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

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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.

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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).

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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

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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)