1

UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA AGRONÓMICA, ALIMENTARIA Y DE BIOSISTEMAS

GRADO EN BIOTECNOLOGÍA

DEPARTAMENTO DE BIOTECNOLOGÍA–BIOLOGÍA VEGETAL

STUDY ON THE RELEVANCE IN PATHOGENESIS OF THE PERCEPTION OF AMINO ACIDS AND DERIVATIVES IN THE PHYTOPATHOGENIC

BACTERIA PSEUDOMONAS SYRINGAE PV. TOMATO

ESTUDIO DE LA RELEVANCIA EN PATOGÉNESIS DE LA PERCEPCIÓN DE AMINOÁCIDOS Y DERIVADOS EN LA BACTERIA FITOPATÓGENA

PSEUDOMONAS SYRINGAE PV. TOMATO

TRABAJO FIN DE GRADO

Autora: Alba Díaz Bárcena

Tutora: Emilia A. López Solanilla Cotutora: Saray Santamaría Hernando

Junio de 2019

ii

UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA

AGRONÓMICA, ALIMENTARIA Y DE BIOSISTEMAS

GRADO EN BIOTECNOLOGÍA

STUDY ON THE RELEVANCE IN PATHOGENESIS OF THE PERCEPTION OF

AMINO ACIDS AND DERIVATIVES IN THE PHYTOPATHOGENIC BACTERIA

PSEUDOMONAS SYRINGAE PV. TOMATO

(ESTUDIO DE LA RELEVANCIA EN PATOGÉNESIS DE LA PERCEPCIÓN DE

AMINOÁCIDOS Y DERIVADOS EN LA BACTERIA FITOPATÓGENA

PSEUDOMONAS SYRINGAE PV. TOMATO)

TRABAJO FIN DE GRADO

Alba Díaz Bárcena

MADRID, 2019

Directoras:

Emilia A. López Solanilla. Profesor Titular de Universidad.

Dpto. de Biotecnología–Biología Vegetal. ETSIAAB. UPM

Saray Santamaría Hernando. Investigador post-doctoral.

Centro de Biotecnología y Genómica de Plantas. UPM-INIA

iii

ESTUDIO DE LA RELEVANCIA EN PATOGÉNESIS DE LA PERCEPCIÓN DE

AMINOÁCIDOS Y DERIVADOS EN LA BACTERIA FITOPATÓGENA PSEUDOMONAS

SYRINGAE PV. TOMATO

Memoria presentada por Alba Díaz Bárcena para la obtención del título de Graduado en Biotecnología por la Universidad Politécnica de Madrid

Fdo: Fdo:

VºBº Tutora y Directora del TFG VºBº Cotutora y Co-Directora del TFG

Emilia A. López Solanilla

Dpto. de Biotecnología–Biología Vegetal

Escuela Técnica Superior de Ingeniería

Agronómica, Alimentaria y de Biosistemas

Madrid, 25 de Junio de 2019

Saray Santamaría Hernando

Centro de Biotecnología y

Genómica de Plantas (CBGP)

UPM-INIA

iv

AGRADECIMIENTOS

Durante este último año de carrera he sido consciente de que era el momento en el que todo aquello

que había aprendido en los cursos anteriores culminaba en la realización de unas prácticas en las que mi

rutina daría un giro radical y dejaría las clases para incorporarme en un grupo de trabajo en un

laboratorio. Al principio uno no llega a ser del todo consciente de la exigencia y la dedicación que

acompaña un trabajo de investigación, pero según te adaptas a la rutina te das cuenta de que la única

forma de llevar un proyecto así a cabo es echándole horas, ganas y, especialmente, contando con un

grupo de personas que trabajan contigo codo con codo para ayudarte en cada obstáculo que te

encuentres.

Es por esto por lo que quería aprovechar este pequeño espacio para agradecer el apoyo incondicional

recibido este año por parte de todos mis compañeros del laboratorio 285.

En primer lugar, Emilia, no sé como agradecerte la dedicación y atención constante que me has

brindado durante todo el curso. No solo a nivel profesional considero que hayas estado siempre al pie

del cañón, sino que a nivel personal el trato no ha podido ser mejor. Realmente el grado de confianza y

de cercanía con la que nos acoges y trabajas con nosotros es admirable y no me cabe ninguna duda de

que si las cosas funcionan tan bien en este laboratorio es por el clima tan familiar que generas. Sólo

puedo decirte: GRACIAS!, por ser como eres y por tratarme tan bien.

Saray, Paul… Lo primero que quiero deciros es que como bien sabéis, gran parte de este trabajo es

vuestro y que, realmente, no puedo estar más agradecida por todo el tiempo que habéis dedicado a

explicarme, enseñarme y aconsejarme. Pero por supuesto, os quiero dar especialmente las gracias por

ser como sois, por ser dos personas llenas de vitalidad que transmiten energía de forma constante, por

las risas, los vaciles, el bullying a la chufi, los análisis de moda diarios (Saray) y todos los casos resueltos

por mi constante afición de sentirme Holmes (Paul). En el poco tiempo que os conozco habéis

conseguido ganaros toda mi confianza y es que realmente, sois dos ejemplos a seguir para mí. Claris… tú

iniciaste como yo este año la locura de embarcarte en este laboratorio y menos mal! Menos mal que he

podido conocerte, exprimirte, achucharte y darme cuenta de la bellísima persona que eres. Eres alegría

y amor en estado puro y quería darte las gracias por contagiarme siempre con esa súper sonrisa tuya.

Titi… a ti qué te voy a decir a estas alturas! Si estoy aquí y he tenido la oportunidad de trabajar con esta

gente maravillosa ha sido gracias a ti. Si pensaba que nuestra amistad no podía ser más fuerte este año

me he reafirmado porque tenerte en mi rutina ha sido lo mejor del mundo. Sabes lo mucho que te

quiero. Sandri…aunque hayamos coincidido un tiempo muy escaso, también te quería dar las gracias por

hacer que siempre todo estuviese en su sitio y por estar siempre tan pendiente de todos.

Por último, gracias Evi e Ito por estar siempre ahí para cada consejo y cada conversación requerida. A los

huevetes y a las fufas por alegrarme cada día en la universidad y hacer de agrónomos algo así como mi

casa. Gracias a los truñas por esos momentos de desconexión tan necesarios en el Rincón de Juan. Y

cómo no, gracias a mis padres por haberme apoyado siempre de forma incondicional en cada decisión

tomada y en cada paso dado no solo durante este año, sino durante toda mi vida.

v

CONTENTS

Table index ................................................................................................................................... vi

Figure index .................................................................................................................................. vii

Abbreviations .............................................................................................................................. viii

Abstract ......................................................................................................................................... xi

Chapter I – Introduction and objectives........................................................................................ 1

Chapter II – Material and methods ............................................................................................. 10

LBD sequence and protein structure determination ........................................................... 10

Strains, plasmids and culture conditions .............................................................................. 10

DNA amplification and purification ....................................................................................... 11

DNA extraction and quantification ....................................................................................... 11

Mutants construction............................................................................................................ 11

Swarming motility assays ...................................................................................................... 11

Swimming motility assays ..................................................................................................... 12

Quantitative capillary chemotaxis assays ............................................................................. 12

Bioscreen assays ................................................................................................................... 12

Plants assays ......................................................................................................................... 12

Protein expression ................................................................................................................ 13

Western blot assays .............................................................................................................. 13

Protein purification ............................................................................................................... 13

Fluorescence-based thermal shift assays ............................................................................. 14

Statistical analysis ................................................................................................................. 14

Chapter III - Results ..................................................................................................................... 15

Chapter IV - Discussion ................................................................................................................ 23

Chapter V - Conclusions .............................................................................................................. 27

Chapter VI - References............................................................................................................... 28

Appendix ..................................................................................................................................... 33

vi

TABLE INDEX

Table 1 - Bacterial strains used in this study ............................................................................... 10

Table 2 - Plasmids used in this study ........................................................................................... 10

Supplementary Table 1 – Primers used in this study .................................................................. 33

vii

FIGURE INDEX

Figure 1 - Chemotaxis pathway in Escherichia coli ....................................................................... 3

Figure 2 - Schematic representation of the most common MCP topology .................................. 4

Figure 3 - Structural diversity of the different LBD types that were analyzed from all the

sequences matching the chemoreceptor signalling domain available in the Pfam 31.0 database

....................................................................................................................................................... 5

Figure 4 - Amino acid alignment between PscB and PsPto_1061 LBD´s chemoreceptors (A) and

between PscE and PSPto_3379 LBD´s chemoreceptors (B) ....................................................... 15

Figure 5 - Growth curve of Pspto and PsPto_1061 in KB medium and M9 minimal medium .... 16

Figure 6 - Swimming and swarming assays… .............................................................................. 17

Figure 7 - Chemotactic response of PsPto and PsPto_1061 towards amino acids ..................... 18

Figure 8 - Tomato virulence and colonization assays. ................................................................ 19

Figure 9 - Western Blot analysis of the protein expression levels of the recombinant LBDs

PsPto_1061 (A) and PsPto_3379 (B) under different culture conditions ................................... 20

Figure 10 - SDS-PAGE gel electrophoresis of purified His6-tagged PsPto_3379 LBD ................. 20

Figure 11 - Differences of melting temperature (ΔTm) of PsPto_3379 LBD in the presence of

GABA, L-Homoserine and L-Phenylalanine ................................................................................ 22

viii

ABBREVIATIONS

μg: microgram

μL: microliter

μM: micromolar

4HB: four-helix bundle

°C: celsius degrees

acyl-HSL: acyl-homoserine lactones

Ap: ampicillin

pb: base pairs

C: carbon

c-di-GMP: cyclic diguanylate

Cb: carbenicillin

CCW: counterclockwise

CheA: cytoplasmic histidine kinase

CheW: receptor kinase coupling protein

CheY: response regulator protein

C-terminal: carboxylic terminal

CW: clockwise

CZB: chemoreceptor zinc binding

D-Asp: D-Aspartate

dCache: double-Cache

DSF: differential scanning fluorimetry

GABA: gamma-aminobutyric acid

h: hours

HAMP: Histidine kinases, Adenylate cyclases, Methyl-accepting Proteins

HBM: helical bimodular

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IPTG: Isopropil-D-1-thiogalactopyranoside

ITC: isothermal titration calorimetry

KB: King's B

Km: kanamycin

L-Ala: L-Alanine

L-Arg: L-Arginine

L-Asn: L-Asparagine

L-Asp: L-Aspartate

L-Cit: L-Citrulline

L-Gln: L-Glutamine

L-Glu: L-Glutamate

L-His: L-Histidine

L-Homoserine: L-HS

L-Ile: L-Isoleucine

L-Leu: L-Leucine

L-Lys: L-Lysine

L-Met: L-Methionine

L-Phe: L-Phenylalanine

L-Pro: L-Proline

L-Ser: L-Serine

L-Trp: L-Tryptophan

LB: Luria Bertani

LBD: ligand binding domain

MA: methyl-accepting

MCP: methyl-accepting chemotaxis protein

mg: milligram

x

mL: milliliter

mM: millimolar

N-terminal: amino terminal

N: nitrogen

NCBI: National Center for Biotechnology Information

NIT: nitrate and nitrite sensing

OD: optical density

PCR : polimerase chain reaction

Psa: Pseudomonas syringae pv. actinidae

PsPto: Pseudomonas syringae pv. tomato

Rf: rifampicin

RH: relative humidity

s: seconds

sCache: single-Cache

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM: standard error of the mean

Sm: streptomycin

T3SS: type III secretion system

TCA: tricarboxylic acid cycle

Tm: melting temperature

TM: transmembrane

TS: thermal shift

w/v: weight/volume

xi

ABSTRACT

Pseudomonas syringae pv. tomato DC3000 (PsPto) is the causal agent of bacterial speck of

tomato, an important agricultural disease. PsPto displays a short initial epiphytic phase

followed by an aggressive pathogenic phase in plant apoplast. As other plant-pathogens, PsPto

senses different plant signals in order to activate motility mechanisms and to colonize plant

tissues through natural openings or via mechanical wounds. One of the signal perception

mechanisms in bacteria is mediated by the function of Methyl-accepting Chemotaxis Proteins

(MCPs) specialized in the binding to the putative chemical signal. Amino acids levels in plant

change during bacterial infection. Moreover, these compounds have been shown to play an

important role in plant defense. Therefore, the recognition of these changes seems to be

decisive for PsPto success.

In this work the functional characterization of PsPto_1061 and PsPto_3379 chemoreceptors

has been approached. In vivo assays using a mutant strain in PsPto_1061 MCP have allowed us

to determine the relevance of this chemoreceptor not only in the perception of amino acids,

but also in virulence process. PsPto_1061 mutant strain was impaired on the perception of L-

Homoserine, L-Tryptophan, L-Asparagine, L-Methionine and L-Phenylalanine and showed less

virulence than the wild type strain. These results suggest that bacteria exploit amino acid

perception to optimize virulence. After several attempts it was not possible to obtain the

mutant strain in PsPto_3379 chemoreceptor. Therefore, the in vivo functional characterization

of this MCP could not be carried out. Recombinant expression and purification of PsPto_3379

LBD has been optimized which would allow in vitro assays to determine its ligand binding

profile. Preliminary thermal shift assays performed with the purified recombinant LBD

revealed that it does not bind GABA, L-Homoserine and L-Phenylalanine.

1

CHAPTER I - INTRODUCTION AND OBJECTIVES

Pseudomonas syringae pv. tomato DC3000

The phyllosphere of terrestrial plants is considered one of the most important ecological niches

for the development of phytopathogenic bacteria. Plant pathogenic bacteria colonize plant

surfaces and can multiply epiphytically and saprophytically before entering the plant tissues,

where initially find mesophyll cells surrounded by an intercellular space called apoplast (Lindow

and Brandl, 2003). Drastic variations in weather conditions, limited nutrients availability and

direct exposure to UV irradiation make the plant surface a hostile environment for

phytopathogenic bacteria. Bacteria have evolved mechanisms such as biofilm formation or

surfactants production to counteract this changing environment and ensure their survival in the

leaf surface (Lindow and Brandl, 2003). In contrast to many fungal pathogens that have the

ability to degrade the plant cuticle and the cell wall, bacteria cannot directly penetrate the leaf

epidermis. Bacteria enter the leaf tissues through natural openings such as stomata,

hydathodes and lenticels or via mechanical wounds (Melotto et al., 2008).

Pseudomonas syringae is a gram-negative hemibiotrophic pathogen that presents two different

lifestyles: an initial epiphytic phase upon the arrival to the plant surface and a pathogenic phase

multiplying in the apoplast after entering the plant tissue (Hirano and Upper, 2000).

P. syringae causes economically important diseases in a wide range of plant species. However,

there are different strains, commonly grouped into pathovars, which exhibit a high degree of

host specificity infecting a limited number of plant species (Xin and He, 2013).

Pseudomonas syringae pv. tomato DC3000 (PsPto) has been considered as a model for the

study of plant-bacterial interactions since is the causal agent of the bacterial speck of tomato

(Solanum licopersicum), but it is also able to infect the model plant Arabidopsis thaliana (Xin

and He, 2013). For this reason, PsPto infection mechanisms have been deeply studied and it is

known that the main virulence determinants in this strain are the type III secretion system

(T3SS) effectors (Cunnac et al., 2011). In addition to these effectors, a phytotoxin called

coronatine is synthesized during infection in order to facilitate bacterial invasion through

stomata and to promote bacterial multiplication and chlorotic-lesion induction in plant tissues

(Xin and He, 2013).

Although many strains of P. syringae are well-adapted to the epiphytical lifestyle, PsPto is a

weak epiphyte and if it fails to enter inside the apoplast, PsPto populations die in less than 48

hours (Boureau et al., 2002). However, PsPto adapts to the apoplast environment where is able

to activate and express different nutrient assimilation pathways involved in the metabolism of

organic acids and amino acids that are abundant in this medium (Rico and Preston, 2008).

2

During the first steps of leaf invasion, PsPto needs to perceive different plant and

environmental signals to activate motility mechanisms and respond to favorable or unfavorable

stimuli in the environment (Cuppels, 1988). The ability to respond to these stimuli not only

enhances the survival in the epiphytic phase, but also increases the opportunities to efficiently

invade the host apoplast, and thus the success of infection (Haefele and Lindow, 1987;

Santamaría-Hernando et al., 2018). Therefore, this initial stage of the interaction seems to be a

key point for the design of interfering strategies to restrict the infection.

Perception, chemotaxis and MCPs

The most common mechanisms for the recognition of plant and environmental signals are one-

and two-component systems and chemoreceptor-based signaling cascades (Ulrich and Zhulin,

2005; Laub and Goulian, 2007; Wuichet and Zhulin, 2010). Chemotaxis is the ability of motile

organisms to sense concentration gradients of different chemicals in the environment and

respond to changes by modulating their motile behaviour (running towards or away) (Adler,

1966).

Most studied chemotaxis systems regulate flagellar or type IV pili motility, whereas others

control different cellular functions such as biofilm formation, development or cell-cell

interactions (Wuichet et al., 2007). The canonical model to study the flagellar-mediated

chemotaxis pathway is Escherichia coli because of its simplicity considering the small number of

genes and proteins that participate in the process. Some of the core proteins that regulate E.

coli chemotaxis are highly conserved: 245 out of 450 bacterial and archaeal genomes present

homologues to E. coli chemotaxis proteins (Wuichet and Zhulin, 2010). However, although the

general control mechanism is conserved, there is a great variety of signaling proteins that

participate in the chemotactic process that are only conserved in some bacterial species

(Szurmant and Ordal, 2004).

Chemotaxis is based on the recognition of ligands by methyl-accepting chemotaxis proteins

(MCPs), which variate among the different bacterial species. In the absence of ligand, MCPs

interact with cytoplasmic histidine kinase CheA through the receptor kinase coupling protein

CheW and stimulates CheA’s autophosphorylation, which phosphorylates the response

regulator protein CheY. When phosphorylated, CheY interacts with the flagellar motor and

controls flagellum rotation (Figure 1). This causes the flagella to rotate clockwise (CW) which

allows the cell to reorient randomly into a different direction (tumble). On the other hand,

counterclockwise rotation status (CCW) enables the bacteria to move in a single direction for

an extended period of time (smooth swimming). Upon ligand binding, MCPs suffer a

3

conformational change that inhibits CheA activity, producing a signaling cascade that alters

flagellum rotation to a CCW mode, and thus changing bacterial movement (Ferrández, 2002;

Manson, 2018).

A key aspect of chemotaxis is the ability to detect compound gradients. This is achieved

through a precise detection of changes in compound concentration and a chemical memory of

previously received stimulus. Measurement of concentration changes is MCP-dependent, whilst

in chemical memory there are other proteins involved. These proteins are the

methyltransferase CheR and methylesterase CheB, that avoid the saturation of the molecular

machinery by reversible methylation of the MCP, and phosphatase CheZ, which catalyzes CheY

dephosphorylation (Wuichet and Zhulin, 2010).

Figure 1. Chemotaxis pathway in Escherichia coli. Reactions in red represent the molecular response to

an attractant ligand, showing a decrease in CheA and CheY phosphorylation that causes a CCW rotation.

Reactions in pink represent the molecular response to a not attractant ligand, showing an increase

phosphorylation pattern and a CW rotation (Sampedro et al., 2015).

MCPs differ drastically in abundance, sequence and topology among different bacterial species

depending on their lifestyle. The number of MCPs per genome depends on three principal

parameters: the stability of the habitat, the ability of the bacteria to interact with other living

organisms and its metabolic versatility. Bacteria that explore different habitats or that are

capable to establish complex relationships are those that usually present a higher number of

MCPs encoded in their genomes (Matilla and Krell, 2018).

4

The average number of chemoreceptor proteins in bacteria is 14 per genome, but the number

differ widely from bacteria such as E. coli that presents 5 MCPs and others like Pseudomonas

aeruginosa and PsPto, with 26 and 49 MCPs, respectively (Lacal et al., 2010; Sampedro et al.,

2015).

MCPs are constituted by three structural and functional modules: a sensory domain or ligand

binding domain (LBD) located in the N-terminal (amino terminal) region, which binds the ligand

directly or mediated by an auxiliary protein (Matilla and Krell, 2018; Rico-Jiménez et al., 2013),

a HAMP linker cytosolic domain and a methyl-accepting (MA) signaling domain in the C-

terminal (carboxylic terminal) region that interacts with CheA initiating the signaling cascade

(Sampedro et al., 2015).

While the cytoplasmic signaling domain is highly conserved among species, there are large

differences at the levels of protein topology and LBD type. Although a transmembrane (TM)

topology with an extracellular LBD is predominant (Figure 2), some chemoreceptors either are

completely cytosolic, lack a LBD, or are membrane anchored with the LBD located on the

intracellular space (Ortega et al., 2017).

Figure 2. Schematic representation of the most common MCP topology. The ligand binding domain is extracellular and is connected to the membrane (shaded in gray) by two TM regions. The linker HAMP domain is cytosolic as well as the signaling domain, which is composed by two methylation regions (MH1, MH2) and the signaling sub-domain itself (Alexander and Zhulin, 2006).

The LBD domain is characterized by a low degree of conservation that seems to be the result of

the rapid evolutionary pressure to allow the recognition of different ligands and, consequently,

to ensure bacterial adaptation to distinct environments (Mowbray and Sandgren, 1998;

Alexander and Zhulin, 2007).

Despite the high structural variability of LBDs, specificity of MCPs relays precisely on their LBD

structure. The latest classification regarding structural LBD diversity was developed by Ortega

and colleagues (Ortega et al., 2017). From all the different LBDs found in that study, only three

superfamilies were abundant in chemoreceptors and represent more than the 80% of the total

(four-helix bundle: 4HB, Cache and PAS: ), while others like CZB (chemoreceptor zinc binding),

5

protoglobin or NIT (nitrate and nitrite sensing) represent a 3% each (Figure 3).

Figure 3. Structural diversity of the different LBD types that were analyzed from all the sequences

matching the chemoreceptor signalling domain available in the Pfam 31.0 database. Bound ligands are

shown in red. Different chain colours indicate the domain was experimentally shown to be dimeric. PDB

accession numbers are shown in parentheses (Ortega et al., 2017).

The four-helix bundle domain (4HB) is the most abundant LBD in chemoreceptors and it has

been identified as an extracytoplasmic sensory module found in the principal signal

transduction families of bacteria and archaea (Ulrich and Zhulin, 2005). E. coli possess five well

studied chemoreceptors, four of them (Tar, Tsr, Trg and Tap) present this type of LBD as a

sensory module (Parkinson et al., 2015). This domain superfamily also includes the double 4HB

domain, also known as helical bimodular domain (HBM), which contains two different ligand

binding modules (Matilla and Krell, 2018).

The Cache superfamily is the second most abundant and includes the single-Cache domain

(sCache) and the double-Cache domain (dCache) (Ortega et al., 2017). dCache domain presents

a bimodular arrangement and is composed by a membrane-distal module and a membrane-

proximal module (Pineda-Molina et al., 2012). The studies conducted to date indicate that the

ligands bind to the distal module whereas the proximal module might be involved in signal

transmission (Glekas et al., 2012).The domains included in these two superfamilies are mostly

located as extracytoplasmic LBDs of transmembrane chemoreceptors and generally participate

in chemotaxis-related processes (Ortega et al., 2017) However, the third most abundant LBD

domains, the PAS domains, mostly form part of cytosolic chemoreceptors and are involved in

aerotaxis processes (Collins et al., 2014).

6

Despite the recent advances in the knowledge of the structural diversity of LBDs, it has been

demonstrated that even those LBDs with similar structural topology, can recognize different

ligands and also the same ligand can be recognized by structurally different LBDs (Tajima et al.,

2011). For this reason it is not possible to establish a general rule that relates LBD topology and

MCP function and therefore, functional annotation of MCPs always require experimental

approaches (Matilla and Krell, 2018). In addition, for most of bacterial sensor proteins, its

cognate ligand remains unknown.

During the last years, several in vivo and in vitro approaches to analyze quantitative and

qualitative chemotaxis behaviour and identify signal molecules have been developed.

In vivo approaches include chemoattraction capillary assays. This approach is based on the first

capillary assay developed by Adler (Adler, 1966) which consists on the generation of a chemical

gradient of a compound that can be metabolized by bacteria in situ. Over the years, the

experimental procedure of this assay has been improved making it suitable to detect not only

nutrient-related molecules, but also other ligands. Currently, is performed using a 96-well-

microtiter plate to carry out multiple and simultaneous assays. This system allows a high-

throughput capillary assay (Sampedro et al., 2015).

In vitro approaches are based on the measurement of thermal stability of recombinant LBDs in

the absence or presence of putative signal molecules (Thermal Shift, TS). The theoretical basis

on which these assays are based is the fact that ligand binding to a protein typically increases

its thermal stability. Binding of a ligand can be quantified by calculating the temperature at

which half of the protein is unfolded and the other half remains in its native state (melting

temperature, Tm). When binding occurs, the protein is more stable and its Tm increases

(Fernández et al., 2015; Chiu and Prenner, 2011).

Currently this determination is carried out using the differential scanning fluorimetry assay

(DSF) (Fernández et al., 2015). In DSF analysis a fluorescent compound with affinity for

hydrophobic parts of the protein is added to the protein sample and exposed to a temperature

gradient. As a result of the temperature increase, the protein will unfold and the hydrophobic

regions will be more exposed, then the fluorophore will bind more easily and changes in

fluorescence can be detected (Senisterra et al., 2012). When binding to a ligand occurs, stability

is enhanced and temperature required for unfolding increases, so the detected signal is

emitted later.

In vivo approaches are useful to identify putative chemoeffectors but in vitro methods are

needed to characterize the corresponding LBD and establish its ligand binding profile. Despite

thermal shift constitutes a promising technique to perform high throughput ligand assays,

results must be always corroborated with a most robust method called: Isothermal titration

7

calorimetry (ITC) (Krell, 2008) that allows to unambiguously determine the ligands that bind to

the recombinant LBD.

Thanks to these technologies it has been possible to characterize a number of MCPs from

different bacterial species, which have been shown to respond to a wide diversity of chemical

molecules, including sugars, amino acids, purines, Tricarboxylic Acid Cycle (TCA) intermediates

and polyamines among others.

The role of amino acids during plant-bacteria interaction

Previous studies have demonstrated the importance of amino acids perception for plant-

associated bacteria (Halvorson, 1972). Amino acids have biological importance in bacterial

physiology and metabolism and in addition, they are abundant in the apoplastic fluid of plants

(Rico and Preston, 2008).

Apoplastic fluid provides a rich medium that promotes the activation of metabolic pathways of

nutrient assimilation in some phytopathogenic bacteria like PsPto (Rico and Preston, 2008).

Many of the apoplastic compounds are assimilated by bacteria as carbon (C) and nitrogen (N)

sources; however, these compounds are also implicated in the regulation of virulence-related

processes such as the expression of type III secretion system, the principal pathogenesis

determinant in PsPto (McCRaw et al., 2016).

While in some species of Pseudomonas such as the saprophytes P. putida or P. fluorescens it

has been observed that the genes that encode permeases that incorporate both sugars and

amino acids are very numerous, in PsPto and other strains of P. syringae, the amino acids

transporters are scarce (Buell et al., 2003). It has been previously reported that L-Aspartate (L-

Asp), L-Asparagine (L-Asn), L-Glutamate (L-Glu). L-Glutamine (L-Gln), L-Arginine (L-Arg) and γ-

aminobutyric acid (GABA) amino acids are used by P. syringae as carbon and nitrogen sources

(Rico and Preston, 2008). The other proteinogenic amino acids either could not be analyzed or

did not seem to be assimilated by the bacteria. Interestingly, it has been observed that there is

a direct relationship between the content of amino acids in the apoplast and the level of use by

the bacteria, being the most abundant the most assimilated (Rico and Preston, 2008).

Different experimental procedures have demonstrated that during bacterial infection, the

chemical profile of metabolites produced by the plant changes (Ward et al., 2010; O’Leary et

al., 2016). It has been observed that amino acids content in the apoplast increases as bacterial

colonization proceeds (O’Leary et al., 2016). A possible explanation may be the increase of

protease activity, as it has been reported for the pathogen-related serine protease P69B in

tomato leaves, a protein that also directly participates in the plant defense response (Solomon

and Oliver, 2001).

8

Other studies point out that certain amino acids increase noticeably in the apoplast fluid since

they have a regulatory role in defense or because they are substrates for the biosynthesis of

defense-related compounds. As example, the non-proteinogenic amino acid GABA at high

concentrations presents an inhibitory effect in the expression of the type III secretion system

genes in PsPto (McCraw et al., 2016). Another case is L-Phenylalanine (L-Phe), which

concentration increases in plants at the beginning of infection to activate the biosynthesis of

phenylpropanoids, compounds that directly participate in plant defense (Yu et al., 2013). As a

mechanism to counteract the possible negative effects in bacterial metabolism and virulence,

an increased expression of GABA and phenylalanine catabolic genes has been observed in P.

syringae epiphytic and apoplastic populations (Yu et al., 2013; McCraw et al., 2016).

Perception of amino acids by bacteria

Several chemoreceptors that specifically recognize amino acids have been identified in

different bacterial species. In P. aeruginosa PAO1 PctA, PctB and PctC chemoreceptors are

responsible for the detection of 20 L-amino acids. All of them present a d-Cache domain as

ligand binding domain. PctA-LBD directly binds 18 proteinogenic amino acids (except for L-Asp

and L-Glu) and also binds other non-proteinogenic amino acids such as L-Citrulline (L-Cit) and L-

Homoserine (L-HS) (Kuroda et al., 1995; Rico-Jiménez et al., 2013; McKellar et al., 2015). On the

other hand, PctB-LBD binds five proteinogenic amino acids L-Arg, L-Lysine (L-Lys), L-Alanine (L-

Ala), L-Methionine (L-Met) and L-Glu and PctC-LBD binds only two amino acids that are also

recognized by PctA (L-Histidine (L-His) and L-Proline (L-Pro)) but also binds GABA with high

affinity.

P. putida KT2440 presents two MCPs with a d-Cache type LBD, McpA chemoreceptor binds

directly 12 proteinogenic amino acids and McpG binds GABA with the highest affinity observed

to date (Reyes-Darias et al., 2015; Corral-Lugo et al., 2016). P. fluorescens Pf0-1 presents three

d-Cache LBD type MCPs orthologous to P. aeruginosa amino acids chemoreceptors (Oku et al.,

2012).

Regarding plant pathogenic bacteria, only a limited number of studies on chemotaxis and

chemoreceptors have been carried out. P. syringae pv. actinidae (Psa), a strain phylogenetically

related to PsPto, presents five Cache LBD containing chemoreceptors (PscA to PscE).

PscA, PscB and PscC present a d-Cache LBD and are homologs to P. aeruginosa PctA, PctB and

PctC, respectively. Their binding profiles were stablished by a fluorescence-based thermal shift

assay. PscA binds L-Asp, D-Asp and L-Glu. PscB binds L-Asn, L-Gln, L-Phe, L-Met, L-Ala, L-

Isoleucine (L-Ile), L-Leucine (L-Leu), L-Serine (L-Ser), L-Tryptophan (L-Trp) and the non-

proteinogenic amino acid L-HS. PscC binds L-Pro, L-Ile and the non-proteinogenic amino acid

9

(GABA) (McKellar et al., 2015). Interestingly, the binding profiles were different from those of

their P. aeruginosa homologues.

PscD chemoreceptor presents a s-Cache LBD and was characterized by combining fluorescence

thermal shift assays and isothermal titration calorimetry (ITC). The results proved that

recognizes C2 and C3 carboxylate ligands, showing a great affinity for glycolate.

PscE chemoreceptor also presents a s-Cache LBD domain, however, it could not be

characterized since the recombinant LBD domain was completely insoluble under all conditions

tested, so the ligand profile of this MCP remains unknown (Brewster et al., 2016).

Our laboratory is interested in identifying, characterizing and studying at a functional level the

chemoreceptors of PsPto, to acquire a detailed knowledge of the perception mechanisms

during the initial stages of plant colonization.

Due to the relevance of amino acids during plant-bacteria interaction and, since other bacterial

chemoreceptors involved in their perception had already been studied, this work was designed

as part of the group research line focused on amino acids perception.

Among the homologues to the 5 previously studied Psa chemoreceptors, PsPto-PscA MCP has

been already characterized (Cerna-Vargas et al., under review) and it presents the same ligand

binding profile as PscA in Psa, specifically recognizing L-Glu, D-Asp and L-Asp. It has been

determined that this MCP exerts a double function by mediating not only chemotaxis

processes, but also other functions such as biofilm formation and virulence-related processes.

PsPto-PscC is the objective of research of other member of the group.

PsPto homologues to PscB (PsPto_1061) and PscE (PsPto_3379) had been selected as object of

study in this project due their possible involvement in amino acids perception.

The general objective of this study was the analysis of the function of two putative amino acid

chemoreceptors in PsPto in order to study their contribution during the plant-bacteria

interaction. To tackle this general aim the following specific tasks were proposed:

1. Generation of mutants of PsPto for PsPto_1061 and PsPto_3379 MCPs.

2. Functional characterization of PsPto_1061 and PsPto_3379 MCPs mutants through

chemotaxis and tomato virulence assays.

3. Expression and purification of the recombinant LBDs PsPto_1061 and PsPto_3379 and

characterization of their ligand binding profiles.

In order to carry out these objectives, in vitro and in vivo assays were performed. To accomplish

these approaches, the mutant strain PsPto_1061 and the expression vectors containing the

respective LBDs (which had been previously generated in the laboratory) were provided as

starting material to develop this work.

10

CHAPTER II - MATERIAL AND METHODS

2. 1. LBD sequence and protein structure determination

Sequences were obtained from NCBI database (https://www.ncbi.nlm.nih.gov/). Sequence

alignments were performed using ClustalW 2.0.12

(https://www.ebi.ac.uk/Tools/msa/clustalo/). LBDs were identified as the regions between

transmembrane segments predicted by TMHMM Server v. 2.0

(http://www.cbs.dtu.dk/services/TMHMM/). Topology of LBDs was obtained by using Swiss-

model server (http://swissmodel.expasy.org/).

2. 2. Strains, plasmids and culture conditions

Bacterial strains and plasmids used in this study are described in Table 1 and Table 2

respectively.

Table 1. Bacterial strains used in this study

Bacteria Strain Genotype Reference

E. coli

DH5α supE44 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA1

endA1 gyrA96 thi‐1 relA1 Hanahan,

1983

BL21 (DE3) F‐ omp hsdSB gal tonA Invitrogen

CC118λpir Δ (ara-leu), araD, ΔlacX74, galE, galK, phoA20, thi-1, rpsE, rpoB, argE (Am), recA1, 7~pir phage

lysogen

Herrero et al., 1990

Pseudomonas syringae pv. tomato

DC3000

PsPto Wild- type strain RfR

Cuppels, 1986

PsPto_1061 PSPTO_1061::pKNG101, SmR Laboratory

collection

Table 2. Plasmids used in this study

Plasmid Relevant characteristics Reference

pDESTTM

17 His6-tagged (N-terminal) protein expression vector, CbR

Invitrogen

pDEST™17: 1061 His6-tagged 1061 protein expression vector, CbR Laboratory collection

pDEST™17: 3379 His6-tagged 3379 protein expression vector, CbR Laboratory collection

pGEM-T easy® Subcloning vector, ApR

Invitrogen

pGEM-T easy®: 3379 PsPto3379::pGEM-T easy, ApR

This study

pKNG101 R6K origin sacB marker exchange vector; mob+, SmR

Kaniga et al., 1991

pKNG101:3379 PsPto3379::pKNG101, SmR

This study

http://swissmodel.expasy.org/

11

E. coli strains were grown in Luria-Bertani (LB) medium (Sambrook et al., 1989) at 37 °C. PsPto

strains were cultivated in King´s B (KB) medium (King et al., 1954) at 28 °C. When required,

medium was supplemented with the appropriate antibiotics at the following concentrations

(μg/ml): kanamycin 50, ampicillin 100, carbenicillin 100, rifampicin 25 and streptomycin 50.

Bacterial growth was monitored at OD600nm using a Jenway 6300 spectrophotometer.

2. 3. DNA amplification and purification

DNA fragments were amplified using PCR Supermix High Fidelity and its correspondent primers

(Supplementary Table 1). DNA fragments were purified from agarose gels using the GeneJet™

Gel Extraction Kit (Thermo Fisher Scientific, MA, USA).

2. 4. DNA extraction and quantification

Plasmid DNA from cell cultures grown in LB with appropriate antibiotic was purified with

NucleoSpin® Plasmid kit following the manufacturer’s instructions (Macherey-Nagel GmbH &

Co.KG, Düren, Germany). DNA extracted and purified was quantified using a NanoDrop ND-

1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA).

2. 5. Mutants construction

To generate the PsPto_3379 mutant (PsPto_1061 mutant was previously obtained in the lab),

an internal 454-bp fragment of PsPto_3379 was amplified by PCR from PsPto genome. The

resulting fragment was cloned in pGEM-T easy (Invitrogen) and transformed in E. coli DH5α by

heat shock. Blue-white screening for positive colonies was performed using LB Ap100, X-gal (80

mg/mL) and IPTG (0.5 mM) plates.

Plasmid was digested with XmaI at the restriction sites incorporated by the primers and the

resulting fragment was cloned in pKNG101 vector. The resulting plasmid pKNG101:3379 was

introduced in E. coli CC118λpyr and subsequently transferred to PsPto by electroporation.

Single crossover integrants were selected based on resistance to Sm50 and confirmed by PCR.

2. 6. Swarming motility assays

Swarming assays were performed according to Santamaría-Hernando et al., 2018. Briefly, PsPto

strains were grown at 28 °C for 24 h on KB agar under dark conditions. Cells were resuspended

in KB medium to an OD600nm of 1. Five microliters of the bacterial suspension were spotted onto

soft KB agar (0.5% agar, w/v). Plates were incubated for 16 h at 28 °C and 80% relative humidity

(RH) under dark conditions. Six replicate plates were prepared and swarm colonies were

photographed.

12

2. 7. Swimming motility assays

Swimming assays were performed in semisolid LB (0.3% agar, w/v). PsPto and PsPto_1061

strains were grown in fresh LB medium with the respective antibiotics and inoculated with a

toothpick in the surface of the medium. 16 hours after incubation at 28 °C and 80% RH under

darkness conditions, the generated ring was quantified.

2. 8. Quantitative capillary chemotaxis assay

Capillary assays were carried out following a modified version of the capillary assay developed

by Sampedro et al., 2015. Overnight cultures were diluted to an OD600 of 0.05 in KB and grown

at 28 °C with orbital shaking. At an early stationary phase of growth (OD600nm = 0.25), the

cultures were washed twice by centrifugation at 1750 x g for 3 minutes and resuspended in

HEPES 10 mM pH=7 to an OD600nm of 0.25. Then, 230 μL were placed in each well in a 96 well

plate. One-microliter capillaries were heat sealed and filled with the compound to test solved in

water and immersed into the bacterial suspensions for 30 minutes. Capillaries were removed,

rinsed with sterile water and the content expelled into 1 mL of NB medium (Fathy and Persley,

1983). Serial dilutions were plated onto NB agar medium with the appropriate antibiotics and

the number of colony forming units was determined. In all cases, data were corrected with the

number of cells that swam into capillaries containing water.

2. 9. Bioscreen assays

Bacterial growth was monitored using a Bioscreen C device at OD600nm at 28 °C during 24 hours

with an initial OD600nm of 0.14. Bacterial populations (PsPto and PsPto_1061) were grown in a

rich medium (KB) or in a minimal medium (M9 2x) (Harwood and Cutting, 1990).

2. 10. Plants assays

PsPto and Pspto_1061 strains were grown at 28 °C for 24 h in KB plates with the respective

antibiotics. Cells were resuspended in MgCl2 and washed twice by centrifugation at 5000 x g for

5 minutes. Cells were adjusted to an OD600nm of 0.1 (108 cfu/mL).

Silwet L-77 was added at a final concentration of 0.02% (vol/vol). Three-week-old tomato

plants (Solanun lycopersicum cv Moneymaker) were immersed in the bacterial suspension for

30 s and incubated in a growth chamber at 25 °C, 60% RH and a daily light period of 12 h.

Six days after infection, symptoms were recorded and bacterial populations in the apoplast

were determined by sampling five 1-cm diameter leaf discs per plant. Discs were homogenized

and drop-plated on KB agar plates with the corresponding antibiotics.

13

2. 11. Protein expression

E. coli BL21 (DE3) strains transformed with pDEST™17:1061 or pDESTTM17:3379 vectors were

grown in LB medium at 37 °C until they reach an OD600nm of 0.6. Bacterial cultures were

fractionated in volumes of 10 mL and subjected to different temperature conditions (18 and 28

°C) and different IPTG (protein expression inductor) concentrations (0 mM, 0.1 mM, 0.5 mM

and 1mM). Samples from all treatments were collected by centrifugation at 10000 x g for 30

minutes and then examined by SDS-PAGE Coomassie-stained gels in order to determine the

best conditions for expression.

Once the best conditions of temperature and IPTG concentration were estimated, a large-scale

protein expression assay was carried out. E. coli BL21 strains containing the expression vectors

were grown at 37 °C in 2 L Erlenmeyer flasks containing 400 mL of LB medium supplemented

with carbenicillin. Once the culture reached an OD600nm of 0.5, protein expression was induced

by adding 1 mM IPTG and incubated at 18 °C for PsPto_3379 LBD and 28 °C for PsPto_1061 LBD

overnight. Cells were harvested by centrifugation at 10000 x g for 30 minutes at 4 °C. Cell

pellets were stored at -80 °C.

2. 12. Western blot assays

Clarified cell lysates were subjected to SDS-PAGE and then transferred to a PVDF membrane.

Membranes were blocked and washed following standard procedures (Sambrook et al., 1989)

and incubated with mouse anti-His6 antibody (GE Healthcare Europe GmbH, Germany)

overnight at 4 °C. Then, membranes were washed and incubated with anti-mouse IgG-

peroxidase conjugated antibody (Sigma-Aldrich, MO, USA) at room temperature. The Western

blot was developed by addition of Immobilon™ Western Chemiluminiscent HRP substrate

(Millipore, MA, USA).

2. 13. Protein purification

Protein purification was performed as described by McKellar et al., 2015 with slight

modifications. Briefly, cell pellets were resuspended in 40 mL of lysis buffer (50 mM sodium

phosphate, 300 mM NaCl, 10 mM imidazole and pH 7.5) supplemented with benzonase (1U),

lysozyme (10 mg/mL) and protease inhibitor cocktail (Roche). Cells were lysed by sonication

and centrifuged at 20,000 x g for 1 h at 4 °C. Supernatant was loaded on a 1 mL bed volume of

TALON metal affinity resin (Agarose Beads Technologies, Madrid, Spain) and gently mixed at 4

°C for 1 h. Sample was washed with buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7)

containing 20 mM imidazole and eluted with a 50 mM to 150 mM imidazole gradient in (50 mM

sodium phosphate, 300 mM NaCl, pH 7) buffer. Purity of the His-tagged proteins was checked

14

by SDS-PAGE Coomassie-stained gels. Protein concentration was quantified using the Bradford

assay (Bio-Rad Laboratories, CA, USA). The fractions containing the purified protein were then

dialyzed against two different buffer solutions containing: 5 mM TrisHCl, 5 mM PIPES, 5 mM

MES, 10% glycerol, 150 mM NaCl, pH 7.6 (Buffer 1) and 50 mM sodium phosphate, 200 mM

NaCl, 10% glycerol, pH 7.6 (Buffer 2).

2. 14. Fluorescence-based thermal shift assays

Thermal shift assays were carried out as described by McKellar et al., 2015 using a Roche Light-

Cycler 480 Real-Time PCR instrument. Each 20 μL standard assay contained 1 μM protein, 10

mM compound and SYPRO Orange (Sigma- Aldrich) at 5 x concentration.

During the assay, samples were heated from 20 °C to 80 °C at a rate of 1.2 °C min−1. SYPRO

Orange presents affinity for hydrophobic parts of the protein. As the protein unfolds due to the

increase in temperature, hydrophobic parts of the protein are exposed, allowing the binding of

the fluorescent dye.

The first derivative values (−dF/dt) from the raw fluorescence data were used to determine the

melting temperature (Tm). Binding was determined by analyzing the difference in the protein

unfolding temperature in the presence and absence of ligand.

2. 15. Statistical analysis

All experiments were performed at least three times. Data obtained from the assays was

subjected to statistical analysis. Student’s t-test was used to establish significant differences

between samples. Differences were considered significant at P value < 0.05. All analyses were

performed using the statistical software package SPSS 25.0 (SPSS, Chicago, Illinois).

15

CHAPTER III – RESULTS

3.1. PsPto genome presents homologous sequences to Psa amino acid chemoreceptors.

A sequence alignment was performed to determine the identity between Psa chemoreceptors

PscB and PscE and their respective homologues in PsPto (1061 and 3379). PSPTO_1061

chemoreceptor showed a 97% LBD sequence similarity with Psa. PscB. PSPTO_3379 showed a

98% LBD sequence similarity with Psa PscE (Figure 4).

Figure 4. Amino acid sequences alignment between PscB and PsPto_1061 LBD´s chemoreceptors (A)

and between PscE and PsPto_3379 LBD´s chemoreceptors (B). Clustalw Omega was used to perform the

alignment. Amino acids marked in red are those that present a mismatch in sequence alignment.

3. 2. Construction of PsPto_3379 mutant.

Internal fragment of PsPto_3379 was cloned into pGEM-T easy vector. Then, fragment was

restricted from this plasmid and successfully cloned into the compatible sites of pKNG101

vector. Both constructions were confirmed by PCR and restriction (data not shown). Several

attempts to transfer the resulting plasmid to PsPto in order to obtain single crossover

A

B

16

cointegrates were performed. However, we were unable to obtain PsPto_3379 mutant strain.

PsPto_1061 mutant strain was already available in the laboratory collection and was used for in

vivo assays.

3. 3. Growth of PsPto_1061 mutant strain is not altered in rich and minimal mediums.

Growth of PsPto and PsPto_1061 in KB and M9 minimal medium was monitored at 28 °C for 24

h using a Bioscreen C device at OD600nm.

No significant differences in growth were observed between the wild type and the mutant

strain when growing in KB medium (Figure 5). In M9 minimal medium a slightly higher growth

of PsPto_1061 mutant was observed, although growth rates for both strains were similar over

time (Figure 5).

Figure 5. Growth curve of PsPto and PsPto_1061 in KB medium and M9 minimal medium. Curves in blue

and black show PsPto growth in KB and M9 respectively. Curves in orange and green show PsPto_1061

growth in KB and M9 respectively. Data represent the means and standard errors of three replicates.

3. 4. PsPto and PsPto_1061 mutant show similar swimming motility while morphology of

swarm colonies is different.

In order to discard that mutation in the gene coding for PsPto_1061 MCP affected the overall

motility of PsPto, we conducted swimming and swarming motility assays.

Swimming is a flagellum-dependent movement that consists on the displacement through a

semisolid framework (agar concentration < 0.3%). Swarming consist on a multicellular surface

migration (0.3-1.0% agar) that requires both flagellum and surfactants production (Sampedro

et al., 2015).

17

Results in Figure 6 showed that swimming motility of PsPto_1061 mutant was identical to that

observed for the wild type strain since the halo diameter produced by the expansion of

bacteria was similar (Figure 6A). Both strains presented swarming motility, however the

morphology of swarm colonies was different in the mutant strain with respect to that of the

wild type (Figure 6B).

Figure 6. Swimming and swarming assays. (A) Photographs of representative swimming plates of PsPto

and PsPto_1061. Bacteria were inoculated in a semisolid LB medium (agar 0.3%) and incubated for 16

hours at 28 °C and 80% RH under darkness conditions. (B) Photographs of representative swarming

colonies of PsPto and PsPto_1061. Bacteria were inoculated in soft KB agar (0.5% agar). Plates were

incubated for 16 h at 28 °C and 80% RH under dark conditions.

3. 5. PsPto_1061 mutant is impaired in the perception of certain amino acids.

Previous works carried out with a PsPto_1061 LBD homolog in Psa (PscB) indicated that this

MCP was implicated in the binding of L-HS, L-Trp, L-Met, L-Phe, L-Gln and L-Asn (McKellar et al.,

2015).

Initially, to analyze the chemotactic response of PsPto wild type strain to these amino acids we

conducted chemotaxis capillary assays. For all the selected amino acids two concentrations

were tested (1 and 5 mM). We observed significant chemoattraction towards all the amino

acids tested. Maximum response to L-HS and L-Phe was observed at 1 mM, however for the

other amino acids further assays with increasing concentrations of the compounds are required

to determine the maximum response (Figure 7).

To determine whether the chemotactic response to these amino acids is mediated by

PsPto_1061 MCP we conducted chemotaxis capillary assays with the PsPto_1061 mutant.

A B

18

Figure 7. Chemotactic response of PsPto and PsPto_1061 towards amino acids. (A) Response to L-

homoserine (L-HS) and L-tryptophan (L-Trp), (B) L-methionine (L-Met) and L-phenylalanine (L-Phe), (C) L-

glutamine (L-Gln) and L-asparagine (L-Asn). Data have been corrected with the number of cells that swam

into buffer containing capillaries. The means and standard errors of three replicates are shown. Asterisks

indicate significant differences according to Student’s t-test.

Chemotaxis to L-Phe at both concentrations tested was impaired in the mutant strain, whereas

the chemotactic response to L-HS, L-Trp, L-Met and L-Asn was significantly reduced. No

significant differences were observed for L-Gln with respect to the wild type strain (Figure 7).

3. 6. PsPto_1061 mutant is less virulent than wild type strain in tomato plants.

To determine whether the mutation of PsPto_1061 MCP affected virulence, tomato plants

were inoculated with the wild type and the mutant strain. At 6 days post inoculation, leaves

infected with the wild type presented more severe symptoms since both chlorotic and necrotic

spots became visible both on the beam and on the underside of the leaf. Leaves infected with

PsPto_1061 also had chlorotic spots, but no necrotic spots were observed on the leaf surface

(Figure 8A). Quantification of bacterial populations inside the leaves showed no statistically

significant differences between the two strains (Figure 8B). However, a trend of increased

colonization could be observed in those inoculated by the wild type strain.

B A

C

19

Figure 8. Tomato virulence and colonization assays. (A) Disease symptoms in representative leaves 6-dpi

(B) Bacterial population 6-dpi. The means and standard errors of three replicates are shown. Asterisks

indicate significant differences according to Student’s t-test.

3. 7. Recombinant LBDs from PsPto_1061 and PsPto_3379 MCPs were expressed at different

conditions.

In order to identify the putative ligands of the selected chemoreceptors and to study its binding

properties, expression and purification of the recombinant LBDs were carried out. The

expression vector pDESTTM17 with the respective LBD sequence was already available in the

laboratory collection and was used for in vitro assays (pDESTTM17:1061 and pDESTTM17:3379).

Small-scale expression assays under different temperature conditions (18 °C and 28 °C) and

IPTG concentrations (0, 0.1, 0.5 and 1.0 mM) were carried out to determine the best conditions

to obtain the highest concentration rates of recombinant protein.

As shown in Figure 9A, PsPto_1061 recombinant LBD reached a maximum expression level at

28 °C and 0.5-1.0 mM IPTG concentration. On the other hand, PsPto_3379 recombinant LBD

expression was maximum at 18 °C and 1 mM IPTG concentration (Figure 9B).

A B

A B

PsPto

PsPto_1061

PsPto PsPto_1061

A B

20

Figure 9. Western Blot analysis of the protein expression levels of recombinant LBDs PsPto_1061 (A)

and PsPto_3379 (B) under different culture conditions. Recombinant protein presence in clarified lysates

was detected by Western Blot with specific mouse anti-His6 antibody.

3. 8. PsPto_3379 was purified using the 6x his purification tag. His6-tagged PsPto_3379 recombinant LBD was purified from the soluble fraction using a

chelating agarose beads bulk resin. Fractions obtained during purification were analyzed by

SDS-PAGE gel electrophoresis (Figure 10 A-B).

Figure 10. SDS-PAGE gel electrophoresis of purified His6-tagged PsPto_3379 LBD. Purified proteins were visualized with Coomassie blue after separation in a 12.5% SDS-PAGE gel. Size was determined according to molecular size marker (M). Protein fractions were collected corresponding to: 1-5. Flowthrough fractions obtained using a lysis buffer containing 10 mM imidazole. 6. Washing fraction obtained using a buffer containing 20 mM imidazole. 7. Elution fraction obtained using a buffer containing 50 mM imidazole. 8.1- 8.3. Elution fractions obtained using a buffer containing 100 mM imidazole. 9. Elution fraction obtained using a buffer containing 150 mM imidazole. All buffers contained 300 mM NaCl, 50 mM sodium phosphate, pH 7.

A B

A

B

21

The purest protein fractions of this protein (Figure 10B: lanes 8.1, 8.2, 8.3 and 9) were eluted at

100 and 150 mM imidazole. This purification was performed twice with similar results.

Protein concentration of these fractions estimated by Bradford assay was about 12-20 μM for

the first purification. The fractions containing the purified protein were unified in a single

sample and dialyzed during 24 hours at 4 °C against a buffer containing: 5 mM TrisHCl, 5 mM

PIPES, 5 mM MES, 10% glycerol, 150 mM NaCl, pH 6.8. When sample was collected protein was

precipitated, so the ligand binding thermal shift assay could not be performed at this time.

Assuming that this was due to a problem in the chemical composition of the dialyzing buffer or

in the pH used, we decided to carry out a second purification and a subsequent adjusted

dialysis protocol. Two buffers with different composition were tested: A buffer with the same

composition used in the previous dialysis (Buffer 1) and a buffer containing 50 mM sodium

phosphate, 10% glycerol and 200 mM NaCl (Buffer 2). Considering the isoelectric point of the

protein, both of them were adjusted to a pH 7.6. The total volume of purified protein was

distributed between these two buffers.

After 24 hours of dialysis, the resulting protein sample from both dialysis buffers was collected.

No precipitation was observed in any of the two samples. Protein concentration was estimated

by Bradford assay. The results indicated a protein yield of 1.8 μM in buffer 1 and 4.4 μM in

buffer 2.

Despite obtaining a good level of protein expression in the small-scale assay for PsPto_1061

LBD (Figure 9A), we were not able to carry out its purification. After the purification steps, it

was not possible to detect the presence of the protein in the obtained fractions by SDS-Gel

electrophoresis (data not shown).

3. 9. Thermal shift assays revealed that PsPto_3379 LBD was unable to bind GABA, L-

Homoserine and L-Phenylalanine.

As it has been previously presented, our attempts to construct the PsPto_3379 mutant strain

were not successful and therefore we could not contrast the implication of this chemoreceptor

in the chemoattraction to specific amino acids described for the wild type strain. In order to get

some insights regarding the identification of the ligands recognized by PsPto_3379 LBD, a

thermal shift ligand binding assay was performed with the purified protein samples obtained.

The previous study performed with Psa chemoreceptors did not allow the study of the binding

capacity of PscE, the homologous protein to PsPto_3379, due to the unsuccessful protein

purification (Brewster et al., 2016). Therefore we decided to start this analysis checking the

binding capacity of PsPto_3379 to three amino acidic molecules described to have a relevant

role during PsPto plant infection process: L-HS, L-Phe and the amino acidic derivative GABA.

22

Thermal Shift assays were carried out against GABA 1 and 5 mM, L-HS 5 mM and L-Phe 5 mM. It

was not possible to test more compounds and more concentrations due to a limitation in the

amount of protein. Thermal Shift assays were carried out following the methodology explained

above.

Results showed a denaturation curve for the protein sample obtained after the dialysis against

Buffer 1, which indicates that the protein was folded, however, in the sample dialyzed against

Buffer 2, a temperature melting curve was not obtained.

No significant Tm increases (∆Tm > 2°C) were observed by the addition of any of the amino

acids tested in the thermal shift assay (Figure 11). This result indicates that PsPto_3379 LBD did

not bind any of these compounds at the concentrations tested.

Figure 11. Differences of melting temperature (ΔTm) of PsPto_3379 LBD in the presence of GABA, L-Homoserine and L-Phenylalanine. Red line represents the threshold to consider binding between the receptor and the ligand tested (∆Tm > 2°C). Error bars represent standard error of the mean (SEM).

23

CHAPTER IV - DISCUSSION The objective of this project was to get insights into the role of two PsPto chemoreceptors in

the perception of amino acids and derivatives. Given that previously studies reported early

changes in apoplastic amino acids levels associated with defense and bacterial interactions

(Ward et al., 2010; O´Leary et al., 2016), the perception of these compounds by PsPto during

the infection process may be crucial for its success.

The two selected chemoreceptors (PsPto_1061 and PsPto_3379) harbor double and single

Cache structural motives respectively in its LBD, which have been previously described to be

involved in the perception of these type of molecules in other bacteria (Ortega et al., 2017).

PsPto_1061 MCP is homologue to the Psa chemoreceptor PscB, showing a 97% of identity; and

PsPto_3379 MCP is homologue to the Psa chemoreceptor PscE, showing a 98% of identity.

Neither of the two Psa homologues had been characterized at a functional level. In fact, PscE

has never been studied at any level due to the impossibility of being obtained as a

recombinant protein. Likewise, PscB ligand binding profile has been studied in vitro through

thermal shift assay but, neither in vivo studies or confirmation of binding by ITC have been

performed (Mckellar et al., 2015; Brewster et al., 2016). Changes in amino acids´s sequences

(especially if such changes occur in the binding site) can completely change the profile of

recognized ligands (Tajima et al., 2011). For this reason, despite the high sequence similarity,

differences in the perception of amino acids between PsPto chemoreceptors and their

homologues could not be ruled out.

To characterize PsPto_1061 MCP, a series of in vivo assays were carried out with a mutant

strain affected in this chemoreceptor (PsPto_1061). We confirmed that the mutation did not

compromise bacterial growth and overall motility capacity. Despite swimming movement did

not appear to be affected in the mutant strain, the described swarming motility was different

as compared to that of PsPto wild type strain. Medium used to carry out both motility assays

(swimming and swarming) contains hydrolyzed proteins among their nutrient sources. It could

be hypothesized that the lack of a chemoreceptor involved in amino acids perception alters

the regulation of motility traits underneath swarming ability. This later type of motility

requires not only the flagellar function, but also the production of certain surfactants (Berti et

al., 2007). Moreover, swarming motility and surfactant production have been previously

described to be essential for full virulence (Kanda et al., 2011). Therefore, a tight regulation of

this processes mediated by perception mechanisms might be considered. However the specific

role of PsPto_1061 MCP function in the regulation of swarming motility requires further

experiments.

24

Chemotaxis assays against the amino acids perceived by the homologue PscB were performed

with PsPto and PsPto_1061 strains in order to characterize PsPto_1061 LBD function.

PsPto showed chemoattraction to L-Asn, L-Gln, L-Trp, L-Met, L-Phe and L-HS. PsPto_1061 did

not seem to be affected in L-Gln perception while a reduction in chemotaxis respect to that of

wild type is observed in the presence of the other amino acids tested. Interestingly, the

greatest affinity described for the different ligands tested with the Psa PscB chemoreceptor

was observed for L-Gln (McKellar et al., 2015). This may indicate that specific changes in the

amino acids sequences between PscB and 1061 LBDs could be responsible for the difference

observed between the two chemoreceptors. Further analyses through specific in silico

structural tools are required to establish the relevance of the changes over the putative

domains involved in binding.

It has been previously described in the literature that PsPto uses some of the amino acids

present in the apoplastic fluid as C and N sources (Rico and Preston, 2008). Two of the

described amino acids with this metabolic role are L-Gln and L-Asn. After confirming a positive

chemotactic response to these compounds, we can establish that PsPto is attracted to L-Asn

and L-Gln in order to incorporate them as energy sources. Therefore perception of these

amino acids could be considered a relevant trait to stablish the infection.

With respect to the rest of the amino acids tested, no descriptions about their role as C or N

sources have been reported. Since the apoplastic composition changes during the infection

process, the perception of such changes by PsPto may be important for its survival. Some

defense pathways such as phenylpropanoids and indoles biosynthesis are overexpressed

during infection and the precursors for the synthesis of these compounds are L-Phe and L-Trp

respectively (Yu, et al., 2013; Glawischnig et al., 2004). Therefore, the changes in concentration

of these two amino acids might be considered a hallmark of defense during the interaction

which is perceived by the bacteria. Moreover, the expression of Phe catabolic pathways has

been described to be induced in epiphytic populations of P. syringae B728a (Yu et al., 2013).

Therefore, it cannot be ruled out that PsPto also uses this amino acid as nutrient source

diminishing its concentration during the interaction and causing a detrimental effect in plant

defense pathway. A similar picture has been previously described in the case of the amino acid

derivative GABA, which regulates plant defense response (McCraw et al., 2016).

Likewise, PsPto also showed chemoattraction to L-HS. This molecule is an important precursor

of acyl-homoserine lactones (acyl-HSL), which are synthesized by the bacteria as signaling

molecules to modulate quorum sensing (Parsek et al., 1999). This process allows cell to cell

communication and leads to a coordinate response at the level of gene regulation under

certain conditions (Parsek et al., 1999). L-HS also seems to play an important role in plant

25

fitness because it stimulates plant growth and transpiration (Palmer et al., 2014). Therefore,

PsPto chemoattraction to this molecule may support the incorporation of HS with the

subsequent positive effect on the synthesis of bacterial signaling and the negative effect on

plant development. A detailed analysis of the production of acyl-homoserine lactones (acyl-

HSL) in the PsPto_1061 mutant strain compared to wild type strain is required to contrast this

hypothesis.

To corroborate the ligand binding profile, in vitro assays based on thermal shift and ITC are

required. However, difficulties during protein expression of PsPto_1061 LBD did not allow us to

perform a purification and a subsequent thermal shift assay against the amino acids tested in

the chemotaxis assays. In order to solve the low expression rate obtained, several approaches

will be carried out like the use of different expression vectors, the expression in different E. coli

backgrounds or the ad-hoc gene synthesis in order to obtain a codon optimization in the

sequence.

Virulence assays with the PsPto_1061 mutant strain showed a lower virulence ability

highlighted by the reduction of symptoms caused by this strain in tomato leaves. However, a

significant effect on plant colonization was not detected. It has been previously described that

bacterial colonization and symptoms production can be uncoupled in PsPto (Ferreiro et al.,

2018). These observations, made in mutants affected in global regulators of virulence, suggest

that the expression of different virulence determinants at different stages of infection is tightly

coordinated to orchestrate symptoms production and bacterial colonization. The observation

of this uncoupled effect in PsPto_1061 strain suggests the involvement of amino acid

perception in the regulation pathways of virulence. Taking into account the above mentioned

role of the amino acid perceived by this chemoreceptor during plant defense, it can be

hypothesized that bacteria exploits amino acid perception to optimize virulence. In fact, a

recent research in our lab shows the involvement of PsPto-PscA chemoreceptor in the control

of c-di-GMP levels upon amino acid perception. The levels of this cellular second messenger in

PsPto control the expression of several virulence determinants (Cerna-Vargas et al., under

review; Romling et al., 2013).

The second chemoreceptor under study in this work is PsPto_3379. Unfortunately, the

generation of mutant strain was not possible after several attempts. Therefore, we couldn´t

carry out the functional characterization. This could be due to a low recombinant rate between

the wild sequence and the internal fragment cloned in the suicide vector pKNG101. For this

reason other central fragment of the LBD sequence should be selected to recombine with

PsPto chromosome in future works. However the recombinant LBD was successfully purified.

Results obtained after performing a thermal shift assay against a selected number of amino

26

acids showed no binding activity, however this approach was very limited due to the quantity

of available protein. A throughput analysis, after a high scale purification rate, using multi-cell

plates with compounds of different chemical nature could help the characterization of the

binding profile while the mutant strain is obtained. It cannot be discarded that this

chemoreceptor which harbors a s-Cache LBD type, may bind compounds of different nature as

it has been described for other MCPs of this type (Ortega et al., 2017).

27

CHAPTER V – CONCLUSIONS The aim of this work was to obtain information regarding the role of PsPto_1061 and

PsPto_3379 chemoreceptors in the perception of amino acids during PsPto infection.

Functional and biochemical approaches carried out with these two MCPs lead us to obtain the

following conclusions:

1. PsPto chemoreceptors 1061 and 3379 are homologous to the Pseudomonas syringae pv.

actinidae chemoreceptors PscB and PscE, respectively.

2. PsPto_1061 mutant is affected in chemotaxis towards L-Asparagine, L-Homoserine, L-

Tryptophan, L-Methionine and L-Phenylalanine amino acids. However, and in contrast to the

PscB homolog, PsPto_1061 seems not to be involved in L-Glutamine perception.

3. PsPto_1061 mutant is affected in swarming motility and virulence suggesting a role of

amino acid perception in the regulation of pathogenicity.

4. Optimized conditions to purify PsPto_3379 recombinant LBD have required the adjustment

of pH and composition of dialysis buffer.

5. PsPto_3379 MCP does not bind GABA, L-Homoserine and L-Phenylalanine at the

concentrations tested as revealed by thermal shift assays.

28

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