuselloviridae
Cystoviridae
Inoviridae
Leviviridae
Microviridae Corticoviridae
Myoviridae
Schematic figure
Small, no envelope
Complex capsid,
internal lipid membrane
Protein shell and
inner lipoprotein
vesicle
Icosahedral capsid
Lipid envelope
Filaments or rods
Rods, lipid envelope
Straight, rigid rods
Characteristics
Contractile tail
Long, noncontractile tail
Short tail
No capsid, lipid
envelope
Fuselloviridae
Pleomorphic
Lemon-shaped,
lipid envelope,
spikes at one pole
Modified from (Ackermann, 2003, Ackermann & DuBow, 1987a), http://www.ncbi.nlm.nih.gov/ICTVdb/index.htm
Order
Caudovirales
Table 2. Classification of bacteriophages
L2
SSV1
dsDNA
SIRV1
MS2
φ6
fd
TTV1
PRD1
φX174
PM2
T7
Example
T4
λ
dsDNA
dsDNA
ssRNA
dsRNA
ssDNA
dsDNA
dsDNA
ssDNA
dsDNA
dsDNA
Nucleic acid
dsDNA
dsDNA
2.2.3. Phage evolution
As a group, viruses are believed to be ancient and originate from the time before
the divergence of the three domains of life - Archaea, Bacteria, and Eucarya
(Hendrix, 1999, Hendrix et al., 2003). Tailed dsDNA phages infect cyanobacteria
and other Gram-negative bacteria but have not been described for Archaea, which
suggests that the emergence of this virus group succeeded the divergence of
Archaea and Bacteria but preceded the split of cyanobacteria from other Gramnegative bacteria. This gives a time estimation of 3–3,5 billion years (Hambly &
Suttle, 2005).
The molecular mechanisms driving evolution may be either vertical or horizontal.
Vertical mechanisms create variation trough mutations (nucleotide exchanges,
deletions, and inversions), after which the new variant is inherited to the next
generation. Horizontal mechanisms transfer the genetic information (ranging from
single genes to complete functional units) within the whole population, and even
through species barriers, to form novel combinations ( Brüssow et al., 2004,
Woese, 2000).
The genomes of modern phages are highly mosaic (Brüssow & Desiere, 2001,
Juhala et al., 2000, Pedulla et al., 2003) and according to the present understanding,
they constitute a common gene pool that is mixing constitutively by horizontal
gene transfer (Campbell, 2003). As stated by the most popular hypothesis on phage
evolution, “the modular theory”, each phage genome is composed of multiple
functional units, e.g. head genes or tail genes. Each of these units is represented by
several alleles, “modules”, which are exchanged by homologous or, less often, nonhomologous recombination between different phage genomes ( Brüssow et al.,
2004, Hendrix, 2002). Most such recombination events disrupt an essential gene or
regulatory function and result into non-viable phage that is rapidly eliminated.
Sometimes, however, the recombination produces a viable combination that has a
selective advantage over the donor genomes, resulting into a “new” phage.
The Pittsburgh phage group has introduced “a moron accretion hypothesis” to
explain how phage genomes have gradually increased in size by sequential addition
of new genes. The term “moron” (for “more DNA”) describes a genetic element
containing a gene preceded by a transcription promoter sequence and followed by a
transcription terminator, thus including all the information needed for the gene to
be transcribed. Recently added morons are often recognized by a G+C –content
that clearly differs from that of the surrounding genes. A moron that benefits its
new host then gradually becomes fully integrated into the phage genome and its
regulatory circuit (Hendrix, 2002, Hendrix et al., 2003, Hendrix et al., 2000, Juhala
et al., 2000).
2.2.4. The impact of phages on bacterial evolution
Bacteriophages share a long co-evolution with their hosts and have had a
fundamental influence on bacterial evolution and diversification ( Brüssow et al.,
2004, Weinbauer & Rassoulzadegan, 2004). The struggle for survival between
20
phage and bacteria has provoked “an arms race”, where bacteria have developed
defense mechanisms like restriction enzymes or changes in their phage-receptor
molecules. Phages have then circumvented these by chemically modified
nucleotides and avoidance of restriction enzyme recognition sequences or mutated
adhesins, respectively (Comeau & Krisch, 2005, Weitz et al., 2005).
Bacteriophages can promote the bacterial diversity in several ways. In the lytic
state they may do it by “killing the winner” – that is, infecting and lysing the most
abundant bacterial species, thus allowing less competitive species to co-exist
(Weinbauer & Rassoulzadegan, 2004). Phages are also the main mediators in the
horizontal evolution of prokaryotes (Canchaya et al., 2003). Phage-mediated gene
transfer may occur either via (generalized) transduction or lysogenic conversion. In
generalized transduction, the fragments of host DNA are accidentally packaged
into phage head and delivered into a new host cell ( Brüssow et al., 2004,
Weinbauer & Rassoulzadegan, 2004). Lysogenic conversion is a feature of
temperate phages. Since the integration of the phage genome into the bacterial
chromosome may be harmful for the bacterium (important genes or regulatory
elements may be interrupted and the prophage constitutes a risk of induction to the
lytic cycle), the prophage would be rapidly eliminated unless it encodes functions
that increase the fitness of the lysogen. Among the commonly encountered
lysogenic conversion genes are genes that confer immunity and exclusion to
superinfection by similar or closely related phages, or genes whose products
enhance the survival of the lysogen in its ecological niche. For pathogenic bacteria
this means genes enhancing bacterial virulence, like different toxin genes and genes
promoting bacterial adhesion, invasion, or resistance to human serum or
phagocytosis ( Brüssow et al., 2004, Canchaya et al., 2003, Wagner & Waldor,
2002).
On the evolutional time scale, the nature of prophage genomes in bacterial
chromosomes is ephemeral. Mutations and deletions accumulate gradually in the
genes coding for phage lytic functions, resulting in a defective prophage that can no
longer be induced. Often, most of the original prophage genome is lost, and only
the remnants of the phage sequence remain in the bacterial genome. In such cases
the prophage origin of the remaining genes may be difficult to interpret ( Brüssow
et al., 2004, Canchaya et al., 2003).
2.2.5. Phage receptors
The first step in bacteriophage infection is the specific attachment of the phage to
the surface components of the host cell. This attachment often takes place in two
stages: a temperature-independent reversible step is followed by an irreversible step
which is independent on temperature (Lindberg, 1973). The primary adhesin of
tailed phages is usually the tail fiber. Extensively studied examples of such
adhesins are T4 gp37, the large subunit of the distal tail fiber (Oliver & Crowther,
1981, Tétart et al., 1998), T7 gp17, whose trimers form the tail fibers (Steven et al.,
1988) and the tail fiber protein J of λ (Wang et al., 2000). Some short-tailed
21
phages, like Salmonella phages P22 and SP6 and Shigella phage Sf6, have tail
spikes instead of fibers (Baxa et al., 1996, Chua et al., 1999, Scholl et al., 2002).
Filamentous phages fd, M13 and f1 use their minor capsid protein g3p to attach to
E. coli cell (Bennett & Rakonjac, 2006, Lubkowski et al., 1999, Martin & Schmid,
2003) and lipid containing phages PM2 and PRD1 utilize spikes that are located at
their vertices (Grahn et al., 1999, Huiskonen et al., 2004, Kivelä et al., 2002).
Presumably every structure exposed on the bacterial surface may serve as
bacteriophage receptor. For Gram-negative bacteria, this means carbohydrates,
outer membrane proteins, or pilus and flagellum structures (Heller, 1992, Koebnik
et al., 2000, Lindberg, 1973, Wright et al., 1980). LPS O-antigen is used e.g. by
P22 (Baxa et al., 1996), Sf6 (Chua et al., 1999), and V. cholerae phage K139
(Nesper et al., 2000). Of these, both P22 and Sf6 have tail spikes possessing
endorhamnosidase activity, which cleaves the O-antigen to facilitate the phage
access to the injection site. The core region of LPS serves as receptor for phages
like T3 and T4 (Prehm et al., 1976), T7 (Lindberg, 1973) and P. aeruginosa phage
φCTX (Yokota et al., 1994).
Many clinical isolates of E. coli are covered by a polysaccharide capsule, or K
antigen. The structures of K antigens are highly diverse, and more than 80 K
serotypes are known (Whitfield & Roberts, 1999). E. coli K antigens are utilized as
receptors by several phages, like PK1A to E (Gross et al., 1977), K1F (Scholl &
Merril, 2005, Vimr et al., 1984), K5 (Gupta et al., 1982) and K1-5, which is
exceptional in having two different tail fiber proteins that allow it to recognize both
K1 and K5 type capsules (Scholl et al., 2001). Capsule-specific phages often have
capsule-degrading enzymatic activities associated with their tail spikes or fibers,
the examples of which are the endosialidases of K1- specific phages (Kwiatkowski
et al., 1982, Pelkonen et al., 1989). Except for serving as the receptor for
bacteriophages, the polysaccharide capsule may also function as a barrier against
phage infection, as shown recently for T7 (Scholl et al., 2005).
The outer membrane proteins (OMPs) of Gram-negative bacteria serve to maintain
the membrane structural integrity or function in solute and protein translocation.
The transport proteins include non-specific porins (for transport of small,
hydrophilic solutes), specific porins (for transport of larger molecules, e.g.
oligosaccharides or nucleosides), and high affinity transporters (for transport of
nutrients that are present in low concentrations). Most these protein groups are also
utilized as bacteriophage receptors (Bonhivers et al., 1998, Koebnik et al., 2000).
The E. coli major OMP, OmpA, is used by several phages (Morona et al., 1984,
Morona et al., 1985, Schwarz et al., 1983), and a ferricrome transporter FhuA is
recognized by T1, φ80, and T5 (Bonhivers et al., 1998, Endriss & Braun, 2004,
Killmann et al., 1995, Plancon et al., 2002). The most extensively studied
bacteriophage, λ, utilizes a maltoporin LamB as its receptor (Randall-Hazelbauer &
Schwartz, 1973, Wang et al., 2000).
Pili of Gram-negative bacteria are generally utilized as receptors by filamentous
phages. E. coli phages fd, M13, and f1 (collectively called Ff phages) recognize the
22
F pilus as their primary receptor, and P. aeruginosa phages Pf1 and Pf3 use the
type IV PAK pilus and the conjugative pilus, respectively (Holland et al., 2006,
Lubkowski et al., 1999). The mannose-sensitive hemagglutinin type IV pilus serves
as receptor for a newly characterized filamentous phage KSF-φ infecting V.
cholerae (Faruque et al., 2005a). The utilization of bacterial pili is not, however,
merely a feature of filamentous phages, as the broad host-range enveloped phage
PRD1, for instance, recognizes the conjugative pilus (Grahn et al., 1999, Olsen et
al., 1974). The flagellum can also function as the bacteriophage receptor, as
exemplified by phage Χ that infects the motile strains of Escherichia, Salmonella,
and Serratia (Samuel et al., 1999) and Aeromonas phage PM3 (Merino et al.,
1990).
The cell wall of Gram-positive bacteria is composed of multilayered peptidoglycan
sacculus (also called murein) decorated with proteins, teichoic acids and neutral
polysaccharides. Some strains also contain a surface layer (S-layer) made of
paracrystalline protein subunits (Delcour et al., 1999). The phage-host interactions
in Gram-positive bacteria are generally less known than those in Gram-negative
bacteria. The phage adsorption to Gram-positive cell wall typically occurs via cell
wall carbohydrates, often teichoic acids (Dupont et al., 2004, Wendlinger et al.,
1996). Carbohydrates have been shown to function as receptors e.g. for
Lactococcus lactis phages KH, bIL170 and φ645 (Dupont et al., 2004, Valyasevi et
al., 1990) and teichoic acids for B. subtilis phage SP50 (Archibald & Coapes, 1976)
and Listeria monocytogenes phages A118 and A500 (Wendlinger et al., 1996).
Other structures may, however, also provide phage adsorption for Gram-positive
bacteria, as B. subtilis phages PBS1 and SPP1 recognize the flagellum and an
integral membrane protein, respectively (Raimondo et al., 1968, Sao-Jose et al.,
2004), and Listeria phage A511 the peptidoglycan moiety (Wendlinger et al.,
1996).
2.3. Applications of phages
2.3.1. Bacteriophage therapy
The early bacteriophage research was largely driven by the desire to use phages to
combat bacterial diseases – the phage therapy. Phages were independently
discovered by Frederick Twort in 1915 and Felix d’Herelle in 1917, after which
d’Herelle dedicated his career in phage therapy research (Stone, 2002, Summers,
1999, Summers, 2001). D’Herelle and other early phage therapy researchers used
phages to cure shigellosis, cholerae and staphylococcal infections. The obtained
results were often promising, but hampered by improper understanding about the
bacteriophage biology. Also the experimental settings used, the crude and
inefficient phage preparations and the lack of proper controls generated
contradiction. The phage therapy research ceased at the advent of antibiotics in the
Western world, but was continued actively in the Former Soviet Union and Eastern
23
Europe, especially Poland (McKinstry & Edgar, 2005, Stone, 2002, Sulakvelidze et
al., 2001, Summers, 2001).
Along with the increasing incidence of antibiotic-resistant bacteria, the interest in
bacteriophage therapy has risen in the Western world again (Merril et al., 2003,
Stone, 2002). As potential therapeutic agents, phages have many advances: The
host specificity of a particular phage is usually very narrow, which minimizes the
side effects of the treatment on the normal flora. The appearance of phage-resistant
mutants is not as common as that of antibiotic resistance, and it is relatively easy to
isolate a phage variant that is capable in infecting the mutated strain. Also, the
development of a new therapeutic phage is cost-effective in comparison with the
development of a new antibiotic ( Brüssow, 2005, Matsuzaki et al., 2005, Skurnik
& Strauch, 2006). The major concerns of phage therapy include safety issues and
pharmacokinetics. As discussed in Chapter 2.2.4., phages may carry genes whose
products promote bacterial virulence. This is more a feature of temperate phages,
thus only lytic phages whose genome has been sequenced should be used for
therapeutic purposes (Brüssow, 2005, Bruttin &
Brüssow, 2005). Phage
pharmacokinetics is still rather poorly understood and more research in this field
needs to be done. Often, in vitro and in vivo situations are substantially different
and a phage that lyses its host bacteria in laboratory conditions may fail to do so in
mammalian environment (Dabrowska et al., 2005, Kasman et al., 2002, Payne &
Jansen, 2003).
In recent years, phages have been successfully used e.g. to rescue mice from
infection by Enterococcus faecium (Biswas et al., 2002) and Staphylococcus
aureus (Matsuzaki et al., 2003). In addition to whole phages, also individual phage
enzymes may be used as therapeutic agents, as demonstrated by Schuch et al.
(2002) who used a phage lysin to cure mice infected with Bacillus anthracis
(Schuch et al., 2002).
2.3.2. Phages in bacterial diagnostics
Another field that has found a wide utilization of bacteriophages is bacterial
diagnostics. At their best, phage-based detection methods may be rapid, specific,
and economical (McKinstry & Edgar, 2005). Phage typing, i.e. studying the ability
of a certain phage collection to propagate on a given bacterial isolate, has long been
used in bacterial identification and subtyping (Baker & Farmer III, 1982,
McLauchlin et al., 1996, Schmieger, 1999). Today, phage typing and other
amplification assays are utilized mostly in combination with modern molecular
typing methods, even though in developing countries they can provide a good
alternative to more expensive diagnostic tools (Alcaide et al., 2003, Gali et al.,
2006, Pai et al., 2005, Rodriguez-Calleja et al., 2006, Rybniker et al., 2006,
Willshaw et al., 2001).
In 1989, Ulitzur and Kuhn patented a detection method based on reporter phages,
i.e. phages whose genomes carry reporter genes, like luciferase or green fluorescent
protein (GFP). Infection of host bacteria by the reporter phage results in luciferase
24
or GFP expression, which can be monitored with appropriate apparatus (for
example, luminometer or fluorescence microscope) (Billard & DuBow, 1998,
Ulitzur & Kuhn, 1989, Ulitzur & Kuhn, 2000). Reporter phages have most
thoroughly been applied in detection and antibiotic susceptibility testing of
mycobacteria (Banaiee et al., 2001, Hazbon et al., 2003, Jacobs et al., 1993, Pai et
al., 2005, Pearson et al., 1996, Sarkis et al., 1995), the median sensitivity of the M.
tuberculosis detection being 106 CFU/ml (Banaiee et al., 2001). Reporter phages
have also been developed for enteric bacteria (Kodikara et al., 1991), Listeria
(Loessner et al., 1996, Loessner et al., 1997) and E. coli (Funatsu et al., 2002,
Waddell & Poppe, 2000). The Listeria reporter was able to detect 500 to 1000
cells/ml and, after enrichment, one cell per g of salad (Loessner et al., 1996).
A slightly different approach, where GFP was fused to capsid proteins of T4 and
PP01, was used to detect E. coli K12 and O157:H7, respectively. This method even
detected bacterial cells that are in viable but nonculturable state, which is a clear
advantage over methods that require phage infection and amplification (Oda et al.,
2004, Tanji et al., 2004). A method where phages labeled with fluorescent dyes
were used to identify and enumerate bacteria in sea water was introduced by
Hennes et al. (1995) (Hennes et al., 1995) and a similar technique has then been
applied in the detection of E. coli O157:H7 (Goodridge et al., 1999a, Goodridge et
al., 1999b) and Microlunatus phospovorus (Lee et al., 2006). The detection limits
in these studies were 102 to 103 cells/ml.
In past few years, diagnostic tools where phage-specific identification is combined
to modern detection methods have been developed. To shorten the required
enrichment time, an immunomagnetic separation step was combined to phage
amplification assay in the analysis of Salmonella and E. coli O157:H7 (Favrin et
al., 2001, Favrin et al., 2003). A system, where bacteriophage infection was linked
to quorum sensing signaling and a bioluminescent bioreporter E. coli cell, was
introduced by Ripp et al. and shown to detect E. coli in pure culture at the
concentration of 1 CFU/ml (Ripp et al., 2006). Neufeld et al. presented a method
where the phage-induced host cell lysis and subsequent release of intracellular
enzymes was monitored by the amperometric detection of enzymatic activity,
reaching sensitivity of 1 CFU/100 ml (Neufeld et al., 2003). A procedure where the
phage amplification was followed by the detection of the phage capsid protein by
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was
applied to detect both E. coli alone and E. coli and Salmonella simultaneously. This
method sensed ca. 5 × 104 cells in less than two hours (Madonna et al., 2003, Rees
& Voorhees, 2005). Recently, Edgar et al. developed a technique where a
biotinylation peptide was displayed on T7 major capsid protein and the
biotinylation of this ‘reagent phage’ inside the target bacteria was detected by
conjugating the phage to streptavidin-coated quantum dots and analyzing the
conjugate with flow cytometry or fluorescence microscopy. This method was able
to detect and quantify E. coli at ca. 10 CFU/ml in one hour assay time (Edgar et al.,
2006).
25
2.3.3. Other applications
Phage display, at its simplest, is a technique where peptides, proteins, or antibody
fragments are expressed on the surface of phage particles (Willats, 2002). The
technology was first developed for filamentous phages in 1985 (Smith, 1985) and
is generally used for the selection of molecules that bind specifically to predetermined targets. Phage display has been widely utilized e.g. in the identification
of receptors and their ligands or proteins that modulate receptor activity (Hartley,
2002, Ladner et al., 2004) and isolation of recombinant antibodies (Azzazy &
Highsmith, 2002, Petrenko & Vodyanoy, 2003). In addition to filamentous phages,
display systems have recently been developed for members of other phage groups,
like λ (Ansuini et al., 2002, Gupta et al., 2003), MS2 (van Meerten et al., 2001) and
T7 (Danner & Belasco, 2001, Gnanasekar et al., 2004, Sokoloff et al., 2000).
The modern molecular biology was largely based on bacteriophage research, and
many enzymes nowadays used in in vitro genetic engineering have phage origin.
Examples of such enzymes are T4 DNA polymerase, ligase and polynucleotide
kinase, which are sold by most (if not all) biotechnology companies. Also
heterologous gene expression systems based on T7 RNA polymerase (Studier &
Moffatt, 1986) are in common use. A more recent application is recombineering,
i.e. the use of enzymatic machinery for generalized recombination encoded by
lambdoid phages. These enzymes catalyze homologous recombination between
DNA sequences having very short homologies and provide a method for molecular
cloning without restriction enzymes or DNA ligase (Thomason et al., 2005).
2.4. Bacteriophages of Yersinia
Yersiniophages were first bought to public attention by Felix d’Herelle, who
treated plague patients in Alexandria, Egypt, in 1925 with an “antiplague phage” he
had isolated few years earlier (Summers, 1999, Summers, 2001). In the following
decades, a number of yersiniophages were isolated, primarily for phage typing
purposes (Baker & Farmer III, 1982, Calvo et al., 1981, Garcia et al., 2003,
Kawaoka et al., 1987, Nilehn, 1969, Nunes & Suassuna, 1978). In these early
publications, however, phages were mainly described by their host specificity, and
more detailed information about these phages is scarce. The first more thorough
characterization of yersiniophages was published in 1982 by Kawaoka et al., who
studied morphology, adsorption kinetics and stability of three phages infecting Y.
enterocolitica (Kawaoka et al., 1982). Interestingly, already in this paper it was
stated that most Y. enterocolitica phages lyse their host cells when grown at 25oC
but not at 37 oC, due to smaller amount of phage receptors on the cell surface at 37
o
C. According to the present understanding, this indicates phages using LPS Oantigen as their receptors. In 1984, Stevenson and Airdrie reported the isolation and
characterization of eight phages infecting Y. rückeri (Stevenson & Airdrie, 1984).
A systematic molecular study of temperate yersiniophages was made in 2000 by
Popp et al., who characterized the morphology, host range, genome size, DNA
26
homology and protein composition of eight phages (Popp et al., 2000). Still today,
the genomic sequences of only three yersiniophages (φYeO3-12, φA1122 and
PY54) are known. The main features of these phages are briefly discussed below.
2.4.1. φYeO3-12
φYeO3-12 was isolated from the sewage of Turku, Finland, in 1989 based on its
ability to infect Y. enterocolitica serotype O:3 (YeO3) (Al-Hendy et al., 1991). It
uses the LPS O-antigen as its receptor and is able to infect and proliferate on E. coli
strains expressing the YeO3 O-antigen gene cluster. φYeO3-12 is a lytic phage
belonging to Podoviridae and is closely related to E. coli phages T3 and T7 (I). The
genome of φYeO3-12 is a 39 600 bp long linear, double-stranded DNA molecule
having 58 putative genes that all are transcribed from the same DNA strand
(Pajunen et al., 2001). The overall identity between φYeO3-12 and T3 genomes is
about 84%, and the genome organization of these two phages is colinear (Pajunen
et al., 2002).
2.4.2. φA1122
φA1122 infects most isolates of Y. pestis and is used as a diagnostic agent by the
Centers for Disease Control and Prevention (CDC, the U.S.A.) in the identification
of Y. pestis. The φA1122 genome is a 37,555 bp long DNA molecule having 51
predicted gene products. The genome is colinear with the genomes of T7, T3 and
φYeO3-12 and the genome-wide identities with T7 and T3 are about 89% and 73%,
respectively. Even though the general similarity of φA1122 gene products to their
counterparts in T7 is much higher than to T3, there is a stretch of 9,188 bp (coding
about half of the morphogenetic functions) having 99.8% identity with T3. The
genomes of T7, T3, φYeO3-12 and φA1122 are thus mosaics in respect for each
other, and it has been proposed that T3 is a host range mutant of a recombinant
between two yersiniophages, one resembling φYeO3-12 and the other φA1122
(Garcia et al., 2003).
2.4.3. PY54
PY54 is a temperate phage belonging to Siphoviridae. It was isolated from Y.
enterocolitica serotype O:5 and infects Y. enterocolitica strains belonging to the
non-pathogenic biogroup 1A and the pathogenic serotype O:5,27 (Popp et al.,
2000). The PY54 genome is a linear, 46,339 bp DNA molecule having 67 plausible
genes, 55 of which are transcribed rightwards and 12 leftwards on the genetic map
(Hertwig et al., 2003b). In the lysogenic state the PY54 genome is not integrated
into the bacterial chromosome, instead, it is replicated as a linear plasmid having
covalently closed hairpin ends. The phage genome and the plasmid prophage are
almost 50% permuted in such a way that the phage cohesive ends are in the middle
of the plasmid (Hertwig et al., 2003a). The strategy for PY54 to establish and
maintain its prophage state is highly similar to E. coli phage N15, which is closely
27
related to lambdoid phages. However, the sequence similarity of these two phages
is fairly limited outside the genomic regions involved in the conversion of the
phage DNA into the plasmid prophage and the prophage maintenance and
immunity (Hertwig et al., 2003b).
2.4.4. Applications of yersiniophages
The early literature about yersiniophages was mainly focused on isolating phage
collections and developing schemes for phage typing (see above). The culturebased methods for Yersinia diagnostics are time-consuming and often unreliable
(Fredriksson-Ahomaa & Korkeala, 2003, Gomes-Solecki et al., 2005) and phage
typing is, in combination with other methods, still used in the diagnostics of Y.
pestis (Anisimov et al., 2004, Garcia et al., 2003) and Y. enterocolitica (SoltanDallal et al., 2004).
After the pioneering work by d’Herelle, not many reports about phage therapy in
Yersinia infections have been published, at least in English. Damasko et al. (2005)
studied the bactericidal activity of enterocoliticin, a phage tail-like bacteriocin
isolated from Y. enterocolitica serotype O:7,8 (Strauch et al., 2001), against Y.
enterocolitica in cell culture and mice. In cell culture, enterocoliticin reduced the
titer of bacteria adhering to the surface of eukaryotic cells, but had no antibacterial
effect against bacteria that had invaded the cells. In the mouse model,
enterocoliticin was not able to prevent the Y. enterocolitica infection (Damasko et
al., 2005). Somewhat similar results were obtained when Y. enterocolitica –specific
phages φYeO3-12 and PY100 were used in phage therapy experiments in mice
(Skurnik & Strauch, 2006). In the G. Eliava Institute of Bacteriophage, Tbilisi,
Georgia, an active research on yersiniophages that could be used for sanitation,
prevention and treatment of the infection is carried out (Darsavelidze et al., 2003).
However, only limited amount of data about these studies is available in the
Western world, partly because of language restrictions.
Yersiniophages have also been utilized as tools in basic microbiology research.
Shaw et al. (1983) used an Y. pestis –specific mutant of E. coli phage T6 to follow
the internalization of Y. pestis to macrophages (Shaw et al., 1983). In the Skurnik
laboratory, phages φYeO3-12 and φR1-37 (III), specific to YeO3 LPS O-antigen
and outer core, respectively, and φ80-81, specific to Y. enterocolitica O:8 (YeO8)
O-antigen, have had an important role in studying the molecular biology of
Yersinia LPS biosynthesis (Al-Hendy et al., 1991, Skurnik et al., 1995, Skurnik &
Zhang, 1996, Zhang & Skurnik, 1994).
28
3. AIMS OF THE PRESENT STUDY
This PhD thesis work originates from a project to construct a reporter
bacteriophage that would be used as a diagnostic tool for detecting Y. enterocolitica
serotype O:3 bacteria in food and clinical samples. Phage φYeO3-12 that has a
strict host specificity to serotype O:3 strains as it uses the LPS O-antigen as its
receptor (Al-Hendy et al., 1991) was chosen as a starting point for the construction
of the reporter phage. For this purpose, a detailed molecular genetic analysis of the
phage genome became necessary. In the course of the research, the aims of the
studies expanded to the molecular, genetic and biological characterization of Y.
enterocolitica –specific phages φYeO3-12 and φR1-37 and to the analysis of the
receptor of Y. pestis –specific phage φA1122. The detailed aims were:
•
To characterize the biological properties of φYeO3-12
•
To analyze the φYeO3-12 genome and identify the non-essential genes in
order to be able to manipulate the phage genetically
•
To construct a reporter bacteriophage starting from φYeO3-12 to be used in
bacterial diagnostics
•
To characterize the biological and genetic features of φR1-37
•
To identify the cellular receptor of φA1122
29
4. MATERIALS AND METHODS
The detailed description of the materials and methods used in the present study is
given in the original publications I-IV.
4.1. Bacterial strains, phages and plasmids
The bacteriophages and transposons used in this study are described in Table 3,
bacterial strains in Table 4 and plasmids in Table 5. Unless otherwise stated, the
Yersinia incubations were done at room temperature (RT) and E. coli was cultured
at +37oC. Tryptic soy broth (TSB) medium (Oxoid) and Luria Broth (LB)
(Sambrook & Russel, 2001) were used for bacterial liquid cultures. Soft-agar
medium included additionally 0.4 % (w/v) agar (Biokar Diagnostics). Luria agar
(LA) (Sambrook & Russel, 2001) was used as solid medium for bacteria and
lambda agar (Tryptone 10 g l-1, NaCl 2.5 g l-1, agar 15 g l-1) for phage plates. Solid
and liquid media were supplemented with antibiotics when required. Large-scale
preparations of bacteriophages were purified by glycerol density gradient
ultracentrifugation (Sambrook & Russel, 2001).
Table 3. Bacteriophages and transposons used in this study
Phage/Transposon
Description
φYeO3-12
Τ7+
Y. enterocolitica serotype O:3 specific, wild type
isolated from sewage
Wild type from F.W. Studier
Τ3+
Wild type from F.W. Studier
φR1-37
Y. enterocolitica serotype O:3 specific, wild type
isolated from sewage
The reference phage used by the Centers for Disease
Control and Prevention to identify Y. pestis
φYeO3-12 with deletion in gene 0.7
φYeO3-12 with lacZ’ insertion in gene 5.5; 16 237
(TTAAA)
φYeO3-12 with lacZ’ insertion in gene 0.45; 1 747
(CAGGG)
φYeO3-12 with lacZ’ insertion in gene 5.5; 16 269
(GACAG)
φYeO3-12 with lacZ’ insertion in gene 1.3; 7 830
(CCGCG)
φYeO3-12 with lacZ’ insertion in gene 1.1; 6 475
(ATGGC)
φYeO3-12 with lacZ’ insertion in gene 1.3; 7 680
φA1122
ΔPK
φ::lacZ1
φ::lacZ2
φ::lacZ3
φ::lacZ4
φ::lacZ5
φ::lacZ6
30
Source
/reference
(Al-Hendy
et al., 1991)
Ian
Molineux,
Texas
Ian
Molineux,
Texas
(Skurnik et
al., 1995)
(Garcia et
al., 2003)
I
II
II
II
II
II
II
φ::lacZ7
φ::lacZ8
φ::lacZ10
φ::lacZ11
φ::lacZ12
φ::lacZ13
φ::lacZ14
φ::lacZ15
φ::lacZ16
φ::lacZ17
φ::lacZ18
φΔ888-2449
φΔ1681-1741
φΔ7801-7823
φΔ11141-11200
φ::lucFF1.2
φ::lucFF5.3
φ::lucFF1
Transposon lacZ’Mu(NotI)
Transposon lucFFMu
(φ::lacZ61) (ATGAC) and gene 3.7; 11 300 (φ::lacZ62)
(AGTGC)
φYeO3-12 with lacZ’ insertion in gene 4.5; 13 523
(φ::lacZ71) (AGAAG) and gene 5.5-5.7; 16 529
(φ::lacZ72) (TACCT)
φYeO3-12 with lacZ’ insertion in gene 1.3; 7 799
(ACATC)
φYeO3-12 with lacZ’ insertion in gene 3.5; 11 206
(AACTG)
φYeO3-12 with lacZ’ insertion in gene 0.7; 2 772
(GACGC)
φYeO3-12 with lacZ’ insertion in gene 1.6; 8 623
(TACTG)
φYeO3-12 with lacZ’ insertion in gene 4.3; 13 322
(AAACA)
φYeO3-12 with lacZ’ insertion in gene 0,45; 1 682
(ATTGG)
φYeO3-12 with lacZ’ insertion in gene 0.7; 2 423
(AGGCT)
φYeO3-12 with lacZ’ insertion in gene 3.5; 11 134
(ACCAT)
φYeO3-12 with lacZ’ insertion in gene 1.1; 6 528
(GCGGT)
φYeO3-12 with lacZ’ Insertion at nt position 888 and
deletion of nt 888 to 2449 (genes 0.3-0.7)
φYeO3-12 with deletion of nt 888 to 2449 (genes 0.30.7)
φYeO3-12 with deletion of nt 1681 to1741 (in gene
0.45)
φYeO3-12 with deletion of nt 7801 to 7823 (in gene
1.3)
φYeO3-12 with deletion of nt 11141 to 11200 (in gene
3.5)
Like φ::lacZ1; lacZ replaced with lucFF
Like φ::lacZ5; lacZ replaced with lucFF
φΔ888-2449 with lucFF inserted in between genes 6.3
and 6.5; 17 988 (CTTAA)
Promoterless lacZ’, NotI sites close to transposon ends
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
This work
This work
This work
(Vilen et al.,
2003)
Promoterless lucFF
H. Vilen and
H. Savilahti,
unpublished
For transposon insertion mutants, the interrupted gene and the first base pair after the 5 bp
duplication are indicated. The duplicated sequence is shown in parenthesis. Deletion mutants are
named by indicating the base pairs that are deleted. For φΔ888-2449, the nucleotide numbering of
wt φYeO3-12 is used.
31
Table 4. Bacterial strains used in this study
Bacterial strain
Characteristics
Source
/reference
Yersinia enterolitica
6471/76 (YeO3)
Serotype O:3; wild-type patient isolate
(Skurnik,
1984)
(Skurnik,
1984)
(Skurnik et al.,
1995)
(Biedzka-Sarek
et al., 2005)
(Biedzka-Sarek
et al., 2005)
(Portnoy et al.,
1981)
(Zhang et al.,
1997)
(Skurnik,
1985)
III
6471/76-c (YeO3-c)
YeO3-R1
YeO3-c-OC
YeO3-OC-R
8081 (YeO8)
8081-R2
467/73
467/73-φR1-37-R
3229
3229-φR1-37-R
14779/83
14779/83-φR1-37-R
18425/83
18425/83-φR1-37-R
Serotype O:3; virulence plasmid-cured derivative
of 6471/76
Serotype O:3; spontaneous O-antigen negative
derivative of 6471/76-c
Serotype O:3. Δ(wzx-wbcQ). Outer core negative
derivative of 6471/76-c
Serotype O:3. Δ(wzx-wbcQ). Outer core and Oantigen negative derivative of 6471/76-c
Serotype O:8
Serotype O:8; O-antigen negative derivative of
8081
Serotype O:9. Human stool isolate from patient.
Sensitive to φR1-37.
Spontaneous phage φR1-37 resistant derivative
of 467/73
Serotype O:50. Human stool isolate. Sensitive to
φR1-37.
Spontaneous phage φR1-37 resistant derivative
of 3229
Serotype O:5. Human stool isolate. Sensitive to
φR1-37.
Spontaneous phage φR1-37 resistant derivative
of 14779/83
Serotype O:25,26,44. Human stool isolate.
Sensitive to φR1-37.
Spontaneous phage φR1-37 resistant derivative
of 18425/83
Yersinia
pseudotuberculosis
PB1
Serotype O:1b
1
Serotype O:1a; lcr-
43
Serotype O:3; lcr-
32
Serotype O:4a; lcr-
R708Ly
Serotype O:9 reference strain, isolated from mole
in Japan. Sensitive to φR1-37.
32
(Skurnik &
Toivanen,
1991)
III
(Skurnik, 1985,
Skurnik &
Toivanen,
1991)
III
(Skurnik, 1985,
Skurnik et al.,
1995)
III
(Porat et al.,
1995)
(Samuelsson et
al., 1974)
(Samuelsson et
al., 1974)
(Samuelsson et
al., 1974)
(Tsubokura &
Aleksic, 1995)
YPIII
Serotype O:3
R708Ly-R
R708Ly-φR1-37-R
Spontaneous rough derivative of R708Ly
Spontaneous phage φR1-37 resistant derivative
of R708Ly
Serotype O:1b; O-antigen negative derivative of
PB1, kmR
Spontaneous φA1122 resistant derivative of
PB1Δwb
Spontaneous φA1122 resistant derivative of
PB1Δwb
Spontaneous φA1122 resistant derivative of
PB1Δwb
PB1Δwb
PB1Δwb-R4
PB1Δwb-R7
PB1Δwb-R12
(Bölin et al.,
1982)
III
III
IV
IV
IV
IV
Yersinia pestis
KIM D27
Lcr+, pgm-, pst+
KIM D27Nar
EV76-c
NalR; Lcr+, pgm-, pst+
Virulence-plasmid cured derivative of EV76
Yersinia frederiksenii
IP23047
Serotype O:3
Elisabeth
Carniel, Institut
Pasteur
Yersinia mollaretii
IP22404
Serotype O:3
Elisabeth
Carniel, Institut
Pasteur
Yersinia kristensenii
IP22828
Serotype O:3
Elisabeth
Carniel, Institut
Pasteur
Yersinia intermedia
821/84
Serotype O:52,54. Human stool isolate
(Skurnik &
Toivanen,
1991)
III
821/84-φR1-37-R
Escherichia coli
JM109
DH10B
C600
HB101
Spontaneous phage φR1-37 resistant derivative
of 821/84
F´ traD36 proA+B+ lacIq Δ(lacZ)ΔM15/ Δ(lacproAB) glnV44 e14- gyrA96 recA1 relA1 endA1
thi hsdR17
F- mcrA Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15
ΔlacX74, deoR, recA1 endA1 araΔ139 Δ(ara,
leu)7697 galU, galK λ- rpsL nupG λ- tonA
thi thr leuB tonA lacY supE
F-Δ(gpt-proA)62 leuB6 glnV ara-14 galK2 lacY1
Δ(mcr-mrr) rpsL20 (Strr) xyl-5 mtl-1 recA13
33
(Garcia et al.,
1999)
III
(Ben-Gurion &
Hertman, 1958,
Portnoy &
Falkow, 1981)
(YanischPerron et al.,
1985)
Life
Technologies
(Appleyard,
1954)
(Boyer &
RoullandDussoix, 1969)
IJ511
ΔlacX74 galK2 galT22 supE44 hsdS
IJ512
F42 (F’ lac) derivative of IJ511
IJ855
ara Δ(lac-proAB) thi supD hsrD
IJ1419
lacY1 or Δ(lac)6 supE44 galK2 galT22 λ- rfbD1
metB1 mcrA1 hsdR2 rpoC319 (tsnB)
lacY1 or Δ(lac)6 supE44 galK2 galT22 λ- rfbD1
metB1 mcrA1 hsdR2 rpoC320 (BR3)
F+LAM-, dut-1, ung-1, thi-1, spoT1, relA1/
pCJ105 (CmR)
thi thr leuB tonA lacY supE recA::RP4-2Yc::Mu-Km (λpir)
IJ1420
CJ236
Sm10λpir
Ian Molineux,
Texas
Ian Molineux,
Texas
Ian Molineux,
Texas
Ian Molineux,
Texas
Ian Molineux,
Texas
E. coli Genetic
Stock Center
(Simon et al.,
1983)
Shigella sonnei
IJ286
D2371-48
(Hausmann et
al., 1968)
Salmonella enterica
serovar Typhimurium
His515
Δ(his-rfb)
TV-163
rfaL
(Nikaido et al.,
1967)
(Beckmann et
al., 1964,
Subbaiah &
Stocker, 1964)
Table 5. Plasmids used in this study
Plasmid
Characteristics
pUC18
ampR, Cloning vector
pAY100
ampR tetR; O-antigen gene cluster of YeO3
cloned in pBR322
clmR tetR; Mobilizable cloning vector
pTM100
pAY100.1
pBR322
ampR; Derivative of pAY100 with the
tetracycline-resistance marker inactivated
ampR tetR; Cloning vector
pRK2013
kmR; Helper-plasmid for conjugation
pBAD33oT
clmR; oriT from pTM100 cloned into pBAD33
pCSS810
clmR kmR; lucFF in E. coli – B. subtilis shuttle
vector
ampR; YeO3 outer core gene cluster cloned in
pRV7
34
Source
/reference
(YanischPerron et al.,
1985)
(Al-Hendy et
al., 1991)
(Michiels &
Cornelis, 1991)
(Oyston et al.,
2003)
(Bolivar et al.,
1977, Sutcliffe,
1979)
(Ditta et al.,
1980)
Pacot-Hiriart et
al.,
unpublished
data
(Lampinen et
al., 1992)
(Skurnik et al.,
p34S-Km
pBR322
kmR; ampR; Cloning vector
pCVD442
ampR,kmR, Suicide vector
pBAD33oT-g1.3
clmR; φYeO3-12 gene 1.3 cloned into
pBAD33oT
clmR; E. coli C600 ligA cloned into pTM100
clmR; φYeO3-12 gene 3.5 cloned into
pBAD33oT
clmR; φYeO3-12 promoter 2.5 and gene 3.5
cloned into pBAD33oT
clmR; reporter plasmid, a pTM100 derivative.
The lucFF gene from pCSS810 cloned between
φYeO3-12 genes 9 and 10 downstream of φ10
promoter
clmR; outer core gene cluster of YeO3 cloned in
pTM100
ampR; the upstream region of O-antigen gene
cluster of YPIII cloned in pUC18
ampR; the up- and downstream regions of the
YPIII O-antigen gene cluster cloned in pUC18
ampR, kmR; the kanamycin resistance gene of
p34S-Km cloned between the up- and
downstream regions of the YPIII O-antigen gene
cluster in pUCwbdel
ampR, kmR; Suicide vector carrying the PvuII
fragment of pUCwbGB including the kanamycin
resistance gene and the up- and downstream
regions of O-antigen gene cluster of YPIII
Like pMP300; the orientation of the insert in
pTM100 is opposite
Like pMP100; the insert contains an additional
translation terminator after lucFF
clmR; reporter plasmid, a pTM100 derivative.
The lucFF gene from pCSS810 was cloned
between φYeO3-12 genes 10 and 11.
pEcligA
pg3.5long
pprg3.5
pMP300
pRV16NP
pUCwbup
pUCwbdel
pUCwbGB
pCVDwbGB
pMP100
pMP200
pSK110
1995)
(Dennis &
Zylstra, 1998)
(Donnenberg
& Kaper,
1991)
II
II
II
II
II
III
IV
IV
IV
IV
This work
This work
This work
4.2. Biological characterization of bacteriophages
4.2.1. Host specificity, efficiency of plating, growth curve and fitness-analysis (I,
II, III)
The host range of phages was determined by pipetting droplets of phage dilutions
on bacterial lawn and observing the formation of a clear lysis zone. The efficiency
of plating (EOP) was analysed by plating the phage dilution with the bacterial
suspension on top agar plates (Sambrook & Russel, 2001).
35
One-step growth curves were determined as described earlier (Birge, 1988).
Bacterial cells in mid-exponential growth phase were infected with the phage,
samples were taken at time intervals and the PFU/ml was titrated both immediately
and after the release of intracellular phages with chloroform.
Fitness-analysis was performed as previously described for T7 (Rokyta et al.,
2002). The bacteria were infected with the phage and the number of intracellular
phages was titrated from samples taken at 0 and 45 minutes.
4.2.2. Antisera (I)
Polyclonal antisera against φYeO3-12 was obtained by sequential immunization of
three rabbits with the purified phage. The ability of the antisera to recognize
φYeO3-12 was tested by enzyme immunoassay (EIA) using microtiter plates
(Nunc) coated with the phage.
The capability of the strongest antiserum to neutralize the φYeO3-12 infection was
tested by incubating the serum and the phage for 10 minutes prior to titrating the
PFU/ml.
4.2.3. Receptor characterization and adsorption analysis (III, IV)
To characterize the receptor of φR1-37, YeO3-R1 (Table 2) cells were immobilized
on nitrocellulose membrane and treated with periodate, Proteinase K or SDS
sample buffer. The ability of digoxigenin (DIG) -labeled φR1-37 to recognize the
immobilized bacteria was then studied according to the instructions of the DIG
Protein Labelling Kit (Roche).
For the phage adsorption assay, φA1122 was incubated with the bacteria for five
minutes, after which the cells together with the bound phage were centrifuged
down and the phage remaining in the supernatant was titrated. The effect of
periodate and Proteinase K on the phage receptor was tested by incubating the cells
with the reagent for two or three hours, respectively, prior to the adsorption assay.
4.2.4. LPS isolation and analysis (III, IV)
LPS was isolated and analysed by deoxycholate-polyacrylamide gel electrophoresis
(DOC-PAGE) as described earlier (Skurnik et al., 1995, Zhang & Skurnik, 1994).
4.3. Structural characterization of bacteriophages
4.3.1. Electron microscopy (I, III)
The phages were sedimented with ultracentrifuge and stained with phosphotungstic
acid. Stained particles were examined with Philips EM300 (I) or JEOL (Tokyo,
Japan) JEM-1200 EX (III) electron microscope. For infection samples, YeO3-R1
cells were incubated with φR1-37 for two minutes prior to examination.
36
4.3.2. SDS-PAGE, Western-analysis and N-terminal sequencing of structural
proteins (I, III)
SDS-PAGE of purified φYeO3-12 and φR1-37 particles was conducted using a
Hoefer SE 600 device (Amersham Pharmacia Biotech) according to manufacturers
instructions. For Western-analysis, the proteins were blotted to a nitrocellulose
membrane and identified with the polyclonal anti-φYeO3-12 serum (above).
For the determination of amino-terminal amino acid sequences, the protein bands
were blotted to a Problott membrane (Applied Biosystems) and the sequences of
the immobilized proteins were determined with an Applied Biosystems 477A protein sequencer equipped with an Applied Biosystems 120A PTH (phenylthiodantoin)-amino acid analyzer using standard parameters provided by the manufacturer.
The sequence data was analyzed with the European Bioinformatics Institute Fasta3
search (http://www.ebi.ac.uk/fasta33/), the National Center for Biotechnology
Information (NCBI) BLAST program (www.ncbi.nlm.nih.gov/blast/Blast.cgi) and
the European Molecular Biology Open Software Suit (EMBOSS) version 2.6.0.
4.4. Molecular biology techniques
4.4.1. General DNA techniques (I, II, III, IV)
Standard DNA techniques were performed (Ausubel et al., 1987, Sambrook &
Russel, 2001), and enzymes were used as recommended by the suppliers. Phage
DNA was extracted as described for bacteriophage lambda (Sambrook & Russel,
2001). DNA sequencing reactions were performed with ABI PRISMTM BigDyeTM
Terminator Cycle Sequencing v2.0 or v3.1 Ready Reaction Kit and analyzed with
ABI 377 DNA analyzer as recommended by the manufacturer. The sequence data
was analyzed with the Genetics computer Group (GCG) suite of programs (version
10, Accelrys, San Diego, CA), EMBOSS and NCBI BLAST program. For DNA
hybridization, the DNA was labelled with DIG using the DIG-High Prime DNA
Labelling and Detection Starter Kit II (Roche Molecular Biochemicals).
4.4.2. In vitro transposon mutagenesis (II)
The MuA transposase-catalyzed DNA transposition reaction was performed as
described previously (Vilen et al., 2003) using lacZ’-Mu(NotI) transposon as donor
DNA and φYeO3-12 DNA as target DNA. Mutant phage clones were identified on
indicator plates by visual inspection, and transposon insertion sites were localized
by restriction analysis, PCR-based analysis and sequence determination.
4.4.3. mRNA isolation and analysis (II)
Total RNA from YeO3-c cells infected with wild type φYeO3-12 or its derivatives
(Table 1) was isolated with Bio-Rad AurumTM Total RNA Mini Kit, and an extra
treatment with RQ1 DNAse (Promega) was performed when necessary. For RTPCR, the cDNA synthesis was done with Amersham Pharmacia Biotech Ready-ToGo You-Prime First-Strand Beads and the following PCR was done with
37
DyNAzyme DNA polymerase using conditions recommended by the manufacturer.
The mRNA and RT-PCR samples were analysed with electrophoresis by standard
procedures.
4.4.4. Analysis of modified nucleotides (III)
The nucleotide composition of the φR1-37 genome was determined by hydrolyzing
the phage DNA to deoxynucleosides by the method of Crain (Crain, 1990) and
analyzing the hydrolysate for nucleosides and dinucleotides by LC-MS/MS (PE
Sciex API 365 triple quadrupole LC/MS/MS System equipped with two PE Series
200 Micro pumps and a PE Series 200 autosampler).
4.5. Luminescence measurements and the construction of the reporter phage
4.5.1. Luminescence measurements (II)
The luminescence was measured from bacterial suspension samples with Luminova
1254 luminometer (Bio-Orbit) using d-luciferin, pH 5.0 (Labsystems) as a
substrate.
4.5.2. Construction of the reporter phage
To construct the reporter phage by homologous recombination, plasmids where
lucFF was cloned in between φYeO3-12 genes 9 and 10 (pMP100 and pMP200) or
genes 10 and 11 (pSK110) were made (Table 5) The construction of these plasmids
followed the scheme described for pMP300 (II). For homologous recombination to
occur, cultures of YeO3-c carrying the plasmids were infected with φYeO3-12.
In order to obtain pure reporter phage preparations, a direct cloning approach was
utilized. For this purpose, the lacZ’ inserts of transposon mutants φ::lacZ1 and
φ::lacZ5 (Table 3, II) were replaced with lucFF gene to generate phages
φ::lucFF1.2 and φ::lucFF5.3, respectively (Table 3). For the cloning, standard
techniques were used and the recombinant phage DNA was electroporated to
bacterial cells like described for transposon mutagenesis (II). The expression of
active luciferase during phage infection was verified by measuring the light
production and the stability of the reporter phages was studied by plating the
lysates on top-agar plates, isolating several plaques and measuring their
luminescence production. To confirm the results of stability measurements, DNA
was isolated from φ::lucFF1.2 and φ::lucFF5.3 lysates and the genomic region
containing the lucFF gene was analysed with PCR.
The in vitro transposon mutagenesis approach was utilized to create luciferase
reporter starting from the deletion mutant φΔ888-2449 (Table 3, II). The MuA
transposon reaction was done using lucFF-Mu (Table 3) as donor and φΔ888-2449
DNA as recipient, otherwise the reaction and the subsequent electroporation and
phage plating were performed as described (II). The reporter phage φ::lucFF1
(Table 3) was isolated based on its ability to produce light during the infection of
YeO3-c. The ability of φ::lucFF1 to detect YeO3-c cells in pure culture was tested
38
by infecting cells at different concentrations with different PFU of φ::lucFF1 and
measuring the luminescence at multiple time points. The stability of the mutant was
determined as above.
5. RESULTS AND DISCUSSION
5.1. Characterization of φYeO3-12
φYeO3-12 was isolated from the sewage of Turku, Finland, in 1989 (Al-Hendy et
al., 1991). It infects Y. enterocolitica serotype O:3 and other Yersinia strains
containing 6-deoxy-L-altrose in their LPS. In addition, it is able to infect and
proliferate on E. coli strains expressing the YeO3 O-antigen gene cluster. The
spontaneous phage-resistant derivatives of YeO3 have lost the O-antigen. (AlHendy et al., 1991, Al-Hendy et al., 1992).
5.1.1. φYeO3-12 is related to T3 and T7 (I)
The electron microscopy showed that φYeO3-12 is a member of the family
Podoviridae (I, Figure 1). The genome size, ca. 40 kb, was typical for this type of
phages (I, Figure 2). As shown by the one-step growth curve (I, Figure 3), the
eclipse and latent periods were 15 and 25 minutes, respectively, and the burst size
was ca. 140 PFU per infected cell. The antiserum raised against φYeO3-12 was
able to inhibit the φYeO3-12 infection (I, Figure 6) and, interestingly, T3 was also
neutralised by the serum. T7 infection was not inhibited, which indicated that
φYeO3-12 is more closely related to T3 than to T7. This hypothesis was supported
by the N- terminal amino acid sequencing of φYeO3-12 structural proteins (I, Table
3) and the growth of φYeO3-12 on bacterial strains that are restrictive to T7 but not
to T3.
5.1.2. Transposon insertions in the early genomic region of φYeO3-12 cause
growth defects on Y. enterocolitica (II)
Even though the φYeO3-12 genome had been sequenced (GenBank accession no.
AJ251805) and found to be closely related to T3 and T7 (Pajunen et al., 2002,
Pajunen et al., 2001), there were still open reading frames without obvious role.
The in vitro transposon mutagenesis was carried out in order to study further the
function of the phage genome and, especially, to identify nonessential genes. In this
assay, 17 insertion mutants were obtained, in which five insertions were located in
the early and 14 in the middle region of the phage genome (II, Figure 1).
The transposon mutants having the insertion in the early genomic region,
φ::lacZ14, φ::lacZ2, φ::lacZ15, φ::lacZ11 and φ::lacZ18 (Table 3), had lowered
EOP on Y. enterocolitica compared with E. coli and other enterobacteria (II, Table
4). For φ::lacZ11, the growth problem was also seen in the fitness-analysis (II,
39
Figure 2). The growth defects were shown to be due to the inserted transposon and
not the interruption of the individual genes, since the corresponding deletion
mutants plated normally on YeO3-c. The mRNA analysis showed that in the
insertion mutants, the expression of gene 1, coding for the phage RNA polymerase
(RNAP), was delayed (II, Figure 3), which was considered the probable cause of
the growth problems.
The effects of the interruption of the early genes 0.45 and 0.7 became visible when
a luciferase reporter strain YeO3-c/pMP300, indicating the activity of the phage
RNAP, was infected with the aforementioned mutants (II, Figure 4 A, B and C).
The gene 0.45 mutants produced slightly more light than the wild type phage,
implying that gp0.45 might have a role in the regulation of the phage growth cycle.
This protein is unique to φYeO3-12 (Pajunen et al., 2001) and this is the first
indication about its role. The functions of gp0.7, on the contrary, are well known,
one of which is the shutoff of the host transcription (Hesselbach & Nakada, 1977,
Marchand et al., 2001). It is thus not surprising that the mutants having defective
gene 0.7 showed an increased RNAP expression.
5.1.3. φYeO3-12 DNA ligase and lysozyme are needed for growth on Y.
enterocolitica (II)
The transposon mutants having the insertion in the middle region of the φYeO3-12
genome showed heterogenous phenotypes. The interruption of genes 1.1., 1.6 and
5.5. had no obvious effect on the phage phenotype. However, the phage mutants
with the transposon integrated in genes 1.3 and 3.5, coding for DNA ligase and
lysozyme, respectively, had a clearly slower growth rate on YeO3-c even though
they grew normally on E. coli, as indicated by the fitness-analysis (II, Figure 2).
The reduction of the growth rate was due to the inactivation of the genes and not
the transposon insertion itself, since the corresponding deletion mutants showed a
similar phenotype (II, Figure 2) and the slow-growing phenotypes were
complemented with the corresponding wild type genes (II, Figure 5). In addition,
the growth defects of the gene 1.3 mutants were partially complemented by
heterologous expression of E. coli DNA ligase [which is known to complement T7
gp1.3 (Rokyta et al., 2002)] and the expression of the phage lysozyme in trans was
able to revert the growth rate of the gene 3.5 deletion mutant to the level
comparable with wild type φYeO3-12 (II, Figure 2).
In the above-mentioned luciferase reporter assay, the gene 1.3 insertion mutants
showed an increase in the RNAP activity (II, Figure 4 D). As the corresponding
deletion mutant produced light at a roughly same level than the wild type phage, it
is possible that the effect was mainly caused by the transposon insertion itself and
not the interruption of the DNA ligase gene. The lysozyme mutants, instead,
showed a significant increase in the light production (II, Figure 4 E), indicating that
in these mutants the regulation of RNAP activity was lost. This is an expected
finding, since T7 lysozyme is known to regulate the phage RNAP activity (Huang
et al., 1999, Zhang & Studier, 1997).
40
In conclusion, this part of the study identified genes that are nonessential for
φYeO3-12, at least in laboratory conditions. However, the genes coding for DNA
ligase and lysozyme were needed for growth on the normal host of the phage, Y.
enterocolitica, even though they were not required on E. coli. These genes may
thus be considered as evolutionary factors important in adaptation of φYeO3-12 to
grow on Yersinia. The molecular mechanisms behind the different requirements for
phage growth on E. coli and Yersinia are not, however, understood.
5.2. Characterization of φR1-37
Phage φR1-37 was isolated in 1990 from the incoming sewage of Turku, Finland,
using the virulence-plasmid cured, O-antigen negative strain YeO3-R1 (Table 4) as
host (Skurnik et al., 1995). The ability of the phage to infect the O-antigen positive,
virulence-plasmid negative strain YeO3-c was dependent on growth temperature in
such a way that the strain was resistant when grown at 22 oC and sensitive at 37 oC.
The virulence-plasmid positive strain YeO3, interestingly, was resistant to phage
φR1-37 irrespective of the growth temperature, whereas rough forms of both
virulence plasmid positive and negative YeO3 were sensitive at both temperatures.
It was thus concluded that the phage receptor is as structure which is blocked by
the abundant O-antigen expression or YadA [Yersinia adhesin A, an outer
membrane protein which is expressed by the virulence plasmid at 37ºC (Bölin et
al., 1982, Bölin et al., 1985, El Tahir & Skurnik, 2001)] combined to less abundant
O-antigen. Since φR1-37 resistant mutants were missing the LPS outer core, it was
hypothesized that this structure might serve as the phage receptor (Skurnik et al.,
1995).
Besides the host range and the postulated receptor, no information about φR1-37
was available. The aim of this project was to characterize further the biological,
structural and genomic features of this phage.
5.2.1. Biological and structural features of φR1-37 (III)
The electron microcopy showed that φR1-37 is a large member of the family
Myoviridae (II, Figure 1). In the one-step growth curve (III, Figure 2), the eclipse
and latent periods were 40 and 50 minutes, respectively, which were followed by a
rise period of 20 minutes. The average burst size of φR1-37 was ca. 80
PFU/infected cell. The presence of φR1-37 related DNA sequences on bacterial
chromosomes was tested by hybridizing the DIG-dUTP labelled φR1-37 DNA to
the genomic DNA of 128 bacterial strains (not shown). The hybridization produced
no positive signals, indicating a strictly lytic life cycle.
SDS-PAGE of φR1-37 structural proteins showed four major protein bands, sp69,
sp46, sp31 and sp24 (III, Figure 6). Of these, sp46 was the most abundant, which
makes it the likely major capsid protein. The N-terminal amino acid sequencing of
these proteins revealed that the N-termini of sp69 and sp46 were almost identical,
41
suggesting that sp69 may be the minor capsid protein. The comparison of the
amino acid sequences to databanks did not discover significant similarities to any
known protein sequences, indicating that close relatives of φR1-37 have not been
described.
5.2.2. The φR1-37 receptor (III)
There was already an indirect indication that Y. enterocolitica serotype O:3 LPS
outer core might serve as the phage receptor (see above), however, it could not be
ruled out that the receptor would be composed of some protein structure, either
alone or in combination with LPS. To unequivocally determine the φR1-37
receptor, YeO3-R1 cells were immobilized on membranes that were subjected to
different treatments. The ability of the phage to bind the immobilized cells was
then tested. Since the treatment with Proteinase K had not effect on the phage
binding but periodate (which degrades carbohydrates) abolished it (III, Table 2), it
was concluded that the receptor is composed of LPS and does not contain protein
structures.
As the final confirmation, plasmid PRV16NP (Table 5), carrying the gene cluster
for YeO3 outer core biosynthesis, was transferred to several φR1-37 resistant
Yersinia and E. coli strains. All the strains harboring this plasmid became sensitive
to the phage, proving that the YeO3 outer core is the receptor for φR1-37.
A somewhat surprising finding was that even though the spontaneous φR1-37
resistant Y. enterocolitica and Y. intermedia derivatives (Table 4) had lost the LPS
outer core, two separate phage resistant isolates of Y. pseudotuberculosis serotype
O:9 (R708Ly-R and R708Ly-φR1-37-R) did not have the O-antigen (III, Figure 3).
It thus seems that the sugar residues forming the YeO3 outer core (shown in Figure
1) are part of O-antigen of Y. pseudotuberculosis O:9. However, the sugar
composition and structure of the LPS of this strain are not yet known.
5.2.3. The size and composition of the of φR1-37 genome (III)
The size of φR1-37 genome was determined by digesting the phage DNA with
restriction enzyme and analyzing the products by pulse-field electrophoresis
(PFGE). The analysis gave a size estimation of 270 kb, which is comparable with
the 280 kb of P. aeruginosa phage φKZ, the largest bacteriophage genome whose
nucleotide sequence has been determined (Mesyanzhinov et al., 2002).
While doing the restriction digestions to φR1-37 DNA, we noticed that Acc65I
failed to digest the DNA even though its isoschizomer KpnI digested it into > 10
fragments. Since Acc65I is known to be inhibited by DNA methylation, we
reasoned that the phage DNA might be chemically modified and set up a study to
analyse its chemical composition. For this, the DNA was enzymatically hydrolyzed
to nucleosides, which were analysed by LC-MS/MS. The phage genome was found
to contain three normal deoxynucleosides dA, dC, and dG (III, Figure 5). However,
a highly interesting finding was that instead of T, there was a peak in the
42
chromatogram whose molecular weight and retention time were identical to those
of dU. Until this, the only known viruses having dU-DNA were B. subtilis phages
PBS1 and PBS2 (Takahashi & Marmur, 1963).
The GC-content of the φR1-37 DNA, as estimated from the peak area of the
individual nucleosides in the chromatograph, was 36 %. The proportions of
individual nucleosides were 31 % for dA, 33 % for dU, 18 % for dG, and 18 % for
dC.
5.2.4. Sequencing of the φR1-37 DNA (III, this work)
The sequencing of the φR1-37 genome was initiated by cloning fragments of the
phage DNA to plasmid pBR322 (Table 5) using E. coli strain CJ236, devoid of
dUTPase and uracil-DNA glycosylase (Table 4), as a host. In this strain, the baseexcision repair (BER) pathway was inactivated and the dU-DNA remained
undamaged (Lindahl et al., 1977, Sung & Mosbaugh, 2003, Taylor & Weiss, 1982).
For sequencing, both the plasmid clones and the phage genomic DNA were used as
templates.
At the moment, ca. 76 kb of the phage genome has been sequenced. The
preliminary sequence analysis showed that the GC-content was 33 %, which is in
acceptable agreement with the value obtained by the LC-MS/MS.
In the sequence analysis, genes coding for structural proteins sp24 and sp46 were
identified (accession numbers AJ972879 and AJ972880, respectively). However, it
not yet known whether sp46 and sp69 are produced from the same or different
genes. Some deduced protein sequences of φR1-37 showed similarity to known
proteins, the examples of which are shown in Table 6. An interesting feature was
the presence of four stretches of sequence with high similarity to φKZ
(Mesyanzhinov et al., 2002). As might be expected, though, most φR1-37 open
reading frames (ORFs) produced no hits in databanks.
5.3. Identification of the φA1122 receptor
φA1122 is a T7-related bacteriophage that infects most isolates of Y. pestis (Garcia
et al., 2003). It is used as a diagnostic phage by CDC and can differentiate Y. pestis
and Y. pseudotuberculosis by infecting both strains at 37oC but only Y. pestis at
20oC. In this work, we aimed to characterize the φA1122 receptor by studying the
phage adsorption to bacteria with different LPS structures and testing whether the
degradation of cell surface proteins or carbohydrates abolishes the phage
recognition.
43
44
Sulfolobus acidocaldarius
Anabaena sp. (strain PCC 7120)
Conserved domain
240 aa
228 aa
59 aa
168 aa
DNA polymerase I (875 aa)
DNA double-strand break repair
rad50 ATPase (886 aa)
Guanylate kinase (199 aa)
Reverse transcriptase (RNA directed DNA polymerase)
Q8Z0I7, (Kaneko et al., 2001)
pfam00078,
http://www.ncbi.nlm.nih.gov/St
ructure/cdd/cddsrv.cgi?uid=pfa
m00078&version=v2.06
BAE49464 (Matsunaga et al.,
2005)
CAE14316, (Duchaud et al.,
2003)
AF399011, (Mesyanzhinov et
al., 2002)
AF399011, (Mesyanzhinov et
al., 2002)
AF399011, (Mesyanzhinov et
al., 2002)
AF399011, (Mesyanzhinov et
al., 2002)
P95690, (Datukishvili et al.,
1996)
O33600, (Elie et al., 1997)
P05472, (Wilson & Meacock,
1988)
Accession number, Reference
Plasmid maintenance system
83 aa
Magnetospirillum
9e-09
magneticum
antidote protein (95 aa)
Unnamed protein; similar to a
383 aa
Photorhabdus luminescens
4e-57
putative tail fiber protein of a
subsp. laumondii TTO1
prophage (493 aa)
a
Only one hit for each sequence is presented. bThe size of the similar region indicates the size of the deduced φR1-37 protein sequence. cE-value
of 1e-03 was considered as the limit for similarity.
1e-05
9e-09
5e-06
1e-05
1e-09
φKZ
194 aa
Sulfolobus acidocaldarius
8e-52
φKZ
989 aa
ORF 178; RNA Polymerase
(1450 aa)
ORF 182 (664 aa)
4e-10
φKZ
230 aa
ORF 050 (731 aa)
1e-06
φKZ
210 aa
2e-04
E-valuec
Kluyveromyces lactis
Organism
ORF 025 (716 aa)
Probable DNA-directed RNA
polymerase (982 aa)
Size of the
similar regionb
65 aa
Table 6. Proteins showing similarity to deduced φR1-37 protein sequences
Protein (Size) a
5.3.1. The φA1122 receptor is a LPS core structure present in Y. pestis and Y.
pseudotuberculosis but not in Y. enterocolitica (IV)
The adsorption assay showed that the φA1122 adsorption to Y. pseudotuberculosis
serotypes O:1a, O:1b, O:3 and O:4a was dependent on temperature in such a way
that the phage was able to bind the cells at 37oC but not at 20oC (IV, Figure 1). The
rough derivative of Y. pseudotuberculosis O:1b (BP1Δwb), however, adsorbed the
phage as efficiently at both temperatures. This indicates that the Y.
pseudotuberculosis O-antigen expression sterically blocks the phage receptor. The
finding that periodate but not Proteinase K destroys the receptor (IV, Figure 2)
implicates that the receptor consists of carbohydrates, most likely, LPS. The
essential nature of the receptor, as shown by the result that the phage-resistant
derivatives of BP1Δwb still adsorbed the phage (IV, Figure 4), further supported
the hypothesis that the LPS core is the phage receptor.
The φA1122 receptor was not present in any of Y. enterocolitica strains studied
(IV, Figure 3). The core structures of different Yersinia species are rather similar
and specially theYeO8 core resembles that of Y. pestis and Y. pseudotuberculosis
(IV, Table 2), indicating the highly specific nature of phage-host recognition.
Based on the core structures of Y. pestis and YeO8, it might be assumed that the
free hydroxyl group in the C2 position of the second heptose is required for the
efficient adsorption of φA1122.
5.3.2. The φA1122 receptor is blocked by the heterologous expression of Y.
enterocolitica O:3 outer core (IV)
To further characterize which outer core sugar residues are needed for the φA1122
adsorption, the potential of Y. enterocolitica serotype O:3 O-antigen and outer core
to inhibit the phage binding was studied. The heterologous expression of YeO3 Oantigen on Y. pestis had no effect on φA1122 adsorption, in contrast to outer core
which blocked it completely (IV, Figure 6). When expressing the YeO3 outer core
in BP1Δwb, two clones with different expression levels were obtained (IV, Figure
7). Of these, the strain expressing the outer core at higher level was completely
resistant to φA1122, whereas the less expressing strain adsorbed the phage
moderately and was infected with it (IV, Figure 8, not shown).
The expression of YeO3 outer core thus inhibited the phage adsorption to both Y.
pestis and Y. pseudotuberculosis. In the future, the LPS structure of the overexpressing strain BP1Δwb/pRV16NP#1 will be determined. This will reveal the
sugar residues blocked by the outer core, thus indicating the residues required for
the phage attachment.
5. 4. Reporter phages
The starting point of this PhD project was to construct a reporter bacteriophage by
inserting the luciferase gene from Photinus pyralis (firefly) to the genome of
45
φYeO3-12 and to use this reporter in the diagnostics of Y. enterocolitica O:3. In the
course of the work, several strategies were utilized to achieve this objective, the
most extensively studied examples of which are presented below.
5.4.1. Construction of the reporter phage by homologous recombination
The phage lysates resulting from homologous recombination between φYeO3-12
and plasmids pMP100, pMP200 and pSK110 (Table 5) contained phages having
lucFF in their genome. This was shown by the production of luminescence during
infection of the host cells (not shown). However, it was not possible to isolate the
luminescent phages, possibly because of the small number and slower growth of
the recombinant phages compared to the wild type φYeO3-12.
5.4.2. Phages φ::lucFF1.2 and φ::lucFF5.3
Phage φYeO3-12 derivatives φ::lacZ1 and φ::lacZ5 (Table 3) were chosen as
starting points for constructing the luciferase reporters due to their stability, their
ability to grow well on YeO3-c and the intensive blue color that was formed
around the plaques on indicator plates (II). It was thought that in these mutants,
lacZ’ was inserted in such a genomic location where the insert did not impede the
essential funcions of the phage and was expressed with high efficiency.
The resulting phages φ::lucFF1.2 and φ::lucFF5.3 (Table 3) produced luminescence during infection of YeO3-c (not shown). However, these mutants grew
poorly on YeO3-c, as indicated by their very small plaque size on top-agar plates.
In addition, the luminescent phenotype of the mutants disappeared during
sequential plaque purifications, indicating highly unstable nature. The PCR
analysis of the genomic regions containing the inserts confirmed that the lucFF
gene was rapidly lost from the genomes of both of these mutants. Since the lacZ’
mutants were stable but the corresponding lucFF mutants were not, it was thought
that the insertion of the longer lucFF gene (1.7 kb, compared to 0.45 kb for lacZ’)
resulted to genome that was too long to be packaged efficiently, thus leading to
instability.
5.4.3. Phage φ::lucFF1
To avoid problems in packaging the elongated genome of the reporter phage, a
strategy where the luciferase gene was inserted in the genome of the deletion
mutant φΔ888-2449 by MuA catalysed in vitro transposon mutagenesis was
followed. After screening 113 candidates, one light-producing mutant was found
and named φ::lucFF1. In this reporter, lucFF was inserted between the phage genes
6.3 and 6.5 (Table 3).
To measure the sensitivity of φ::lucFF1 in Y. enterocolitica detection, dilutions of
YeO3-c culture were infected with different PFU of the reporter phage and the light
production was followed at fixed time intervals. An example of these
measurements is shown in Figure 2.
46
6000
5000
RLU
4000
3000
2000
1000
0
0
20
40
60
80
Time/min
Figure 2. The sensitivity of φ::lucFF1 reporter phage in Y. enterocolitica serotype O:3 detection.
YeO3-c dilutions were infected with φ::lucFF1 and the light production was followed with
luminometer. YeO3-c CFU per sample was 1.2 ×107 („), 5.9 ×106 (●), 1.2 ×106 (○), 5.9 ×105 (▼)
and 2.4 ×105 (◊). In each sample, there was 2.8 ×107 PFU φ::lucFF1. RLU, relative light units.
As the outcome of the sensitivity measurements made, the detection limit of
φ::lucFF1 settled to ca. 5×105 CFU. It thus seems that the sensitivity of this
reporter is not as high as reported for other reporter phages (see Chapter 2.3.2.) and
is not good enough for delicate diagnostic purposes. Moreover, the stability studies
indicated that the luciferase gene is lost from the φ::lucFF1 genome with
substantial ease (not shown).
To conclude, all the attempts to construct a reporter phage from φYeO3-12 resulted
in unstable mutants that rapidly lost the inserted luciferase gene. Even though the
sensitivity of the present reporters might be improved by e.g. adding a strong phage
promoter before the lucFF gene, it is unlikely to obtain a reporter phage that is
stable enough for large-scale production.
47
6. SUMMARY
φYeO3-12 is a member of Podoviridae that belongs to the T7-group of phages, T3
being its closest relative. The phage has a strict host specificity to Y. enterocolitica
serotype O:3 (YeO3). The specificity is determined by the interaction between the
tail fiber adhesin and LPS O-antigen, and the phage is able to infect other
Enterobacteriaceae carrying the YeO3 O-antigen gene cluster. This work
characterized the basic biological properties of φYeO3-12 and identified nonessential regions in the phage genome. The phage genes coding for DNA ligase and
lysozyme were shown to be needed for growth on YeO3 but not on E. coli,
indicating that they are evolutionary factors contributing to the adaptation of
φYeO3-12 to utilize Y. enterocolitica as its host.
The information about the non-essential regions of the φYeO3-12 genome was
exploited in the construction of a reporter phage that could be exploited in Yersinia
diagnostics. The obtained reporter phages were, however, too unstable for largescale production and utilization.
φR1-37 is a Y. enterocolitica O:3 specific phage that uses LPS outer core as its
receptor. In this work, φR1-37 was shown to be a large member of Myoviridae. The
phage genome is 270 kb dsDNA molecule, in which thymidine is replaced by
deoxyuridine. The only other organisms known to have such an exceptional DNA
are B. subtilis phages PBS1 and PBS2. The N-terminal amino acid sequences of the
φR1-37 structural proteins revealed no significant similarities to known viral
proteins, indicating that close relatives of φR1-37 have not been characterized.
Interestingly, the deduced protein sequence of the partially sequenced φR1-37
genome showed similarity to four open reading frames of P. aeruginosa phage
φKZ. This is a member of Myoviridae with a size comparable to that of φR1-37.
φA1122 is a T7-related bacteriophage that infects all but two clinical isolates of Y.
pestis. It is used as the reference phage in the Y. pestis diagnostics by the Centers
for Disease Control and Prevention. However, the receptor that the phage
recognizes on the cell surface was not known. In this work, the LPS core of Y.
pestis and Y. pseudotuberculosis was identified as the φA1122 receptor.
To conclude, this thesis provides new information about the biology and genetics
of three bacteriophages infecting the genus Yersinia. A special attention was paid
in the understanding the phage-host recognition and other factors determining the
host specificity. In addition, the utilization of φYeO3-12 in bacterial diagnostics
was evaluated.
48
ACKNOWLEDGEMENTS
49
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