pedro miguel azevedo veloso improving derived listeria phage

Escola de Engenharia

Pedro Miguel Azevedo Veloso

Improving derived Listeria phage endolysins properties at low temperatures

Outubro 2014

Escola de Engenharia

Pedro Miguel Azevedo Veloso

Improving derived Listeria phage endolysins

properties at low temperatures

Tese de Mestrado Mestrado em Bioengenharia

Trabalho realizado sob a orientação de Doutor Leon D. Kluskens

Outubro 2014

É autorizada a reprodução parcial desta tese apenas para efeitos de investigação, mediante

declaração escrita do interessado, que a tal se compromete,

Universidade do Minho, __ /__ /____

Assinatura:_________________________________________________

Aknowledgements

Agradecimentos

x

Agradecimentos

A vida é uma longa caminhada e, apesar de este ser apenas mais um capítulo que agora se encerra,

dela fazem parte várias pessoas. Pessoas essas que de forma direta ou indireta me ajudaram a

conseguir alcançar objetivos, metas. É a essas pessoas que expresso, de seguida, os meus sinceros

agradecimentos por me terem apoiado ao longo deste projeto.

Em primeiro lugar quero agradecer ao meu orientador, Doutor Leon Kluskens, por me ter dado a

oportunidade de estar presente num projeto científico ambicioso, por todo o conhecimento e

conselhos transmitidos, por ter permitido aumentar o meu gosto pela área da biologia molecular

assim como pela disponibilidade que sempre demonstrou ao longo de todo este projeto.

Agradecer à Graça, que desde o primeiro dia me ajudou de forma incansável, demonstrando sempre

enorme disponibilidade, espírito de entreajuda, apoio e compreensão mesmo nos momentos mais

difíceis. Obrigado pelo conhecimento e experiência transmitidos.

Agradecer ao Hugo, que apesar de ter entrado um pouco mais tarde no projeto, me ajudou com

todo o seu conhecimento, pragmatismo e criatividade ajudando a contornar obstáculos que, por

diversas vezes, se colocaram no caminho.

Aos meus colegas de laboratório: Luís, pelos conselhos prestados, boas conversas e momentos de

bom humor; à Catarina pela sua grande simpatia; ao José pelas boas conversas tecnológicas que

tivemos. À Marta e Patrícia, colegas de mestrado, pela sua prontidão e disponibilidade em ajudar.

Aos meus amigos de sempre, Pedro, Rui, João e Maria João pelo apoio prestado e pela boa

disposição em todos os momentos.

À Ana, meu ombro amigo, meu porto de abrigo, minha conselheira. Obrigado teu apoio incondicional

e preciosos conselhos transmitidos durante este capítulo da minha vida.

E por fim, o mais importante, à minha família, mãe, pai, irmã e avós. Obrigado por todo o esforço

que fizeram para que conseguisse alcançar os meus objetivos pessoais e académicos. Obrigado por

todo o apoio nos bons e maus momentos. Obrigado por me terem incutido desde sempre bons

valores humanos. Espero um dia conseguir retribuir-vos tudo aquilo que me deram. Ser-vos-ei

eternamente grato!

Abstract

Resumo

xiv

Abstract

Listeria monocytogenes is a Gram-positive opportunistic pathogen that can grow in a wide variety of

conditions and is responsible for listeriosis, a potential fatal disease, associated to the ingestion of

contaminated food. The concerns about the upsurge of widespread reported cases, combined with

emerging antibiotic-resistance amongst pathogenic bacteria, such as L. monocytogenes, demand for

the development of novel preservation techniques that ensure the safety of food products.

Endolysins, which originate from virulent bacteriophages, are responsible for the hydrolysis of the

covalent bonds in peptidoglycan layer of the host cell. These enzyme properties represents a good

alternatively approach against Gram-positive foodborne pathogens without altering the organoleptic

properties of food products.

However, in most of the cases, the activity and stability of naturally occurring enzymes is significantly

lower than the biotechnological industry needs. Besides, there is a lack of research advances in lytic

activity improvements of endolysins in food storage conditions.

The experimental work developed in the scope of this thesis aimed at directing endolysin activity

towards refrigeration temperatures against L. monocytogenes through the use of directed evolution

strategies – error-prone PCR and cryodrilling.

Different attempts were done for the isolation of listerial phages from livestock industries effluents

and consequently identification and improvement of lytic activity of its derived endolysins.

An in silico analysis of two different lysins – Ply500 and Ply511 – were performed to provide

contextualization about their structure and domains. Although both proteins possess modular

structure, Ply511 has a central catalytic domain and a not well characterized binding domain which

contrasts to Ply500 domain organization. Protein expression in large and micro-scales of wild-type

proteins was successfully done and confirmed by performing antibacterial tests against L.

monocytogenes 5725.

To enhance the activity of endolysins against L. monocytogenes cells, modified endolysins were

constructed by amplifying their sequences using error-prone PCR technique and cloning into pQE-30

vector. The cloned vectors were transformed in E. coli JM109 competent cells, however no colonies

were obtained.

At the same time, using PlyP100 endolysin, a second approach based on the biotic interaction

between phage-host at successively temperature was done to promote phage adaptation and

consequently enzymatic evolution.

xv

Resumo

Listeria monocytogenes é um agente patogénico oportunista Gram-positivo, responsável por provocar

listeriose, doença potencialmente mortal associada ao consumo de alimentos contaminados. A

preocupação inerente à sua capacidade de sobreviver numa grande variedade de condições, o

crescente número de surtos da doença e o aumento da resistência a antibióticos obrigam a que

novas estratégias de conservação e preservação dos alimentos sejam desenvolvidas.

Endolisinas derivadas de fagos são enzimas responsáveis pela lise das células do hospedeiro. Uma

vez que não alteram as propriedades organoléticas dos alimentos, o uso destas enzimas representa

uma boa alternativa na eliminação de agentes patogénicos Gram-positivos.

Contudo, a baixa atividade e estabilidade das enzimas no seu estado natural torna-se incompatível

com as necessidades industriais.

Este trabalho experimental visou o melhoramento das propriedades líticas das endolisinas a

temperaturas de refrigeração recorrendo a técnicas de evolução direta – error-prone PCR e

cryodrilling.

Numa primeira abordagem foram efectuadas tentativas para o isolamento de fagos de Listeria a

partir de efluentes de indústria pecuária com posterior identificação e melhoramento das

propriedades líticas das respetivas endolisinas. No entanto, as tentativas não foram bem-sucedidas.

Foi efetuada a análise bioinformática das duas diferentes endolisinas – Ply500 and Ply511 – para

se obter informações precisas sobre a sua estrutura. Apesar das duas proteínas possuírem uma

estrutura modular, Ply511 apresenta um domínio catalítico central com função desconhecida e por

conseguinte, pouco caracterizado, relativamente à organização modular de Ply500. A expressão das

respetivas endolisinas wild-type em larga e micro escalas foi efetuada com sucesso e confirmada

através de testes antibacterianos contra L. monocytogenes 5725.

Para melhorar a atividade lítica das respetivas endolisinas, as sequências foram amplificadas por

error-prone PCR, clonadas no vetor pQE-30 e transformados em células competentes E. coli JM109.

No entanto, não foram obtidas quaisquer colónias.

Ao mesmo tempo, usando a endolisina PlyP100, uma nova abordagem baseada no princípio de

interação biótica entre fago-hospedeiro, foi efetuada a temperaturas sucessivamente mais baixas de

forma a promover a evolução/adaptação do fago e consequentemente da endolisina.

xvi

Index

xviii

Index

Agradecimentos .......................................................................................................................... x

Abstract ..................................................................................................................................... xiv

Sumário ....................................................................................................................................... x

List of abbreviations .................................................................................................................. xxiv

List of figures .......................................................................................................................... xxviii

List of tables ............................................................................................................................. xxiv

CHAPTER 1 – Introduction and Background .............................................................................. 1

1. Listeria monocytogenes and listeriosis .................................................................................... 3

1.1. Adaptation mechanisms ................................................................................................ 4

1.2. Antimicrobial resistance ................................................................................................. 4

2. Bacteriophages as potential means to battle listeriosis ............................................................ 5

2.1. Listeria phages .............................................................................................................. 7

3. Endolysins ............................................................................................................................. 9

3.1. Different types of endolysis ............................................................................................ 9

3.2. Endolysin structure ...................................................................................................... 10

4. Strategies for endolysins improvement ................................................................................. 11

4.1. Phage adaptation “cryodrilling” .................................................................................... 12

4.2. Directed evolution: site-directed mutagenesis Vs. random mutagenesis ........................ 12

4.3. Site-directed mutagenesis ............................................................................................. 13

4.3.1. Domain swapping .............................................................................................. 14

4.4. Random mutagenesis for in vitro directed enzyme evolution ......................................... 15

4.4.1. Chemical mutagenesis....................................................................................... 15

4.4.2. Mutator strains .................................................................................................. 16

4.4.3. Site-saturanting mutagenesis ............................................................................. 16

4.4.4. Error-prone PCR ................................................................................................ 17

5. Advances in molecular engineering of endolysins .................................................................. 18

5.1. Advances using site-directed mutagenesis .................................................................... 19

5.1.1. Advances using domain-swapping ..................................................................... 19

5.2. Advances using random-mutagenesis .......................................................................... 20

6. Main goals of this work ........................................................................................................ 21

xix

CHAPTER 2 – Materials and Methods ..................................................................................... 25

1. Bacterial strains, endolysins and plasmids ........................................................................... 25

2. Listeria phage isolation ........................................................................................................ 26

2.1. Non-lysogenic strains selection ..................................................................................... 26

2.2. Effluent samples and phage detection .......................................................................... 27

2.3. Phage isolation and propagation ................................................................................... 27

3. Protein production ............................................................................................................... 28

3.1. Large-scale production ................................................................................................. 28

3.1.1. Culture and induction ........................................................................................ 28

3.1.2. Cell lysis............................................................................................................ 28

3.1.3. Purification ........................................................................................................ 28

3.1.4. Polyacrylamide gel electrophoresis (SDS-PAGE) ................................................. 29

3.1.5. Protein quantification ......................................................................................... 29

3.2. Micro-scale protein production experiments .................................................................. 30

3.3. Host preparation and antibacterial assays ..................................................................... 30

4. Bioinformatic tools ............................................................................................................... 31

4.1. In silico analysis of bacteriophage endolysins ................................................................ 31

4.2. Primers design ............................................................................................................. 32

5. Cloning ................................................................................................................................ 32

5.1. Polymerase chain reaction (PCR) techniques ................................................................ 33

5.1.1. Error-prone PCR ................................................................................................ 33

5.1.2. Colony-PCR ....................................................................................................... 33

5.2. Plasmid extraction and digestion .................................................................................. 34

5.3. Ligation ........................................................................................................................ 35

5.4. Agarose gel electrophoresis .......................................................................................... 35

6. Transformation .................................................................................................................... 35

6.1. Competent cells ........................................................................................................... 36

6.1.1. Chemio-competent cells .................................................................................... 35

6.1.2. Electrocompetent cells ...................................................................................... 36

6.2. Transformation and plasmid replication ........................................................................ 36

6.2.1. Heat-shock transformation ................................................................................. 36

6.2.2. Electroporation .................................................................................................. 37

xx

7. Directed evolution screening assay ....................................................................................... 37

7.1. Mutant libraries ............................................................................................................ 37

7.2. Screening tests ............................................................................................................. 37

7.2.1. Low temperature tests ....................................................................................... 38

7.2.2. High salt conditions ........................................................................................... 38

8. Cryodrilling .......................................................................................................................... 39

CHAPTER 3 – Results and Discussion ..................................................................................... 41

1. Background ......................................................................................................................... 45

2. Listeria phage isolation......................................................................................................... 45

3. In silico analysis of bacteriophage endolysins........................................................................ 46

4. Protein expression preliminary tests ..................................................................................... 48

4.1. Large-sclae protein production ...................................................................................... 48

4.2. Micro-scale protein expression and preliminary tests ..................................................... 48

4.3. Lytic activity – Low temperatures vs. Room temperatures .............................................. 51

5. Cloning ................................................................................................................................ 52

5.1. Gene amplification ........................................................................................................ 52

5.2. Plasmid linearization and ligation .................................................................................. 54

5.3. Transformation ............................................................................................................. 54

6. Cryodrilling .......................................................................................................................... 56

CHAPTER 4 – Conclusions and Future Perspectives ................................................................ 59

1. Conclusions and future perspectives ........................................................................................ 63

Bibliography .......................................................................................................................... 66

Annexes ............................................................................................................................... 76

xxiv

List of abbreviations

BCA - Bicinchoninic acid assay

CaCl2 – Calcium chloride

CBD – Cell-Binding Domain

CEB – Centre of Biological Engineering

DLA – Double-layer agar

DNA – Desoxyribonucleic Acid

ds – Double-stranded

ECD – Enzymatic Catalytic Domain

ECDC – European Centre Disease Prevention and Control

EFSA – European Food Safety Authority

ep – Error-prone

FDA – U.S. Food and Drug Administration

GAD – Glutamate Decarboxylase System

GlcNAc – N-acetylglucosamine acid

GRAS – Generally Recognized as Safe

ICTV – International Committee on the Taxonomy of Viruses

IPTG – Isopropyl β-D-1-thiogalactopyranoside

LB – Lysogeny Broth

mDAP – meso-2,6-diaminopimelic acid

MurNA – N-acetylmuramic acid

PBS – Phosphate Buffered Saline

PCR – Polymerase Chain Reaction

PEG – Polyethyleneglycol

PG – Peptidoglycan

PPI – Protein-protein interactions

RCA – Rolling-circle

rT – Room temperature

RM – Random Mutagenesis

RNA – Ribonucleic Acid

xxv

SDM – Site-Directed Mutagenesis

SDS-PAGE - Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

ss – Single-stranded

SSM – Site-Saturating Mutagenesis

Tm – Melting temperature

Tris – Tris(hydroxymethyl)aminomethane

TAE – Tris-Acetate-EDTA

TSB – Tryptic Soy Broth

xxvi

List of figures

Chapter 1

Figure 1 – Phage life cycle: lytic and lysogenic. Phage attaches to host and injects genetic material.

In lytic cycle the genomic material is transcripted and replicated by replication and protein synthesis

mechanisms of the host cell used to assemble new phage particles and subsequent release by

cellular lysis. In the lysogenic cycle the phage genome integrates chromosomal DNA of the host

without cell death Adapted from Campbell (2003). ...................................................................... 6

Figure 2 - Representation of peptidoglycan differences between Gram-positive (left) and Gram-

negative (right) cells and different type of endolysins mode of action. 1) Glycosidases; 2)

Muramidases; 3) lytic transglycosidases; 4) Amidases; 5), 6), 7) e 8) Endopeptidases. Withdrawn

from Oliveira et al., (2012) ......................................................................................................... 10

Figure 3 – Overview of the QuickChange Lightning site-directed mutagenesis method. ................ 14

Figure 4 – Diagram scheme comparing the most conventionally random mutagenesis procedures to

ep-RCA evidencing the complexity and time-consuming of each method. ..................................... 18

Chapter 2

Figure 1 - Representation scheme of pQE30 cloning vector used to clone the endolysins sequences

of ply500 and ply511 ................................................................................................................. 25

Figure 2 - 96-wells plate scheme of antibacterial assay against L. monocytogenes 5725 using Ply500

expressed in micro-scale conditions. Protein expression was tested for 24 and 48 h. Endolysin 68

was used as positive control against outer membrane permeabilzed of P. aeruginosa cells. Negative

controls are present in dashed lines wells. Blue wells represents protein expression in 4mL tube;

yellow wells the protein expression in micro-scale; green wells protein expression of 200 µL collected

from 4 mL expression tube and pink wells represent the protein production in 1 mL collected from

four wells. ................................................................................................................................. 31

Figure 3 - 96-wells plate scheme of antibacterial assay against L. monocytogenes 5725 using Ply500

expressed in micro-scale conditions. Protein expression was tested for 24 and 48 h. Endolysin 68

was used as positive control against outer membrane permeabilzed of P. aeruginosa cells. Negative

controls are present in dashed lines wells. Blue wells represents protein expression in 4mL tube;

yellow wells the protein expression in micro-scale; green wells protein expression of 200 µL collected

xxvii

from 4 mL expression tube and pink wells represent the protein production in 1 mL collected from

four wells. ................................................................................................................................. 38

Chapter 3

Figure 1 – SDS- PAGE electrophoresis gel 12%, stained with Coomassie Blue, for Ply511 (A) and

Ply500 (B) proteins. M, Protein Ladder (10-250 kDa) from NEB; F1, 2, 3, and 4, fractions 1, 2, 3

and 4 of eluted proteins; W, wash; FT, flow through. The proteins were eluted with elution buffer

containing 250 mM imidazole concentration. .............................................................................. 48

Figure 2 - Antibacterial assays of Ply500 endolysin against normal and permeabilized L.

monocytogenes 5725 cells. The endolysins were expressed in 4 mL broth and cell lysis was done by

sonication (A). Protein expression was done in 5 wells (200 µL each well) then joined (1 mL) and

sonicated (B). Endolysins were expressed in 200 µL of a 96-wells plate (C). Also protein expression

was induced in 4 mL broth (D). Next 200 µL were transferred to 96-wells. Cell lysis was done by

chloroform vapors. In all the cases the expression conditions were 16°C during 24 h or 48 h with

1.5 mM IPTG concentration. E. coli JM109 wild-type was used as negative control. ..................... 49

Figure 3– Kinetics of antibacterial assays of Listeria phage endolysins Ply500 and Ply511 at

refrigeration temperatures (A) and room temperatures (B). Negative control was done using 180 µL

bacterial suspension mixed with 20 µL of PBS. ........................................................................... 51

Figure 4. – PCR of amplified protein genes using temperature gradient between 55-65°C. Gradient

temperatures from left to right: 55°C, 57°C, 60°C, 62°C, 65°C. Agarose gel 1% concentration,

stained with Sybr Safe and run at 90 V. Legend: M – DNA 1 kb ladder. ....................................... 52

Figure 5 – Amplified endolysins sequences by ep-PCR. The generated products possess 1020 bp

(plyP500) and 1176 bp (plyP511) and were then double digested by using selected restriction

enzymes and purified. Agarose gel 1% concentration, stained with Sybr Safe and run at 90 V. Legend:

M – 1 kb ladder. ........................................................................................................................ 53

Figure 6 – Double digestion of pQE-30 cloning vector in agarose gel 0.7% concentration in order to

promote better separation of digested plasmid The gel was stained with Sybr Safe and run at 90 V.

Legend: M – DNA 1 kb ladder. ................................................................................................... 54

Figure 7 – Representative illustration pQE-30 vector cloned with ply500 (A) and ply511 (B) using

Vector NTI software. The ligation generates two plasmids with different lengths – 4039 bps and 4465

bps, respectively. Both genes are cloned between BamHI and SalI restriction sites. ..................... 55

xxviii

Figure 8 – Colony-PCR results after “heat-shock” transformation using NZY5α E. coli competent

cells. Only pQE-30 + ply500 ligation were assembled by competent cells. Agarose gel 1%

concentration, stained with Sybr Safe run 90 V. Legend: M – DNA 1 kb ladder. ........................... 56

Figure 9 – PCR of amplified endolysins sequences derived from co-evolved (wells 1-5) and evolved

(wells 6-10) adapted phages. Amplified products were visualized by 1% agarose gel, stained with

Syber Safe, at 90 V. The marker (M) is 1 kb ladder. .................................................................... 57

Figure 10 – Example of sequencing chromatogram of adapted plyP100 endolysin derived from co-

evolutionary adapted phage. The putative mutations are highlighted. PlyP100CE1_Fw and

PlyP100CE_Rv are the primers used for gene sequencing. ........................................................ 59

xxix

List of tables

Chapter 2

Table 1 - L. monocytogenes pathogen strains, references and serovars used for phage isolation. . 40

Table 2 - Designed primers for endolysins sequences amplification. ply500 (primers 1 e 2) e ply511

(primers 3 e 4). Underlined are shown the restriction sites for enzymes BamHI and SalI. Melting

temperatures also are present in this table. ................................................................................ 26

Table 3 – PCR conditions for amplification of ply500 and ply511 endolysins sequences in Error-

Prone PCR and Colony-PCR. The volumes were calculated for 50 µL of final reaction volume. .... 34

Table 4 – Used primers for amplification of the PlyP100 endolysins derived from phage adapted

assays. This primers were already available in the group primers collection. Underlined are shown

the restriction sites for enzymes NcoI and BamHI, respectively.. ................................................. 39

Table 5 – PCR conditions for amplification of plyP100 from phage adapted endolysins. The volumes

were calculated for 30 µL of final reaction volume. The initialization step at 95°C during 10 minutes

was used in order to promote the release of phage DNA. ............................................................ 40

Chapter 3

Table 1 – Putative binding and catalytic domains for Ply500 and Ply511 endolysins. ................... 47

Table 2 – Amplified plyP100 sequences of the 5 different phage plaques isolated from Co-Evolution

(CE) and Evolution (E) populations. Once amplified the sequences were aligned and compared to the

original plyP100. Point mutations are observable at specific positions. Legend: A – adenine, T –

thymine, G – guanine, C – cytosine, * – absence of nucleotide. ................................................. 58

Chapter 1

Introduction and

Background

CHAPTER 1 INTRODUCTION AND BACKGROUND

3

1. Listeria monocytogenes and listeriosis

The genus Listeria is an important group of Gram-positive and anaerobic bacteria with low G+C

content and it is closely related to Bacillus, Clostridium, Enterococcus, Streptococcus and

Staphylococcus [1]. It can be isolated from various sources, such as soil, water, effluents and human

and animal faeces [2]. Currently it is divided into six species: L. monocytogenes, L. ivanovii, L.

seeligeri, L. innocua, L. welshimeri, and L. grayi.

L. monocytogenes it is classified as psychrotolerant organism as its optimal growth temperature can

vary between 30 and 37ᵒC. It is considered to be highly pathogenic causing listeriosis, a foodborne

illness that can induce death (20-30% of fatality rate) especially in risk groups such as elderly,

pregnant women and other people with impaired immune system. In this case invasive listeriosis

can be triggered causing meningitis, meningoencephalitis, endocardiditis,septicemia, premature

births, neonatal listeriosis, stillbirths or misbirths [3][4][5] . Non-invasive listeriosis can cause gastro-

intestinal infections and mainly affects healthy individuals.

It is associated with the ingestion of contaminated food mainly in a wide variety of ready-to-eat foods

such as milk, seafood, fish products, meats and meat products [6]. However, the consumption of

soft cheese and seafood is the main responsible for the most human listeriosis cases reported [7].

Since 1960’s decade the listeriosis has become more widespread which has been correlated to the

increased use of refrigerators, consumption of processed foods and extension of shelf-life food

products. According to most recent reports from the European Food Safety Authority (EFSA) and the

European Centre Disease Prevention and Control (ECDC) the number of cases with listeriosis

infections in Europe is on the increase from 0.1 cases per 100.000 in 2000 to 0.3 cases per

100.000 in 2006. It also refers to Germany, Ireland, Lithuania, the Netherlands, Spain and United

Kingdom (UK) as the countries with highest number of infections in the same period [8].

However the increasing demand of consumers for clean-label products in most recent years also has

led to a greater scientific investigation, which consequently increased the characterization of this

pathogen becoming a well-studied model.

Not all strains of L. monocytogenes are equally capable to cause disease in humans and only four

serovars – 1/2a, 1/2c, 1/2b and 4b – of the 13 identified for this strain are responsible for 98% of

the reported listeriosis cases. The strains with 4b serovar are responsible for most food-borne

outbreaks of listeriosis and for the sporadic cases which can suggest that this serovar can possess

unique mechanisms of virulence [9].


4

1.1. Adaptation mechanisms

In food industry usually low temperatures, low pH conditions and high salt concentrations are the

common conditions in order to preserve food products. However, L. monocytogenes has the ability

to generate adaptation mechanisms to maintain its activity, stability and growth, making it difficult to

control this pathogen in the food industry [10]. At low temperatures the mechanisms consists mainly

in changing the membrane composition increasing unsaturated and shortening fatty acids chains

[11], production of cold shock proteins [11][12] and accumulation of solutes as cryoprotectants

[13][14]. For survival under acidic stress the induction of specific proteins (GroEL, ATP synthase and

transcriptional regulators) [15], mechanism of pH homeostasis achieved by proton transport chain,

the glutamate decarboxylase system (GAD) [16] and the two-component regulatory system LisR/

LisK [17] are the main mechanisms of adaptation in low pH conditions. The pathogen also possess

osmoadapation mechanisms such as the induction of salt shock proteins, accumulation of

compatible solutes such as glycine betaine, proline betaine, acetyl carnitine, carnitine, ϒ-

butyrobetaine as osmoprotectants. Furthermore, this bacteria has the ability to change the

expression levels of transcription genes under adverse conditions through the association of the

alternative sigma factor with RNA polymerase core [10].

1.2. Antimicrobial resistance of L. monocytogenes

The current treatment for listeriosis for immunocompromised patients is based on the application of

high doses of β-lactam antibiotics (ampicillin or penicillin) alone or in association with aminoglycoside

(gentamicin). For allergic people to β-lactam antibiotics the treatment consists in the combination of

trimethoprim with sulfonamide [18].

Usually L. monocytogenes is susceptible to a wide range of antibiotics, however, some recent studies

reported an increasing antimicrobial resistance to one or more relevant antibiotics in environmental

isolates [19][20][21]. In animals the use of antimicrobials can lead to new potential development of

antimicrobial-resistant zoonotic foodborne bacterial pathogens, such as L. monocytogenes, that can

subsequent be transmitted to humans as food contaminants.

Furthermore, spontaneous mutations of this foodborne pathogen agent or gene transfection between

antibiotic-resistant bacteria and L. monocytogenes cells may contribute to the increasing of

antimicrobial resistance and subsequent spreading in environment and food.


5

2. Bacteriophages as a potential means to battle listeriosis

Bacteriophages or phages are viruses that infect specific bacterial cells and represent one of the

most abundant biological entities in nature and have been recognized by their potential use as

therapeutic agents in alternative to antibiotics [22]. The history of phages started over a century ago

(1986) when Ernest Hankin observed bacteriocidal activity against Vibrio cholera from filtered and

collected water from Ganges and Jumma River, in India. Almost 20 years after the first observations

by Hankin, two investigators, Frederick Twort, an English bacteriologist, and the French-Canadian

microbiologist Felix d’Herelle reintroduced the subject and independently identified filterable and

transmissible viral particles responsible for bacterial lysis. However, only d’Herelle continue to pursuit

this findings and named the viral particles as bacteriophages [23][24].

Phages are omnipresent and accidentally consumed through the ingestion of food or water, they are

presumed to be safe as undesirable effects have not been detected. Their different genome sizes,

from 17 kpb to 0.5 Mbp and the high frequency of novel genes found in newly characterized phage

genomes proof that bacteriophages are genetically extremely diversified. This genetic diversification

represent a great advantage in infection role as the ability of phages to evolve and circumvent the

defense mechanisms of the host is higher [25].

Bacteriophages are constituted by a protein or lipoprotein coat, called capsid, with different shapes

and sizes that encloses nucleic acid genome which can be single-stranded (ss) or double-stranded

(ds), circular or linear, DNA or RNA. The International Committee on the Taxonomy of Viruses (ICTV)

is responsible for the phage classification that is based on the properties of the virion as morphology

and type of nucleic acid form. Currently, over 5,500 phages are known, divided and recognized by

ICTV into one order and 14 families, among which 96% are tailed (belongs to Caudovirales order)

and possess double-stranded DNA (dsDNA) enclosed in icosahedral symmetry heads and three

different tails lengths subdivided in Myoviridae (25%) with contractile tails, Siphoviridae (61%) with

noncontractile and long tails and Podoviridae (15%) with short tails [26]. The remaining phages are

“cubic”, filamentous or pleomorphic and possess ssDNA, ssRNA or dsDNA as genome and represent

4% of phage population.

As a virus, phages are obligatory parasites and needs to infect the host cells. To be able to infect

host cells, phages attach to the cell through specific receptors in membrane surface using

mechanisms that depends of the virion morphology. The most usual mechanism consists in tail

contraction and enzymatic degradation of small portion of cell membrane allowing injection of genetic

material. Using the replication mechanisms of the host the synthesis of new phage particles occurs


6

which are released by the action of produced lytic proteins responsible for cell lysis (lytic cycle).

Phage infection follows the lysogenic cycle, when phage infection leads to the integration of genetic

information into the chromosome of the bacterial host without cell death. In lysogenic cycle the phage

genome will assume a quiescent state called prophage coexisting in a stable form with the host. The

two possible life cycles of phages can be observed in Figure 1.

Fig. 1 – Phage life cycle: lytic and lysogenic. Phage attaches to host and injects genetic material. In lytic cycle the genomic material is transcripted and replicated by replication and protein synthesis mechanisms of the host cell used to assemble new phage particles and subsequent release by cellular lysis. In the lysogenic cycle the phage genome integrates chromosomal DNA of the host without cell death Adapted from Campbell et al., (2003) [27].


7

2.1. Listeria phages

The first reports of specific L. monocytogenes phages date from 1940 and 1960. Currently more

than 500 Listeria specific phages have been isolated and characterized mainly in the course of phage

typing studies.

Usually the described Listeria-specific bacteriophages possess dsDNA genomes with sizes between

30-65 kb but a few possess larger genomes (124-140 kb). All Listeria phages belong to Caudovirales

order featuring long noncontractile tails (Siphoviridae family) or with complex contractile tails

(Myoviridae family). However, no Listeria specific podoviruses have ever been isolated which can be

related to the low diversity of morphological structure of this bacterial cells [28] [29].

Listeria bacteriophage genomes contain one module responsible for the encoding of structural

proteins, another responsible for encoding DNA functions as recombination, replication and repair.

It also contain a lysis cassette which contains holin and endolysin genes. A lysogeny control region

with the integrase gene is present in case of the temperate phages, which is responsible for mediate

integration of the phage genome into the chromosomal host genome and excision in induction

conditions. In fact many Listeria phages are temperate and most of them are capable of generalized

transduction [28].

There is already some application of Listeria phages in food industry although this application as

biocontrol of bacteria require some specific characteristics: phage must be strictly virulent, feature a

broad host-range, unable to perform generalized transduction, does not affect the pathogenicity or

virulence of the host and also does not integrate its genome on the genetic material of the host [30].

The well characterized Listeria phage P100 is already used as a biocontrol agent product and it

acquired the Generally Recognized as Safe (GRAS) status by U.S. Food and Drug Administration

(FDA). Furthermore P100 phage has been showing some interesting properties, displaying high

efficacy by removing Listeria contaminations from fish and cooked ham and Listeria biofilms in steel

surfaces [31][32][33].

There are also other FDA-approved anti-Listeria products such as a six-phages cocktail. This cocktail

was allowed to reduce Listeria occurrence in food production facilities of fresh cut produce and

melons [34]. Some other studies also showed interesting lytic properties of A511 phage against

Listeria contaminations on various ready-to-eat food samples providing up to 5 log reduction of this

foodborne pathogenic cells [35].

Despite showing many advantages, the use of lytic phages as biocontrol agents in food industry

remains unclear, especially in European countries where it remains uncertain whether phages can


8

be considered as processing aids or as decontaminants or additives [36]. Furthermore, the lack of

studies about phage application in food industry performed in specific preparation, processing and

storage conditions is considered to be an obstacle for its application in this field [22].

An alternative way to phage application consists in the use of its recombinant encoded peptidoglycan

hydrolases (endolysins) which can be easier to approve than virus-based food additive. Endolysins

have no effect on the original organoleptic and texture properties on food and act as an innocuous

substance for human consumption, making these type of enzymes a good candidate for control of

foodborne pathogens [37].

3. Endolysins

The name endolysin was first coined in 1958 with the aim to designate a proteinaceous lytic

substance synthesized inside bacterial cells during phage multiplication and infection stage [38].

Endolysins are dsDNA bacteriophage encoded lytic enzymes that target the integrity of the cell wall

and degrade its main constituent, peptidoglycan (PG), causing cell lysis and the consequent release

of the bacteriophage progeny [39][40].

During phage infection lysins are produced and accumulated in the cytoplasm, however as they do

not have signal sequences to be translocated through the cell membrane this movement is controlled

by a holin. The holin is typically expressed in the late stages of the lytic infection cycle, forming a

pore in the cell membrane allowing cytoplasmic lysins to access the peptidoglycan, causing cell lysis

[41][42][43].

Endolysins are also capable to digest the cell wall when applied exogenously, especially in Gram-

positive organisms, such as L. monocytogenes, and can lyse the cell wall of healthy and uninfected

cells, originating the “lysis from without” phenomenon [39][44]. It proved its ability to be used as

alternative antimicrobial agents [40].

Numerous studies have been demonstrating the potential application of phage derived endolysins as

biocontrol agents in foods which has been considered a good alternative over other antimicrobial

agents. Their high specificity for the target pathogen leaves the natural and desired flora of the food

products untouched. Due to their proteinacious nature endolysins are noncorrosive and

biodegradable [45].

These applications of lysins as biocontrol agents against foodborne diseases have been recently

reviewed [46]. The first and more obvious approach in control strategy consists in the application of

purified endolysins to food products.


9

Some studies have reported the staphylococcal phage endolysin LysH5 [47] rapidly showed great

lytic activity in pasteurized milk, reducing bacterial numbers below the detection level within 4 h. Also

streptococcal lysins B30 [48] and Ply700 [49] and the Clostridium butyricum phage ΦCTP1 [50]

lysin reported to be high lytic activity in milk and milk products. The three Listeria phage endolysins

Ply118, Ply511 and PlyP35 exhibit high thermoresistance which is a very important characteristic

for products that undergo heat treatment such as pasteurized milk products [51].

Another approach is the production and release of this endolysins in starter organisms in

fermentation processes. This alternative has been reported for derived Listeria and Clostridium phage

endolysins, although no studies of application in food has been yet demonstrated [52][53][54].

In conclusion, phage endolysins represent a good alternative for the control of foodborne pathogens.

However some tests are required to verify their stability on other food products and consumer safety.

Physiochemical conditions where these type of biocontrol agents act, must be first evaluated as well.

Furthermore, it has to be economically attractive possible to justify an investment of it application in

food industry [55].

3.1. Different types of endolysins

The cell wall is responsible for maintaining the shape and physical integrity, protecting against

mechanical damage and osmotic rupture. Peptidoglycan is the main constituent and it is composed

of several chains of N-acetylmuramic acid and N-acetylglucosamine residues, linked together by β-

1,4-glycosidic bonds, connected to a short stem tetrapeptide side chains [40][55].

Adjacent tetrapeptides may be cross-linked by an interpeptide bond (in Gram-negative bacteria) or

by an interpeptide bridge (in Gram-positive bacteria) [40].

In Gram-negative bacteria the cell wall is relatively thin (10 nm) and is composed by single layer of

PG surrounded by an outer membrane. In these organisms, lactyl ether connects the glycan

backbone to a peptide side chain that contains mostly L- and D-amino acids [56].

In Gram-positive bacteria, the cell wall is thick (15-80 nanometers), consisting of several layers of

PG. Running perpendicular to the PG sheets is a group of molecules called teichoic acids which are

unique for these organisms. In this case the cell wall it is considerably more diverse in length and

composition coinciding with many different peptide arrangements among PG.

Peptidoglycans may vary the amino in position 3 of the tetrapeptide, defining two types of PGs: meso-

2,6-diaminopimelic acid type (mDAP-type) in Gram-negative and some Gram-positive species (i.e. L.

monocytogenes) and a l-Lysine type (Lys-Type) typical for Gram-positive species [55][57].


10

Therefore, endolysins can be divided in five classes according to their enzymatic specificity.

Glycosidases (Fig.2, target 1) cleave the glycan component at the reducing end of N-

acetylglucosamine (GlcNAc) [58] or at the reducing end of N-acetylmuramic acid (MurNAc) [59].

Muramidases/lysozymes (Fig. 2, target 2) share the same glycan target as the lytic transglycosidases

(Fig. 2, target 3), however both form different products. The 1,6-anhydro bond is formed instead of

the muramic acid residue, due to absence of water that is important for correct function of

transglycosidase activity [60]. N-acetylmuramoyl-L-alanine amidases (Fig. 2, target 4) cut the amide

bond between N-acetylmuramic acid residues and L-amino acid residues. These types of endolysins

are responsible for the strongest destabilization effect in the peptidoglycan layer. Endopeptidases

(Fig. 2, target 5, 6, 7 and 8) cleave the peptides moieties attacking LD- and DD-bonds.

3.2. Endolysin structure

Generally, the sizes of phage endolysins are between 25 and 45 kDa. The only exception is the 114

kDa streptococcal bacteriophage C1 endolysin, PlyC, as it is a multimeric lysin with globular structure

[61]. Endolysins can vary in their molecular structure, modular or globular, and in their domain

orientations.

Fig. 2 - Representation of peptidoglycan differences between Gram-positive (left) and Gram-negative (right) cells and different type of endolysins mode of action. 1) Glycosidases; 2) Muramidases; 3) Transglycosidases; 4) Amidases; 5), 6), 7) e 8) Endopeptidases. Withdrawn from Oliveira et al., (2012) [55]


11

The typical feature for all Gram-positive phage endolysins is their two-domain structure also called

modular structure which is composed by an enzymatic catalytic domain (ECD) at the N-terminal and

the cell wall binding (CBD) domain at the C-terminal [39][41][44]. A few endolysins which have a

modular organization display an inverted structure, with the ECD at the C-terminal and CBD at the

N-terminal.

Gram-negative derived phage endolysins usually presents a globular structure with only ECD.

The evolutionary explanation for the occurrence of CBD domains only in Gram-positive endolysins is

based on previous studies by Fischetti et al., (2008) [41] and Loessner et al., (2002) [62] which say

that CBD domain binds irreversibly to the cell wall preventing the lysis of surrounded cells before the

new phage particles release. Thus it can be said that phages are induced to produce right amounts

of enzymes to induce the cell lysis. This phenomenon is not verified in Gram-negative bacteria

because the presence of the protective outer membrane inhibits the external lysin treatment.

The ECD function consists in the cleavage of the bacterial cell wall while the CBD is responsible for

the substrate recognition after cell binding. The main advantage of modular organization is that it

enables the specificity binding and the enzyme activity in an independent way, enabling the

substitution of either domain with other domain from another enzyme [63][64]. Furthermore, some

interesting studies have demonstrated that some endolysins acquired more than a single ECD, such

as endopeptidase/amidase lysin from the phage phi11 and endopeptidase/muramidase from

Streptococcus agalactiae bacteriophage B30 Hermoso (2003) [65] and Pritchard (2004) [59].

4. Strategies for endolysins improvement

Natural evolution results in a large number of enzyme variants which exhibit their specific function

and efficiency adjusted only to perform under physiological activities. Therefore, naturally occurring

enzymes often lack features necessary for biotechnology industry applications. In this field this study

pretends to accelerate the evolutionary process of a well-adapted endolysin to the most adversal

conditions in food industry more specifically at low temperatures and high salt and low pH conditions.

The strategies consists in phage adaptation at successively low temperatures, also known as

cryodrilling and one of the most used random mutagenesis technique, error-PCR. However some

other methods for directed evolution are presented in this section in order to individually compare

the advantages of each technical approach.


12

4.1. Phage adaptation – “Cryodrilling”

Interactions in many host-parasites are influenced by biology and environment conditions. The

pathogenic parasites impose selection for resistant hosts which in turn impose selection for infective

parasites resulting in rapid antagonist coevolution.

According to the Red Queen hypothesis these biotic interactions give rise to continual natural

selection for adaptation and counter-adaptation playing an important role in molecular evolution and

consequently in population dynamic [66].

There are two types of antagonistic coevolution: the “arms race” dynamics, leading to broader

resistance range in the host against a greater number of parasite genotypes and an increased

variability of host allowing the infection of most host genotypes; and the fluctuating selection

dynamics, favoring hosts that resists to the most common parasite phage genotypes, not contributing

for directional evolution of host range.

Coevolutionary dynamics between hosts and parasites is increasingly investigated using experimental

evolution. The most used model for antagonistic coevolutionary studies are bacteria and their lytic

phages.

Pseudomonas fluorescens SBW25 and its lytic phage ϕ2 is the most studied model of bacteria-

phage evolution given deeper knowledge about this process and coevolution interactions. Although

it is important to diversify this coevolutionary studies to other host-parasite dynamics with particularly

importance in human health as they can be applied as biocontrol agents [67][68][69].

4.2 Directed evolution: site-directed mutagenesis Vs. random mutagenesis

Two contradictory tools can be used on a molecular level: rational protein design and directed

evolution. The first method requires extensive information about relationships between sequence

structures, mechanisms and function of the enzyme. Directed evolution concept was first introduced

in 1967 [70]. It is a laboratory in vitro process responsible for the generation of proteins, enzymes,

metabolic pathways and entire genomes with desired properties under different conditions, such as

extreme temperatures, pH and salinity [71]. Directed evolution implements an iterative Darwinian

optimization process, whereby the fittest variants are selected from an ensemble of random

mutations [72]. Despite the advances to date on the industrially enzymes, the most challenging part

of the directed evolution experiment consists in the development of generic screening or selection

tools, identifying novel enzymes activities more efficiently [73][74].


13

Our understanding of protein function has been enhanced significantly since the early 1980s, due to

the advent of site-directed mutagenesis (SDM) and random mutagenesis (RM).

The use of SDM for the generation of mutations at specified sites within a known polynucleotide

sequence is an extremely common process nowadays, providing a very powerful tool for the

manipulation of sequences and structures.

However, there are certain limitations associated with this technique such as lack of sufficient

structural information or the need to know which sites within a molecule should be investigated in

detail.

At that point, RM has proved to be very useful to identify functional sites within a protein or nucleic

acid sequences. Moreover, RM can provide the opportunity to screen proteins, which has previously

been determined, for structurally important residues or sites, without introducing the selective bias

typical of directed approach. RM also allows the study of regulatory nature and structural features of

nucleic acid sequences, such as promoters, enhancers, ribosome binding sequences, transcription

termination sequences, structures and functions of transfer and ribosomal RNA, the processes

involved in viral assembly, in the primary transcript splicing and the initiation of nucleic acid

replication. When combining RM technique with later measurements large and useful amounts of

important information can be obtained that suggest targets in order that a site-specific or random

oligonucleotide approach can be posteriorly directed [75].

4.3 Site-directed mutagenesis

SDM has a variety of applications and is an important tool in molecular biology that has revolutionized

the study of gene regulation and protein structure-function relationship [76]. The SDM techniques

can be grouped into two major categories: polymerase chain reaction (PCR)-based or non-PCR-based

[77].

The first, also known as single-primer, uses an oligonucleotide complementary to part of single-strand

DNA. The primer contain an internal mismatch to promote the desired mutation. However, because

of the several limitations of this method, low and variable mutants were obtained, which forced the

use of suitable screening approaches to identify mutants [75].

The second strategy, denominated by “cassette mutagenesis” refers to the replacement of a region

of interest with a synthetic mutant fragment generated by annealing complementary oligonucleotides

[78] or by hybridization and ligation of a number of oligonucleotides [79]. The main disadvantages

of this method is that in most of the cases, it needs at least two rounds of primer-based mutagenesis


14

to introduce suitable restriction enzyme sites into templates and it is not appropriate for routine

mutagenesis [80].

Several approaches of SDM technique have been published. However this method generally are labor

intensive or technically difficult. To overcome this difficulties QuickChange Lightning Site-Directed

Mutagenesis (Fig. 3) is a commercial Kit by Stratagene used that allows site-specific mutations. The

main advantage of its usage is that eliminates the need for subcloning and for single-stranded DNA

rescue. Furthermore, does not require specialized vectors, unique restriction sites, multiple

transformations or in vitro methylation treatment steps. Also a single kit allows rapid, efficient and

accurate mutagenesis of small and large plasmids.

Fig. 3 - Overview of the QuikChange Lightning site-directed mutagenesis method.

4.3.1 Domain swapping

Domains are the most important functional units in proteins and a significant proportion of protein-

protein interactions (PPI). Domain swapping is a mechanism for forming oligomeric assemblies. In

domain swapping, a secondary or tertiary element of a monomeric protein is replaced by the same

element of another protein. Domain swapping can range from secondary structure elements to whole

structural domains [81]. It also represents a model of evolution for functional adaptation by

oligomerisation of enzymes that have their active site at subunit interfaces [82].


15

The modular architecture of phage lysins of Gram-positive background and the knowledge

accumulated in recent years from available crystal structures and bioinformatics allows the creation

of a protein with multiple modules of a single origin and also any desired amino acid sequence and

function [83][84][85].

4.4 Random Mutagenesis for in vitro directed enzyme evolution

Random Mutagenesis (RM) is a powerful tool for generating enzymes, proteins and entire metabolic

pathways or even entire genomes with improved properties. RM mechanisms can be divided into five

different categories: (i) transitions, involving substitution of a purine by another, or a pyrimidine by a

second pyrimidine, (ii) transversions, involving substitution of a purine nucleotide by a pyrimidine, or

vice versa, (iii) deletions, in which one or more nucleotides are deleted from a gene, (iv) insertions,

one or more extra nucleotides are incorporated into a gene, and (v) inversions, involving the 180º

rotation of a double-stranded DNA segment [86].

There are many methods by random mutagenesis that can generate genetic diversity such as treating

DNA or entire bacteria with chemical mutagens [87], passing cloned genes using mutator strains

[88], error-prone PCR (ep-PCR) [89], site-saturating mutagenesis (SSM) [90] and finally by using

rolling circle error-prone PCR (ep-RCA) [91].

4.4.1 Chemical mutagenesis

In vitro chemical mutagenesis is one of the most used techniques for the generation of random

mutagenesis in DNA and it does not involve the introduction of heterologous DNA or the manipulation

of interesting DNA by recombinant methods [86][87].

Despite of several chemicals have been tested, the majority of the compounds cannot be used due

to damage caused in the cell [87]. Consequently only five of the best-understood treatments are

currently widely employed in generating randomly distributed single-point mutations in vitro: nitrous

acid, sulfurous acid, hydroxylamine, hydrazine and mil-acid hydrolysis [75]. The main advantage of

chemical mutagenesis is associated with the simple usage and the low cost processing. On the other

hand, the main disadvantage is its inefficient control of the mutation rate and limitation to amino

acid substitutions [86].


16

4.4.2 Mutator strains

Another useful random mutagenesis method is the bacterial mutator strain method that introduces

random point mutations into whole genes. The most popular mutator strain is Escherichia coli XL1-

Red which lacks the DNA repair pathways, MutS, MutD and MutT resulting in a ≈ 5000-fold random

mutation rate higher than the wild type strain [91]. Transformation of the mutator strain and recovery

of the mutant from the transformant are the two main steps for the using mutator strain protocol.

The main advantages of this method are its simplicity and absence of a ligation step, and it can

incorporate a wide variety of mutations such as substitutions, deletions and frame-shift mutations.

The several steps of the growth, plasmid isolation, transformation and re-growth, of this process

originates a progressively sickness of the mutator strain. Additionally this method generates a low

mutation frequency under standard conditions and requires a long cultivation period (longer than

24h) for introduce multiple mutations [86].

4.4.3 Site-saturating mutagenesis

Site-saturation mutagenesis (SSM) is another powerful technique for protein optimization due to its

simplicity and efficiency in which a single amino acid can be substituted to any other 19 possible

substituents [92][93]. As a result, the mutagenesis product is a collection of clones, each having a

different codon in the targeted position, so it is called “saturated”. Whole plasmid single-round PCR

it is a technique that makes SSM method more efficient [90]. This method uses two complementary

primers containing a mutant codon with partial overlap in the 5’ region that improve the amplification

efficiency by decreasing formation primers dimers. This amplification method requires the parental

DNA which is then degraded by using methylation-dependent endonuclease digestion. As a result a

circular, nicked and containing mutated gene vector is produced that can be transformed directly in

E. coli cells [92].

The advantage of this procedure is that eliminates time-consuming subcloning, ligation and single-

stranded DNA rescue. The main disadvantages refers to only two nucleotides can be replaced at a

time and it does not work well with large plasmids (>10 kB).

This method was already successfully applied to generate improved activity of proteins under study

[94][95][96].


17

4.4.4 Error-prone PCR

Error-Prone PCR (ep-PCR) is the most used method to generate random mutagenesis into a defined

DNA sequence that is too long to be chemically synthesized. It consists in reducing the fidelity of the

DNA polymerase which can introduce random mutations during the PCR process [97]. In ep-PCR

process the fidelity of the DNA polymerase is modulated by alteration of the conditions of reaction

buffer, increasing the error rate. The conditions are the addition of Mn2+ for reducing base pair

specificity [98]; increase Mg2+ concentration for stabilizing no complementary base pairs [99];

unbalanced dNTP stoichiometry in order to achieve misincorporation; increase the concentration of

DNA polymerase and altering the extension time [100].

After PCR, three steps are required to clone the library of genes into a host strain: digestion of the

product with restriction enzymes, separation of the fragments by agarose gel electrophoresis and

ligation into a vector, which makes it a very time consuming method [101].

In addition, the success of this method depends of the mutational bias of DNA polymerase, because

most part of low-fidelity polymerases used nowadays show strong mutational preferences that can

favor the substitution of certain nucleotides instead of others, causing the reduction of library diversity

[86].

Studies by Vanherck et al., (2005) [102] combined to different low-fidelity DNA polymerases, Taq

polymerase and Mutazyme, which exhibit opposite mutational spectra. This strategy should permit

generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal

mutagenesis frequencies through ep-PCR and controlling the concentration of template in the

shuffling reaction while taking into account the GC content of the target gene.

Other studies by Asano et al., (2005) [103], Edmond et al., (2008) [104] and Maeda et al., (2008)

[105] have used the efficiency of ep-PCR method in the modification of enzymes with the aim to

increase their potential in industrial applications.

A variation of traditional ep-PCR is called error-prone rolling circle amplification (ep-RCA) and it is an

in vitro amplification DNA method which amplifies circular DNA by a rolling circle mechanism,

yielding linear DNA composed of tandem repeats of the circular DNA sequence [106]. This technique

does not require specific primers because random hexamers can be used as a universal primer for

any template and does not require a thermal-cycler because the amplification reaction proceeds at

a constant temperature. Furthermore, ep-RCA products can be used directly to transform a host

strain (Fig. 4).


18

Ep-RCA consists of only one DNA amplification step followed by direct transformation of the host

strain and producing mutants with an adequate mutation frequency, about 3–4 mutations per

kilobase, for in vitro evolution experiments without using restriction enzymes, ligases, specific primers

or special equipment such as a thermal-cycler are required. This method will enable random

mutagenesis to become a more commonly used technique.

Fig. 4 - Diagram scheme comparing the most conventionally random mutagenesis procedures to ep-RCA evidencing the complexity and time-consuming of each method.

5 Advances in molecular engineering of endolysins

Despite that some endolysins have been optimized by their natural evolution and selection processes,

in most of the cases there is still potential for improvement of their activity and stability, especially in

complex environments such as certain food matrices [107].

Therefore, protein engineering based on directed evolution approaches can alter some endolysins

properties for specific biotechnology applications.

In this section some studies and advances in molecular engineering of endolysins will be discussed

using site-directed and random mutagenesis approaches


19

5.1. Advances using site-directed mutagenesis

In this case, the enzymatic optimization is done through localized changes in amino acids and then

the effect on lytic activity is examined.

The alteration of conserved amino acids in the streptococcal B30 endolysin CHAP and lysozyme

domains resulted in a sequential loss of activity from each domain [59]. Afterward, Donovan et al.,

(2006) [48] analyzed this dual domain lysin on live bacteria and concluded that the B30 endolysin

CHAP domain was the primary source of activity when lysing “from-without”. Site-directed

mutagenesis and deletion analyses of the Bacillus anthracis phage lysin PlyG were essential in

defining the binding domain and active site residues. These observations provided new knowledge

about the mechanism of specific binding of lysin to B. anthracis and may be useful in establishing

new methods for detection of B. anthracis [108].

Nelson et al., (2006) [61] also examined the streptococcal bacteriophage C1 lysin PlyC, using point

mutations and concluded that subunit PlyCA is responsible for catalytic activity. The active-site

residues were confirmed too. Similarly, site-directed mutations altering histidine codons in the

staphylococcal glycyl-glycine PG hydrolase ALE-1 have been used to define essential amino acids in

the M23 endopeptidase domain [109].

Low et al., (2011) [110] demonstrated the influence of a positive net charge in the peptidoglycan

layer induced by site-directed mutagenesis. The increase of the positive net charge can be favorable

for ECD function independently of its CBD through amino acid replacement. This can be important

for fine-tuning enzyme activity, such as in cases where efficacy of an enzyme is limited by its size.

The already described knowledge of binding domains and active site residues of these endolysins is

very useful when constructing novel fusion constructs for the purpose of making a better antimicrobial

agent.

5.1.1. Advances using domain-swapping

The earliest approaches to alter regulatory properties were created by exchange of the CBDs of

pneumococcal autolysins (LytA) and phage endolysins Cpl-1 [111][112]. Studies by Lopes et al.,

(1997) [64] concluded that the fusion of lactococcal N-acetylmuramidase catalytic domains to

choline-binding domains from pneumococcal endolysin CBD resulted in choline dependence of the

chimeric enzyme. Croux et al., (1993) [113], made the reverse experiment by combining a clostridial

CBD with LytA, thereby rendering the chimera active against choline-containing pneumococcal cell

walls and abolishing its activity against clostridial cell walls.


20

More recently, Donovan et al., (2006) [114], fused full-length and truncated versions of the

Streptococcus agalactiae phage B30 endolysin to mature lysostaphin, yielding enzymes that were

active against both streptococcal and staphylococcal cells.

Similar to the studies with protein chimeras of pneumococcal and clostridial origin, the exchange of

Listeria phage CBDs of different serovar specificity also yielded enzymes with swapped lytic

properties and enhanced activity, which constitute interesting antimicrobial candidates for control of

the pathogen. In the same study it was also possible to combine the binding specificities of different

single CBDs in heterologous tandem CBD constructs, which were then able to recognize the majority

of Listeria strains. Manoharadas et al., (2009) [115], created a fusion of the insoluble S. aureus

phage P68 endolysin with a minor coat protein of the same phage and demonstrated that in certain

cases, modular engineering of endolysins may also solve solubility problems ensuring efficient

production and purification of otherwise insoluble lytic proteins.

5.2. Advances using random-mutagenesis

Random mutagenesis is another approach to improve lytic activity of phage endolysins. One of the

first studies consisted in mutate the gene coding for the S. agalactiae phage endolysin PlyGBS using

two DNA mutagenesis, E. coli mutator strain and ep-PCR. After repeated rounds of mutagenesis and

screening a mutant with 28-fold activity was obtained comparatively to the parental enzyme. It also

resulted in the incorporation of unpredicted sequences at the C-terminus of the generated mutant

endolysin. The screening method in this study selected enzyme mutants with high diffusion capability

such as the C-terminal truncation which is smaller than a molecule lacking a CBD. This methodology

can be applied for identification of endolysin mutants with improved activity under various pH and

salt conditions or certain food products [84]. However, the screening method has to be adapted for

every individual condition.

Studies by Heselpoth et al., [116] involved PlyC endolysin thermostability improvement. In this study,

the random mutagenesis approach was used to improve the thermal stability through the use of ep-

PCR, followed by an optimized screening process. The results suggested the methodology generated

PlyC mutants that retain high activities when compared to wild-type after elevated temperature

treatment.


21

6. Main goals of this work

In recent years, listeriosis has become more widespread due to the increasing consumption of

processed foods and extension of shelf-life food products.

The phage derived endolysins are appointed as the best alternative to antibiotic and phage usage as

biocontrol agents. Furthermore, endolysins have no effect on the original organoleptic and texture

properties on food acting as an innocuous substance for human consumption making these type of

enzymes a good candidate for control of foodborne pathogens such as L. monocytogenes.

However, naturally occurring enzymes often lack features necessary for biotechnology industry

applications. Beyond that, little is known about endolysin function in food storage and processing

conditions - low temperatures, low pH conditions and high salt concentrations. There is also a lack

of knowledge aiming the improvement of derived Listeria phage endolysins using random

mutagenesis techniques.

This study pretends to accelerate the evolutionary process, through the use of directed evolution

strategies, aiming to generate well-adapted endolysins to the most adverse conditions in food industry

such as low temperatures and also, high salt and low pH conditions.

Two different directed evolution strategies will be used in this work: error-PCR, the most used random

mutagenesis technique and cryodrilling which mainly consists in biotic interaction of phage-host

aiming its adaptation at successively low temperatures.

Therefore, this work will have two well-defined objectives:

The first objective is to improve endolysins activity at low temperatures, known as refrigerator

temperatures.

The second objective is to reduce the usage of phage and chemical compounds application in food

industry by using the improved endolysins.

Chapter 2

Materials and Methods


25

1. Bacterial strains, endolysins and plasmids

Criovials of E. coli JM109 containing two different Listeria monocytogenes phage endolysins – pQE-

30-ply500 (accession nº Q37979) and pQE-30-ply511 (accession nº Q38653) – and pQE-30 plasmid

with CBD-P35 sequence were kindly provided by Doctor Mathias Schmelcher from Swiss Federal

Institute of Technology Zurich – Department of Health Sciences and Technology.

The pQE-30 plasmid, present in Figure 1 was used for every cloning step.

E. coli JM109 strain belonging to the Centre of Biological Engineering of University of Minho (CEB)

was used as the expression vector. Cultures of this strains, previously transformed with pQE-30, were

done using Lysogeny Broth (LB, Liofilchem) supplemented with 100 µg/mL ampicillin (AppliChem).

Overnight cultures E. coli BL21 (CEB collection) containing Lys68 sequence (plasmid PET28a-

68gpLys) growth in LB supplemented with tetracycline marker (30 µg/mL).

After growth overnight at 37°C and 120 rpm agitation (Environmental Shaker incubator ES- 20/60),

cultures were diluted 1:100 in fresh medium and incubated in the same conditions until reach

exponential growth phase.

The induction for protein expression was accomplished by adding Isopropyl β-D-1-

thiogalactopyranoside (IPTG) (Sigma-Aldrich).

For Listeria phage isolation twelve bacterial strains were used and are present in Table 1. All the

strains were grown overnight in static incubator at 37°C in Tryptic Soy Broth (TSB) (Sigma-Aldrich)

agar plates and incubated next day in TSB broth maintaining the growth conditions.

Fig. 1 - Representation scheme of pQE30 cloning vector used to clone the endolysins sequences of ply500 and ply511

CHAPTER 2 MATERIALS AND METHODS

26

2. Listeria phage isolation

2.1. Non-lysogenic strains selection

In order to avoid the selection of temperate phages, lysogeny test was performed to verify if the

strains used for phage detection did not possess lysogenic activity – prophages – responsible for

bacterial lysis.

A double-layer agar (DLA) test was used to perform the lysogeny test [117]. This method consists in

two LB broth medium layers supplemented with different agar concentrations, 1.2% and 0.6% for

bottom layer and top agar respectively.

All bacteria, previously grown overnight, were resuspended in 100 µL of NaCl 0.1 mM and then

gently homogenized in 5 mL of top-agar and poured into bottom agar layer of petri plate previously

prepared. This procedure was done for all twelve L. monocytogenes strains listed in table 1. Ten µL

of each Listeria strain in every bacterial lawn was placed. Bacterial lawns plates were dried at room

temperature and incubated overnight.

Next day, by observing the plates, the strains that generate an inhibition halo should be discarded

from phage isolation methodology.

Table 1 - L. monocytogenes pathogen strains, references and serovars used for phage isolation.

Bacterial strain Reference Serotype

Listeria monocytogenes CECT 5725 4c

CECT 5873 1/2a

CECT 911 1/2c

CECT 933 3a

CECT 934 4a

CECT 936 1/2b

CECT 937 3b

CECT 938 3c

CECT 4031 1a

923 4b

994 4ab

strain Scott A 4b


27

2.2. Effluent samples and phage detection

For Listeria phage isolation four different effluent fonts were examined: two local sewage treatment

plan (Braga and Vila Verde); one effluent from local livestock industries and one water sample from

Rio Este (Braga). The samples were stored at 4°C until properly treatment and evaluation.

Throughout this procedure two times concentrated TSB medium was used. Depending of the amount

of large debris present in effluent, the sample was centrifuged between 10-30 min at 9000 g, 4°C.

The supernatant was then filtered through a 0.45 µm and 0.22 µm pore-size filters (VWR) preventing

the passage of remaining bacterial cells.

Each sample (20 mL) was then mixture with equal volume of sterile TSB broth and divided in three

batches (40 mL each). Forty µL of bacterial suspension previously grown overnight was added to

each batch wand incubated for 24 hours at the same temperature and agitation conditions. Each

one of the twelve L. monocytogenes strains were used to form confluent lawn over the surface of the

plate and 10 µL of treated sample was placed in each plate. The plates were dried and then inverted

and incubated overnight. Next day phage the formation of phage plaques and the plaque-forming

unit (PFU) were evaluated.

2.3. Phage isolation and propagation

When isolating phages in environmental samples it is important to realize that the phage populations

may consist of several phage strains, hence there is a need to obtain pure strains since a plaque

might contain more than one type of phage. In order to obtain pure phage strains, different phage

plaques were individually toothpicked to new individual plates with the same bacterial lawns from

which they were initially isolated. The phage was spread across the surface of a new second plate

with a sterile piece of paper and incubated overnight in the same optimal conditions of the host. After

add 4 mL of SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl, pH 7.5) to suspend lysis

zones plates were incubated overnight at 4°C. Then the buffer and top agar were centrifuged at

9000 g for 10 min at 4°C and exposed to chloroform (4 volumes to 1 volume of sample) for bacterial

cells removal and suspension filtered with 0.22 µm pore diameter membrane. For phage

precipitation, 0.5 g of NaCl was added to 10 mL of sample and incubated at 4°C for 1 h and the

centrifuge step was repeated. The supernatant was recovered and mixed with polyethylene glycol

(PEG) (Sigma-Aldrich) (1 g for 10 mL of sample) and incubated overnight. After repeat the

centrifugation step, supernatant was discarded and the pellet resuspended in a low volume of SM

buffer (2-10 mL). Finally, 3 volumes of chloroform were added to 9 mL of sample, vortexed and


28

centrifuged at 3500 g for 15 minutes at 4°C. Supernatant was recovered and filtered using 0.22 µm

pore diameter membrane and stored at 4°C.

3. Protein production

3.1. Large scale production

3.1.1. Culture and induction

Cultures of E. coli JM109 containing the wild-type endolysins genes were cultured as previously

described in section 1 with a final volume of 250 mL broth. Once reach the exponential phase

(OD600nm≈0.6) the culture was induced with 0.5 mM IPTG concentration and incubated (MIR-254-PE

cooled incubator) overnight at 16 °C with 200 rpm (Orbital Shakers MIR-S100-PE) agitation.

3.1.2. Lysis

After the incubation period cultures were centrifuged at 4 °C, 4500 g (SIGMA 3-16K Centrifuge),

during 30 min. The supernatant was discarded and the pellet was resuspended in 10 mL of cold

lysis buffer (20 mM NaH2PO4, 0.5 M NaCl/ NaOH, pH 7.4). After transfer the lysate to a new 50 mL

tube it was exposed to three freezing and thawing cycles by exposing the sample at -80 °C during

20 min and then thawed at room temperature. The sample was then submitted to ten sonication

cycles of 30 seconds of pulse (30 kHz) and 30 seconds of rest (Ultrasonic Processors from Cole-

Parmer). Insoluble cell debris was removed by centrifugation at 4 °C, 4500 g for 30 min.

Supernatant was filtered and collected through two different filters of 0.45 µm and 0.22 µm,

sequentially.

3.1.3. Purification

Collected samples were purified through the use of gravitational His-Trap columns (GE Healthcare)

based on the affinity of immobilized nickel present in the column for the N-terminal poly-histidine tail

present in cloning vector.

To remove another possible proteins, columns were firstly washed using 10 mL of equilibration buffer

(lysis buffer with 25 mM imidazole), followed by the binding buffer (25 mM imidazole), and 10 mL

of wash buffer (lysis buffer with 25mM imidazole) to remove contaminant proteins. Finally the protein

was eluted using 2 mL of elution buffer (300 mM imidazole) and kept at 4 °C for further analysis.

The collected fractions in this procedure were analyzed by SDS-PAGE.


29

3.1.4. Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Produced proteins fractions were visualized by using standard denaturation SDS-PAGE according to

Schagger and von Jagow conditions [118]. Gels with 0.75mm thickness, 3.75% of upper and a 12%

lower stacking and separating acrylamide (composition described in Anexe A, Table A1) were

assembled to perform the electrophoresis.

Samples of 10 µL were mixed with application buffer (x2) (125 mM Tris-HCl pH 6.8 (Biorad), 20%

glicerol (p/v) (Biorad), 1% β-mercaptoetanol (v/v) (Sigma), 0,01% bromofenol blue (p/v) (Biorad),

SDS 4% (p/v) (Biorad)) boiled at 95°C for 5 min and loaded onto the gels together with Protometrics

ladder (National Diagnostics).

Generally proteins were separated for 2 h at 90 V using TGS buffer 1x (25 mM Tris, pH 8,6, 192

mM glycine e 0.1% SDS) in MiniPROTEAN®Tetra system (Bio-Rad). Gels were stained using

Coomassie Blue R-250 solution (0,25% Coomassie Blue R-250 (p/v) (Biorad), 50% methanol (v/v)

(Fisher Scientific) and 10% acetic acid (v/v) (Fisher Scientific)) for 15 min. Background distaining

was done in several wash steps with distilled water in the same agitation conditions above described.

Gels were conserved in distilled water for further observation and analysis. Only eluted fractions with

intense bands of protein were posteriorly dialyzed in phosphate buffered (PBS) (137 mM NaCl, 10

mM phosphate, 2.7 mM KCl, pH 7.4) (1x) using Amicon Ultra 0.5 mL columns (Milipore).

3.1.5. Protein quantification

Total protein concentration was measured through of bicinchoninic acid methodology [119] using

BCA Protein Assay Kit (Thermo Scientific) and following the manufacturer instructions.

For better calculation of protein content, dilutions of 10, 30 and 50 times of protein sample were

initially done using PBS 1X. To measure protein concentration, 200 µL of BCA solution was done

and mixed with 25 µL of each diluted protein sample. The mix was incubated during 30 minutes at

37°C and OD580nm was measured in Synergy 2 Multi-Mode Microplate Reader (Biotek).

Calibration curve was performed using different Bovine Serum Albumin concentrations (0-2000

µg/mL) (Fig. C1 – Annexe C).


30

3.2. Micro-scale protein production experiments

In order to determine optimal conditions, cultures of E. coli JM109 containing Ply500 endolysin gene

in pQE-30 vector, wild-type E. coli JM109 and E. coli BL21 containing plasmid PET28a-68gpLys were

done using the same conditions previously described in section 1.

Next day, beyond the dilution already described in section 1, a dilution of 1:10 was also done in each

well of the 96-wells plate with a final volume of 200 µL. Once reached exponential phase, cultures

were inducted with 1.5 mM IPTG and incubated ate 16 °C (MIR-254-PE cooled incubator) with 250

rpm (Orbital Shakers MIR-S100-PE) agitation for 24 and 48 h.

The decreasing of cellular growth in the first three hours after induction was measured by

spectrophotometry (OD620nm) evaluating the correct protein expression.

After incubation time, cultures present in the different wells of the original microplate were joined up

to 1 mL in new eppendorfs and centrifuged at 6000 g (VWR MicroStar 17R) for 10 min. The pellet

was resuspended in 500 µL of PBS and submitted to sonication as described in 3.1.2.

Also, 200 µL of each original tube culture were collected and placed in new different wells of a

second new plate. In this case microplates were firstly centrifuged at 2255 g (Thermo Scientific

CL31R) for 15 min and the supernatant was carefully decanted. Finally the pellet was exposed for

20 min to chloroform vapors lysing the cells. The pellet was then resuspended using 50 µL of PBS.

3.3. Host preparation and antibacterial assays

After a culture of L. monocytogenes 5725 have grown respecting the conditions described in section

1, centrifugation was done at 4500 g during 15 min. The pellet was resuspended in Tris-HCl buffer

(10 mM Tris, 150 mM NaCl, pH 8.0) and the suspension adjusted to OD620nm ≈ 1.0. Twenty µL of

purified protein were mixed with 180 µL of bacterial suspension.

Endolysin activity against permeabilized of L. monocytogenes 5725 cells (previously submitted to -

20°C for 1 h) were also tested following the instructions already described.

As positive control Lys68 endolysin was tested against P. aeruginosa PAO1 cells with exposed

peptidoglycan. This substrate was prepared, using chloroform to remove outer cell membrane,

following the instructions of Lavigne et al., (2004) [120].


31

Fig. 2 - 96-wells plate scheme of antibacterial assay against L. monocytogenes 5725 using Ply500 expressed in micro-scale conditions. Protein expression was tested for 24 and 48 h. Endolysin 68 was used as positive control against outer membrane permeabilzed of P. aeruginosa cells. Negative controls are present in dashed lines wells. Blue wells represents protein expression in 4mL tube; yellow wells the protein expression in micro-scale; green wells protein expression of 200 µL collected from 4 mL expression tube and pink wells represent the protein production in 1 mL collected from four wells.

4. Bioinformatic tools

4.1. In silico analysis of bacteriophage endolysins

In order to identify putative domains within the Ply500 and Ply511 endolysins deposited in GenBank

(http://www.ncbi.nlm.nih.gov/genbank), an in silico analysis of conducted using the HHpred

webserver (toolkit.tuebingen.mpg.de/hhpred) and Pfam as a database. Enzymatic catalytic domain

(ECD) and cell binding domain (CBD) found with an E-value higher than 1x10-5 with at least 80% of

query coverage were considered as significant.

http://www.ncbi.nlm.nih.gov/genbank


32

Table 2 - Designed primers for endolysins sequences amplification. ply500 (primers 1 e 2) e ply511 (primers 3 e 4). Underlined are shown the restriction sites for enzymes BamHI and SalI. Melting temperatures also are present in this table.

Primers Code Sequence Tm (˚C)

1 pHPL500Fw 5ÁAAAAAGGATCCATGGCATTAACAGAG 3´ 68.4 ˚C

2 pHPL500Rv 5ÁAAAAAGTCGACTTATTTTAAGAAGTATTCTTGCTG 3´ 67.8 ˚C

3 pHPL511Fw 5´ AAAAAAGGATCCATGGTAAAATATACCGTA 3’ 66.4 ˚C

4 pHPL511Rv 5ÁAAAAAGTCGACTTATTTTTTGATAACTGC 3´ 65.2 ˚C

4.2. Primers design

For the isolation and amplification of the endolysin sequences, primers in Table 2 were designed

according to the insertion site of the pQE-30 plasmid. Calculation of melting temperatures and GC

content were calculated using online software Tm Calculator from Thermo Scientific. Melting

temperatures were calculated based on thermodynamic [121] and the salt concentration [122].

The formation of secondary structures (primer dimers, hairpins, self-dimers, hetero-dimers) was

predicted using OligoAnalizer 3.1 (https://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/).

In order to avoid cutting of the ply500 and ply511 and obtain the correct cloning, the restriction map

of that sequences were generated using online software NEBcutter: a program to cleave DNA with

restriction enzymes [123] (http://tools.neb.com/NEBcutter2). BamHI and SalI were selected and

the restriction sites were incorporated in primers sequences generating the correct homology

regions.

5. Cloning

This method involves the use of bacterial plasmid pQE30 as cloning vector in order to obtain many

copies of genes of interest. The strategy consists in placing amplified endolysins genes by error-prone

PCR and insert into the cloning vector previously opened up with restriction enzymes (NEB) BamHI

and SalI. The same restriction enzymes are used to cleave the gene of interest generating sticky-

ends that promote the ligation together on the plasmid using T4 DNA ligase enzyme (KAPA

Biosystems).

https://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/

http://tools.neb.com/NEBcutter2


33

5.1. Polymerase chain reaction (PCR) techniques

5.1.1. Error-prone PCR

T4 DNA polymerase and primers used to amplify Listeria endolysins were purchased from KAPA

Biosystems and Invitrogen, respectively.

The conditions of polymerase chain reaction (PCR) (Bio Rad Mycycler Thermal Cycler) were modified

in order to introduce random mutations into the endolysins sequences. The amplification of wild-type

endolysins sequences was done using the primers present in table 2 and the ep-PCR procedure

conditions are present in table 3. This conditions were modified following some instructions described

by Pritchard et al., (2005) [124].

Once obtained, the amplified products were purified using the DNA Clean & Concentrator KitTM-5

(Zymo Research Corporation). The correct lengths were confirmed by agarose gel.

To promote the ligation between the generated sequences and the plasmid, restriction enzymes

BamHI and SalI were used to create sticky ends on ep-PCR products. The generated digested

sequences were newly purified and the concentrations calculated throughout spectrophotometric

parameters of Nanodrop (Thermo Scientific Nanodrop 1000).

5.1.2. Colony-PCR

This PCR-based technique is used to screen for plasmids containing a desired insert directly from

bacterial colonies without the need of culturing or plasmid purification steps. The insert-specific

primers, present in table 2, generate amplified and known length sequences which correspond to

the correct and wild-type sequences.

Colonies were picked from transformation plate, placed into 50 µL PCR tubes and resuspended in

10 µL sterile H2O. PCR reagents that compose the 50 µL reaction mixture, described in table 3, were

added. Individual transformants were harvested in the initial heating step of PCR and cause the

release of DNA plasmids and is used as template for amplification reaction. The correct amplified

genes were confirmed by running agarose gel.


34

30x

Table 3 – PCR conditions for amplification of ply500 and ply511 endolysins sequences in ep-PCR and Colony-PCR. The

volumes were calculated for 50 µL of final reaction volume.

Error-Prone PCR

Colony PCR ply500 ply511

Buffer A (x10)

DNTP’s (10 mM)

Primer FW (10 µM)

Primer RV (10 µM)

Template DNA

MgCl2 (25 mM)

MnCl2 (25 mM)

DNA Kapa Taq

H20 Nuclease Free

5 µL

1 µL

2 µL

2 µL

1 µL

11 µL

1 µL

1 µL

26 µL

5 µL

1 µL

2 µL

2 µL

2.5 µL

11 µL

1 µL

1 µL

24.5 µL

5 µL

10 µL

2 µL

2 µL

10 µL

-

-

0.2 µL

29.8 µL

PCR conditions

95°C – 3 min.

95°C – 30 s.

65°C – 30 s

72°C – 2 min.

72°C – 2 min.

95°C – 3 min.

95°C – 1 min.

65°C – 1 min.

72°C – 3 min.

72°C – 2 min

5.2. Plasmid extraction and digestion

Overnight culture of E. coli JM109 with CBD-P35 sequence present in pQE-30 vector was grown

conditions previously described. Cell culture was then centrifuged at 9000 g during 3-4 minutes and

medium decanted. Plasmid extraction was done using minipreps kit NucleoSpin Plasmid (Macherey-

Nagel). Once extracted, plasmids were digested by using the two selected restriction enzymes

(BamHI and SalI) during 2 h at 37˚C, removing the CBD-P35 sequence. The plasmid digestion was

confirmed by running agarose gel and the correct band was then extracted by removing the

correspond portion of the gel under UV light with sterile scalpel.

In order to prevent the recirculation of cloning vector by removing the 5’-phosphate, Antarctic

Phosphatase (NEB) was used following the manufacturer instructions - during 15 min at rT; the

enzyme was inactivated by increasing temperature to 70°C for 5 min.

25x


35

5.3. Ligation

For the ligation step KAPA T4 ligase (KAPA Biosystems) was used to catalyze the ligation of error-

prone PCR products with linearized cloning vector (section 5.2) with formation of a phosphodiester

bond between 5’ phosphate and 3’ hydroxyl termini in duplex DNA. The required volumes of cloning

vector for this step were calculated based on this equation below:

𝑛𝑔 𝑖𝑛𝑠𝑒𝑟𝑡 =𝑖𝑛𝑠𝑒𝑟𝑡 (𝑘𝑏)

𝑐𝑙𝑜𝑛𝑖𝑛𝑔 𝑣𝑒𝑐𝑡𝑜𝑟 (𝑘𝑏)× 50 𝑛𝑔 𝑣𝑒𝑐𝑡𝑜𝑟 × 5

According with the equation above, the volume of used amplified products depends on the lengths

and on the ratio (1:5) between the two later, for 50 ng of vector used.

5.4. Agarose gel electrophoresis

To confirm the correct lengths of amplified products 0.7% agarose gel were done achieving a better

band separation and promote the correct band excision from the gel. Generally 1% agarose was used

for the evaluation of correct ligation between amplified products and cloning vector. The agarose gels

were prepared with Tris-Acetato-EDTA (TAE) (Biorad) 1x buffer and the electrophoresis performed at

90 V during 45 min. Samples were loaded with gel loading dye blue 6x (NEB) and 1kb (NEB) was

used as ladder. SYBR Safe (Invitrogen) was used as DNA stain for visualization of DNA in agarose

gel in ChemiDoc XRS (BioRad).

6. Transformation

Transformation is a process in which foreign DNA is introduced into a cell. In some bacterial strains

transformation occurs naturally, however most of them require artificial procedures to be in state of

competence and accept exogenous DNA for a time limited response. In this work E. coli JM109 cells

were used and adapted to be in state of competence – chemical and electro competent cells – and

accept recombinant DNA plasmids through heat shock transformation and electroporation,

respectively. All this procedures were based on described protocols in Current Protocols in Molecular

Biology (2007) [125].


36

6.1. Competent cells

6.1.1. Chemio-competent cells

E. coli JM109 chemo competent cells were prepared based on previous described protocols. The

first bacterial inoculum in LB medium, supplemented with 100 µg/mL of ampicillin, grew overnight

at 37°C with 120 rpm agitation (Environmental Shaker incubator ES- 20/60), the next day, a 1:100

dilution of the first inoculum was done into fresh LB medium until the bacterial growth reach

exponential phase (OD620nm≈0.3) maintaining the same conditions. The culture should be centrifuged

at 3300 g, 4°C (Sigma 3-16k) during 10 min and decant the supernatant.

The pellet was resuspended with ice-cold 0.1 M CaCl2 solution in half of the volume used in the

dilution step and store in ice during 30 minutes and then centrifuged following the same conditions

described in first centrifugation step. In this step, competent pellet is much cloudier and there is a

hole in the pellet which indicates already good competent cells. Resuspend the pellet again with the

same ice-cold CaCl2 solution in the 1/10 of the volume used in dilution step. Then, another

centrifugation step is needed in the same conditions above. The final pellet is resuspended in aliquots

of 1mL of ice-cold 0.1M CaCl2 and stored at -80°C.

6.1.2. Electrocompetent cells

To produce electrocompetent cells a inoculum of one colony from a fresh plate of the strain E. coli

JM109 was done in LB broth and incubate at 37°C, 120 rpm, overnight. Dilution of 1/100 from

initial culture in fresh LB medium and incubate at the same conditions above described until the

culture reach OD600nm≈0.5. The culture was then kept on ice for 15 min before the centrifugation step

at 5000 g at 4°C (Sigma 3-16k) during 10 min and the pellet was resuspended in ice-cold and sterile

glycerol 10% solution. This step was repeated three times with decreasing volumes of glycerol 10%

solution. All the procedure was done in ice to prevent thermal shock and increase the efficiency of

competent cells. The final aliquots (100-200 µL of final volume) were stored at -80°C.

6.2. Transformation and plasmid replication

6.2.1. Heat-shock transformation

In this transformation method 100 µL aliquots of chemi-competent cells were withdrawn from -80°C,

after thaw the cells were mixed with 50 ng of DNA and incubated on ice for 30 min. The heat shock

was done during 40 seconds using a 42°C water bath. After heat shock the cells were placed in ice

for 2 min, then resuspended in 300 µL of SOC broth and incubated at 37°C with 120 rpm agitation


37

about 1 h for recovery and cellular growth. Cells were then plated (100 µL and pellet after

centrifugation) in LB plates with ampicillin (100 µg/mL) and incubated overnight at 37°C.

6.2.2. Electroporation

Electroporation was performed using Gene Pulser XcellTM (Bio-Rad) with 0.1cm ice-cold cuvettes at

1.8kV. 50 µL of chemi-competent cells and 0.5 to 2 µL of DNA were mix into the cuvettes. After

electric pulse the cuvette were removed, supplemented with 1 mL of super optimal broth (SOC) and

incubated at 37°C with 120 rpm agitation for 1 hour for recovery and cellular growth. The cells were

plated (100 µL and pellet after centrifugation) using LB plates with ampicillin.

7. Direct evolution screening assay

This methodology allows one to utilize directed evolution to increase the bacteriolytic activity of

translated endolysins at low temperatures and in the presence at high salt conditions. Random

mutations introduced through the use of an error-prone DNA polymerase generate mutant libraries

that are submitted to an extensive screening procedure used to identify possible best bacteriolytic

mutants compared to wild-type molecules in the same conditions. This procedure must be performed

until the desired properties are obtained.

7.1. Mutant libraries

The first step consists in the generation of the mutant libraries throughout the incorporation of

random mutations in wild-type endolysins sequences using error-prone PCR technique which was

previously described in section 5.1.1. The modified endolysins sequences generated were cloned

and transformed as previously described in sections 5 and 6. Additionally, original sequences

followed the same protocol and used as positive controls of transformation step and during the

screening tests.

7.2. Screening tests

The second step, called screening, selects the best adapted colonies in very specific conditions.

Two conditions were tested in this step, low temperatures and high salt concentrations.


38

For protein production the plated colonies were carefully selected from agar plates with sterilized

toothpick and inoculated in the designated well of the 96-wells. The protein production and cell lysis

were carried in the same conditions as described in section 3.2 using 24 h for protein expression.

Original plate, containing toothpicked mutant colonies, was conserved at -20°C in order to recover

potential candidates and repeat the mutation step until desire properties were obtained.

Fig. 3 – Directed evolution assay. Wild-type ply500 and ply511 endolysins sequences are used for error-prone PCR (1) generating library mutants containing random nucleotide mutations. Mutated gene sequences are cloned into expression vector pQE-30 and transformed into E. coli JM109 competent cells (2). Individual colonies are inoculated into their own specific well of a 96-wells plate. Through an extensive screening process the constructs, individual mutants that are catalytically active after incubation at low temperatures and at high salt conditions are selected and classified as mutants with enhanced kinetic stability (3). This process must be repeated until desired properties are obtained (4).

7.2.1. Low temperature tests

In this test L. monocytogenes 5725 cells were prepared according to 3.3 section by transferring 180

µL of bacterial suspension, previously adjusted to OD620nm ≈ 1.0 with PBS to each well previously filled

with 20 µL of protein. The microplate was immediately placed in the microplate spectrophotometer

previously refrigerated at 4°C and the enzyme kinetics were monitored by measuring the OD620nm every

5 minutes for 30 minutes.

AmpR AmpR

AmpR AmpR


39

7.2.2. High salt conditions

This screening test only differs in the preparation of the Listeria strain compared to the previous by

resuspending the pellets in 10 mM Tris, 150 mM NaCl, pH 8.0 buffer the bacterial and adjust

OD620nm ≈1.

8. Cryodrilling

An alternative strategy to increase endolysin enzymatic activity is by using phages as a mutagenesis

promoter. This strategy is based on phage adaptability to different environmental conditions. The

assay called Cryodrilling consists in the propagation of the phage to successive lower temperatures

(two degrees below the previous temperature). The hypothesis is that by adapting phage to lower

temperatures the endolysin encoded in its genome will also be modified, and be consequently more

active. Listeria phage P100 and L. monocytogenes 5725 host were used in this new evolution

strategy.

Table 4 – Used primers for amplification of the PlyP100 endolysins derived from phage adapted assays. This primers

were already available in the group primers collection. Underlined are shown the restriction sites for enzymes NcoI and

BamHI, respectively.

Primers Code Sequence Tm (˚C)

1 plyP100_Fw 5' TATATACCATGGTAAAATATACCGTAGAGAACA 3' 56.9 ˚C

2 plyP100_Rv 5' TATATACCTAGGTTATTTTTTGATAACTGCTCCTG 3' 58.6 ˚C

This assay was divided in two different approaches: in the first approach, called evolution, only the

Listeria phage P100 was allowed to evolve and the bacterial genotype was held constant; on the

second approach, called co-evolution, both phage and host were allowed to evolve.

Initial L. monocytogenes 5725 culture growth in 10 mL of fresh TSB medium until reach the

OD620nm≈0.3. The host culture was then infected with 107 Listeria phage P100 particles. Cultures were

propagated by serial transfers every 48 h in a static incubator. Each culture transfer was done

decreasing the initial temperature (25°C) two degrees until reach the final temperature of 7°C.

Transfers of the evolving populations involved isolating phage particles by centrifuging at 9000 g for

10 minutes and filter (0.22 µm pore membrane). Transferring 1 mL of phage suspension into fresh

wild-type grown previously. In co-evolved populations the transfer was done by inoculating 10% of

volume of each previous suspension in fresh TSB broth.


40

To confirm the phage presence in every two transfers, phage population was estimated by plating

dilutions of each phage population on to TSB agar plates with a semi-solid overlay bacterial lawn.

At 7 °C, different phage plaques were found in evolved and co-evolved populations and isolated. The

new adapted phage production was done as described in section 2.3.

Table 5 – PCR conditions for amplification of plyP100 from phage adapted endolysins. The volumes were calculated for

30 µL of final reaction volume. The initialization step at 95°C during 10 minutes was used in order to promote the

release of phage DNA.

PCR plyP100

Buffer A (x10) 5 µL

dNTPs (10 mM) 0.75 µL

DNA template 2 µL

KAPA Taq Pol. HF 0.5 µL

Primer Fw (10 µM) 0.75 µL

Primer Rv (10 µM) 0.75 µL

H20 nuclease free 15.15 µL

PCR conditions

95°C – 10 min.

95°C – 3 min.

65°C – 1 min.

72°C – 3 min.

72°C – 2 min.

Before sequencing the plyP100 of adapted phages was amplified by PCR using specific primers

(Table 4) and following the conditions present in Table 5. Correct amplification was confirmed by

agarose gel and PCR- products were then purified. Thereafter the amplified sequences, containing

separately each primer, were submitted for sequencing

30x

Chapter 3

Results and Discussion

CHAPTER 3 RESULTS AND DISCUSSION

45

1. Background

Listeriosis is a foodborne disease that has become widespread in recent years. In order to avoid the

inappropriate usage of antibiotics which can lead to an increase antibiotic resistance bacteria and

due to the limitations of phage usage, phage endolysins are appointed as a good alternative as

biocontrol agents [37]. However, little is known about the enzymatic activity under food processing

and storage conditions, such as refrigeration temperatures, high salts and low pH conditions. In

order to that it becomes important to verify endolysins stability in food matrices as well as in

consumer safety.

The basis of this study was to improve structural stability and lytic properties of endolysins by using

specific directed evolution techniques such as ep-PCR. Directed evolution is defined as a method to

harness natural selection in order to engineer proteins to acquire particular properties that are not

associated with the protein in nature. The main advantage of using directed evolution instead of more

rational-based approaches for molecular engineering relates to the volume and diversity of variants

that can be screened [116].

The first approach consisted in some attempts to isolation of derived listerial phage endolysins for

further characterization and subsequent lytic improvements.

Based on the non-successful phage isolation approach, a second strategy was followed using two

well-known derived L. monocytogenes phage endolysins, Ply500 and Ply511. Some attempts to

improve lytic properties of this two proteins were performed namely by the introduction of random

mutations through the use of ep-PCR.

At the same time an alternatively strategy based on biotic interactions between phage and host was

developed in order to promote the phage adaptation at low refrigeration temperatures and generating

improved lysins. According to the Red Queen hypothesis the adaptation of the host generates counter

adaptation in phages, increasing the population fitness and lead to an evolution of phage particles.

2. Listeria phage isolation

Despite they have a conserved biological function, phage endolysins are greatly enzymatically and

architecturally diverse comprehending 89 different types of organizational architecture and are able

to infect 64 different bacterial genera [126]. However, the tremendously variety and complexity of

PG composition with more than 100 chemotypes has led to an evolutionary pressure which has

forced phages to refine their lytic activity over host cell wall. As a result, phages have acquired a


46

huge diversity of PG hydrolases which may vary in type, number and organization of binding and

catalytic domains [126].

In order to obtain listerial phages and consequently isolate and characterize their derived endolysins,

various isolation attempts were performed using four different effluents from different livestock

industries.

Although no phages were isolated from any effluent sample, the presence of prophage activity during

non-lysogenic strains selection was verified by formation of inhibition halos of L. monocytogenes

strain Scott A against L. monocytogenes CECT 934 lawn.

It is known that L. monocytogenes strain Scott A has been extensively used in as a reference strain

in for efficacy testing of food processing and preservation techniques [127]. Also this strain encodes

temperate phage PSA that can justify the presence of inhibition halos in bacterial lawn [128][28].

This listeriophage integrates in the 3' end of an arginine tRNA gene and when isolated using UV-

induction it is characterized by exclusively infect L. monocytogenes serovar 4b strains [129].

3. In silico analysis of bacteriophage endolysins

Since no phages were isolated it was needed to use two well-known endolysins. Ply500 and Ply511

possess modular structure with cell binding (N-terminal) and catalytic (C-terminal) domains. To

identify putative domains of this endolysins with sequences deposited in NCBI, an in silico analysis

was performed using Pfam database with an E-value higher than 1x10-5 with at least 80% of query

coverage. The results for putative domains for each endolysin are showed in table 1.

The most similar enzymatic catalytic domain for the derived Listeria phage A500 endolysin Ply500

is VANY (Table 1). This domain is described in Pfam database as a carboxypeptidase, responsible

for cleavage of C-terminal residues of D-alanyl-D-alanine in PG possibly due to antibiotic resistance

provided by VanY protein on some Enterococcus strains. VANY domain was identified in endolysins

derived from Gram-positive or Gram-negative phages mainly in Listeria, Bacillus and Escherichia

cells. However, in contradiction with Pfam, Listeria phage endolysin Ply500 is classified as a

carboxypeptidase, cleaving L-alanyl-D-glutamate endopeptidases [25].

Despite that the probability percentages in Table 1 for the putative binding domains of Ply500 are

lower than initially defined query coverage value, it is known that family domain SH3 is commonly

found in phage endolysins and SH3_3 is referred as one of the most common domain type. Pfam

database refers to the SH3 family domain as the responsible for peptide binding that can be found

in proteins that interact with other proteins. Other SH3 related domains are appointed as very


47

commonly found in Listeria phage A500 derived endolysins [1]. This binding domain has been

identified for the recognition of pentaglycine cross bridge in PG which is present in Gram-positive

bacteria.

Table 1 – Putative binding and catalytic domains for Ply500 and Ply511 endolysins.

Ply500 endolysin putative domains

Catalytic domain Conserved domains HHpred Probability (%) HHpred E-value

VANY PF02557 99.9 2.3E-22

Peptidase_M15_4 PF13539 99.5 2.8E-15

Peptidase_M15 PF01427 99.5 1.6E-14

Peptidase_M15_3 PF08291 98.0 1.2E-05

Peptidase_M15_2 PF05951 98.0 2E-05

Binding domain

SH3_4 PF06347 72.8 1

SH3_3 PF08239 69.7 1.4

Ply511 endolysin putative domains

Amidase_2 PF01510 99.9 3.4E-24

Binding domain

DUF3597 PF12200 81.6 0.43

For the Ply511 endolysin, the most putative catalytic domain after in silico analysis is the Amidase_2

with 99.9% of probability and 3.4E-24 of E-value. This domain is characterized by Pfam database as

an amidase responsible for the cleavage reaction of the bond between N-acetylmuramoyl residues

and L-amino acid residues in bacterial cell walls. Amidase_2 is usually located as the central domain

[25].

The most putative binding domain for this protein is the Domain of Unknown Function (DUF) 597

with 81.6% query coverage and 0.43 E-value. Although this domain has no characterized function it

is located at C-terminal and it is supposed to play an important rule as cell binding domain. It is also

known that most listerial phage endolysins, including Ply511, do not directly require wall teichoic

acid as the binding ligand [130]. In this aspect it was found out that the removal of C-terminal CBD

does not affect the attachment of Ply511 endolysin to L. monocytogenes cells PG [131]. Therefore,

a modular architecture (i.e. the presence of CBD) is not always necessary for enzyme activity.


48

4. Protein expression preliminary test

4.1. Large-scale protein production

The ply511 and ply500 sequences encodes two different proteins with distinct molecular weight –

36.48 kDa and 33.43 kDa, respectively. In order to evaluate the correct expression of this two

proteins, culture of 250 mL of E. coli JM109 cells, containing cloned plasmid with wild-type protein

sequences, were done and induced as described in section 3.1 of Materials and Methods. The final

soluble protein concentrations of 315 µM and 106 µM respectively, are in agreement with figures

1A and 1B. The same Figures shows a protein overexpression and also that the majority of interesting

proteins are present in elution of fraction 2 (F2). A residual concentration of unspecified soluble

proteins is visible too and still present in a lower concentration. These results indicate that the

expression system was working correctly and producing folded proteins. The use of L.

monocytogenes 5725 was considered after preliminary antibacterial assays (results not showed) as

described in section 3.3 of Materials and Methods. The lytic activity of the two produced proteins

were tested against five different L. monocytogenes strains – 5725, 934, 936, 911, 923, 5875 –

with the last four belonging to furthermost common serovars reported in the most human listeriosis

cases, 1/2b, 1/2c, 4b and 1/2a, respectively [132].

4.2. Micro-scale protein expression and preliminary tests

The use of ep-PCR as the method of directed evolution generates a higher number of protein variants

or potentially mutants that can be screened. Therefore an optimized process is needed in order to

Fig. 1 – SDS- PAGE electrophoresis gel 12%, stained with Coomassie Blue, for Ply511 (A) and Ply500 (B) proteins. M, Protein Ladder (10-250 kDa) from NEB; F1, 2, 3, and 4, fractions 1, 2, 3 and 4 of eluted proteins; W, wash; FT, flow through. The proteins were eluted with elution buffer containing 250 mM imidazole concentration.


49

select the best candidates. Thus, the use of a 96-wells plate becomes absolutely necessary for the

realization of this procedure wherein each well will contain individually different candidates. As result,

a lower volume (200 µL broth) for cell culture is required – here called micro-scale protein

expression.

Fig. 2 - Antibacterial assays of Ply500 endolysin against normal and permeabilized L. monocytogenes 5725 cells. The endolysins were expressed in 4 mL broth and cell lysis was done by sonication (A). Protein expression was done in 5 wells (200 µL each well) then joined (1 mL) and sonicated (B). Endolysins were expressed in 200 µL of a 96-wells plate (C). Also protein expression was induced in 4 mL broth (D). Next 200 µL were transferred to 96-wells. Cell lysis was done by chloroform vapors. In all the cases the expression conditions were 16°C during 24 h or 48 h with 1.5 mM IPTG concentration. E. coli JM109 wild-type was used as negative control.

As already shown in section 3.1, the protein is overexpressed in large-scale conditions. In a first

approach, the same conditions of protein production (0.5 mM IPTG, 200 rpm agitation, overnight)

were maintained for micro-scale protein expression as well as the use of repeated frozen-thawing

0

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)

Time (h)Control 24h Normal24h frozen 48h Normal48h frozen

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TIme (h)Control 24h Normal

24h frozen 48h Normal

48h frozen

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48h frozen

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48h frozen

A B

C D


50

cycles for cell lysis. However, after performing antibacterial assays against L. monocytogenes 5725

no enzymatic activity was detected suggesting that the protein would not be produced in considerable

concentrations.

The lower volumes in each filled well generates a higher friction which may negatively influence the

agitation conditions as well as IPTG concentration and consequently the incubation time. Efficiency

of the repeated frozen-thawing cycles as cell lysis method was also identified as the one of the most

possible sources for the lack of antibacterial reduction. Besides, the absence of lytic activity may be

related to the difficult attachment of proteins in PG layer. As result, the main objective of this very

important preliminary test mainly consisted in evaluate the sources for the lack of lytic activity and

improve those conditions before proceeding to generate mutant library and screening steps.

To improve protein expression in micro-scale a higher agitation (250 rpm) and inducer concentration

(1.5 mM IPTG) were tested. Two different lysis methods (sonication and chloroform vapors),

incubation times (24 and 48 h) and lytic activity of proteins against normal and permeabilized

(previously frozen at -20°C for 1 h) Listeria cells were also evaluated.

The most immediate conclusion of the results present in Figure 2 is the more effectiveness lytic

activity of Ply500 in permeabilized Listeria cells, 24 h after the addiction of IPTG. Once permeabilized,

endolysins can easily bind to its specific sites of PG causing a better cell lysis. These results contrasts

with the lower lytic activity observed in normal listerial cells.

In 4 mL protein expression (Fig. 2A) a better lytic activity is visible, however, turns out to be normal

since that a higher protein concentration is produced too, especially when comparing to the other

conditions.

Another conclusion is that using 250 rpm agitation instead the initially used (200 rpm) the problem

between broth volume and agitation conditions was solved which can be confirmed by lytic activity

in micro-scale expression which is considerably significant (Fig. 2C). No enzyme activity was detected

in Figure 2D suggesting problems during cell growth or protein production.

The bacterial reduction in the all conditions presented in Figure 2B is very similar to those observed

in Figures 2A and 2C. The slight difference observed may be due to the possible variations in cell

growth and efficiency of cell lysis.

Although the efficiency of cell lysis methods is very similar, the chloroform vapor method seems to

be more adapted for this work once that promotes cell lysis equally in all wells of 96-wells plate which

is corroborated by the significantly lytic activity showed in Figure 2C.


51

4.3. Lytic activity – Low temperatures vs. Room temperatures

Despite the fact that these endolysins reveal good stability and lytic activity in a wide range of higher

temperatures, they have not yet been well characterized at low temperatures [133]. Once determined

the optimal protein expression conditions in micro-scale, the enzymatic activity of wild type endolysins

at food storage temperatures (≈4°C) and at room temperatures (≈25°C) was compared by

performing antibacterial assays against L. monocytogenes 5725. The importance of this test was to

prove the lower lytic activity of natural Ply500 and Ply511 and the need to improve its properties in

food storage conditions.

The reduction of antibacterial activity present in Figure 3 suggests a very similar enzymatic behavior

in the two different conditions. Nevertheless, Ply500 shows better lytic activity in both cases with a

two log reduction in thirty minutes whereas the Ply511 did not show significant activity in either

conditions.

Fig. 3– Kinetics of antibacterial assays of Listeria phage endolysins Ply500 and Ply511 at refrigeration temperatures (A) and room temperatures (B). Negative control was done using 180 µL bacterial suspension mixed with 20 µL of PBS.

The connection factor with these results could be related to the L. monocytogenes 5725 strain which

belongs to serovar 4c. This serovar is included into the non-most common listerial strains detected

in foods and is rarely reported to be implicated in human listeriosis cases [134]. However, Ply500

and Ply511 CBDs are referred to promote a very strong and weak binding for the PG, respectively,

to this listerial serovar, which can suggest the difference of lytic activity [135].

Despite the difference of lytic activity between the two endolysins, these results are not considered

relevant since a high bacterial concentration is still present. As a result the improvement of the

0

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ply500 ply511 Control

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Time (h)

ply500 ply511 Control

A B


52

catalytic activity in specific food storage conditions is required in order to be liable applied in food

processing industry. The optimization mainly consists using two different approaches of directed

evolution – random mutagenesis by ep-PCR and phage-based adaptation mechanisms.

5. Cloning

5.1. Gene amplification

For the improvement of lytic activity of Ply500 and Ply511 endolysins a PCR-based technique, ep-

PCR, was used as first approach. Besides, this technique promotes the insertion of random point

mutations in unknown sequences by imposing imperfect and mutagenic reaction conditions. The

mutated sequences can generate modified proteins with improved lytic properties at specific

conditions of food storage.

Before proceeding to the gene amplification an initial PCR optimization was done in order to

determine the annealing temperatures of designed primers.

Fig. 4. – PCR of amplified protein genes using temperature gradient between 55-65°C. Gradient temperatures used for endolysins sequences amplification – from left to right: 55°C, 57°C, 60°C, 62°C, 65°C. Agarose gel 1% concentration, stained with Sybr Safe and run at 90 V. Legend: M – DNA 1 kb ladder.

Due to the use of KAPA Taq DNA polymerase the annealing temperatures of designed primers are

about 5°C lower than the initially calculated melting temperatures. A temperature gradient between

55-65°C was used to perform a traditional PCR to determine and optimize the annealing

temperatures of primers.

Genes were amplified over the entire temperature gradient (Fig. 4) and the annealing temperature

chosen was 65°C.


53

The ep-PCR was performed amplifying the sequences using the conditions and the primers described

in 5.1.1 and 4.2 sections of Materials and Methods. The generated products are present in Figure

4.2 which shows a good amplification step. The visualized bands correspond to the expected lengths

with a 1020 bp for plyP500 and 1176 bp for plyP511. The generated products were purified for

further utilization in ligation step.

Fig. 5 – Amplified endolysins sequences by ep-PCR. The generated products possess 1020 bp (plyP500) and 1176 bp (plyP511) and were then double digested by using selected restriction enzymes and purified. Agarose gel 1% concentration, stained with Sybr Safe and run at 90 V. Legend: M – 1 kb ladder.

Some optimizations were done in ep-PCR experiments. These alterations mainly consisted in altering

some steps. Addiction of fresh KAPA Taq DNA polymerase after fifteenth amplification cycles for

better amplification of the sequences. Also increasing and decreasing the number of cycles and

variation of the initial template concentration between 3-50 ng/µL were done in order to increase

the error rate [136][137].

According to the mostly used conditions in this experiments (20 cycles and 50 ng/µL of template

DNA), 11 to 21 mutations rate were expected for each template lenght. Furthermore, the increase

of multiple mutations leads to a high number of different enzymes that can be screened. However

when the mutation rate required is too high, the resulting proteins will carry multiple amino acid

changes and could therefore be inactive [136].

M


54

5.2. Plasmid linearization and ligation

Before ligation step, cloning vector pQE-30 was digested using BamHI and SalI restriction enzymes,

according to the section 5.2 of Materials and Methods. The digestion resulted linearized plasmid

(3461 bps) and other bands that may corresponds to CBD-P35 sequence with 1187 bp length (Fig.

6)

Fig. 6 – Double digestion of pQE-30 cloning vector in agarose gel 0.7% concentration in order to promote better separation of digested plasmid The gel was stained with Sybr Safe and run at 90 V. Legend: M – DNA 1 kb ladder.

After linearization the plasmid was extracted from agarose gel, purified and the recirculation

prevented by using Antarctic Phosphatase. The resulting ep-PCR amplified genes (Fig. 5) were cloned

using T4 DNA ligase following the instructions described in section 5.3 of Materials and Methods.

The lengths of generated cloned fragments are 4039 bps for ply500 gene (Fig. 7A) and 4465 bps

for ply511 gene (Fig. 7B).

5.3. Transformation

After ligation step, the cloned vectors were initially transformed by chemical transformation

(described in section 6.2.1 of Materials and Methods) on NZY5α E. coli competent cells (nzytech)

and later on E. coli JM109 chemo-competent (preparation described in section 6.2.2 of Materials

and Methods).

M


55

Fig. 7 – Representative illustration pQE-30 vector cloned with ply500 (A) and ply511 (B) using Vector NTI software. The ligation generates two plasmids with different lengths – 4039 bps and 4465 bps, respectively. Both genes are cloned between BamHI and SalI restriction sites.

In spite of the lower yield of generated colonies, the efficiency of transformation was evaluated by

colony-PCR (section 5.1.2. of Materials and Methods).

The transformation efficiency using NZY5α E.coli chemiocompetent cells was low with only two

successful colonies transformed with pQE-30-plyP500 in contrast with no transformed colonies in

case of cloned plyP511 (Fig. 8).

Successful directed evolution experiments are the integrated results of efficient library construction

followed by a robust high-through-put screening [138]. The lower rate of successful transformed

colonies obtained discarded the chemical transformation procedure.

In order to increase the yield of transformed colonies, electroporation (Materials and Methods,

section 6.2.2) was done as it is described as an extremely high efficiency procedure for E. coli

transformation [139]. However, NZY5α E. coli cells are only chemically competent preventing its

usage for transformation by electroporation.

Therefore, the use of E. coli JM109 electrocompetent cells (Materials and Methods, section 6.1.2)

for electroporation was required.

Despite being considered a highly efficient transformation method no colonies were obtained.

Severall attempts have been made to overcome this problem. The plasmid extraction, ligation and

cloning steps were newly performed as well as newly electrocompetent cells were done however with

no practical results.


56

Fig. 8 – Colony-PCR results after “heat-shock” transformation using NZY5α E. coli competent cells. Only pQE-30 +

ply500 ligation were assembled by competent cells. Agarose gel 1% concentration, stained with Sybr Safe run 90 V. Legend: M – DNA 1 kb ladder.

It is known that high salt concentration influences the electric conductivity, causing loss of cells

viability. In order that different volumes of ligation were also tested however the problem persisted.

The absence of colonies can also be related to the residual presence of manganese ions. This ions,

resulted from ep-PCR, can negatively affect the enzymatic digestion of amplified products and the

activity of T4 DNA ligase during ligation step [138].

Original pQE-30 vector containing original sequences of ply500 and ply511 and pUC19 cloning

vector were unsuccessfully tested as positive control, by transformation into E. coli JM109

electrocompetent cells.

Wherefore the extensive reutilization of electroporation cuvettes may lead to the accumulation of

aluminum oxide that can change the electric parameters of electroporator and negatively influence

the efficiency of transformation.

6. Cryodrilling

Another strategy to improve lytic properties of endolysins is based on biotic interactions influenced

by environment conditions. This interactions between hosts and parasites, such as phages-host

interaction, impose adaptation and counter-adaptation that results in rapid antagonistic coevolution

and population dynamics [67].

A different approach for the improvement of endolysin enzymatic activity is by adapting phage to

successive lower temperatures which acts as mutagenesis promoter. The hypothesis is that by


57

adapting phage to lower temperatures the endolysin encoded in its genome will also be modified,

and be consequently more active at lower temperatures.

The well characterized Listeria phage P100, known to be able to infect and kill a broad host range

of L. monocytogenes strains, and L. monocytogenes 5725 host cells was used in this new evolution

strategy [140].

This assay was divided in two different strategies. The first approach, called evolution, only the

Listeria phage P100 was allowed to evolve and the bacterial genotype was held constant. The second

approach, called co-evolution, both phage and host were allowed to evolve.

Fig. 9 – PCR of amplified endolysins sequences derived from co-evolved (wells 1-5) and evolved (wells 6-10) adapted phages. Amplified products were visualized by 1% agarose gel, stained with Syber Safe, at 90 V. The marker (M) is 1 kb ladder.

Culture transfers were done decreasing the initial temperature (25°C) two degrees until reach the

final temperature of 7°C (section 8 of Materials and Methods).

Due to the biotic interactions between phage and host under decreasing temperatures it was

expected that different adapted phages were generated. Therefore, at 7 °C, 5 phage plaques with

presumable different evolutionary adaptations were found in evolved and co-evolved populations and

then isolated. The specific primers for plyP100, already available in primers group collection, were

used for amplification of the endolysins sequences of newly adapted phages (Fig. 9) which were then

purified. Once amplified and purified, the plyP100 from isolated co-evolutionary and evolutionary

phage plaques were sequenced, aligned and compared to the original plyP100.


58

Table 3 – Sequencing results of amplified plyP100 sequences of the 5 different phage plaques isolated from Co-Evolution (CE) and Evolution (E) populations. Amplified sequences were aligned and compared to the original plyP100. Point mutations are observable at specific positions of the amplified sequences lenghts. Legend: A – adenine, T – thymine, G – guanine, C – cytosine, * – absence of nucleotide.

Sequence position (kb) CE1 882 Original A Fw G Rv A CE2 52 65 73 846 955 Original A * * A A Fw C G T C A Rv A * * A * CE3 846 998 Original A C Fw C C Rv A G CE4 846 941 956 961 970 Original A A * * * Fw C * * * * Rv A A A C C CE5 95 96 109 117 994 Original * * * * * Fw A G T G * Rv * * * * A E1 112 981 Original * A Fw T C Rv * A E32 42 44 72 73 81 925 933 944 958 Original A C * * * A A A A Fw C T T T T A * * A Rv A C * * * * A A * E3 65 Original - Fw G Rv * E4 99 846 941 956 Original C A A * Fw T C * * Rv C A A A E5 111 Original * Fw T Rv *


59

Sequencing results, compiled and presented in Table 3, show at first sight, some point mutations in

the plyP100 sequences derived from co-evolved and evolved phage populations.

However, the high length of plyP100 turns it difficult to sequence and the available primers were

used for sequencing of mutated endolysins sequences from adapted phages. Besides, it is known

that the optimal length for sequencing is about 700 bp which decreases the reliability of the point

mutations present.

The forward primer provides reliable sequencing results in the first 700 bps which contrasts with the

remaining sequence generating undefined peaks and denoting sequencing errors. Nevertheless, this

sequencing errors can be quickly detected once using the same theory applied to reverse primer.

The same but reverse argument may also be applied.

Fig. 10 – Example of sequencing chromatogram of adapted plyP100 endolysin derived from co-evolutionary adapted phage. The putative mutations are highlighted. PlyP100CE1_Fw and PlyP100CE_Rv are the primers used for gene sequencing.

The non-conclusive sequencing results are showed in the chromatogram example of Figure 5.2.

Despite the highlighted putative mutations are present in amplified sequences using plyP100CE_Rv

primer compared to the original sequence, the chromatogram shows undefined peaks for each

mutation. These observations contrasts with no mutations for the sequence amplified using the

plyP100_Fw primer which presents well defined peaks.

For future better amplification and sequencing, the primers must be redesigned and optimized in

order to pair with homology zone in phage genome instead of pairing with plyP100 gene as was the

case.

Chapter 4

Conclusions and

future perspectives

CHAPTER 4 CONCLUSIONS AND FUTURE PERSPECTIVES

63

1. Conclusions and future perspectives The main goal of this work was to improve lytic properties of phage derived endolysins against

foodborne pathogen L. monocytogenes in specific food storage and processing conditions. Due to

the lack of knowledge of endolysins sequences and which type of mutation can improve its lytic

activity represented the most scientific challenge of this study. As result two different approaches of

directed evolution were performed to overcome this problem – ep-PCR and cryodrilling.

Concerning the first stage of this study – phage isolation – no listerial phage was identified

in all used effluents and only the presence of one prophage was detected. This prophage is

within L. monocytogenes strain scott A genome and when excised it is known to act on other

listerial strains with 4b serovar. Future experiments can be focused on its excision and

consequently improvement of lytic properties.

In silico analysis of the Ply500 and Ply511 endolysins revealed a modular structure for both

proteins. However, as can be observed in Ply511 endolysin which possess a central catalytic

domain, the modular structure (i.e. the presence of CBD) is not always necessary for the

lytic activity of the protein. As the function of CBD domain of Ply511 is not known, interesting

future approaches may involve whether its presence or not influences potential

improvements of lytic properties of lysins against a wide range of listerial serovars. Another

possible approach may be related to the modification of endolysin binding domain. Through

the use of specifically designed primers, the proteins sequences would successively be

shortened and an increase or decrease of catalytic properties can be evaluated.

Preliminary antimicrobial assays using wild-type proteins revealed lower catalytic activity at

storage (refrigeration) temperatures emphasizing the need to improve those properties.

Moreover, several limitations (absence of refrigerated spectrophometer) to keep these

temperatures constant were shown, decreasing the reproducibility of these antibacterial

assays. Consequently, further screenings at low temperatures has yet to be extensively

optimized.

The first approach for endolysins lytic improvement – ep-PCR – revealed several limitations

during cloning and transformation steps which did not allowed the fulfillment of the screening

stage. Furthermore, this was a very time-consuming methodology with several interrelated

steps. Future experiments to generate improved catalytic properties of this protein should

use alternative techniques. Site-saturating mutagenesis (SSM) technique is more efficient

and eliminates time-consuming subcloning ligation.

CHAPTER 4 CONCLUSIONS AND FUTURE PERSPECTIVES

64

Regarding the second approach for endolysins improvements – cryodrilling – a few point

mutations in PlyP100 sequence of coevolutionary and evolutionary phage populations were

achieved. Moreover this strategy was less problematical than the first approach. However,

there is no confirmation that mutations improve lytic activity of phage derived endolysins and

therefore no conclusion can be retrieved about efficiency between coevolutionary and

evolutionary phage populations. Further improvements for future works related to phage

adaptation mainly involves the increasing of adaptation time at refrigeration temperatures in

order to promote higher genetic diversity and consequently increasing the probabilities of

isolation of well adapted endolysins. Also new primers design should be done once those

that were used in this study pair within protein sequence

63

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Anexe A – SDS-PAGE gel composition

Table A1 - Components and volumes used for SDS-PAGE gels preparation. The present volumes were used for

preparation of four gels.

Stacking Gel Separating Gel

3,75% 12%

Acrylamide-Bisacrylamide

(30%/0.8% p/v)

2 mL 10mL

Tris-HCl 0,5M pH 6,8 4 mL -

Tris-HCl 1,5M pH 8,8 - 3 mL

H2O ultra-pure 9 mL 9,6 mL

SDS 10% 160 µL 240 µL

TEMED 12 µL 12 µL

PSA 10% 800 µL 1,2 mL

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Anexe B – Endolysins genes

ply500

ATGGCATTAACAGAGGCATGGCTAATTGAAAAAGCAAATCGCAAATTGAATGCTGGGGGA

ATGTATAAAATTACATCGGATAAAACACGAAATGTAATTAAAAAAATGGCAAAAGAAGGT

ATTTATCTTTGTGTTGCGCAAGGTTACCGCTCAACAGCGGAACAAAATGCGCTATATGCA

CAAGGGAGAACCAAACCTGGAGCAATTGTTACTAATGCCAAGGGCGGGCAATCTAATCAC

AACTACGGGGTAGCTGTTGACTTGTGCTTGTATACAAATGACGGAAAAGATGTTATTTGG

GAGTCAACAACTTCCCGGTGGAAAAAGGTTGTTGCTGCTATGAAAGCAGAAGGGTTTAAA

TGGGGCGGAGACTGGAAAAGTTTTAAAGACTATCCGCATTTTGAACTATGTGATGCTGTA

AGTGGTGAGAAAATCCCTGCTGCAACACAAAACACTAATACAAATTCAAATCGTTACGAG

GGTAAAGTCATGATAGCGCACCACTGCTACCGAAAATGGACTTTAAATCATCACCATTCC

GCATGTATAAGGTAGGAACTGAGTTCTTAGTATATGATCATAATCAATATTGGTACAAG

ACATACATTGATGACAAACTTTACTACATGTATAAAAGCTTTTGCGATGTTGTAGCTAAA

AAAGACGCAAAAGGTCGCATCAAAGTTCGAATTAAAAGCGCGAAAGACTTGCGTATTCCA

GTCTGGAATAACATAAAATTGAATTCTGGGAAAATTAAATGGTATGCACCCAATGTAAAA

CTAGCGTGGTACAACTATCGAAGAGGATATTTAGAGCTATGGTATCCGAACGACGGCTGG

TATTACACAGCAGAATACTTCTTAAAATAA

Fig. B1– Endolysin Ply500 sequence derived from Listeria phage A500.

ply511

ATGGTAAAATATACCGTAGAGAACAAAATTATTGCAGGATTACCTAAAGGTAAACTAAAA

GGGGCTAACTTTGTTATTGCTCATGAAACTGCAAATAGCAAGTCTACTATTGACAATGAA

GTAAGCTACATGACTAGGAACTGGAAGAACGCATTTGTAACTCACTTTGTAGGTGGCGGA

GGTAGAGTCGTTCAGGTTGCTAATGTAAACTATGTTTCTTGGGGAGCAGGTCAGTATGCT

AACTCTTATTCCTATGCGCAGGTAGAGTTGTGCCGTACAAGTAATGCAACTACATTTAAG

AAAGACTATGAAGTGTACTGTCAATTACTAGTAGACCTAGCTAAAAAAGCAGGTATCCCT

ATTACACTTGACTCTGGTAGTAAAACTAGTGATAAAGGTATTAAATCCCATAAATGGGTT

GCTGATAAGCTAGGAGGAACAACACACCAAGACCCATATGCTTACTTAAGCTCATGGGGT

ATTAGTAAAGCACAATTTGCTAGTGACTTGGCTAAAGTATCTGGCGGAGGAAACACAGGA

ACAGCGCCAGCTAAACCAAGCACACCAGCACCTAAACCAAGCACACCATCTACTAACCTA

GACAAACTTGGCTTAGTAGACTACATGAACGCTAAGAAAATGGACTCTAGCTACAGTAAC

AGAGATAAGTTAGCTAAACAGTATGGTATTGCTAACTATTCAGGAACAGCTAGCCAGAAC

ACTACACTCCTTAGTAAAATTAAAGGAGGAGCACCTAAACCAAGCACACCAGCACCTAAA

CCTAGTACATCTACAGCTAAGAAAATTTATTTCCCACCAAATAAAGGAAACTGGTCTGTG

TATCCAACAAATAAAGCACCCGTTAAGGCTAATGCTATTGGTGCTATTAACCCTACTAAA

TTCGGAGGATTGACTTACACTATCCAAAAAGATAGAGGAAACGGTGTATACGAAATCCAA

ACAGACCAATTCGGCAGAGTTCAAGTCTATGGTGCACCTAGTACAGGAGCAGTTATCAAA

AAATAA

Fig. B2– Endolysin Ply511 sequence derived from Listeria phage A511.

75

plyP100

ATGGTAAAATATACCGTAGAGAACAAAATTATTGCAGGATTACCTAAAGGTAAACTAAAA

GGGGCTAACTTTGTTATTGCTCATGAAACTGCAAATAGCAAGTCTACTATTGACAATGAA

GTAAGCTACATGACTAGGAACTGGAAGAACGCATTTGTAACTCACTTTGTAGGTGGCGGA

GGTAGAGTCGTTCAGGTTGCTAATGTAAACTATGTTTCTTGGGGAGCAGGTCAGTATGCT

AACTCTTATTCCTATGCGCAGGTAGAGTTGTGCCGTACAAGTAATGCAACTACATTTAAG

AAAGACTATGAAGTGTACTGTCAATTACTAGTAGACCTAGCTAAAAAAGCAGGTATCCCT

ATTACACTTGACTCTGGTAGTAAAACTAGTGATAAAGGTATTAAATCCCATAAATGGGTT

GCTGATAAGCTAGGAGGAACAACACACCAAGACCCATATGCTTACTTAAGCTCATGGGGT

ATTAGTAAAGCACAATTTGCTAGTGACTTGGCTAAAGTATCTGGCGGAGGAAACACAGGA

ACAGCGCCAGCTAAACCAAGCACACCAGCACCTAAACCAAGCACACCATCTACTAACCTA

GACAAACTTGGCTTAGTAGACTACATGAACGCTAAGAAAATGGACTCTAGCTACAGTAAC

AGAGCTAAGTTAGCTAAACAGTATGGTATTGCTAACTATTCAGGAACAGCTAGCCAGAAC

ACTACACTCCTTAGTAAAATTAAAGGAGGAGCACCTAAACCAAGCACACCAGCACCTAAA

CCTAGTACATCTACAGCTAAGAAAATTTATTTCCCACCAAATAAAGGAAACTGGTCTGTG

TATCCAACAAATAAAGCACCCGTTAAGGCTAATGCTATTGGTGCTATTAACCCTACTAAA

TTCGGAGGATTGACTTACACTATCCAAAAAGATAGAGGAAACGGTGTATACGAAATCCAA

ACAGACCAATTCGGCAGAGTTCAAGTCTATGGTGCACCTAGTACAGGAGCAGTTATCAAA

AAATAA

Fig. B3– Endolysin PlyP100 sequence derived from Listeria phage P100.

76

Anexe C – Protein quantification

Fig. C4– Calibration curve of BSA protein used in BCA assay for protein quantification.

y = 1049,3x - 173,85R² = 0,9964

0

500

1000

1500

2000

2500

0,00 0,50 1,00 1,50 2,00 2,50

Pro

tein

co

nc.

(u

g/m

l)

OD (580 nm)

Series1 Linear (Series1) Linear (Series1)

pedro miguel azevedo veloso improving derived listeria phage

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