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Research Collection
Doctoral Thesis
Specific receptor recognition and cell wall hydrolysis bybacteriophage structural proteins
Author(s): Bielmann, Regula
Publication Date: 2009
Permanent Link: https://doi.org/10.3929/ethz-a-005783673
Rights / License: In Copyright - Non-Commercial Use Permitted
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ETH Library
Diss. ETH No 18255
Specific Receptor Recognition and Cell Wall Hydrolysis by
Bacteriophage Structural Proteins
A dissertation submitted to ETH Zurich
for the degree of Doctor of Sciences
presented by
Regula Bielmann Dipl. Natw. ETH
born September 29, 1978 from Rechthalten (FR)
accepted on the recommendation of
Prof. Dr. Martin Loessner, examiner Prof. Dr. Herbert Schmidt, co-examiner
2009
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I
Table of contents
Table of contents ................................................................................................. I
Abbreviations ..................................................................................................... III
Summary..............................................................................................................V
Zusammenfassung ...........................................................................................VII
1. Introduction .............................................................................................. 1
1.1. Listeria ................................................................................................................... 1 1.1.1. Listeria: History, taxonomy, ecology, and growth factors ...................... 1 1.1.2. Listeria monocytogenes – the causative agent of listeriosis.................. 3 1.1.2. Virulence of Listeria: Intracellular infection cycle ................................... 4
1.2. Bacteriophages...................................................................................................... 6 1.2.1. History, taxonomy, and morphology of bacteriophages......................... 6 1.2.2. Phage life cycle...................................................................................... 7 1.2.3. Listeria phages and their application ................................................... 12 1.2.4. Research objectives ............................................................................ 16
2. Material and Methods............................................................................. 17
2.1. Bacterial strains, growth conditions, phage propagation, and phage purification 17
2.2. Molecular cloning................................................................................................. 20 2.2.1. Constructs for recombinant protein expression ................................... 20 2.2.2. Construction of deletion mutant Listeria phages.................................. 20
2.3. Proteomics........................................................................................................... 23 2.3.1. Protein expression and purification...................................................... 23 2.3.2. Polyclonal rabbit-antibodies................................................................. 24 2.3.3. Mass spectrometry .............................................................................. 25 2.3.4. SDS-PAGE, Western blot analysis, visualization of lytic phage
proteins by zymography, and 2D-gel electrophoresis ......................... 25
2.4. Assays ................................................................................................................. 27 2.4.1. Binding assays..................................................................................... 27 2.4.2. “Pull-down” assay ................................................................................ 27 2.4.3. Transmission electron microscopy (TEM) ........................................... 28
2.5. Bioinformatics ...................................................................................................... 28
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II
3. Results .................................................................................................... 29
3.1. Proteomics of different Listeria phages. .............................................................. 29 3.1.1. Profiles of three temperate phages A118, A500, A006 and three
virulent phages P40, P35, and A511................................................... 29 3.1.2. Programmed translational frameshifting in A118 and A500 ................ 32
3.2. Identification of the lytic structural protein (LSP) ................................................. 40 3.2.1. LSP: a common element among Listeria phages ................................ 40 3.2.2. Identification of gp19 as the lytic structural protein (LSP) in A118 ...... 44
3.3. Topological model of the A118 tail tip.................................................................. 46 3.3.1. Antibodies against putative tail and baseplate proteins of A118 ......... 46 3.3.2. Gp18, gp19, and gp20 of A118 play an important role in the early
steps of infection ................................................................................. 46 3.3.3. Transmission electron microscopy (TEM) analysis of Listeria phage
A118.................................................................................................... 49
3.4. Identification of the receptor binding protein (RBP) ............................................. 53 3.4.1. Gp20 of A118 and A500 binds to Listeria cell walls............................. 53 3.4.2. The A118 RBP requires N-acetylglucosamine and rhamnose for
binding................................................................................................. 56
4. Discussion .............................................................................................. 59
5. References.............................................................................................. 67
Publications....................................................................................................... 85
Danksagung ...................................................................................................... 87
Curriculum Vitae ............................................................................................... 89
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III
Abbreviations
aa Amino acid
ATCC American Type Culture Collection
bp Base pairs
CBD Cell wall binding domain
cfu Colony forming units
Cps/ Cps-L Major Capsid protein/ Major Capsid protein-Long
CsCl Cesium Chloride
DNA Deoxyribonucleic acid
ds double stranded
DTT Dithiothreitol
E. coli Escherichia coli
GFP Green fluorescence protein
GlcNAc N-Acetylglucosamine
HCCA hydroxy-alpha-cyanocinnamic acid
ICTV International Committee on Taxonomy of Viruses
IEF Isoelectric focusing
InlA InternalinA
InlB InternalinB
IPTG Isopropyl-β-D-thiogalactopyranosid
kDa kilo Dalton
LB Luria Bertani
LLO Listeriolysin-O
L. monocytogenes Listeria monocytogenes
LSP Lytic structural protein
MW molecular weight
NAM N-Acetylmuramic acid
OD Optical density
ORF Open reading frame
pfu Plaque forming units
RBP Receptor binding protein
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IV
PCR Polymerase chain reaction
PEG Polyethyleneglycol
PlcA Phospholipase C
Ply Phage lysin
PVDF Polyvinylidenfluorid
Rha Rhamnose
RNA Ribonucleic acid
SDS-PAGE Sodium-dodecylsulfate-polyacrylamide-gelelectrophoresis
SLCC Special Listeria Culture Collection
SV Serovar
tal Tail associated lysin
TB Tryptose broth
TBS Tris buffered saline
TEM Transmission electron microscopy
TFA Trifluoro acetic acid
Tmp Tape measure protein
Tris Tris[hydromethyl]aminomethan
Tsh/ Tsh-L Tail sheet protein/Tail sheet protein-Long
WSLC Weihenstephan Listeria Collection
Wt Wildtype
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V
Summary
Adsorption of a bacteriophage to the cell wall of the bacterial host requires
recognition of a cell wall associated receptor by the phage receptor binding
protein (RBP). This recognition event is extremely specific, and high affinity
binding is important for rapid and efficient virus attachment. After adsorption, the
phage-DNA is injected to the host cytoplasm, which requires penetration through
the multilayered peptidoglycan of the Gram-positive Listeria cell wall. For this
purpose, a lytic structural protein (LSP) locally digests the murein during the
infection process. Little is known about the receptor binding and DNA delivery
during the early steps of phage infection of Gram-positive bacteria.
Listeria phage A118 was isolated from Listeria monocytogenes serovar (SV) 1/2.
It features a non-contractile tail of approximately 300 nm in length, an isometric
capsid with a diameter of 61 nm, and belongs to the Siphoviridae family of dsDNA
bacterial viruses, in the order Caudovirales (B1 morphotype). The phage adsorbs
to the SV-specific L-rhamnose and N-acetylglucosamine substituents in the cell-
wall teichoic acids of host cell. Listeria phage A500 exhibits a non-overlapping
and complementary host-range, infecting L. monocytogenes SV 4b. Although the
host range of the two phages is different, they share significant sequence
similarities in the predicted gene products of the late gene cluster.
The identification of the RBP in phages A118 and A500 is reported here. Specific
binding of GFP-RBP fusion proteins to the listerial cells could be demonstrated.
Binding of truncated versions of the putative RBPs suggested that the binding
specificity of the RBP resides in the C-terminal part. Furthermore, the receptor on
the host cell could be identified for the RBP of A118.
It was shown by zymograms that lytic structural proteins are present in all tested
Listeria phages. Whereas the tested temperate phages (A118, A500, and PSA)
revealed a single lytically active band, located directly below the prominent major
capsid protein (Cps), the virulent phage A511 demonstrated two lytically active
bands of different sizes. Peptide fingerprinting and Western blot analysis of the
zone responsible for lytic activity enabled assignment of a lytic activity to a
baseplate protein (LSP) of A118.
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VI
Finally, application of antibodies against several baseplate proteins and
transmission electron micrography (TEM) enabled the proposition of a model of
the A118 tail tip with the arrangement of both RBP and LSP within the baseplate.
This thesis work provides answers to fundamental questions about the biology of
Listeria bacteriophages and will also be useful to develop novel and effective tools
for specific recognition and control of the foodborne pathogen L. monocytogenes.
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VII
Zusammenfassung
Bakteriophagen benötigen zur Adsorption an die Zellwand einer Wirtszelle ein
Protein, welches den Rezeptor erkennt und bindet. Es wird angenommen, dass
dieses Rezeptorbindeprotein (RBP) hoch affin und extrem spezifisch bindet, denn
genau diese Eigenschaften sind wichtig für ein effizientes und schnelles Anhaften
des Virus an die Wirtszelle zu Beginn des Infektionsprozesses.
Das Genom des Phagen wird nach Adsorption an die Zelle in das Cytosol des
Wirts eingeschleust. Hierfür muss die Gram-positive Zellwand der Listerienzelle
durchdrungen werden. Um dies zu bewerkstelligen, besitzt der Phage ein
lytisches Strukturprotein (LSP), welches während des Infektionsprozesses lokal
das Peptidoglykan lysiert. Bislang ist jedoch über die frühen Schritte der
Phageninfektion in Gram-posiviten Bakterien bislang wenig bekannt.
Der Phage A118 wurde aus Listeria monocytogenes Serovar (SV) 1/2 isoliert. Er
hat einen nicht-kontraktilen, ca. 300 nm langen Schwanz und einen
ikosaederförmigen Kopf mit einem Durchmesser von 61 nm und gehört zur
Familie der Siphoviridae von dsDNA-Viren in der Ordnung der Caudovirales (B1
Morphotyp). Der Phage adsorbiert an SV-spezifische Kohlenhydrat-Reste (L-
Rhamnose und N-Acetylglukosamin), die sich in den zellwandassoziierten
Teichonsäuren der Wirtszelle befinden. Ein komplementäres und nicht
überlappendes Wirtsspektrum zu A118 weist Phage A500 auf, der
L. monocytogenes SV 4b infiziert. Obschon das Wirtsspektum von A118 und
A500 unterschiedlich ist, sind sich die beiden Phagen in den späten Genen doch
sehr ähnlich.
Diese Arbeit beschreibt die Identifizierung der rezeporbindenden Proteine (RBP)
der Phagen A118 und A500. Es konnte gezeigt werden, dass GFP-RBP
Fusionsproteine spezifisch an Listerienzellen binden. Durch Bindung von
verkürzten GFP-RBP an die Zellen konnte weiter gezeigt werden, dass die
Spezifität der Bindung im C-Terminus des RBPs liegt. Zusätzlich konnte für das
rezeptorbindende Protein von A118 der Zelloberflächen-Rezeptor identifiziert
werden.
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VIII
Lytische Strukturproteine (LSP) wurden mit Hilfe von Zymogrammen in allen
getesteten Listeria Phagen gefunden. Während drei der untersuchten
temperenten Phagen (A118, A500 und PSA) nur eine lytisch aktive Bande
zeigten, wurden im virulenten Phagen A511 zwei lytisch aktive Banden
unterschiedlicher Grösse gefunden. Gelstücke, die das lytische Protein enthalten,
wurden mittels Peptide Fingerprint und Western blot analysiert. Dies ermöglichte
eine genaue Zuordnung des LSP zu einem Strukturprotein von A118.
Um die Proteine im Phagenpartikel zu lokalisieren wurden Antikörper, die gegen
verschiedene Proteine der Basalplatte aus A118 gerichtet waren, eingesetzt. Mit
Hilfe von Transelektronenmikroskopiebildern gelang es schliesslich ein Modell für
das Schwanzende und die Basalplatte von A118 zu erstellen, wo sowohl das RBP
und das LSP darin zugeordnet werden konnten.
In dieser Arbeit wurden fundamentale Aspekte der Biologie von Listeria-
Bakteriophagen betrachtet. Sie liefert eine Grundlage für die Ausarbeitung von
neuen und effizienten Werkzeugen für eine spezifische Erkennung und Kontrolle
des Krankheitserregers L. monocytogenes.
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1
1. Introduction
1.1. Listeria
1.1.1. Listeria: History, taxonomy, ecology, and growth factors
Listeria was described more than 80 years ago by E.G.D. Murray and J. Pirie,
independently of each other (93, 101). Both named the newly described bacterium
differently. Due to the observation of a characteristic monocytosis in laboratory
rabbits and guinea pigs, Murray named it “Bacterium monocytogenes”. Pirie on
the other hand termed it “Listerella hepatolytica” as he isolated the organism from
veld rodents from South Africa. Because of the identity of the organism, the
current name Listeria monocytogenes was then given in 1940 (100).
The genus Listeria belongs to the phylum Firmicutes in the order Bacilliales and is
closely related to the genera Brochothrix, Bacillus, Staphylococcus,
Streptococcus, Enterococcus and Clostridium (54, 105). They share the
characteristic feature of a G+C-content less than 50% (105, 130).
In recent years, the taxonomic position of Listeria species has been the subject of
much work and debate. The ninth edition of Bergey`s Manual of Systematic
Bacteriology (118) recognized five biochemically distinguishable species, namely
L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri and L. ivanovii, whilst
L. denitrificans, L. grayi and L. murrayi are listed as species incertae sedis.
Recently, the genus Listeria was divided into 6 species: L. monocytogenes,
L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi (105, 115). L. grayi
and L. murrayi are considered not to be sufficiently different from each other and
were merged in one species L. grayi with two subspecies L. grayi ssp. grayi and
L. grayi ssp. murrayi (106). Further, the species L. ivanovii was divided into
L. ivanovii ssp. ivanovii and L. ivanovii ssp. londoniensis (10).
According to the pattern of somatic (O) and flagellar (H) antigens, a total of
17 serovars are known. L. monocytogenes is represented by 13 serovars (1/2a,
1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7) (37), some of which are
shared by L. innocua and L. seeligeri (2).
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2
Bacteria of the genus Listeria are Gram-positive, short (0.4-0.5 x 0.5-2.0 µm),
non-sporeforming rods that are motile at 20-25 °C by means of peritrichous
flagella that provide a tumbling motility (103). On the other hand, at 37 °C they are
not motile, as expression of the flagella is temperature dependent (98). Colonies
of Listeria appear in a characteristic bluish color when illuminated by indirectly
transmitted light (51).
They are aerobic and facultative anaerobic. The temperature limits of growth are
1-2 °C to 45 °C (55), the optimum temperature is between 30-37 °C. Listeria can
tolerate high salt concentrations (up to 10% NaCl) and survive low pH (pH 4.5);
growth is optimally at pH 7 (118). These properties enable them to survive under
extreme conditions. Therefore, it is not surprising that Listeria is widespread in
nature. The bacteria have been isolated from many different environments,
including soil, water, vegetation, sewage, animal feeds, farm environments and
food-processing environments (38, 109, 133, 136-138).
Fig. 1. Transmission electron micrograph of L. monocytogenes (this work).
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3
1.1.2. Listeria monocytogenes – the causative agent of listeriosis
All of the different Listeria species are widespread in the environment, but only
Listeria monocytogenes is considered to be a significant human pathogen.
However, occasional human infections due to L. seeligeri or L ivanovii have also
been reported (21, 53, 88, 108). L. ivanovii is responsible for some cases of
abortion, and L. seeligeri is generally considered nonpathogenic (130). There are
13 serotypes of L. monocytogenes, but almost all clinical cases are due to types
4b, 1/2a, and 1/2b (115). They have been isolated from a broad variety of foods
like milk, cheese (especially soft cheeses) and other diary products, meat and
meat products, poultry and eggs, fish, fish products and seafood, raw vegetables,
and salad (37). Transfer of the organism to the food occurs mostly by secondary
contamination. At high risk are in general ready-to-eat products that are
consumed without final heat treatment.
The disease caused by L. monocytogenes is called listeriosis. Human listeriosis is
typically acquired through ingestion of contaminated food, but other modes of
transmission occur. These include transmission from mother to child
transplacentally or through an infected birth canal and crossinfection in neonatal
nurseries. Human-to-human infections have not been documented (18, 115).
Human disease caused by L. monocytogenes occurs most frequently in women of
childbearing age, infants, and the elderly. The risk of listeriosis is greatest among
certain well defined high-risk groups, including pregnant women, neonates, and
immunocompromised adults but may occasionally occur in persons who have no
predisposing underlying conditions (37).
Unlike infection with other common foodborne pathogens such as Salmonella,
which rarely result in fatalities, listeriosis is associated with a mortality rate of
approximately 30% (41). At least two different forms can appear, mainly
depending on susceptibility of the patient: non-invasive, gastrointestinal infections
usually occur in healthy, immunocompetent people, and are characterized by mild
symptoms such as fever, vomiting, and diarrhea. Whereas invasive infections
mainly affect persons belonging to one of the risk groups and are associated with
severe symptoms such as meningitis or encephalitis, generalized bacteremia or
septicemia, endocarditis, myocarditis or pneumonia (37, 40, 115, 130).
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4
Pregnant women infected with L. monocytogenes only develop fever-like
symptoms, but the fetus can be infected via the placenta, which can result in
abortion, stillbirth or generalized infection of the newborn (37, 115, 130).
1.1.2. Virulence of Listeria: Intracellular infection cycle
Internalization of the bacterium begins with the adhesion to the eukaryotic cell and
subsequent penetration into the host cell. Invasion of nonphagocytic cells involves
a zipper-type mechanism where the host cell surface surrounds the bacterium
until it is complete engulfed. This internalization process differs to the membrane
ruffles characteristic of invasion by Salmonella and Shigella (29, 56, 65, 124). The
infection process has been studied extensively in cells in tissue culture (19). The
entry of Listeria into mammalian cells is triggered by at least two surface proteins
belonging to the internalin family: internalins InlA and InlB (20). The completion of
the Listeria genome sequence revealed a large number of surface proteins, so
that additional bacterial factors are probably involved in the uptake (13, 43). After
phagocytosis, the bacterium is enclosed in a subcellular phagolysosom, a hostile
and toxic environment for most bacteria. The low pH within this organelle
activates listeriolysin-O (LLO), a pore-forming cytolysin that, together with
phospholipase C (PlcA), lyses the membrane and allows L. monocytogenes to
escape into the cytoplasm. The LLO is, in contrast to other members of the same
family of toxins such as streptolysin-O or perfringolysin, optimally active at pH 5.5-
6.0 (corresponding to the internal pH of the vacuole) and less active at higher pHs
(30). This lysis of the vacuole occurs about 30 min after infection. The bacterium
is then able to multiply within the cytosol and through actin-based intracellular
motility it is able to invade the neighboring cell. This spreading from cell to cell is
also mediated by virulence factor ActA. During this process a two-membrane
vacuole is formed, where the bacterium is again released into the cytosol, with
help of LLO and another phospholipase C (PlcB) (130).
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5
Invasion (InlA, InlB)
Escape fromphagolysosom(LLO & PlcA))
Actin recruitmentand replication
Polymerized actin-Polymerization (ActA)
Cell-to-cell spread(Listeriapods)
Lysis of two-membrane vacuole
(LLO, PlcB)
Fig. 2. Infection cycle of L. monocytogenes in host cells (Modified from Tilney et al. 1989
(126))
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6
1.2. Bacteriophages
1.2.1. History, taxonomy, and morphology of bacteriophages
Bacteriophages are viruses that infect bacteria. The name bacteriophage is
derived from bacteria and Greek phagein “to eat”. The history of bacteriophages
started in 1915 when F. W. Twort observed a “glassy transformation” within a
layer of bacterial cocci, which could be induced in colonies of normal appearance
after inoculating with substance of such “glassy” colonies (99). Two years later,
F. d`Herelle described independently a similar phenomenon that was
“antagonistic” to bacteria and that resulted in lysis in liquid culture and death in
discrete patches, that he called plaques (22). He was interested in their biological
nature and claimed the idea of an organized infection agent that is an obligate
intracellular parasite. It was also d`Herelle who proposed this culture as a
therapeutic agent in the preantibiotic era. Early studies dealt with the use of
phages for the control of epidemics but the interest in phage therapy diminished
after the invention of antibiotics in the forties. Nevertheless, phage therapy
continued to be investigated extensively especially in the republic of former Soviet
Union. Today, the use of phages as antimicrobial agent regained attention, as the
increasing antibiotic resistances becomes a serious problem. Besides this,
phages have become important model organisms for molecular biology and tools
for application (see chapter 1.2.3. (123)).
The classification of bacteriophages goes back to Bradley (11, 12). Phages are
divided into 6 groups based on the morphological differences and their differences
in nucleic acids (single and double stranded DNA or RNA). These criteria are still
the basis for the phage classification. According to the International Committee on
Taxonomy of Viruses (ICTV), bacteriophages are classified in one order, 13
families, and 30 genera. At least 96% of all known bacteriophages are tailed and
belong to the order Caudovirales. They are subdivided into 3 families, the
Myoviridae (25%), phages with a contractile tail, the Siphoviridae (61%) with a
non-contractile, flexible tail, and the Podoviridae (14%) with a non-contractile,
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7
short tail. Their genetic material is double stranded DNA (62). The remaining 4%
of the bacteriophage virions are polyhedral, filamentous, or pleomorphic, and a
few have lipid-containing envelopes (Fig. 3) (1).
Filamentous (<4%)
Tailed (96%) Polyhedral (<4%)
Pleomorphic (<4%)
Filamentous (<4%)
Tailed (96%) Polyhedral (<4%)
Pleomorphic (<4%)
Fig. 3. Basic bacteriophage morphotypes (Modified from Ackermann, 2003 (1))
1.2.2. Phage life cycle
Phages are obligate intracellular parasites and multiply within their host. At the
end of the infection cycle they destroy their host cell, except for some filamentous
phages which can cause chronic infections and are therefore constantly released
from the host cell by forming protrusion without destroying the cell (1, 62). Among
the remaining phages two phage life cycle types are distinguished, dividing the
tailed phages into virulent phages and temperate phages. Both types are able to
perform the lytic life cycle but temperate phages are further able to integrate their
genome into the host at specific attachment sites and persist in a prophage stage.
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8
Under particular circumstances they reenter the lytic life cycle by excision of their
genome and replicate within their host.
In both cases, the initial step of phage infection is adsorption to and recognition of
the host. This occurs after a random collision between the host and phage which
is initially non-specific (91). This collision is followed by a specific recognition and
attachment of specialized adsorption structures on the phage, for example tail
fibers or spikes that recognize the receptor on the host cell surface. In Gram-
negative bacteria any surface protein, oligosaccharides, and lipopolysaccharides
can serve as receptor, whereas the situation in Gram-positive bacteria is slightly
different. The more complex peptidoglycan layer, also known as murein, of Gram-
positive bacteria offers a different set of potential binding sites. It consists of
alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (NAM)
residues, linked to each other by peptide cross-bridges between NAM residues
(26, 52). Many phages require additional cofactors for adsorption, such as Ca2+,
Mg2+ or other divalent cations. This attachment of the virus particle requires a
phage receptor binding protein (RBP). This recognition event of the RBP is
extremely specific, and high affinity binding is important for rapid and efficient
virus attachment. This interaction is the underlying principle of phage typing (76).
Most of the information about this interaction in double-stranded DNA phages
stems from research on T-even and lambdoid phages infecting E. coli (47, 48,
135). In contrast, very little is known about the infection process for phages
infecting Gram-positive bacteria. Nevertheless some phages have been
investigated more intensively. In recent years, the genes encoding for RBPs of
Streptococcus thermophilus phages DT1 and MD4, Bacillus subtilis phage phi29,
Lactococcus lactis phages bIL67 and CHL92 of the c2 species, sk1, bIL170, and
p2 of the 936 species, and TP901-1 and Tuc2009 belonging to the P335 species,
have been identified (23, 32, 33, 45, 117, 120, 122, 132).
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9
After the irreversible attachment, the phage genome passes through the tail into
the host cell. Gram-negative bacteria have cell envelopes consisting of an inner
and outer membrane and periplasmic space, in which a peptidoglycan layer is
located. Peptidoglycan is considered to be responsible for the mechanical integrity
of the cell and limits diffusion processes of macromolecules and hence, the major
barrier for phage genome passage (25). The cell envelope of Gram-positive
bacteria only consists of an inner membrane and a thick multilayered cover of
peptidoglycan. These crucial differences of cell wall architecture between Gram-
positive and Gram-negative bacteria must result in distinct infection strategies of
phages. For phages infecting Gram-negative bacteria the infection process is well
understood (4, 42, 68, 71, 92). For Gram-positive bacteriophages, similar
structural proteins with cell wall degrading activity have been identified (61, 125).
Lactococcal phage Tuc2009 was shown to have a tail associated structural
component with cell wall-degrading activity (Tal2009 = tail associated lysin of
Fig. 4. Schematic representation of the lytic life cycle of tailed bacteriophages. Infection
process starts with the specific recognition and the attachment to the host cell. The
phage then ejects its DNA into the host cytoplasm where replication occurs. Phage
progeny are then newly assembled before they are released by lysis of the host.
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10
phage Tuc2009) (61). The protein was identified as gp50, which exhibits high
similarity (45% identity) to the known lytic protein Zoocin A from
Streptococcus equi ssp. zooepidemicus. In addition the C-terminal portion of
Tal2009 is a member of M37 peptidase family that includes endopeptidases that
target the interpeptide bridge of the peptidoglycan layer. It was shown that gp50
undergoes autocatalytic processing at a glycine-rich domain after translation (87).
Transelectron microscopy of immuno-gold-labeled Tuc2009 phages demonstrated
that the lytic structural protein is located at the tail tip of the phage. Based on
these results a putative model of the tail tip was proposed (Fig. 5) (87). TP-901, a
phage related to Tuc2009, also features a tail-associated lytic protein. Likewise, a
virion protein of Staphylococcus aureus phage P68 was shown to exhibit muralytic
activity (61, 90).
Such a virion enzyme that locally degrades the cell wall from the outside is
believed to be common for most dsDNA phages (68, 90). The lytic activity of this
protein is responsible for the “lysis from without” phenomenon, which is the
phenotypic result of adsorption of many phage particles, leading to sudden lysis of
the host cell, without infection of production of viral progeny (24). This lysis differ
to the “lysis from within”, taking place at the end of the infection cycle in order to
release the phage progenies (125).
Upon peptidoglycan degradation, the phage genome is transferred to the host
cell. Once the DNA is in the host cell, strong phage promoters lead to the
transcription of the immediate early genes and the transition from host to phage-
directed metabolism takes place. Products of these genes may protect the phage
genome against modifications or degradation and inhibit host proteins. Further, a
set of middle genes, involved in DNA-replication, is transcribed followed by the
expression of the late genes that are responsible for the structural phage proteins.
Phage particles are then assembled and the replicated genomes are packed into
preassembled icosahedral protein shells that are called procapsids. In most
phages the assembly involves the interaction between specific scaffolding
proteins and the major head structural proteins (Cps). The head expands during
packaging and becomes more stable. At one vertex a portal complex serves as
starting point for head assembly, the docking site for the DNA packaging
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11
enzymes, a conduit for the passage of DNA, and, for Myoviruses and
Siphoviruses, a binding site for the phage tail. The tail on the other side is
assembled separately. The Siphoviridae build up an initiator complex to which one
or more fibers may be attached. The tail’s precisely defined length is determined
Tal2009
RBP (BppL)
Tsh Tmp
Dit
BppA
BppU
Fig. 5. Detail of proposed protein architecture of bacteriophage Tuc2009 tail adsorption
apparatus. Identified proteins are indicated by arrows.Tal2009: tail associated lysozyme;
RBP: Receptor binding protein; Tsh: Tail sheet protein; Tmp: Tape measure protein; Dit,
BppA, BppU: other baseplate proteins. (Modified from Mc Grath et al. (87)).
_________________________________________________________________________
12
by the tail tape measure protein and the completed tail is stabilized by a
terminator protein, which interacts with the completed head. For Myoviridae, the
many components of the contractile tail and the baseplate are also assembled in
a highly ordered fashion (9, 49, 58, 59).
As the final step after assembly, the host cell is lysed in order to release the
phage progeny. This lysis is highly controlled and timed. Tailed phages almost
always use two components: a muralytic enzyme, called lysin or endolysin, which
is capable to cleave the peptidoglycan and a holin, a protein which form pores into
the plasma membrane. These lesions in the membrane allow the endolysins to
access their substrate in order to digest the peptidoglycan, followed by lysis of the
host cell (134, 142).
1.2.3. Listeria phages and their application
Bacteriophages have been applied as therapeutic agents against bacterial
infections in humans and animals. F. d`Herelle realized as first the promising
potential of phage therapy in medicine (31). Phages specifically destroy the
pathogenic agents in human diseases and may contribute to healing. Especially in
recent times when multi-antibiotic resistance is emerging, phages may become a
valuable alternative for treating infectious bacterial diseases (94).
Bacteriophages infecting the genus Listeria were first reported by Schultz in 1945
(116). Until today more than 400 phages were isolated and partly characterized.
These phages infect all different Listeria species except L. grayi, where currently
no infecting phage could be found. Examination of more than 120 Listeria phages
by transelectron microscopy demonstrated a relatively limited diversity. Most of
the phages infecting Listeria were shown to belong to the Siphoviridae family
morphotype B1 (isometric capsid, long non-contractile tail). The remaining phages
were classified as Myoviridae of morphotype A1 (isometric capsid, long,
contractile tail) (77). The genome size of Listeria phages range from 36 to more
than 100 kb. The G+C-content is 34.7 – 40.8 mol%, which corresponds to the
G+C-content of Listeria (63, 81, 107, 144). Further, they are well adapted to their
host and complete lytic cycles at temperatures from 10 °C to 37 °C (77).
_________________________________________________________________________
13
All known Listeria phages are strictly genus specific. The temperate phages
display a narrow host range, infecting bacteria of individual serovar groups, while
the virulent ones can attack strains of all species and serovars, displaying a broad
host range (5, 6, 76, 78, 102, 104). It has been demonstrated that the teichoic
acid substituents N-acetylglucosamine (GlcNAc) and rhamnose are major
determinants of phage adsorption in serovar (SV) 1/2 strains, while GlcNAc and
galactose are important in SV 4 strains (17, 127, 139). In contrast it is assumed
that the virulent phage A511, which is able to infect about 80% of all Listeria
strains, recognizes the peptidoglycan itself as receptor (139).
Listeria phage A118 was isolated from L. monocytogenes SV 1/2 (76). It features
a non-contractile tail of approximately 300 nm in length, an isometric capsid with
Fig. 6. Transmission electron micrograph of bacteriophage A118 infecting a listerial host
cell (This study).
_________________________________________________________________________
14
diameter of 61 nm (145), and belongs to the Siphoviridae family of dsDNA
bacterial viruses, in the order Caudovirales (B1 morphotype). A118 was the first
Listeria phage which was completely sequenced and analyzed in molecular detail
(80); and it represents the prototype of a temperate Listeria phage. The A118
genome contains 72 ORFs, organized in three major, life-cycle specific gene
clusters. Listeria phage A500 exhibits a non-overlapping and complementary
host-range, infecting L. monocytogenes SV 4b (82). Although the host range of
the two phages is different, they share significant sequence similarities in the
predicted gene products with respect to the late gene cluster.
Listeria phages and their components have found many practical applications, not
only as tools in the research laboratories. With respect to foods, the biological
specificity of these viruses can be used to detect and control Listeria. Virulent
Listeria phages were studied for control of L. monocytogenes during food
processing and storage. The use of lytic bacteriophages applied on food showed
significant reduction of bacterial populations by two to five log units of viable
bacteria cells (14, 44, 46, 72).
Due to their specificity, Listeria phages are useful for subtyping of Listeria strains
in epidemiological investigations concerning outbreaks of listeriosis. The
application benefits from the different host spectra of a set of bacteriophages,
resulting in distinct lysis patterns of the strains investigated (69, 76, 78, 146).
Evaluation of an improved phage set for Listeria typing revealed that about 90% of
all the strains tested are typable (128).
This specificity also makes bacteriophages appropriate agents for the detection of
viable Listeria contaminants in food. A prominent example is the construction of a
genetically engineered reporter phage A511::luxAB that expresses a bacterial
luciferase gene during infection and facilitates the detection of the infected
bacteria via measurement of emitted bioluminescence (76, 83, 84). This reporter
phage represents an appropriate agent for the detection of Listeria in foods. (83).
The utilization of this reporter phage in a reliable assay was proven for large scale
screening of L. monocytogenes in foods and environmental samples (84).
Single phage components that are recombinantly expressed can also be used to
improve control and detection of pathogenic host cells. In this context, purified
_________________________________________________________________________
15
endolysins can be used for rapid lysis of listerial cells. The C-terminal cell wall
binding domains (CBDs) of these phage endolysins can be fused to green
fluorescent protein (GFP) for specific detection of Listeria cells in mixed bacterial
populations (81, 114). Additional application of these CBDs includes also the
immobilization of host cells to solid surfaces. For example magnetic beads coated
with CBDs offer the opportunity to develop useful applications, such as recovering
Listeria cells from food samples (64).
Phage endolysins and virulent phages against Listeria exhibit a broad field of
possible applications in food science, in microbial diagnostics or for treatment of
experimental infections. They may also be applied in bio-disinfection of solid
surfaces and equipment in combination with common disinfectants as well as in
biocontrol of pathogenic organisms (73, 75).
_________________________________________________________________________
16
1.2.4. Research objectives
The major aim of this work is to obtain information on different virion associated
proteins. Specifically, the focus will be on the receptor binding proteins (RBP),
which are involved in attachment and host recognition, and the lytic structural
proteins (LSP) that possess cell wall hydrolytic activity.
Phage-encoded lytic proteins able to digest cell wall peptidoglycan recently
received much attention because of their possible uses as antimicrobial agents
against diverse pathogens. Investigations for the identification of the LSP include
activity-based zymogram assays with subsequent allocation to a putative gene
product.
Host recognition is a very specific event and high affinity binding is needed for
efficient phage attachment. The RBP is believed to bind specifically to listerial
host cells. The identification of putative RBPs will be investigated by binding
assays. For this, the putative RBPs are first fused to GFP, and the fusion proteins
are then analyzed for specific decoration of the host cells.
The RBP and the LSP are believed to represent parts of the baseplate. Analysis
of the A118 tail tip will be carried out with help of polyclonal antibodies directed
against putative baseplate proteins. Finally, transelectron microscopy will be
applied to gain more information on the prototype temperate Listeria phage A118.
_________________________________________________________________________
17
2. Material and Methods
2.1. Bacterial strains, growth conditions, phage propagation, and phage purification
E. coli strain XL1-Blue MRF’ (Stratagene, La Jolla, USA) was used for molecular
cloning and expression of recombinant proteins. E. coli strains were cultured in
Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.8% NaCl, pH 7.4)
at 37 °C. For overexpression of recombinant proteins the strains were grown at
30 °C, while for overexpression of GFP-fusion proteins, LB medium containing
0.5% NaCl was used. Plasmid selection was accomplished by addition of
ampicillin (100 μg/ml).
Listeria monocytogenes strain WSLC 1001, WSLC 1042, and WSLC 3009
(SV 1/2c, SV 4b, and SV 5) were used as strains for phage propagation, substrate
cells for zymograms, pull down, and binding assays. Strains 1/2a3 (SV 1/2) and
HLT 2 (SV 1/2) (127) and HLT 2/2 (SV 1/2) (S. Kathariou, personal
communication) were used for binding assays. Concentration of 10 µg/ml for
erythromycin and 1 mg/ml for streptomycin were used for the selection of Listeria
mutants. Listeria strains were grown in Tryptose Broth (TB) (Biolife) at 30 °C.
Bacterial strains used are listed in Table 2.1.
Phages A118 (80), A500 (27), P35 (27), P40 (28), and A006 (27) were
propagated in softagar overlay plates and purified by PEG precipitation and CsCl-
gradient centrifugation as described earlier (80, 145). Isolation and purification of
phage structural proteins was performed as described in Loessner et al. (82).
Phage A511 was propagated using liquid culture method and purified by PEG
precipitation and CsCl-gradient centrifugation as described previously (85, 111).
For protein analysis phages A118, A500, A006, P40, P35, and A511 were used.
Zymogram analysis were carried out with phages A118, A500, A511, and PSA
(144). Phages are listed in Table 2.2.
_________________________________________________________________________
18
Tabl
e 2.
1. B
acte
rial s
trai
ns.
Stra
inR
emar
ksSo
urce
or r
efer
ence
List
eria
mon
ocyt
ogen
es W
SLC
100
1S
V 1
/2c,
wild
type
ATC
C 1
9112
List
eria
mon
ocyt
ogen
es W
SLC
104
2S
V 4
b, w
ildty
peA
TCC
230
74
List
eria
ivan
ovii
WS
LC 3
009
SV
5, w
ildty
peS
LCC
476
9
List
eria
mon
ocyt
ogen
es 1
/2a3
Stre
ptom
ycin
-res
ista
nt d
eriv
ativ
e of
SLC
C 5
764
Tran
et a
l. 19
99
List
eria
mon
ocyt
ogen
es H
LT 2
L.m
onoc
ytog
enes
1/2
a3 Δ
Glc
NA
cTr
an e
t al.
1999
List
eria
mon
ocyt
ogen
es H
LT 2
/2L.
mon
ocyt
ogen
es 1
/2a3
ΔR
haS
. Kat
hario
u, p
erso
nal c
omm
unic
atio
n
List
eria
mon
ocyt
ogen
es W
SLC
100
1::A
118
WS
LC 1
001
lyso
geni
c fo
r A11
8 la
bora
tory
sto
ck
List
eria
mon
ocyt
ogen
es W
SLC
100
1::A
118Δ
18A
118
orf1
8 de
letio
n m
utan
t in
WSL
C 1
001
this
stu
dy
List
eria
mon
ocyt
ogen
es W
SLC
100
1::A
118Δ
19A
118
orf1
9 de
letio
n m
utan
t in
WSL
C 1
001
this
stu
dy
List
eria
mon
ocyt
ogen
es W
SLC
100
1::A
118Δ
20A
118
orf2
0 de
letio
n m
utan
t in
WSL
C 1
001
this
stu
dy
Esc
heric
hia
coli
XL1-
Blue
MR
F'
Ele
ctro
pora
tion-
com
pete
nt c
ells
Stra
tage
ne, L
a Jo
lla, U
SA
WS
LC =
Wei
hens
teph
an L
iste
ria C
olle
ctio
n, D
ATC
C =
Am
eric
an T
ype
Cul
ture
Col
lect
ion,
US
AS
LCC
= S
peci
al L
iste
ria C
ultu
re C
olle
ctio
n, D
_________________________________________________________________________
19
Tabl
e 2.
2. B
acte
rioph
ages
.Ph
age
A11
8A
500
PSA
A00
6P4
0P3
5A
511
Fam
il yS
ipho
virid
aeS
ipho
virid
aeS
ipho
virid
aeS
ipho
virid
aeS
ipho
virid
aeS
ipho
virid
aeM
yovi
ridae
Life
cyc
lete
mpe
rate
tem
pera
tete
mpe
rate
tem
pera
tevi
rule
ntvi
rule
ntvi
rule
ntM
orph
otyp
eB
1B
1B
1B
1B
1B
1A
1C
a psi
d di
amet
er61
nm
62 n
m61
nm
62 n
m56
nm
58 n
m80
nm
Tail
lent
gh30
0 nm
27
4 nm
180
nm28
0 nm
110
nm11
0 nm
180
nmG
enom
e si
ze40
834
bp38
867
bp37
618
bp38
124
bp35
638
bp35
822
bp13
4494
bp
Hos
t ran
gem
ainl
y S
V
1/2
stra
ins
mai
nly
SV
4b
, 6 s
train
s on
ly S
V 4
mai
nly
SV
1/2
st
rain
sm
ost s
train
s of
SV
4, 5
, 6;
~50
% o
f S
V 1
/2
stra
ins
~75
% o
f SV
1/
2 st
rain
s>
80%
of
stra
ins
of
all S
V
Ref
eren
ceLo
essn
er e
t al
. 200
0D
orsc
ht
2007
Zim
mer
et
al. 2
003
Dor
scht
200
7D
orsc
ht e
t al
., in
pr
epar
atio
n
Dor
scht
20
07K
lum
pp e
t al
. 200
8
Dat
abas
e ac
cess
ion
num
berA
J242
593
DQ
0036
37A
J312
240
DQ
0036
42E
U85
5793
DQ
0036
41D
Q00
3638
_________________________________________________________________________
20
2.2. Molecular cloning
2.2.1. Constructs for recombinant protein expression
The genes encoding gp16 (C-terminus), gp17 to gp21 of A118, and gp20 of A500
were amplified by PCR using purified phage DNA as template and a proofreading
polymerase (ProofStart, Qiagen). Primers contained suitable restriction sites for
cloning (Table 2.3). PCR products were digested with the appropriate restriction
enzyme BamHI, PstI, or SacI (Fermentas) and cloned into pQE-30 (Qiagen) or its
derivative pHGFP (81). After successful transformation of E. coli XL1-Blue MRF’
plasmids were reisolated (GenElute Plasmid Miniprep Kit, Sigma) and sequenced.
Same procedure was done for cloning of orf97 and orf102 of A511. Primers used
are listed in Table 2.3.
2.2.2. Construction of deletion mutant Listeria phages
In order to test the functionalities of gp18, gp19, and gp20 in A118, several
deletion mutant phages were created. Temperature dependent integration vector
pKSV7 (119) was used for the deletion of the corresponding genes within the
prophage genome of A118. Flanking regions of orf18, orf19, and orf20 of A118,
respectively were PCR amplified using splicing by overlap extension PCR (143).
The primers contained suitable restriction sites (Table 2.3). PCR products were
digested with the appropriate restriction enzyme BamHI or SacI (Fermentas) and
cloned into pKSV7. After successful transformation of E. coli XL1-Blue MRF’,
plasmids were reisolated (GenElute Plasmid Miniprep Kit, Sigma), sequenced,
and transformed into L. monocytogenes WSLC 1001::A118 carrying the prophage
A118. The plasmid was then forced to integrate into the genome by a temperature
shift to 41 °C. Excision of the plasmid was obtained by a temperature downshift.
The excision of the plasmid can lead to wt-situation but also to the allelic
exchange. Strains were UV induced for 3-4 min (254 nm, 220 µW/cm2) and after
4 h of incubation in the dark plated in serial dilutions on Listeria WSLC 1001 for
plaque formation. Phage lysates were further screened by PCR for deletion of the
corresponding genes. Primers X, Y, and Z listed in Table 2.3 were used.
_________________________________________________________________________
21
Tabl
e 2.
3. O
ligon
ucle
otid
e pr
imer
s us
ed fü
r am
plifi
catio
n of
the
diffe
rent
gen
e pr
oduc
ts.
Am
plifi
catio
nTe
mpl
ate
Prim
erSe
quen
ce (5
`-3`)
OR
F16
C-te
rm A
118
A118
F_A1
18_g
p16C
-term
A
TC A
GG
ATC
CA
T G
AC A
GG
TTC
GAA
AA
A C
R
_A11
8_gp
16C
-term
AT
C A
GA
GC
T C
TT A
TA G
CC
CC
T TT
C C
GT
AAA
ATG
O
RF1
7 A
118
F_A1
18_g
p17
ATC
AG
G A
TC C
AT
GG
C T
AC A
TC A
CT
AGC
ATT
AG
R
_A11
8_gp
17
ATC
AG
A G
CT
CTT
AC
T TA
T AC
A AA
A AG
G A
GA
AAT
CC
O
RF1
8 A
118
F_A1
18_g
p18
ATC
AG
G A
TC C
AT
GAA
TAG
CG
A TA
T TA
T AG
R
_A11
8_gp
18
ATC
AG
A G
CT
CTT
ATT
TC
G C
TC C
TT T
C
OR
F19
A11
8F_
A118
_gp1
9 AT
C A
GG
ATC
CA
T G
TT A
AA T
CT
TGA
TAA
ATG
R
_A11
8_gp
19
ATC
AG
A G
CT
CTT
AG
A AT
A TC
T G
AC C
TC C
C
OR
F20
A118
F_A1
18_g
p20
ATC
AG
G A
TC C
AT
GAC
AAA
TC
A AA
T C
TT T
AA A
TC A
GC
TAT
TR
_A11
8_gp
20
AAC
TG
A G
CT
CTT
AAT
TG
C C
AA
CTT
CG
T AT
A AT
A TC
G T
TG A
F_A1
18_g
p20
TAT
CAA
GAG
CTC
ATG
AC
A A
AT C
AA A
TC T
TT A
AA
TCA
GC
T AT
TR
_A11
8_gp
20
TAT
CAA
CTG
CAG
TTA
ATT
GC
C A
AC T
TC G
TA T
AA T
AT C
GT
TGA
F_A1
18_g
p20C
-term
ATC
AG
A G
CT
CG
T G
GA
AAT
TCT
TCA
AAA
TGA
AAT
TGO
RF2
1 A1
18F_
A118
_gp2
1A
TC A
GG
ATC
CA
T G
AA C
TA T
AA
ACA
GTT
TTA
CG
C A
TA T
GA
TR
_A11
8_gp
21AA
C T
GA
GC
T C
TT A
CC
CTA
AAT
TAC
TTT
CG
A AC
A AT
G C
CG
CO
RF2
0 A5
00A5
00F_
A500
_gp2
0TA
T C
AA G
AG C
TC A
TG A
CT
GAA
AA
C G
TT A
TT C
AT A
AA A
AT G
GT
R_A
500_
gp20
TAT
CAA
CTG
CAG
TTA
TG
T TG
T C
AC C
TC T
TT A
GT
TAA
ATA
AAT
F_A5
00_g
p20C
-term
ATC
AG
A G
CT
CTT
CG
A G
AG A
TT A
AA C
AC T
AA A
TT A
Gfu
ll le
ngth
orf9
7 A5
11A5
11Fw
d_Ba
mH
I_97
ATC
AG
G A
TC C
TT G
GA
AAA
CAC
TAA
CTA
TC
G
Rev
_Sal
I_97
ATC
AG
T C
GA
CTT
ATG
TTC
TC
T TG
T AT
T G
TT T
AG A
Gor
f102
A51
1Fw
d_Ba
mH
I_10
2A
TC A
GG
ATC
CG
A G
GA
GAA
ATT
AC
T A
TG G
CT
CG
T TA
T AA
A AA
A C
AC G
Rev
_Bam
HI_
102
ATC
AG
G A
TC C
TT A
AT T
AT C
TA G
CA
AAA
TAA
Res
trict
ion
site
s ar
e un
derli
ned;
sta
rt/st
op s
ites
are
bold
_________________________________________________________________________
22
Tabl
e 2.
3. (C
ontin
ued)
Del
etio
n m
utan
tor
f18
A11
8A1
18Fw
d_A_
Bam
HI
TCA
AG
G A
TC C
GC
GA
T C
TA A
AC
AG
G A
CR
ev_B
CA
T C
TA T
TT C
GC
TC
C G
GT
AC
C A
TT C
AT
GTA
TTC
AC
C T
AC
Fwd_
CG
TA G
GT
GA
A T
AC
ATG
AA
T G
GT
AC
C G
GA
GC
G A
AA
TA
G A
TGR
ev_D
_Sac
ITC
A AG
A G
CT
CC
T C
CC
GC
T TG
T TT
C
orf1
9 A
118
Fwd_
A_Ba
mH
ITC
A A
GG
ATC
CC
T TG
T G
GG
CG
CR
ev_B
GA
A T
AT
CTG
AC
C T
CC
GG
T A
CC
TA
A C
AT
CTA
TTT
CG
C T
Fwd_
CA
GC
GA
A A
TA G
AT
GTT
AG
G T
AC
CG
G A
GG
TC
A G
AT
ATT
CR
ev_D
_Sac
ITC
A AG
A G
CT
CC
A C
CG
TTC
CC
G
or
f20
A118
Fwd_
A_Ba
mH
ITC
A A
GG
ATC
CG
A A
GC
TG
G C
GG
Rev
_BC
TA A
TT G
CC
AA
C T
TC G
GT
AC
C C
AT
TAG
AA
T A
TC T
GA
CC
Fwd_
CG
GT
CA
G A
TA T
TC T
AA
TG
G G
TA C
CG
AA
G T
TG G
CA
ATT
AG
Rev
_D_S
acI
TCA
AGA
GC
T C
GC
CAC
TTA
GA
C G
G
Prim
ers
used
for s
cree
ning
of d
elet
ion
mut
ant s
trai
ns
orf1
8A11
8A
118 Δ
18Fw
d X
153
73-1
5388
GG
C A
GT
TCG
GC
C A
AG
GR
ev Y
161
19-1
6136
CC
A T
GA
AAA
GC
C C
CT
GC
Rev
Z 1
6922
-169
41C
CG
CC
A G
CT
TCT
AAA
AC
A A
Cor
f19
A11
8A
118 Δ
19Fw
d X
164
27-1
6447
CC
A G
TC A
CA
TA
C A
CT
AG
C C
CG
Rev
Y 1
7282
-173
00C
GT
TAT
ATT
GC
C C
CG
GC
T C
Rev
Z 1
7973
-179
89G
CG
TTA
CC
T C
TG C
CG
CG
orf2
0 A
118
A11
8 Δ20
Fwd
X 1
7504
-175
21G
CA
AG
G T
GC
TG
G T
AC
GG
CR
ev Y
184
61-1
8479
GC
T TT
T G
CT
TGT
GA
T C
CC
GR
ev Z
190
53-1
9068
CC
C C
AT
TCC
AA
C G
CG
G
Res
trict
ion
site
s ar
e un
derli
ned;
sta
rt/st
op s
ites
are
bold
_________________________________________________________________________
23
2.3. Proteomics
2.3.1. Protein expression and purification
In order to produce recombinant proteins the expression was induced in E. coli at
OD600 0.5 with 0.1 mM IPTG for 4 h. Then cells were harvested by centrifugation
directly after the 4 h induction or in the case of GFP-fusion-proteins after
additional overnight incubation at 4 °C, resuspended in buffer A (500 mM NaCl,
50 mM NaHPO4, 5 mM Imidazole, 0.1% Tween 20, pH 8.0) and frozen at -20 °C.
After thawing, cells were disrupted by a French cell press (SLC Aminco),
centrifuged (30’000 x g, 1 h) and the supernatant was sterilized by filtration
(0.2 µM PES membrane, Millipore). Raw extracts of gp97 and gp102 (A511) were
directly assayed. Raw extracts of His-tagged proteins were loaded on a Ni-NTA
column (1 ml His-Trap HP, GE Healthcare) using an ÄKTA Purifier (Amersham).
GFP-fusion proteins were purified on 1 ml Ni-NTA sepharose (High Density Ni-
NTA-Affarose, Interchim, France) in gravity flow columns (BioRad). Elution of
proteins was carried out in buffer B (500 mM NaCl, 50 mM NaHPO4, 250 mM
Table 2.4. Proteins used in this study.Name MW [kDa] RE PP AB BA LAA118 gp16 (C-term) 52 - + + - +A118 gp17 32.4 - + + - +A118 gp18 40.8 - + + - +A118 gp19 38.7 - + + - +A118 gp20 41 - + + - +A118 gp21 14.3 - + + - +GFP-RBP A118 67.7 - + - + -GFP-RBP A500 67.5 - + - + -GFP-RBP A118 (C-term) 50 - + - + -GFP-RBP A500 (C-term) 49.6 - + - + -A511 gp97 131.1 + - - - +A511 gp102 26.4 + - - - +RE: Raw extracts were directly tested for activityPP: Purification of proteins on Ni-NTA AB: Used for immunization (Antibody-production)BA: Binding assaysLA: Tested for lytic activity
_________________________________________________________________________
24
Imidazole, 0.1% Tween 20, pH 8.0). The eluted proteins were dialyzed against
buffer containing 100 mM NaCl, 50 mM NaHPO4, and 0.005% Tween 20, pH 8.0
and stored at -20 °C in 50% glycerol. Proteins produced and used in this study are
listed in Table 2.4.
2.3.2. Polyclonal rabbit-antibodies
Polyclonal rabbit-antibodies were generated at the Institut für Labortierkunde,
University of Zurich, Switzerland. Proteins gp16 C-term, gp17, gp18, gp19, gp20,
and gp21 of A118 were purified on Ni-NTA columns and dialyzed against PBST
(120 mM NaCl, 50 mM NaHPO4, 0.1% Tween 20, pH 8.0). For each antigen one
rabbit was used for immunization (six rabbits in total). Aliquots of 200 µg antigen,
diluted in 500 µl PBST, were used for each immunization and booster injection.
Immunization followed a standard immunization protocol (Table 2.5.). Obtained
sera (α-gp16 C-term; α-gp17; α-gp18; α-gp19; α-gp20; α-gp21) were analyzed by
Western blot for their immune reaction against the corresponding antigens. Each
tested immune antisera showed reactivity after 14 weeks. Sera were further
directly used for Western blots or were ProteinA purified (ProteinA-antibody
purification Kit, Sigma) for TEM.
Table 2.5. Standard immunization protocol for rabbits.Day 0 Pre-immune serum; 1st immunization with
Freund's Complete AdjuvantWeek 4 1st booster injection (with Freund's Incomplete
Adjuvant)Week 6 Control bleed (10 ml)Week 8 2nd booster injection with Freund's Incomplete
Adjuvant)Week 10 Standard bleed (50 ml)Week 12 3rd booster injectionWeek 14 Final bleed (antisera)
_________________________________________________________________________
25
2.3.3. Mass spectrometry
Phage structural proteins were separated by a horizontal discontinuous SDS-
PAGE (ExcelGel gradient 8-18% PAA-gels, GE Healthcare, Germany). Samples
were diluted in reducing sample loading buffer (4% SDS, 100 mM Tris-HCl, 0.02%
Servablue G-250, 0.02% Bromophenolblue, 1% DTT stock solution 6.3 M). Gels
were run with 200 V - 600 V, 25 mA, and 15 W at 12 °C and stained for 30 min in
Coomassie blue R-350 (GE-Healthcare). Unstained protein marker (Fermentas)
was used as a molecular weight marker. Protein bands were excised, cut in small
pieces and washed twice with 100 µl of 100 mM NH4HCO3/50% acetonitrile, and
with 50 µl acetonitrile. Supernatants were discarded. The individual protein
species were proteolytically digested (“in gel” digestion) with 15 µl trypsin (10
ng/µl trypsin (Promega, sequencing grade modified) in 10 mM TrisCl, 2 mM
CaCl2, pH 8.2). Supernatant was removed after incubation overnight at 37 °C and
gel pieces were extracted twice with 100 µl in 0.1% TFA/50% acetonitrile. Eluted
supernatants were pooled and vacuum dried. Peptides were dissolved in 15 μl
0.1% TFA. 10 μl of the samples were desalted by using a ZipTip C18 and mixed
1:1 with matrix solution (5 mg/ml 4-HCCA in 0.1% TFA, 50% acetonitrile).
Remaining sample after ZipTip was dried, dissolved in 0.1% formic acid and
transferred to autosampler vials for LC/MS/MS. All MS/MS samples were
analyzed using Mascot (Matrix Science, London, UK; version 2.1.04). Scaffold
(version Scaffold-01_06_17, Proteome Software Inc., Portland, USA) was used to
validate MS/MS based peptide and protein identifications (60). Probabilities were
assigned by the Protein Prophet algorithm (96).
2.3.4. SDS-PAGE, Western blot analysis, visualization of lytic phage proteins by zymography, and 2D-gel electrophoresis
Sodium-dodecylsulfate-polyacrylamide-gelelectrophoresis (SDS-PAGE) was
performed as described previously (66, 111). 14% Tris/Tricin SDS-PAGE were
performed according to Schagger and Jagow (113), run for about 4-5 h at 100 V
in a Mighty Small II SE250/SE260 chamber (Hoefer), and Coomassie stained.
Molecular mass of the proteins was determined using prestained molecular mass
_________________________________________________________________________
26
marker (Fermentas). Protein samples for electrophoresis were diluted in SDS-
loading buffer and boiled for 10 min. For Western blot analysis proteins were
separated by SDS-PAGE and transferred to a PVDF membrane (Immobilon-P
Transfer Membranes 0.45 μm, Millipore) with transfer buffer (25 mM Tris, 192 mM
glycine, 20% methanol, pH 8.3). Membranes were blocked with 10% skimmed
milk in TBST (10 mM TrisCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 1 h, and
incubated with serum containing polyclonal rabbit antibodies (α-gp16 C-term; α-
gp17; α-gp18; α-gp19; α-gp20; α-gp21, and α-Ply118 (39) at a dilution of 1:5000
in 3% skimmed milk) 1 h at room temperature, or alternatively overnight at 4 °C.
Membranes were washed with TBST and incubated for 30 min with HRP-labelled
secondary goat α-rabbit IgG antibody (Calbiochem, VWR, Switzerland) and
washed with TBST. Chemiluminescent signals (Chemiluminescence Blotting
Substrate, Roche) of bound antibodies were detected using a Kodak Image
Station IS2000R (Carestream Health, New Haven, USA).
Zymogram analysis was performed as described previously (70, 125). Briefly,
protein samples were separated on a 12% SDS-PAGE containing ~1012 heat
inactivated Listeria substrate cells per 5 ml resolving gel for detection of
bacteriolytic activity. Substrate cells were prepared as follows: TB medium was
inoculated with an overnight culture of Listeria, cells were grown until late
exponential phase (OD600 0.6-0.8) and inactivated by steaming (100 °C, 10 min).
The zymogram was incubated for 30 min in distilled water at room temperature,
then transferred into regeneration buffer containing 25 mM Tris (pH 7.3) and 0.1%
Triton X-100 and further incubated overnight at 15 °C. Peptidoglycan hydrolase
activity was detected as a clear zone. Molecular weight of active proteins in
zymogram was estimated using prestained protein marker (Fermentas).
For 2D-gel electrophoresis 20 μl of diluted phage sample was boiled for 10 min
with 0.2% SDS, treated with 1 U/µl BenzonNuclease (Merck) for 30 min at room
temperature and mixed 1:10 with buffer containing 8-10 M urea, 2% CHAPS,
50 mM DTT, 0.2% ampholytes (Bio-Lyte 3-10; BioRad) and 0.001% Bromophenol.
IEF-strips (pH 4-7, 7 cm) (BioRad) were passively rehydrated with the sample
overnight at 20 °C and then focused using BioRad IEF system under
recommended standard conditions (50 mA/Gel). Strips were stored at -70° C until
_________________________________________________________________________
27
use. After thawing for 15 min the strips were equilibrated twice for 10 min in
equilibration buffer (6 M urea, 2% SDS, 2% DTT, 0.375 M TrisCl, 20% glycerol,
pH 8.8) and casted into a SDS-PAGE for the second dimension. Gels were either
silverstained (95) or in case of 2D-zymograms renaturated overnight in buffer
containing 25 mM Tris (pH 7.3) and 0.1% Triton X-100.
For identification of bands with muralytic activity, gel pieces (Tris/Tricin-gel) of
phage protein profiling were excised, cut into small pieces, equilibrated with 4 μl
SDS-loading buffer and applied in a slot of the stacking gel on top of a zymogram.
Treatment of the zymogram after the run was carried out as described above.
2.4. Assays
2.4.1. Binding assays
The binding of GFP-RBP fusion proteins was tested similar to the procedures as
described by Loessner et. al. (2002). Before incubation with Listeria cells, the
purified GFP-RBP fusion proteins were centrifuged for 1 h with 30’000 x g at 4 °C.
0.5 ml of exponentially growing Listeria cells were centrifuged and resuspended in
120 µl SM buffer (50 mM TrisCl, 0.55% NaCl, 0.2% MgSO4 7H2O pH 7.4),
supplemented with 1 mM CaCl2 and 20 µl GFP-RBP and incubated for 1 h at
room temperature. Known SV-specific cell wall binding proteins (CBD-118 and
CBD-500) served as positive and negative controls (81). Cells were washed twice
with SM buffer and binding to the listerial cell wall was tested and observed by
fluorescence microscopy.
2.4.2. “Pull-down” assay
100 μl of phage suspensions (107 pfu/ml) were pre-incubated with 5 μl of the
different antisera (α-gp16 to α-gp21) and the corresponding pre-immune sera for
1 h at 30 °C. Samples were incubated for 10-15 min with 0.5 ml of an overnight
culture Listeria WSLC 1001 (SV 1/2). As controls, samples without preliminary
antisera incubation, were incubated with either Listeria WSLC 1001 (SV 1/2) or
Listeria WSLC 1042 (SV 4b). Cells were then centrifuged with 12’000 x g and
_________________________________________________________________________
28
washed twice in PBST (pH 8.0). After dilution of the pellet in 1 ml SM Puffer, 10 μl
of a 10-2 dilution was plated out with new host cells, incubated overnight at 30 °C
and plaques were counted.
2.4.3. Transmission electron microscopy (TEM)
CsCl-purified phage particles (10 µl of 1012 pfu/ml) were mixed with 60 μl TBT
(20 mM Tris, 50 mM NaCl, 10 mM MgCl2) incubated with either 10 μl (for α-gp16,
α-gp18, and α-gp20) or 20 μl (for α-gp17, α-gp19, and α-gp21) of ProteinA
purified antisera and filled up with MQ-water to a total volume of 120 μl. Sample
without antisera was used as control. After incubation overnight at 4 °C the
samples were centrifuged for 15 min at 100’000 x g (Beckman, Airfuge Air-Driven
ultracentrifuge), 100 μl of the supernatant were carefully removed and the
remaining pellet was washed with 100 μl ½ TBT. These steps were repeated
twice. The phages present in the pellet were then either directly prepared for TEM
or further incubated with the secondary 5 nm gold conjugate goat α-rabbit IgG
antibody (British Biocell, Plano). Negative stain and sample preparation were
done with 2% uranyl acetate or 2% ammoniummolybdate solution and adsorption
on carbon-coated G400 Hex-C3 grids (Science Services, Munich, Germany)
(121). The samples were observed in a Philips CM100 at 100 kV acceleration
voltage (FEI Company, Hillsboro, USA) equipped with a TVIPS Fastscan CCD
camera (Tietz Systems, Gauting, Germany) or Tecnai G2 Spirit at 120 kV,
equipped with an EAGLE CCD camera (FEI).
2.5. Bioinformatics Nucleotide and amino acid sequence analysis and interpretation were performed
using VectorNTI (Version 10.3, Invitrogen). Pairwise sequence alignments were
done using the BLASTn and BLASTp programs available at the NCBI website (3).
Multiple sequence alignments were conducted by ClustalW
(http://www.ebi.ac.uk/Toolx/clustalw) (67). Sequences of A118 (AJ242593), A500
(DQ003637), PSA (AJ312240), and A511 (DQ003638) were retrieved from
Genbank.
_________________________________________________________________________
29
3. Results
3.1. Proteomics of different Listeria phages.
3.1.1. Profiles of three temperate phages A118, A500, A006 and three virulent phages P40, P35, and A511
Structural proteins of the six viruses were separated by horizontal SDS-PAGE
(Fig. 7). All phages exhibited relatively specific protein profiles. The individual
bands were analyzed by mass spectrometry. Peptide fingerprints permitted
allocation of many of the bands to predicted phage gene products. The portal
protein, major capsid protein (Cps) and tail sheet protein (Tsh) were identified in
all analyzed phages. Within the different profiles, the most abundant proteins were
identified as Cps and Tsh. All detected gene products of analyzed members of the
Siphoviridae family, namely A118, A500, A006, P40, and P35, were assigned to
the late gene cluster encoding for structural proteins. The Myovirus A511 displays
homologies to staphylococcal phage 812 (36, 63). We observed similar
degradation products of predominant structural components. For example, Tsh
and Cps were found in several bands. Furthermore, the identified gp145 of A511
is not located in the late gene cluster. Correspondingly, the homologous protein of
bacteriophage 812 was detected as well. Therefore, the presence of gp145 is not
unexpected in the mature virion.
Although the protein profile of phage A118 has been studied before (145),
additional structural proteins could be identified. Other identified gene products,
such as putative head associated proteins, include gp8 (14.6 kDa), gp9
(13.8 kDa), gp11 (15.1 kDa), and the portal protein (55.3 and 56.5 kDa). The tail
tape measure protein (Tmp), with a calculated molecular weight of 186 kDa, was
found in a band of lower molecular weight (Fig. 7). Furthermore two proteins
(gp17 and gp20), with predicted molecular mass of 30.9 kDa and 39.2 kDa,
respectively, were assigned as putative tail or baseplate proteins (80). Gp18 and
gp19, two additional putative baseplate proteins were not directly identified in the
_________________________________________________________________________
30
Fig. 7. Protein profiles of different Listeria phages. SDS-PAGE analysis of different
phages structural protein contents. Based on MALDI-MS peptide fingerprints, assignment
of gel bands to predicted gene products is shown. Abbreviations: Tmp: Tape measure
protein, Tsh/Tsh-L: Tail sheet protein/Tail sheet protein long; Cps/Cps-L: Major capsid
protein/Major capsid protein long. Unstained protein marker (Fermentas) indicates the
molecular weight in kDa (Continued next page).
A00
6
Tmp
porta
l
gp17
gp16
Cps
Cps
Tsh
Tsh
(Cps
)
116
66 45 35 25 18.4
14.4
gp18
/Cps
-L
A500
Tmp
porta
l
gp19
gp8/
11/(9
)
Tsh
Cps
Tsh-
L
gp19
(Cps
)
116
66 45 35 25 18.4
14.4
A11
8
gp20
/Cps
-Lgp
20/C
ps-L
Tmp
porta
l
gp8,
gp1
1gp
8, g
p11
gp9
gp9
Tsh
Tsh
Tsh,
Cps
Tsh,
Cps
gp17
gp17
Cps Tsh-
LTs
h-L
Tsh-
L
116
66 45 35 2525 18.4
18.4
14.4
14.4
_________________________________________________________________________
31
Fig. 7. Protein profiles of different Listeria phages. (Continued).
P40
Tmp
porta
l
gp15
gp16
Tsh
gp17
gp7
Cps
gp10
116
66 45 35 25 18.4
14.4
P35
Tmp
porta
l
gp15
gp16
gp17
Cps
/Tsh
116
66 45 35 25 18.4
14.4
A51
1
Tsh
Cps
gp10
6
gp97
, gp1
06gp
104
porta
l
gp10
8C
ps, T
shgp
106,
gp1
03gp
88C
ps, g
p106
gp10
2, g
p145
gp10
5
gp14
5gp
94
116
66 45 35 25 18.4
14.4
_________________________________________________________________________
32
A118 protein profile by mass spectrometry, but could be identified in the A500
profile (145). In this band, one can assume the presence of A118 gp18 (39.4 kDa)
and gp19 (37.2 kDa) at the same designated bands compared to A500, since
A118 and A500 share high homologies within the late gene cluster and show
other similarities such as their morphology and protein profile (145).
Regarding the protein profiles of the recently identified phage P40 and P35, the
relatedness between these two became apparent. Only the Tsh protein of P40
(MW: 34.7 kDa) migrates faster than the Cps (MW: 32.3 kDa), whereas in P35
Tsh (35 kDa) and Cps (32.9 kDa) form one single band. Comparison of their
genomes revealed not only similar organization of the late gene cluster but also
high identities among individual gene products, as e.g. the Cps, Tmp, and the
portal protein.
3.1.2. Programmed translational frameshifting in A118 and A500
Both the major capsid protein (Cps) and the major tail protein (Tsh) are
represented by two proteins of different size in phage A118 and A500.
Bioinformatical analysis indicated that in both proteins a programmed translational
(ribosomal) frameshift (-1 in Cps and +1 in Tsh) at the 3’ end of the analogous
genes could result in the synthesis of a larger polypeptide species. Such recoding
events may result in two products of different sizes, sharing the same N-termini
but varying in the length of their C-termini. The calculated molecular weight of
these proteins corresponds to the observed bands on the gels. To provide
evidence for the actual existence of the elongated products, and to determine the
location and type of frameshift involved, mass spectrometry was employed.
MALDI-MS peptide fingerprints of Cps-L and Tsh-L were generated, and the
determined masses of the individual tryptic polypeptide fragments were compared
with the deduced masses for Cps-L and Tsh-L in both phages (Table 3.1). The
analysis yielded total fragment coverage of 73% and 56.5% for Cps-L, 77.5% and
68% for Tsh-L in A118 and A500, respectively. Protein coverage is shown in Fig.
9.
_________________________________________________________________________
33
MALDI-MS enabled identification of the peptides spanning the potential
frameshifting sites, and therefore also permitted determination of the location and
modus of the shift. Fig. 8 shows that in both phages the frameshift in cps occurs
at a location close to the 3’ end of the gene, at the mRNA sequence GCG GGA
AAC (corresponding to coordinates 6071-6079 and 6048-6056 of the A118 and
A500 genomes, respectively). The ribosome apparently slips from the GGA
glycine codon one nucleotide into the 5’ direction (underlined) and continues from
the overlapping glycine codeon GGG in the -1 frame until it reaches the stop
codon at position 6248 in A118 and 6214 in A500. Thus, Cps-L contains in both
phages most of the sequence of Cps (299 (A118) and 278 (A500) residues), with
53 extra amino acids (aa) from the alternate frame added onto the C-terminus.
With respect to tsh mRNA, the slippery sequence AAA CCC UGA (corresponding
to coordinates 8173-8181, and 8153-8161 of the A118 and A500 genomes,
respectively) is also located at the end of the gene. In contrast to the frameshift in
cps, the ribosome slips one nucleotide position in the 3’ end (underlined), and
continues translation in the +1 frame ending at position 8440 (A118) and 8420
(A500) respectively. The shift in Tsh-L results to an addition of 87 aa in A118 and
A500, whereas the Tsh consist of 144 aa (A118) and 145 aa (A500).
_________________________________________________________________________
34
A
Cps
Cps-L
289865
298891
325972
307918
3431026
289865
298891
325972
307918
3431026
-1 frameshift
A F S A V Q P K A G N * GCG TTC TCT GCT GTT CAA CCA AAA GCG GGA AAC TAA
G K L M A A R S G GGG AAA CTA ATG GCG GCG CGG TCG GGT
K T D S A P I K D F S V M T V A E L AAA ACT GAT AGC GCG CCG ATT AAA GAC TTT TCA GTT ATG ACA GTA GCA GAA TTG
K E E L A N R N I E F A S N A K K A AAA GAA GAG CTT GCG AAT AGA AAT ATC GAA TTT GCA AGT AAT GCG AAA AAA GCG
E L V A L L E G S E * GAG TTA GTT GCG CTG TTG GAA GGT AGT GAG TGA
D E T P T V T K P * GAT GAA ACA CCT ACG GTT ACA AAA CCC TGA
P E E S P S S V E CCT GAG GAG AGC CCG TCC AGC GTC GAA
V G H N T I T V K V G E T F T I N A GTG GGC CAC AAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT
S V L P V G A S Q E V T Y T S S N P TCT GTA TTG CCA GTG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA
P K A K I N S V G T G E G V A E G T CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA
A N I T V A S K E S T S I N K V V Q GCA AAC ATA ACA GTT GCA TCT AAA GAA AGT ACT TCT ATC AAC AAA GTA GTA CAA
V T V E A A D * * GTA ACA GTA GAA GCA GCA GAT TAA TAA
+1 frameshift
D E T P T V T K P * GAT GAA ACA CCT ACG GTT ACA AAA CCC TGA
P E E S P S S V E CCT GAG GAG AGC CCG TCC AGC GTC GAA
V G H N T I T V K V G E T F T I N A GTG GGC CAC AAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT
S V L P V G A S Q E V T Y T S S N P TCT GTA TTG CCA GTG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA
P K A K I N S V G T G E G V A E G T CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA
A N I T V A S K E S T S I N K V V Q GCA AAC ATA ACA GTT GCA TCT AAA GAA AGT ACT TCT ATC AAC AAA GTA GTA CAA
V T V E A A D * * GTA ACA GTA GAA GCA GCA GAT TAA TAA
+1 frameshift
Tsh
Tsh-L
B136406
144431
171512
153458
189566
207620
225674
136406
144431
171512
153458
189566
207620
225674
Fig. 8. Programmed translational frameshift near the 3’ ends of the genes results in synthesis
of two different length products for Cps and Tsh major structural proteins in the two Listeria
phages A118 and A500. A) The -1 frameshift in cps of A118 is shown, leading to an extended
version of the Cps. B) The +1 frameshift is shown in the sequence encoding for the Tsh of
phage A118 (Continued next page).
_________________________________________________________________________
35
C
Cps
Cps-L
272814
277828
308921
290867
326975
-1 frameshift
Tsh
Tsh-L
D137409
145434
172515
154461
190569
208623
226677
V V P V A G N * GTA GTT CCA GTT GCG GGA AAC TAA G K L M A A R S V E T D S GGG AAA CTA ATG GCG GCG CGG TCG GTT GAA ACT GAT AGC A P I Q D F S T M T V A E L K E E L GCG CCG ATT CAA GAC TTT TCA ACT ATG ACA GTA GCA GAA TTG AAA GAA GAG CTT V T R N I E F A S N A K K A E L V A GTG ACT AGA AAT ATC GAA TTT GCA AGT AAT GCG AAA AAA GCG GAG TTA GTG GCG L L E G S D * CTG TTG GAA GGT AGT GAT TGA
+1 frameshift D E T P K V T K P * GAT GAA ACG CCT AAG GTA ACA AAA CCC TGA
P E E S P S S V T CCT GAG GAG AGC CCG TCC AGC GTT ACA
V D H D T I T V K V G E T F T I N A GTG GAC CAC GAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT S V L P A G A S Q E V T Y T S S N P TCT GTA TTG CCA GCG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA P K A K I N S V G T G E G V A E G T CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA A N I T V A S K E S P S I N K V V Q GCA AAC ATA ACT GTC GCA TCT AAA GAA AGT CCT TCT ATC AAC AAA GTA GTG CAA V T V E A A D * * GTA ACA GTA GAA GCA GCA GAC TAA TAA
C
Cps
Cps-L
272814
277828
308921
290867
326975
-1 frameshift
Tsh
Tsh-L
D137409
145434
172515
154461
190569
208623
226677
V V P V A G N * GTA GTT CCA GTT GCG GGA AAC TAA G K L M A A R S V E T D S GGG AAA CTA ATG GCG GCG CGG TCG GTT GAA ACT GAT AGC A P I Q D F S T M T V A E L K E E L GCG CCG ATT CAA GAC TTT TCA ACT ATG ACA GTA GCA GAA TTG AAA GAA GAG CTT V T R N I E F A S N A K K A E L V A GTG ACT AGA AAT ATC GAA TTT GCA AGT AAT GCG AAA AAA GCG GAG TTA GTG GCG L L E G S D * CTG TTG GAA GGT AGT GAT TGA
+1 frameshift D E T P K V T K P * GAT GAA ACG CCT AAG GTA ACA AAA CCC TGA
P E E S P S S V T CCT GAG GAG AGC CCG TCC AGC GTT ACA
V D H D T I T V K V G E T F T I N A GTG GAC CAC GAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT S V L P A G A S Q E V T Y T S S N P TCT GTA TTG CCA GCG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA P K A K I N S V G T G E G V A E G T CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA A N I T V A S K E S P S I N K V V Q GCA AAC ATA ACT GTC GCA TCT AAA GAA AGT CCT TCT ATC AAC AAA GTA GTG CAA V T V E A A D * * GTA ACA GTA GAA GCA GCA GAC TAA TAA
+1 frameshift D E T P K V T K P * GAT GAA ACG CCT AAG GTA ACA AAA CCC TGA
P E E S P S S V T CCT GAG GAG AGC CCG TCC AGC GTT ACA
V D H D T I T V K V G E T F T I N A GTG GAC CAC GAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT S V L P A G A S Q E V T Y T S S N P TCT GTA TTG CCA GCG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA P K A K I N S V G T G E G V A E G T CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA A N I T V A S K E S P S I N K V V Q GCA AAC ATA ACT GTC GCA TCT AAA GAA AGT CCT TCT ATC AAC AAA GTA GTG CAA V T V E A A D * * GTA ACA GTA GAA GCA GCA GAC TAA TAA
Fig. 8. (Continued). (C) and (D) Similar to A118 the Cps-L of phage A500 results through
a -1 frameshift whereas the Tsh-L of phage A500 is produced through a +1 frameshift.
_________________________________________________________________________
36
10 20 30 40 50 60 MGFNPDTTTM QSAKTGSIPI NISEQIITGV KNGSAAMKLA KAVPMTKPEE EFTFMSGVGA 70 80 90 100 110 120 FWVDEAERIQ TSKPTFTKAK MRSKKMGVII PTTKENLNYS VTNFFSLMQA EIVEAFYKKF 130 140 150 160 170 180 DQAVFTGVES PYNWNILKSA TDASNLVEET ANKYDDLNEA IGLIEAEDLE PNGIATIRKQ 190 200 210 220 230 240 RVKYRSTKDG NGMPIFNTAT SNGVDDVLGL PIAYTPKYTF GDKDISELVG DWNQAYYGIL 250 260 270 280 290 300RGVEYEILTE ATLTTVADET GKPLNLAERD MAAIKATFEV GFMVVKDEAF SAVQPKAG KL 310 320 330 340 350 MAARSGKTDS APIKDFSVMT VAELKEELAN RNIEFASNAK KAELVALLEG SE 10 20 30 40 50 60 MRIKNAKTKY SVAEIVAGAG EPDWKRLSKW ITNVSDDGSD NTEEQGDYDG DGNEKTVVLG 70 80 90 100 110 120 YSEAYTFEGT HDREDEAQNL IVAKRRTPEN RSIMFKIEIP DTETAIGKAT VSEIKGSAGG 130 140 150 160 170 180GDATEFPAFG CRIAYDETPT VTKP EESPSS VEVGHNTITV KVGETFTINA SVLPVGASQE 190 200 210 220 230 VTYTSSNPPK AKINSVGTGE GVAEGTANIT VASKESTSIN KVVQVTVEAA D
A
B
10 20 30 40 50 60 MGFNPDTTTM QSAKTGSIPI NISEQIITGV KNGSAAMKLA KAVPMTKPEE EFTFMSGVGA 70 80 90 100 110 120 FWVDEAERIQ TSKPTFTKAK MRSKKMGVII PTTKENLNYS VTNFFSLMQA EIVEAFYKKF 130 140 150 160 170 180 DQAVFTGVES PYNWNILKSA TDASNLVEET ANKYDDLNEA IGLIEAEDLE PNGIATIRKQ 190 200 210 220 230 240 RVKYRSTKDG NGMPIFNTAT SNGVDDVLGL PIAYTPKYTF GDKDISELVG DWNQAYYGIL 250 260 270 280 290 300RGVEYEILTE ATLTTVADET GKPLNLAERD MAAIKATFEV GFMVVKDEAF SAVQPKAG KL 310 320 330 340 350 MAARSGKTDS APIKDFSVMT VAELKEELAN RNIEFASNAK KAELVALLEG SE 10 20 30 40 50 60 MRIKNAKTKY SVAEIVAGAG EPDWKRLSKW ITNVSDDGSD NTEEQGDYDG DGNEKTVVLG 70 80 90 100 110 120 YSEAYTFEGT HDREDEAQNL IVAKRRTPEN RSIMFKIEIP DTETAIGKAT VSEIKGSAGG 130 140 150 160 170 180GDATEFPAFG CRIAYDETPT VTKP EESPSS VEVGHNTITV KVGETFTINA SVLPVGASQE 190 200 210 220 230 VTYTSSNPPK AKINSVGTGE GVAEGTANIT VASKESTSIN KVVQVTVEAA D
A
B
Fig. 9. Complete amino acid sequences of Cps-L and Tsh-L polypeptides of phages A118 and A500. Fragments found by peptide mass fingerprinting (MALDI-MS)
(Table 3.1) are indicated in bold letters. Amino acid residues resulting from the
frameshift are underlined. A) Amino acid sequence of A118 Cps-L. B) Amino acid
sequence of A118 Tsh-L. (Continued next page).
_________________________________________________________________________
37
10 20 30 40 50 60 MADLTTKLAN LIDPEVMGPM ISAKLPKAIK FGKIAPIDNS LEGQPGSEIT VPKYKYIGDA 70 80 90 100 110 120 QDVAEGAAID YSALETESVK HGIKKAGKGV KLTDESVLSG YGDPVEEAQK QIRMAIASKV 130 140 150 160 170 180 DNDILEEALT TTLEVKGAIN IGLIDKIENT FTDAPDAIED ESITTTGVLF LNYKDTAKLR 190 200 210 220 230 240 EEAAGSWTKA SQLGDDLLVK GAFGELLGWE IVRTKKLADG NALAVKAGAL KTFLKRNLLA 250 260 270 280 290 300ESGRDMDHKL TKFNADQHYA VALVDETKAV KVVPVAG KLM AARSVETDSA PIQDFSTMTV 310 320 330 AELKEELVTR NIEFASNAKK AELVALLEGS D 10 20 30 40 50 60 MARIKNAKTK YFVAEIVDGV GEPVWKRLSK WITNVSDDGS DNTEEQGDYD GDGNEKTVVL 70 80 90 100 110 120 GYSEAYTFEG THDREDEAQN LIVAKRRTPE NRGIMFKIEI PDTETAVGKA TVSEIKGSAG 130 140 150 160 170 180 GGDATEFPAF ACRIAYDETP KVTKP EESPS SVTVDHDTIT VKVGETFTIN ASVLPAGASQ 190 200 210 220 230 EVTYTSSNPP KAKINSVGTG EGVAEGTANI TVASKESPSI NKVVQVTVEA AD
C
D
10 20 30 40 50 60 MADLTTKLAN LIDPEVMGPM ISAKLPKAIK FGKIAPIDNS LEGQPGSEIT VPKYKYIGDA 70 80 90 100 110 120 QDVAEGAAID YSALETESVK HGIKKAGKGV KLTDESVLSG YGDPVEEAQK QIRMAIASKV 130 140 150 160 170 180 DNDILEEALT TTLEVKGAIN IGLIDKIENT FTDAPDAIED ESITTTGVLF LNYKDTAKLR 190 200 210 220 230 240 EEAAGSWTKA SQLGDDLLVK GAFGELLGWE IVRTKKLADG NALAVKAGAL KTFLKRNLLA 250 260 270 280 290 300ESGRDMDHKL TKFNADQHYA VALVDETKAV KVVPVAG KLM AARSVETDSA PIQDFSTMTV 310 320 330 AELKEELVTR NIEFASNAKK AELVALLEGS D 10 20 30 40 50 60 MARIKNAKTK YFVAEIVDGV GEPVWKRLSK WITNVSDDGS DNTEEQGDYD GDGNEKTVVL 70 80 90 100 110 120 GYSEAYTFEG THDREDEAQN LIVAKRRTPE NRGIMFKIEI PDTETAVGKA TVSEIKGSAG 130 140 150 160 170 180 GGDATEFPAF ACRIAYDETP KVTKP EESPS SVTVDHDTIT VKVGETFTIN ASVLPAGASQ 190 200 210 220 230 EVTYTSSNPP KAKINSVGTG EGVAEGTANI TVASKESPSI NKVVQVTVEA AD
C
D
Fig. 9. (Continued). C) Amino acid sequence of A500 Cps-L. D) Amino acid sequence
of A500 Tsh-L.
_________________________________________________________________________
38
Table 3.1: Peptide mass fingerprinting (MALDI-MS) of tryptic fragments Cps-L of A118Fragment MW (exp) MW (calc) Δ MW
[aa] [Da]a [Da]b [%]c Corresponding aa-sequence d
15-31 1769.91 1770.00 0.00 TGSIPINISEQIITGVK 69-78 1150.59 1150.65 0.00 IQTSKPTFTK 86-94 959.47 959.56 0.01 MGVIIPTTK119-138 2356.08 2356.20 0.00 KFDQAVFTGVESPYNWNILK 120-138 2228.00 2228.10 0.00 FDQAVFTGVESPYNWNILK139-153 1549.65 1549.73 0.01 SATDASNLVEETANK154-178 2744.17 2744.36 0.01 YDDLNEAIGLIEAEDLEPNGIATIR218-241 2823.16 2823.36 0.01 YTFGDKDISELVGDWNQAYYGILR224-241 2111.94 2112.04 0.00 DISELVGDWNQAYYGILR242-269 3033.30 3033.56 0.01 GVEYEILTEATLTTVADETGKPLNLAER276-286 1227.59 1227.64 0.00 ATFEVGFMVVK287-299 1347.56 1347.69 0.01 DEAFSAVQPKAGK305-314 1003.48 1003.54 0.01 SGKTDSAPIK308-325 1951.90 1952.00 0.01 TDSAPIKDFSVMTVAELK332-340 993.46 993.50 0.00 NIEFASNAK341-352 1258.62 1258.69 0.01 KAELVALLEGSE
Peptide mass fingerprinting (MALDI-MS) of tryptic fragments of Tsh-L of A118
Fragment MW (exp) MW (calc) Δ MW[aa] [Da]a [Da]b [%]c Corresponding aa-sequence d
10-25 1692.05 1691.83 0.01 YSVAEIVAGAGEPDWK 30-55 2860.46 2860.13 0.01 WITNVSDDGSDNTEEQGDYDGDGNEK 56-73 2045.25 2044.96 0.01 TVVLGYSEAYTFEGTHDR 97-108 1286.87 1286.68 0.01 IEIPDTETAIGK116-132 1599.91 1599.69 0.01 GSAGGGDATEFPAFGCR133-161 3128.91 3128.56 0.01 IAYDETPTVTKPEESPSSVEVGHNTITVK162-190 2993.87 2993.51 0.01 VGETFTINASVLPVGASQEVTYTSSNPPK193-214 2075.34 2075.06 0.01 INSVGTGEGVAEGTANITVASK
_________________________________________________________________________
39
Table 3.1. (Continued).
Peptide mass fingerprinting (MALDI-MS) of tryptic fragments of Csp-L of A500
Fragment MW (exp) MW (calc) Δ MW[aa] [Da]a [Da]b [%]c Corresponding aa-sequence d
8-24 1799.05 1798.94 0.01 LANLIDPEVMGPMISAK 34-53 2065.28 2065.08 0.01 IAPIDNSLEGQPGSEITVPK 56-80 2615.48 2615.24 0.01 YIGDAQDVAEGAAIDYSALETESVK 92-110 2037.13 2036.97 0.01 LTDESVLSGYGDPVEEAQK120-136 1903.13 1902.99 0.01 VDNDILEEALTTTLEVK147-174 3117.75 3117.52 0.01 IENTFTDAPDAIEDESITTTGVLFLNYK179-189 1247.71 1247.64 0.01 LREEAAGSWTK190-200 1158.70 1158.64 0.01 ASQLGDDLLVK201-213 1446.86 1446.77 0.01 GAFGELLGWEIVR217-226 971.61 971.55 0.01 LADGNALAVK237-244 859.52 859.46 0.01 NLLAESGR284-304 2269.09 2269.25 0.01 SVETDSAPIQDFSTMTVAELKEELVTR
Peptide mass fingerprinting (MALDI-MS) of tryptic fragments of Tsh-L of A500
Fragment MW (exp) MW (calc) Δ MW[aa] [Da]a [Da]b [%]c Corresponding aa-sequence d
11-26 1807.89 1807.93 0.00 YFVAEIVDGVGEPVWK 31-56 2859.99 2860.13 0.00 WITNVSDDGSDNTEEQGDYDGDGNEK 57-85 3255.33 3255.58 0.01 TVVLGYSEAYTFEGTHDREDEAQNLIVAK 98-109 1272.62 1272.67 0.00 IEIPDTETAVGK117-133 1613.74 1613.70 0.00 GSAGGGDATEFPAFACR134-141 936.43 936.47 0.00 IAYDETPK142-162 2269.13 2269.16 0.00 VTKPEESPSSVTVDHDTITVK163-191 2965.32 2965.48 0.01 VGETFTINASVLPAGASQEVTYTSSNPPK
a. Expected molecular w eight determined from the observed molecular mass of the protonated ions.b. Molecular w eight of the corresponding fragment calculated from the deduced aa sequence.c. Dif ference betw een the expected and the calculated molecular w eights.d. Amino acid residues resulting from the frameshift are indicated in bold letters
_________________________________________________________________________
40
3.2. Identification of the lytic structural protein (LSP)
3.2.1. LSP: a common element among Listeria phages
To assess whether different Listeria phages posses a structural component with
muralytic activity, zymogram assays were performed with heat inactivated
substrate cells of SV 1/2 (WSLC 1001) or SV 4b (WSLC 1042), according to the
host range of the analyzed phage. For that purpose the temperate phages A118,
A500, and PSA and the virulent phage A511 were propagated and purified. Phage
proteins were separated under denaturing conditions. Upon renaturation a single,
transparent ~30-kDa band appeared in the case of A118, A500, and PSA,
whereas for A511 two bands (~24 kDa, ~36 kDa) with muralytic activity appeared
(Fig. 10 B). The same phage proteins were simultaneously separated on a classic
SDS-PAGE (Fig. 10 A) and compared to the corresponding zymograms. The LSP
in A118, A500, and PSA is directly located below the major capsid protein (Cps).
Because the Cps overlaid the LSP, a direct allocation to a designated band was
not possible for these phages.
2D-gel electrophoresis with protein sample of phage A500 was performed to
improve separation of the zone responsible for the muralytic activity (Fig. 11) (82).
The combination of a high-resolution IEF in immobilized pH gradients with
molecular sizing in either SDS gel or zymogram rendered it possible to correlate
these two gels. As control served the same phage protein sample separated by
molecular sizing. After renaturation of the zymogram, a muralytic band appeared
only in control lane (Fig. 11-2). In further experiments we were able to show that
the urea used in 2D-separation irreversibly affected the ability of the LSP to
renature, excluding this alternative strategy (data not shown). Therefore, we opted
for another alternative method in which the renaturation capacity is conserved (no
urea) and thus activity based detection remains possible. Phage proteins were
separated by SDS-PAGE on 14% Tris/Tricin gels. The protein profile of PSA
showed an improved separation within the 30 kDa size region including the major
capsid protein (Cps) and the frameshifted tail protein (Tsh-L) (144). This led to the
_________________________________________________________________________
41
Fig.
10.
LSP
of
List
eria
pha
ges
A11
8, A
500,
PSA
, an
d A
511.
Pha
ge p
rote
ins
in d
enat
urat
ing
SD
S b
uffe
r w
ere
subj
ecte
d to
zym
ogra
phy
on S
DS
-PA
GE
gel
s w
ith e
mbe
dded
sub
stra
te c
ells
(B) a
nd w
ere
rena
tura
ted
over
nigh
t. Zo
nes
with
mur
alyt
ic a
ctiv
ity w
ere
visi
ble
as c
lear
lysi
s zo
nes,
and
cou
ld b
e co
rrel
ated
to th
e C
oom
assi
e st
aine
d co
rres
pond
ing
prot
ein
prof
iles
of th
e ph
ages
(A).
Cps Tsh
Cps Tsh
Cps Tsh
CpsTsh
A
B
Cps
-L
Tsh-
L
A
B
A
B
A
B
A118
A50
0P
SA
A51
1
_________________________________________________________________________
42
IEF (pH 4-7)+ -
+
- A500
IEF (pH 4-7)+ -
1 2
12% SD
S-PAGE
A500
Fig. 11. Improved separation of A500 phage proteins by 2D-gel electrophoresis. Two dimensional separation of phage proteins using IEF in immobilized pH gradient gels
pH 4-7 in the first dimension (left to right) and molecular sizing in SDS-PAGE (1) or
zymogram (2) in the second dimension (top to bottom). Phage A500 proteins served as
control.
detection of new protein species in this region (Fig. 12 A). The bands were
excised from the gel and these gel pieces reloaded onto a zymogram gel for
confirmation of lytic activity. The band responsible for lytic activity in phage PSA
was then subjected to mass spectrometry. However, due to the low abundance of
the protein from the gel piece and high contamination with peptides of Cps, a
direct assignment of the LSP to a structural protein was not yet possible.
As shown in Fig. 10, zymograms of A511 proteins showed two distinct lytically
active bands. In contrast to the temperate phages A118, A500, and PSA, the
corresponding proteins are not located immediately next to any major protein
band, which simplified identification and separation. As performed for the LSP of
PSA, the band(s) suspected to harbor the lytic activity were excised from SDS-
PAGE gels and reloaded onto a zymogram. Protein samples from gels were then
analyzed by MS-based peptide fingerprinting. The results indicated that the lower
_________________________________________________________________________
43
A511 SDS-PAGE SDS-PAGE zymogram
Cps
Tsh
gp106
gp102gp145
3
2 1
45
1 2 3 C
32 1
Cps
PSA SDS-PAGE SDS-PAGE zymogram
Tsh-L
1 2 3 4 5 CA511 SDS-PAGE SDS-PAGE zymogram
Cps
Tsh
gp106
gp102gp145
3
2 1
45
1 2 3 C
32 1
Cps
PSA SDS-PAGE SDS-PAGE zymogram
Tsh-L
1 2 3 4 5 C
Fig. 12. Zymogram based detection of LSP from phage PSA (A) and A511 (B). A)
The band excised from first dimension SDS-PAGE was loaded onto a zymogram gel.
Only the protein from the band loaded in lane 1 showed a zone of lytic activity, whereas
neither the second identified small band between Cps and Tsh-L (lane 2), nor the band
corresponding to Tsh-L (lane 3) showed muralytic activity. PSA phage proteins loaded in
lane C served as positive control. B) The A511 virion contains two cell wall hydrolases.
Phage proteins were first separated by 12% SDS-PAGE and Coomassie stained. Gel
pieces corresponding to the indicated bands (left panel; 1-5) were excised and reloaded
onto a 12% zymogram gel containing Listeria host cells as substrate (right panel; lanes 1-
5). A511 proteins served as positive control (lane C). After renaturation, the proteins from
bands loaded in lanes 1 and 4 showed lytic activity, and were identified by mass
spectrometry.
_________________________________________________________________________
44
band contains a mixture of gp102 and gp145, and the upper band consists of
truncated form of gp106. Gp145 is unlikely to represent a putative LSP, because
orf145 is not located in the late genes cluster encoding the structural proteins, but
putatively encodes an “early protein” of unknown function. Gp106 was found in
several bands of different mass, possibly due to proteolytic cleavage or
degradation and was therefore not further analyzed. In agreement with the
activity-based zymogram results, BLAST analysis suggested a possible lysozyme
activity for gp102. Gp102 was recombinantly produced but did not reveal any
muralytic activity (data not shown). Interestingly, a strong homology to conserved
lysozyme domains was also revealed for gp97 (MW: 131 kDa), which, however
could not be identified among the lytic bands from zymogram. To test whether this
gp97 displays a lytic activity it was recombinantly produced and tested in lysis
assays and zymogram. No activity could be detected (data not shown).
3.2.2. Identification of gp19 as the lytic structural protein (LSP) in A118
Phage A118 was analyzed for lytic structural proteins by zymogram which
revealed a lytic protein of about 30 kDa in mass as shown in Fig. 10. To correlate
the LSP to specific phage proteins, the zymogram was compared to Coomassie
stained SDS-PAGE of A118 (Fig. 13 A / B). The putative LSP is located
immediately below the band comprising the major capsid protein (Cps). It was
found that the lytic principle of a single protein species was conserved. Therefore,
fractions out of several 14% Tris/Tricin gels were removed, combined in a single
tube, and reloaded on a second gel. Content of 8 gel pieces per slot were
reloaded on a 12% SDS-PAGE and analyzed by Western blot using the antisera
against the baseplate proteins (α-gp16 (C-term Tmp), α-gp17, α-gp18, α-gp19, α-
gp20, and α-gp-21) and α-ply118 antiserum as negative control (39). α-gp19
revealed a weak signal in the Western blot analysis (Fig. 13 C). The same sample
was analyzed by zymogram to ensure the presence of this lytic protein and we
found its lytic activity to be conserved (data not shown). In addition the same
equivalents were subjected to mass spectrometry where gp19 was identified in
homogenized gel pieces (Fig. 13 D), indicating that gp19 is responsible for the
_________________________________________________________________________
45
lytic band in zymograms. However, to test whether recombinantly produced gp19
displays lytic activity against listerial host cell, the protein was tested in zymogram
and lysis assays but no activity could be observed.
A B C
16 17 18 19 20 21 Ply118
1 MLNLDKWGNT LFDSNKYQQF NANMEKLEKD SLAKDVDINA41 TNNRIDNVVL EAGGNNITEV VDARTSKNGQ VYSTLNSRLN81 GDYSAIASDL AESNALLQTV NEENKVLKSK LDELYGNSAS 121 NIEYYVSSTN GNDVTGTGAI DAPFKTIQKA VNMVPKVKVG 161 GFIYIFCEPG QYNEDVVVQS FSGAECFYIQ PTNLATIDPT 201 TGQTGFFVKS ILFSGIMFQC VVQGLNSMST AVNNNSTVIQ 241 FARCWYGTVT KCRFDTNLKA TNITTVQYNQ SRGNCYSNYF 281 KNQNIIMSSE YMGHALFAST NTCEATSNVG LKAASGGILV 321 KSGTPVLNAT TAELKQAGGQ IF
D
~30 kDa
Fig. 13. Identification of gp19 as the LSP in A118. Phage A118 proteins in denaturating
SDS buffer were subjected to zymography on SDS-PAGE gels with embedded substrate
cells (B). The region with muralytic activity was visible as clear lysis zone, and could be
compared to the Coomassie stained protein profile of A118 (A). C) Western analysis with
different antisera (indicated by numbers) of gel pieces displaying lytic activity in
zymograms. Fragments found by peptide mass fingerprinting of gp19 are indicated in bold
letters (D).
_________________________________________________________________________
46
3.3. Topological model of the A118 tail tip
3.3.1. Antibodies against putative tail and baseplate proteins of A118
Both the RBP and the LSP are believed to be integral parts of the baseplate,
whose structural proteins are encoded by the late gene cluster. Polyclonal rabbit-
antibodies were raised against six distinct gene products encoded by these late
genes. Specifically the C-terminal part of the tape measure protein (Tmp; gp16),
and gp17 to gp21. These six antisera were tested for specific binding to phage
structural proteins. Purified phage particles were separated by SDS-PAGE and
Western blotted using the different antisera. Individual protein bands were
recognized and labeled by specific antibodies, which correlated well to the results
obtained by peptide fingerprinting (Fig. 14). Apart from the α-gp16 (C-terminal part
of Tmp) that bound to a protein with lower molecular mass than the full-length
Tmp with a calculated molecular mass of about 186 kDa. The faint band
generated by α-gp21 did, however, not correlate to the calculated molecular mass
of gp21 (MW: 12.4 kDa).
3.3.2. Gp18, gp19, and gp20 of A118 play an important role in the early steps of infection
Besides the binding to the protein profile the antisera generated were tested for
binding to specific structural proteins that are important in phage attachment
and/or infection. The infectivity of phage particles after pre-incubation with serum
was tested using a pull down assay. Phages pre-incubated with antiserum were
mixed with Listeria host cells SV 1/2 (WSLC 1001). Adsorbed phages in the pellet
were plated on suitable host cells. Phages incubated with the antisera were
compared to controls challenged with the corresponding pre-immune sera. All
counts were normalized to 100%. As controls, pull down of untreated phages on
either SV 1/2 (WSLC 1001) or SV 4b (WSLC 1042), to which A118 cannot adsorb,
were performed (Fig. 15). Antibodies α-gp16, α-gp17, and α-gp21 had no effect
_________________________________________________________________________
47
Fig. 14. Western blot analysis of the A118 protein profile using antibodies generated against several baseplate proteins. Numbers indicate the antisera used
for immunoprobing (A). Position of Cps is marked by blue lines. Calculated molecular
weight of the different gene products are indicated in table below the lanes. Protein
profile of A118 is shown on in panel B.
gp20
/Cps
-L g
p18
(A50
0)
port
al
gp8,
gp1
1gp
9
Tsh
Tsh,
Cps
gp17
Cps
Tsh-
L
66 45 35 25 18.4
14.4
gp19
(A50
0)
1617
1819
2021
186
AB
30.9
39.4
37.2
39.2
12.4
MW
(cal
c) [k
Da]
of g
ene
prod
ucts
Wes
tern
blo
tana
lysi
s
_________________________________________________________________________
48
0
50
100
150
200
anti-Tmp(C-term)
anti-gp17 anti-gp18 anti-gp19 anti-gp20 anti-gp21 w/o Ab w/o Ab
SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 4b
% p
fu in
pel
let
Fig. 15. Gp18, gp19, and gp20 of A118 play an important role in the early steps of infection. A118 phage particles, pre-incubated with either antisera against gp16 to gp21
and corresponding pre-immune sera were tested for their ability to attach and infect
Listeria SV 1/2 host cells in a pull down assay. Plaque forming units (pfu) of adsorbed
phages were determined in%. Pre-immune sera were normalized to 100%. Adsorbed
phages were counted as plaques. Pull down of untreated A118 with SV 1/2 and SV 4b
cells served as negative control.
on phage infectivity. In contrast, α-gp18, α-gp19, and α-gp20 completely inhibited
infectivity, indicating that their binding partners gp18, gp19, and gp20 play vital
roles in the early recognition and infection process.
To support this finding, different A118 deletion mutants were constructed. The
genes encoding for gp18, gp19, and gp20 were deleted in the WSLC 1001::A118
strain. Each of the prophage was then induced by U.V. light and corresponding
lysates were tested for presence of infective phage particles. Compared to
_________________________________________________________________________
49
induced wildtyp WSLC 1001::A118, none of the three tested induced lysates of
1001::A118Δ18, 1001::A118Δ19, or 1001::A118Δ20 showed infective phage
particles. Detection of the mature phage virions in the lysates was only possible
by PCR and not by Western blot analysis or by transelectron microscopy.
3.3.3. Transmission electron microscopy (TEM) analysis of Listeria phage A118
To determine whether the antibodies generated against the putative baseplate
proteins bind to the phage particles, transmission electron micrography (TEM)
was performed (Fig. 16). For all tested primary antibodies, the gold conjugated
secondary antibodies located to the baseplate (Fig. 16 A). Since antibodies have
two antigen-binding sites, these antibodies were able to crosslink phages with
each other. This property could be used to better locate the antibody binding site
(Fig. 16 B / C). Antibody α-gp16 (C-terminus Tmp) bound in the interconnection of
tail tube and tail tip. Binding of α-gp19 (LSP) was restricted to the lower baseplate
ring. Antibody α-gp20 crosslinked phages at the upper baseplate ring. It appeared
that α-gp17 linked phages at two different positions within the phage baseplate:
one directly located below gp16 at the interconnection of tail to tail tip and the
other between the upper and lower baseplate. This suggested that gp17 may form
an inner core of the tail tip and is accessible from different sites. α-gp18 was able
to bind at the center of the lower baseplate ring. Crosslinkage by α-gp21
demonstrated that the connection of upper and lower baseplate ring is at least
partly made up of gp21. Since each of the different antibodies bound in a
characteristic pattern, it was possible to allocate all of the tested putative
baseplate proteins. Based on the data obtained by TEM, a model of the A118 tail
tip could be generated. The proposed model is presented in Fig. 17 and is
compared to detail TEM micrographs of the tail tip in side and bottom view.
_________________________________________________________________________
50
Fig. 16. Transmission electron micrographs of A118. A) TEM of immunogold-labeled
A118 baseplate proteins. (Continued next page).
_________________________________________________________________________
51
50 nm50 nm
50 nm50 nm
50 nm50 nm50 nm
50 nm50 nm
50 nm50 nm
50 nm50 nm50 nm
α-gp19(LSP)
α-gp20(RBP)
α-gp21α-gp16(C-term)
α-gp17 α-gp18
α-gp17 α-gp18 α-gp21
α-gp16 (C-term)
α-gp19(LSP)
α-gp20(RBP)
B
C
Fig. 16. Transmission electron micrographs of A118. (Continued). B) TEM of A118
particles following incubation with α-gp16 to α-gp21 antibodies. C) Proposed crosslink is
indicated with an arrow. Scale bars correspond to 50 nm.
_________________________________________________________________________
52
15 n
m
300
nm (t
ailt
o he
adju
nctio
n)
20 nm
Tsh / Tsh-L
gp16 (C-term; Tmp)
gp20 (RBP)
gp19 (LSP)
gp17
gp18
gp21
gp20 (RBP)
gp19 (LSP)
11 nm
A
B
C
Fig. 17. TEM analysis and proposed protein architecture of the A118 tail tip. Results
of the antibody-bindings were summarized by a schematic model of the phage tail tip
showing anatomical features and dimensions (C). Proposed model of the tail tip
apparatus is compared to TEM pictures in side view (A) and bottom view (B).
_________________________________________________________________________
53
3.4. Identification of the receptor binding protein (RBP)
3.4.1. Gp20 of A118 and A500 binds to Listeria cell walls
Due to the non-overlapping and complementary host range of Listeria phages
A118 and A500, putative RBP genes were assumed to show no or only partial
sequence homology with each other. Furthermore, based on the genomic location
and on comparison to other known RBPs in Gram-positive bacteria, the putative
RBP in phage A118 and A500 is located in the late gene cluster (33, 132).
Therefore, the putative baseplate and tail fiber proteins of both A118 and A500
(gp17 to gp22 in both phages) were compared to each other and analyzed. The
gp17 (50% identity 138/271; e-value = 2e-68), gp18 (60% identity; e-value = 8e-
118), and gp19 (34% identity 117/344; e-value = 6e-41) proteins revealed high
sequence similarities and were therefore unlikely to represent candidates for the
putative RBP. However, bioinformatic analyses suggested that gp20, gp21, or
gp22 represent the RBP. While there was no significant similarity found for gp21
and gp22, the similarity between gp20 of A118 and A500 was restricted to the N-
terminal part. The amino acid sequences of gp20 were also compared to gene
products of PSA, another phage displaying the same host range as A500. The
various homologies of the putative RBP proteins from A118, A500, and PSA were
indicated in Fig. 18. The alignments revealed that gp15 of PSA is 96% identical in
the C-terminal part over 121 amino acids (e-value 4e-42) to gp20 of A500. All
three proteins showed an identity of 50-62% over 56-59 amino acids (e-values:
9e-11 and 2e-11) in the core part of the proteins. To determine the specificity of
the putative receptor binding proteins of A118 and A500, labeling and localization
studies were performed. For this, purified GFP-tagged proteins were mixed with
exponentially growing Listeria cells of SV 1/2 (WSLC 1001) and SV 4b (WSLC
1042) and after incubation, specific binding was analyzed under the fluorescence
microscope (Fig. 19 A / B). Known SV-specific cell wall binding proteins (CBD-118
and CBD-500) served as positive and negative controls. GFP-RBP A118 (gp20 of
A118) bound to SV 1/2 but not to SV 4b cells, whereas GFP-RBP A500 (gp20 of
A500) bound to cells of SV 4b but not to SV 1/2 (Fig. 19 B1 / B3). Interestingly,
the two truncated versions of the putative RBP of phages A118 and A500
_________________________________________________________________________
54
displayed the same SV restricted binding pattern as observed for the full length
proteins (Fig. 19 B2 / B4). Nevertheless the fluorescence microscope images
showed that compared to full-length versions, the labeling of the truncated
proteins fused to GFP occurred at discrete spots only, and the overall intensity of
the decoration by the truncated proteins was lower.
PSA gp15
A500 gp20
A118 gp20 N C1/2
4b
N C
N C
0 160 220 357 aa
Φ infects SV
4b
proposed RBP
Fig. 18. Alignments of 3 putative RBPs of Listeria phages A118, A500, and PSA. Color bars and vertical lines indicate homologies. Red: identity of 54% (86/157; e-value =
7e-40)/ orange: A118 to A500: identity of 50% (28/56; e-value = 9e-11), PSA to A500:
62% (37/59, e-value = 2e-11)/ yellow: identity of 96% (117/121, e-value = 4e-42)/ white:
no similarity.
_________________________________________________________________________
55
SV 1/2 SV 4b
1
2 4
3
CGFPCGFP GFP-RBP A500 (C-term)
GFP-RBP A500
ProteinA Binding to SV 1/2
Binding to SV 4b
Φ A118
-
-
++
+
-++
B
(3)
(4)
RBP length(aa, w
/o GFP)
198
355
0 157 220 355 aa
C GFP-RBP A118 (C-term)
GFP-RBP A118 GFP C
GFP
-
-
++
+
(1)
(2)201
357
0 156 220 357 aa
Φ A500 ++-
Fig. 19. Identification of gp20 as the receptor binding protein (RBP) in A118 and A500. A) Putative RBPs of A118 and A500 (Full length and N-terminally truncated
version) were fused to GFP and tested for binding to Listeria cells of SV 1/2 and SV 4b.
Lengths of the different RBPs (w/o Gfp) are indicated in aa (Gfp not in scale). B) SV
specific binding by the putative RBP of A118 and A500. Fluorescence microscopy images
of SV 1/2 or 4b cells labeled with full-length GFP-RBP A118 (1) or GFP-RBP A500 (3).
Binding of the N-terminally truncated versions GFP-RBP A118 (C-term) (2) and GFP-RBP
A500 (4) to either SV 1/2 or 4b cells.
_________________________________________________________________________
56
3.4.2. The A118 RBP requires N-acetylglucosamine and rhamnose for binding
Listeria strains resistant to A118 plaque formation were used to study the capacity
of binding of the putative RBP protein in the absence of bacteriophages. In a
previous study, the receptor molecules for A118, N-acetylglucosamine (GlcNAc)
and rhamnose (Rha), were identified (139). Further it was shown that A118 was
unable to attach and to infect SV 1/2 ΔGlcNAc (HLT 2/2, (127)) or SV 1/2 ΔRha
(HLT 2, S. Kathariou, personal communication) compared to SV 1/2 parental
strain (1/2a3 (57)) (127). In order to confirm that phage A118 and its putative RBP
use the same cell wall ligand, the binding of GFP-RBP was studied. The ability of
GFP-RBP A118 to bind to SV 1/2 ΔGlcNAc and SV 1/2 ΔRha Listeria strains was
analyzed (Fig. 20). The two mutant strains, ΔGlcNAc and ΔRha, were not labeled
by GFP-RBP A118 (Fig. 20 B / C). GFP-RBP A118 displays the same binding
pattern as A118 to the tested strains, confirming the role of gp20 as the RBP in
Listeria phage A118.
_________________________________________________________________________
57
GFP-RBP A118
Φ A118 (B) ΔGlcNAc
Infection
(C) ΔRha
(A) Parental stra
inSV 1/2
A
B C
+ - -
+ - -
+ - -
Attachement
Fig. 20. Binding of A118 RBP to phage resistant strains of SV 1/2. GFP-RBP A118
was incubated with SV 1/2 parental strain (A), SV 1/2 ΔGlcNAc (B), and SV 1/2 ΔRha (C).
_________________________________________________________________________
58
_________________________________________________________________________
59
4. Discussion
In this study, the lytic structural protein (LSP) and the receptor binding protein
(RBP) of Listeria phage A118 have been identified and localized. The data
allowed the proposal of a topological model of the phage A118 tail tip.
Gp19 most likely represents the LSP in phage A118, as shown by zymogram
data. The protein profile of A118 showed no signal when immunoprobed with α-
gp19, compared to the corresponding lytic band on the zymogram (about 30 kDa).
However, a band was observed at the predicted full-length gp19. Only when the
protein concentration was increased eightfold, gp19 could be identified in the
region responsible for the lytic zone in zymograms by mass spectrometry and
Western blot analyses. This finding suggests that mainly the full-length gp19 is
incorporated in the mature phage particle, and that gp19 is rare in assembled
virion particles. The LSP might be present only in low copy numbers within the
phage tail or baseplate, or may be activated only upon infection. The latter is
supported by the finding that recombinantly expressed full length LSP (gp19)
showed no lytic activity against Listeria host cells (data not shown). However,
judging from the size of the zymogram lysis zone, the active form of gp19
possesses a strong lytic activity. Processing of phage structural proteins is a
common phenomenon (50). It was demonstrated that Tal2009 of L. lactis phage
Tuc2009 can undergo auto-proteolytic cleavage at a glycine-rich region, which
detaches this lytic activity from the rest of the protein. Both processed and
unprocessed forms of Tal2009 are present in the mature phage particle and were
shown to be located at the tip of the tail (87, 131). Incorporation of an inactive
precursor protein or only few copies of the active protein might be essential as a
self-protection strategy of the phage. Indeed, carrying highly muralytic proteins
may have negative effects on the interaction of phage with host cells.
Consequently, the initial steps of the phage infection cycle would be impaired by
formation of uncoordinated lesions into host cell wall to which the phage is
adsorbed. The data obtained by mass spectrometry, however, exclude a C-
terminal truncation of gp19. Thus, cleavage of the N-terminal part might be the
activation mechanism for the LSP in A118. The exact cleavage site could not be
_________________________________________________________________________
60
determined by N-terminal sequencing, due to the low abundance of the active
protein. The copy number and location of the LSP within the tail tip in A118 differs
compared to the lactococcal phage Tuc2009 (61, 87). Whereas in the case of
Tuc2009 the LSP (Tal2009) form a fiber structure at the tip of the phage tail (87),
gp19 of A118 was shown to form the entire lower baseplate ring (Fig. 17).
However, the activation of gp19 (A118) remains to be elucidated.
It could be shown that the presence of a LSP is a common feature among the
Listeria phages tested. It was previously described that all Siphoviridae infecting
Gram-positive hosts tested, contain murein hydrolases in their virions (86, 90,
129). This was observed not only for the described Listeria phages, but also for
other phages investigated during this study, such as P35, B054, B025, A006 (data
not shown). Nevertheless, zymograms of these phages displayed only weak
bands.
Interestingly, in SDS-PAGE the LSP was often located closely to the dominant
band, corresponding to the major capsid protein (Cps). Why the molecular mass
of Cps and LSP are so similar is still unknown, but this seems to be common in
many phages (I. Molineux, personal communication). However, this finding might
be coincidental.
In case of A511, two lytically active proteins of different sizes (~26 kDa and
~36 kDa) were identified by zymogram analysis. The size of the upper band is in
perfect agreement to the molecular weight of the native endolysin Ply511 (MW:
36.5 kDa). It was previously shown that lysozyme e of phage T4, the endolysin
responsible for “lysis from within”, can also be found associated to the phage
particle (average 0.5 molecule/phage particle), but actually plays no role in the
infection process (35). Another example where the association of the endolysin to
the mature phage particle was also demonstrated is Pseudomonas phage ФKZ
(89). PRD1 is a lipid-containing virus and its morphology differs from the other
described phages, but it also carries the protein responsible for host cell lysis and
liberation of progeny phages (110). T4, ФKZ, and PRD1 are infecting Gram-
negative bacteria. However, Western blot analysis of the A511 proteins, using a
Ply511-crossreacting antibody (39), indicated that Ply511 is probably not
associated to the phage (data not shown). Although it was possible to identify
_________________________________________________________________________
61
proteins within the bands allocated to the size of the lytic bands, it is believed that
another protein might be responsible for this effect. Gp97 displays strong
homology to conserved lysozyme domains, and is therefore likely the protein
responsible for the activity observed within zymograms.
In the protein profile of A511, gp97 (131 kDa) was found to be allocated to a
protein band, together with gp106 (128 kDa), with a molecular weight of around
100 kDa. Zymogram analyses revealed no muralytic band at this position. This
suggests that a full length and inactive gp97 is incorporated in the mature virion.
In both A118 and A511, the band comprising the full length gp19 (A118) or gp97
(A511) did not correlate to the smaller lytic band found in zymograms. Processing
to a smaller and active form, as it is likely for gp19 of A118, might also be possible
for gp97 of A511. Unfortunately, the identification of gp97 was not possible on the
zymograms. This might be again due to the low concentration of the LSP, as
suspected for gp19 in A118. Further, the in vitro expression of full length A511
gp97 in E. coli failed, suggesting that gp97 might have detrimental effects on the
bacterial cell. However, the actual role of gp97 in A511 remains to be elucidated.
Throughout the process of identifying gp19 as the LSP in A118, it was noticed that
urea, used in the first dimension isoelectric focusing of 2D-gel electrophoresis,
irreversibly affected the ability of the lytic active protein to refold. The actual
mechanism how the activity is inhibited remains unclear. However, it was shown
to exhibit residual activity when the zone with the assumed LSP was cut out of a
gel and subsequently reloaded onto a zymogram. SDS-treatments remained
reversible even after several sequential denaturation and staining steps. This
characteristic was essential for the isolation and identification of gp19 as the LSP
of A118.
In this study, gp20 in A118 was identified as the RBP. Moreover, comparison to
A118 related phages, such as A500 and PSA, enabled the identification of a RBP
in these phages. The GFP-RBP-fusion proteins of A118 and A500 were able to
bind SV-specific Listeria cells and moreover display the same binding pattern, as
the phage host range (Fig. 19). The genes encoding the putative RBPs are
_________________________________________________________________________
62
modularly organized and the binding specificity resides in the C-terminal domain.
This finding is supported by the fact that N-terminally truncated GFP-fusion
proteins bound to host cells as well, although binding was less pronounced and
occurred in a spot-like, localized fashion, compared to the full-length proteins.
However, lack of the N-terminus may cause improper folding of the truncated
protein. Further, the N-terminal domain of the RBPs of A118 and A500 could be
responsible for proper phage assembling, stabilization of the baseplate, and may
be involved in a strong protein-protein interaction with other phage tail proteins as
shown for the T-even phages (47). Such a modular organization in genes
encoding RBPs was previously described for phage DT1 and MD4 (32).
The A118 phage receptors are sugar residues in the teichoic acid of the cell wall,
namely N-acetylglucosamine (GlcNAc) and rhamnose (Rha) (139). The data from
this study confirms that GFP-RBP A118 (gp20) binds to the same substituents.
The binding was restricted to the parental strain of SV 1/2 but not to ΔGlcNAc or
ΔRha mutant strains. Resistance to A118 was previously shown by lack of either
of the two sugar components within the listerial cell wall teichoic acids (127). As a
result, it can be concluded that Rha and GlcNAc are necessary for phage
attachment.
Taking into consideration the binding specificity of gp20 of both, A118 and A500,
and the binding to the phage receptor substituents of gp20 of A118, these
proteins are believed to be responsible for attachment and host range
determination in these Listeria phages.
The LSP and the RBP are believed to represent structural components of the
phage baseplate in mature viruses. The baseplate proteins are encoded in a
region located between ORF16 (Tmp) and ORF 23/24 (lysis cassette
Ply118/Hol118) in the late gene cluster of the A118 genome. The use of
polyclonal antibodies against these gene products and subsequent TEM analysis
allowed us to propose a model of the A118 tail tip. Gp16 to gp21 were allocated to
a designated location within the tail tip. Gp16 is believed to be the Tail tape
measure protein (Tmp) (80), which determines the tail length and is located in the
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63
tail tube (49, 58). It is localized by α-gp16, (directed against the C-terminal Tmp
part). The fact that the C-terminal domain is so easily accessible for the antibodies
suggests that it might also be involved in tail sheet to baseplate connection.
Labeling by Tmp-antibodies was also found in a mutant lactococcal phage,
lacking the double baseplate structure and therefore allowing access of the
antibodies to the Tmp (131). The precise role of the Tmp at this interconnection
between tail and baseplate remains to be elucidated. No evidence was found for
Tmp processing, as shown to be the case for other phages (49, 97, 144).
As demonstrated by phage pull down using antisera, not only α-gp19 (LSP) and
α-gp20 (RBP) were able to neutralize A118 phages, but also α-gp18. Since
attachment and receptor recognition are often described as a two-step process
(91), it cannot be excluded that gp18 is also involved in receptor recognition.
Furthermore, the ability of α-gp18 to neutralize phage adsorption may be due to a
sterical obstruction, disabling recognition and binding to the major cell wall
receptor by the RBP (gp20). No putative function could be assigned to gp18
based on BLAST analyses.
The importance of the three proteins gp18, gp19, and gp20 in the mature virion
has also been suggested by introducing deletions in any of the three protein
genes. Lysates of the mutant prophages harbored no infective phage particles.
Unfortunately, it was not possible to obtain evidence for the actual presence of
mutant phages in the lysates (via transmission electron microscopy) because of
concentration effects. Interestingly, induced numbers of wildtype A118 prophage
were also too low to be detected. The inability of the mutant phages to infect did
not allow propagation to higher titers. Therefore, it remains unclear whether the
inability to infect is due to a phage assembly defect, or due to the lack of a crucial
element that is important in host recognition. Further investigations are needed to
assign functionalities to all of the baseplate proteins.
_________________________________________________________________________
64
Finally, the protein profiles of six Listeria phages were compared and analyzed by
mass spectrometry in order to allocate additional protein bands consisting of
minor proteins to predicted gene products (27, 79). The major proteins of five of
the analyzed phages (A511, P35, A500, A118, and A006) have already been
characterized (27, 63, 145). The protein profile of phage P40 was newly
characterized. Related phages display a similar protein profile, therefore A118
and A500, or P35 and P40 were observed as being similar.
Interestingly, programmed translational frameshifts were identified in A118 and
A500. Both utilize +1 as well as -1 programmed translational frameshifting for
generating Cps and Tsh proteins with different length C-termini. The obtained
data showed that the mode of the translational frameshift in both phages is
identical (28). Considering the icosahedral symmetry of a phage capsid, the
possible role of the C-terminally modified Cps was explained for PSA (144). PSA,
similar to the newly tested phages A118 and A500, features a capsid structure
with a triangulation number T = 7 (16). These capsids consist of a total of 420
protein subunits, which are organized in 12 pentameric and 60 hexameric ring
structures, the capsomeres (140). The ratio of Cps and Cps-L in PSA is explained
by the different ratio of these subunits and is in perfect agreement with the
experimentally determined ratio. Ribosomal frameshift within Tsh might also play
a role in correct assembly of the tail. For B. subtilis phage SPP1, it was shown
that the tail morphology was altered, when only one of the two tail proteins was
expressed (7). In bacteriophage λ, a frameshift controls production of two proteins
with overlapping sequences, gpG and gpGT, that are required for tail assembly
(74). The correct relative amounts of proteins for virion assembly seems to be
crucial; the head and tail proteins of phage λ are produced in very different
amounts as a result of different translation efficiencies (112). It has been shown
for phage T4 that a correct ratio of different structural proteins is crucial to achieve
efficient phage production (34). Consequently, programmed translational
frameshifts seem to be important for the biological role of the products, since they
are widespread among phages and strongly conserved (8, 141). The use of such
ribosomal frameshifts was described as a recoding event specified in the
sequences of phages and insertion sequence (IS) elements (8) and might refer to
_________________________________________________________________________
65
a common ancestor or lateral transfer of genes (8, 15). Generation of N-terminal
identical proteins could also ensure best fit for developed phage assembly
strategies. Nevertheless, these translational frameshifts in the structural proteins
were identified in representative members of several hundreds known Listeria
phages, suggesting a universal mechanism rather than an unusual finding.
Control of L. monocytogenes has become an important issue in the food industry
in recent years. The use of bacteriophages for specific recognition and elimination
of this human pathogen offers powerful tools and alternative approaches. So far,
research mainly focused on the application of whole phages or phage endolysins.
These methods have been shown to be very effective in reduction or elimination
of Listeria in food (44, 46, 64, 81). The results presented here describe virion
associated components, namely the LSP and RBP in Listeria phage A118. Both
RBP and LSP appear to harbor significant potential for a number of applications.
For example, it is believed that the RBP, similar to the CBDs of endolysins (64),
can be elegantly used for detection and immobilization of Listeria. The binding of
RBPs is strictly restricted to the corresponding phage sensitive strains, therefore
displaying a higher specificity as CBDs do. This correlates well with a finding that
the CBD-ligands differ to the phage receptors on the surface of the listerial cell
(81).
These are the first identifications of baseplate components with designated
functions in Listeria phages. The morphology and distinct functional assignments
identified in representing members of Listeria phages suggests universal
horizontal exchange of such genetic elements, as they are universally located at
similar positions. Therefore, the results of this report may be extrapolated to other
phages infecting Firmicutes. Evaluation on the application possibilities and
characterization of these functional proteins will provide for interesting future
studies.
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66
_________________________________________________________________________
67
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Publications
Bielmann R., R. Lurz, R. Calendar, M.J. Loessner. 2009. Identification and
Localization of the Lytic Structural Protein (LSP) Receptor Binding Protein (RBP)
in Listeria monocytogenes Bacteriophage A118.
(In preparation).
Dorscht, J., R. Bielmann, M. Schmelcher, Y. Born, M. Zimmer, R. Calendar, J. Klumpp, and M. J. Loessner. 2009. Comparative genomics and proteomics of
Listeria bacteriophages reveals an extensive mosaicism and programmed
translational frameshifting as common elements.
(In preparation).
Klumpp, J., J. Dorscht, R. Lurz, R. Bielmann, M. Wieland, M. Zimmer, R. Calendar, and M. J. Loessner. 2008. The terminally redundant, nonpermuted
genome of Listeria bacteriophage A511: a model for the SPO1-like myoviruses of
gram-positive bacteria. J Bacteriol 190:5753-65.
Szathmary, R., R. Bielmann, M. Nita-Lazar, P. Burda, and C. A. Jakob. 2005.
Yos9 protein is essential for degradation of misfolded glycoproteins and may
function as lectin in ERAD. Mol Cell 19:765-75.
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Danksagung
Ein herzlicher Dank geht als erstes an Prof. Martin Loessner, der mir die
Möglichkeit gegeben hat, meine Forschungsarbeit in seiner Gruppe zu
absolvieren. Sein stets offenes Ohr, die zahlreichen Besprechungen und
Anregungen und sein Vertrauen in mich und diese Arbeit haben massgeblich zum
Gelingen dieser Dissertation beigetragen.
Bedanken möchte ich mich auch bei Prof. Herbert Schmidt für das Übernehmen
des Korreferats.
Vielen Dank an Rudi Lurz für die phänomenalen Bilder am Elektronenmikroskop
und die interessante und sehr erfolgreiche Zeit, die ich am Max-Planck Institut in
Berlin verbringen durfte. Richard Calendar gebührt ein Dank für das perfekte
Aufreinigen der verschiedenen Phagen, die für die Protein-analysen eingesetzt
wurden.
Ein spezieller Dank geht an meine Kollegen Yannick Born, Yves Briers, Simone
Dell`Era, Jeannette de Vries, Dominik Doyscher, Fritz Eichenseher, Marcel
Eugster, Lars Fieseler, Susanne Günther, Steven Hagens, Monique
Herensperger, Thomas Huber, Jochen Klumpp, Kwang-Pyo Kim, Rainer
Lehmann, Miluse Mares, Patricia Romero, Barbara Schnell, Uschi Schuler-
Schmid, Markus Schuppler, Timo Takala und Markus Zimmer.
Die stete Unterstützung bei „Pipettier-Problemen“ und das gute Arbeitsklima
waren von unschätzbarem Wert. Auch die unzähligen Znüni-Kuchen, die den
Laboralltag massgeblich versüsst haben, und die besondere Atmosphäre bei
Laborausflügen und -events, BQM/DVD/Grill-Feierabenden sind positiv zu
erwähnen. Vielen Dank!
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Weiter geht ein grosser Dank an meine Studenten Thomas Luchsinger und
Manuel Kradolfer und meine Studentin Anna-Maria Gabryjonczyk für ihre
motivierte und tolle Arbeit während den absolvierten Forschungsprojekten.
Danke auch allen anderen Mitarbeitern, Freunden und Kollegen am Institut für
Lebensmittelwissenschaften für ihre Hilfsbereitschaft.
Erwähnen möchte ich ausserdem die Personen, die ausserhalb der direkten
Forschungstätigkeit für mich wichtig waren und mich massgeblich beeinflusst
haben. Der wohl grösste Dank geht an meine Freunde und Freundinnen, die mich
während der ganzen Zeit begleitet, gestützt und gestärkt haben. Neben den
Aufmunterungen und wichtigen Entscheidungshilfen in allen Lebenslagen, auch
danke für sportliche Badmintonabende, abkühlende Tauchevents, plaudernde
Joggingrunden, gemütliches Beisammensein und eine spannende und
unvergessliche Studienzeit.
An dieser Stelle möchte ich mich auch bei Patrice Tscherrig bedanken. Seine
grosse Unterstützung, nicht nur im Hinblick auf diese Arbeit, war und ist für mich
von grösster Bedeutung.
Mein herzlichster Dank gebührt meinen Eltern Lilly und Paul Bielmann. Nicht nur,
dass ich ohne sie nicht da wäre wo ich jetzt bin, sie haben mich stets unterstützt
und es mir immer ermöglicht mein Leben nach meinen Vorstellungen zu gestalten
und zu leben.
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Curriculum Vitae
Regula Bielmann Born, 29th of September, 1978 in Freiburg, Switzerland Citizen of Rechthalten (FR), Switzerland 2004 – 2009 Ph.D. student, Food Microbiology Laboratory Institute of Food Science
and Nutrition, Swiss Federal Institute of Technology (ETH), Zürich 2004 Internship in the Laboratory of Dr. E. Chevet, Department of Surgery,
McGill University, Montreal, Canada 2001 – 2004 Studies in Biology at ETH Zürich Diploma thesis in the Institute of Microbiology ETH Zürich 1999 – 2001 Basic studies in Biology at the University of Freiburg (CH) 1994 – 1999 Degree in Elementary Education, Primarlehrerseminar Freiburg (CH) 1991 – 1994 Secondary school, Plaffeien (FR) 1985 – 1991 Elementary school, St. Silvester (FR)