Universidad Autónoma de Madrid Departamento de Biología
Facultad de Ciencias
‘STRUCTURE AND FUNCTION OF THE COMPONENTS OF THE CORE OF T7 BACTERIOPHAGE, A DNA TRANSLOCATION
COMPLEX’
María del Mar Pérez Ruiz
Madrid, 2019
Universidad Autónoma de Madrid Departamento de Biología
Facultad de Ciencias
Memoria presentada para optar al grado de Doctora por:
María del Mar Pérez Ruiz Licenciatura en Biología
Madrid, 2019
Directores:
Prof. José López Carrascosa
Centro Nacional de Biotecnología Dra. Ana Cuervo Gaspar
Centro Nacional de Biotecnología
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The work described in this thesis was performed in the Structure of Macromolecular Assemblies laboratory of the Macromolecular Structure Department at the National Centre for Biotechnology (CNB-CSIC) in Madrid under the supervision of Prof. José López Carrascosa and Dra.
Ana Cuervo Gaspar. It was financially supported by an FPI grant (BES-2015-073615) from the Spanish Economy Ministry.
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RECONOCIMIENTOS
Quiero agradecer a mi director de tesis José L. Carrascosa por darme la oportunidad de trabajar en su laboratorio y a mi co-directora de tesis Ana Cuervo, por permitirme aprender tanto de ella. Me llevo mucho aprendido de ambos. Gracias.
También tengo que agradecer la colaboración de muchas personas que han hecho posible la realización de este trabajo. En primer lugar, a Rocío Arranz, Francisco Javier Chichón y Rafael Nuñez del servicio de cryo-microscopía electrónica del CNB. A Cristina Patiño del servicio de microscopía electrónica del CNB, y al servicio de microscopía electrónica del CBM.
Al servicio de ultracentrifugación analítica del CIB, a Juan Román Luque, a Carlos Alfonso y a Germán Rivas. Al servicio de Proteómica del CNB, a Miguel Marcilla.
A Mark van Raaij del CNB por proporcionarnos el vector de expresión de gp16.
A Juha Huiskonen por darme la oportunidad de realizar una estancia en su laboratorio y aprender un poco más sobre ‘mismatch symmetry’. Gracias a Vahid Abrishami por su ayuda en el procesamiento durante mi estancia.
A Luis Alberto Campos por los experimentos de dicroísmo circular.
Por último, y no por ello menos importante, a todos los compañeros de servicios del CNB que hacen que la vida en el laboratorio sea un poco más fácil: viajes, instrumentación, compras, personal, servicios generales y almacén.
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INDEX
LIST OF ABBREVIATIONS ... XIII AMINO ACID ABBREVIATIONS ... XV ABSTRACT ... XVII RESUMEN ... XVIII
1. INTRODUCTION ... 1
1.1. OVERVIEW ON BACTERIAL MACROMOLECULAR TRANSPORT COMPLEXES AND PUNCTURING DEVICES... 2
1.2 BACTERIOPHAGES ... 4
1.2.1 Bacteriophage structure ... 5
1.2.2 Bacteriophage life cycle and assembly ... 8
1.2.3 Bacteriophage infection ... 10
i. Host receptor recognition ... 10
ii. Degradation of peptidoglycan ... 11
iii. Examples of genome delivery ... 12
iv. Models for DNA transport: chemiosmotic and difference of pressure ... 13
1.3 THE T7 BACTERIOPHAGE ... 14
1.3.1. T7 bacteriophage structure ... 14
1.3.2 T7 assembly pathway ... 16
1.3.3 The T7 infection process and the core complex ... 17
1.4 3D RECONSTRUCTION OF MACROMOLECULES USING SINGLE PARTICLE CRYO-ELECTRON MICROSCOPY (CRYO-EM):THE RESOLUTION REVOLUTION ... 20
1.4.1 A brief introduction of cryo-EM... 21
1.4.2 Sample preparation, data acquisition and processing ... 24
i. Sample preparation ... 25
ii. Vitrification ... 26
iii. Data acquisition ... 27
iv. Processing of cryo-EM data ... 27
1.4.3 Model building, refinement and validation in cryo-EM density maps ... 28
i. De novo model building ... 29
ii. Model optimization and refinement ... 30
iii. Model validation ... 30
2. AIMS AND OBJECTIVES ... 35
3. MATERIALS AND METHODS ... 39
3.1 SAMPLE PREPARATION ... 39
3.1.1 Plasmids and strains ... 39
3.1.2 Protein expression and purification ... 39
3.2 CRYO-EM SAMPLE PREPARATION AND DATA ACQUISITION ... 41
3.3 IMAGE PROCESSING AND MAP CALCULATION ... 42
3.4. MODEL BUILDING AND COORDINATE REFINEMENT ... 42
3.5 SEDIMENTATION VELOCITY ASSAYS (SV) ... 42
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3.6. DYNAMIC LIGHT SCATTERING ASSAYS (DLS) ... 43
3.7. ESTIMATION OF MOLAR MASS OF THE CORE PROTEINS FROM HYDRODYNAMIC MEASUREMENTS ... 43
3.8. LIPOSOMES PREPARATION ... 44
4. RESULTS ... 47
4.1. INTERNAL CORE PROTEINS OF T7 BACTERIOPHAGE ASSEMBLED IN VITRO ... 47
4.1.1. The core protein gp15 ... 47
Biochemical characterization of gp15 protein ... 47
Biophysical characterization of gp15 protein ... 48
a) Sedimentation velocity assay (SV) of gp15 protein ... 49
b) Dynamic light scattering assay (DLS) of gp15 protein ... 49
Structural characterization of gp15 protein ... 50
4.1.2. The core protein gp16 ... 58
Biochemical characterization of gp16 protein ... 58
Biophysical characterization of gp16 protein ... 60
a) Sedimentation velocity assay (SV) of gp16 protein ... 60
b) Dynamic light scattering assay (DLS) of gp16 protein ... 61
Structural characterization of gp16 protein ... 61
4.1.3. Assembly in vitro of the core complex gp15-gp16 ... 62
Structural characterization of the gp15-gp16 core complex ... 65
4.2 STRUCTURAL CHARACTERIZATION OF CORE PROTEINS OF T7 BACTERIOPHAGE INSIDE THE CAPSID: A MISMATCH SYMMETRY QUESTION ... 78
5. DISCUSSION ... 87
5.1 GP15 ISOLATED PROTEIN FORMS TUBULAR OLIGOMERS IN VITRO ... 88
5.2 GP16 ACTS AS A CHAPERONE AND PROMOTES THE FOLDING OF THE FULL SEQUENCE OF GP15 TUBULAR PROTEIN ... 91
5.3 GP16 SHOWS A CONSERVED TRANSGLYCOSYLASE ACTIVE SITE ... 94
5.4 THE STRUCTURE OF CORE COMPLEX GP15 AND GP16 IN THE VIRAL PARTICLE ... 98
5.5 A MODEL FOR GP15-GP16 COMPLEX ASSEMBLY ... 101
5.6 PERSPECTIVES AND FUTURE WORK ... 103
6. CONCLUSIONS ... 107
CONCLUSIONES ... 108
BIBLIOGRAPHY ... 111
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FIGURES AND TABLE INDEX
FIG. 1.THE SECRETION SYSTEMS (TYPES I-VI) IN GRAM-NEGATIVE BACTERIA.. ... 3
FIG. 2.THE BACTERIAL PHAGE TAIL-LIKE COMPLEXES.. ... 4
FIG. 3.THE TRIANGULATION NUMBER OF ICOSAHEDRAL CAPSIDS... 6
FIG. 4.CAUDOVIRALES: DOUBLE-STRANDED DNA(DSDNA) TAILED PHAGES.. ... 7
FIG. 5.PHAGE LIFE CYCLES:THEY CAN HAVE LYTIC OR LYSOGENIC LIFE CYCLES.. ... 8
FIG. 6.SCHEME OF THE ASSEMBLY PROCESS OF TAILED PHAGES.. ... 9
FIG. 7.THE T7 BACTERIOPHAGE STRUCTURE... 15
FIG. 8.SCHEME OF THE T7 BACTERIOPHAGE ASSEMBLY PATHWAY.. ... 17
FIG. 9.MODEL OF T7 EJECTION PROCESS DIVIDED INTO FIVE MAIN STEPS. ... 18
FIG. 10. TYPICAL WORKFLOW OF CRYO-EM TECHNIQUE.. ... 25
FIG. 11.WORKFLOW OF ATOMIC MODEL DETERMINATION FROM NEAR-ATOMIC RESOLUTION DATA. 29 FIG. 12.THE GP15 PROTEIN PURIFICATION PROCESS. ... 48
FIG. 13. STUDY OF THE OLIGOMERIZATION STATE OF PURIFIED GP15 PROTEIN IN SOLUTION AT CONCENTRATION OF 1 MG/ML BY SEDIMENTATION VELOCITY ANALYSIS.. ... 49
FIG. 14.DYNAMIC LIGHT SCATTERING ASSAY OF GP15 PROTEIN.. ... 50
FIG. 15.NEGATIVE STAINNING CHARACTERIZATION OF GP15 PROTEIN.. ... 51
FIG. 16.CRYO-EM ANALYSIS OF GP15 PROTEIN ... 52
FIG. 17.THE WORKFLOW OF GP15 PROTEIN STRUCTURE DETERMINATION PROJECT. ... 54
FIG. 18.STRUCTURE OF THE T7 BACTERIOPHAGE GP15 CORE PROTEIN.. ... 55
FIG. 19.SECONDARY STRUCTURE CHART OF PURIFIED GP15 PROTEIN.. ... 56
FIG. 20.MONOMER-MONOMER INTERACTIONS IN TUBULAR CORE PROTEIN (GP15). ... 57
FIG. 21.ELECTROSTATIC POTENTIAL OF GP15 CORE PROTEIN... 58
FIG. 22.THE GP16 PROTEIN PURIFICATION PROCESS. ... 59
FIG. 23.STUDY OF THE OLIGOMERIZATION STATE OF PURIFIED GP16 PROTEIN IN SOLUTION AT 1 MG/ML CONCENTRATION BY SEDIMENTATION VELOCITY ANALYSIS.. ... 60
FIG. 24.DYNAMIC LIGHT SCATTERING ASSAY OF GP16 PROTEIN AT 1 MG/ML.. ... 61
FIG. 25.10%SDS-PAGECOOMASSIE STAINED OF PURIFIED GP16 PROTEIN (~144 KDA). ... 62
FIG. 26.SDS-PAGE OF GP15-GP16 COMPLEX PURIFICATION PROCESS IN 10%-40%(V/V) GLYCEROL GRADIENT.. ... 63
FIG. 27.ANALYTICAL ULTRACENTRIFUGATION (AUC) BY SEDIMENTATION VELOCITY (SV) OF THE CORE PROTEINS. ... 65
FIG. 28.NEGATIVE STAINING CHARACTERIZATION OF GP15-GP16 CORE COMPLEX.. ... 66
FIG. 29.CRYO-EM ANALYSIS OF GP15-GP16 CORE PROTEINS. ... 67
FIG. 30.THE WORKFLOW OF GP15-GP16 CORE COMPLEX STRUCTURE DETERMINATION PROJECT ... 70
FIG. 31.STRUCTURE OF THE T7 BACTERIOPHAGE GP15-GP16 CORE COMPLEX.. ... 71
FIG. 32.SECONDARY STRUCTURE CHART OF PURIFIED GP15 PROTEIN. ... 72
FIG. 33.SECONDARY STRUCTURE CHART OF GP16 PROTEIN. ... 73
FIG. 34.STRUCTURAL ANALYSIS OF GP15-GP16 COMPLEX. ... 75
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FIG. 35. DOCKING OF T7 BACTERIOPHAGE GP16 FOLDED DOMAIN WITH THE E. COLI SLTY LYTIC
TRANSGLYCOSYLASE ACTIVE DOMAIN.. ... 76
FIG. 36.BINDING OF GP15 AND GP16 TO LIPOSOMES. ... 77
FIG. 37.CRYO-EM ANALYSIS OF T7 BACTERIOPHAGE CORE COMPLEX INSIDE THE CAPSID. ... 79
FIG. 38.THE WORKFLOW OF T7 BACTERIOPHAGE CORE COMPLEX STRUCTURE DETERMINATION PROJECT TO SOLVE A TYPE OF MISMATCH SYMMETRY. ... 81
FIG. 39.REFINED 3D MODEL OF T7 BACTERIOPHAGE CORE COMPLEX PLUS THE TAIL... 82
FIG. 40.DISORDER PREDICTION SEQUENCE OF GP15 BY DISOPRED2. ... 89
FIG. 41.DISORDER PREDICTION SEQUENCE OF GP16 BY DISOPRED2. ... 93
FIG. 42.ATOMIC STRUCTURE OF GP15-GP16 CORE COMPLEX (LIGHT BLUE, PURPLE RESPECTIVELY) WITH A FRAGMENT OF NAG-NAM BACTERIA PEPTIDE. ... 95
FIG. 43.3D MODELS OF LYTIC TRANSGLYCOSYLASE PROTEINS.. ... 97
FIG. 44.DOCKING OF THE 3D RECONSTRUCTION OF THE CORE COMPLEX ASSEMBLED IN VITRO (GP15 IN LIGHT BLUE, GP16 IN PURPLE) INTO THE 3D MODEL OF BACTERIOPHAGE T7 DURING DNA EJECTION OBTAINED BY CRYO-ET BY HU ET AL.,2013. ... 98
FIG. 45.3D RECONSTRUCTION OF PODOVIRIDAE BACTERIOPHAGES. ... 99
FIG. 46.3D MODEL OF T7 BACTERIOPHAGE CORE AND TAIL COMPLEXES.. ... 101
FIG. 47.SCHEME OF THE PROPOSED ASSEMBLY MODEL OF GP15 AND GP16 CORE COMPLEX DURING INFECTION. ... 103
TABLE 1CRYO-EM DATA COLLECTION, REFINEMENT AND VALIDATION STATISTICS ... 83
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LIST OF ABBREVIATIONS
2D Two dimensional
3D Three dimensional
Å Ángstrom
aa Amino acid
ATP Adenosine Triphosphate AUC Analytical ultracentrifugation CCD Charge Coupled Device
CMOS Complementary Metal Oxide Semiconductor Cryo-EM Cryo-electron microscopy
C (S) Sedimentation Coefficient Distributions CTF Contrast Transfer Function
C-terminal Carboxyl terminus
DD Direct Detector
DDD Direct Detection Device DNA Deoxyribonucleic acid DLS Dynamic Light Scattering
dsDNA Double-stranded Deoxyribonucleic acid ssDNA Single-stranded Deoxyribonucleic acid E. coli Escherichia coli
EM Electron microscopy
FSC Fourier Shell Correlation
FT Flow-Through
gp Gene product
HBD Helical bundle domain
IM Inner membrane
IMAC Immobilized-Metal Affinity Chromatography
IMC Inner membrane component
IPTG Isopropyl 𝛽-D-1-thiogalactopyranoside
kb kilobase
kDa kilodalton
LB Luria-broth, Luria-Bertani medium
LPS Lipopolysaccharide
M Molar
MFC Membrane Fusion Component
mM milimolar
M&M Material and methods
ml mililiter
MW Molecular weight
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nm nanometer
NMR Nuclear Magnetic Resonance N-terminal Amino terminus
OD Optical Density
OM Outer membrane
PE Phosphoethanolamine
PG Peptidoglycan
Phosphatydilglycerol
PMSF Phenylmethane Sulfonyl Fluoride
R Hydrodynamic radius
RBP Receptor Binding Proteins
RNA Ribonucleic acid
RT Room temperature
S Sedimentation Coefficient
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis SEC Size exclusion chromatography
SEC-MALS Size exclusion chromatography coupled to multiangle light scattering
SV Sedimentation velocity
T Triangulation number
TEM Transmission electron microscopy T1SS-T6SS Types I to VI secretion systems TMP Tape measure protein
Tris Tris (hydroxymethyl) aminomethane
v/v Volume per volume
µm micrometer
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AMINO ACID ABBREVIATIONS
SYMBOL
AMINO ACID 3-LETTERS 1-LETTER
Alanine Ala A
Arginine Arg R
Apartic Acid Asp D
Asparagine Asn N
Cysteine Cys C
Glutamic Acid Glu E
Glycine Gly G
Glutamine Gln Q
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Tyrosine Tyr Y
Threonine Thr T
Tryptophan Trp W
Valine Val V
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ABSTRACT
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ABSTRACT
Transport of the viral DNA genome trough cell membranes is still one of the most intriguing processes in bacteriophage life cycle. In most of the bacterial viruses this process is carried out by the tail machinery, which shares structural similarities with bacterial secretion systems. However, some viruses present a tail, which is too short to puncture the bacterial cell wall. T7 bacteriophage, a member of the Podoviridae family, presents a short and non-contractile tail and it is an interesting system to study how these viruses puncture the double membranes of E. coli Gram-negative bacteria. In order to enlarge the tail complex, it has been postulated that during infection, an internal head complex, the core, is translocated trough the tail channel and assembles a tubular structure in the periplasm that allows the viral DNA translocation.
In this work, we have cloned and purified gp15 and gp16 core proteins. Both proteins were able to interact in vitro and we have solved the structure of two different core assemblies, gp15 alone and in complex with gp16, by cryo-electron microscopy (cryo-EM) at near atomic resolution (3.64 and 3.18 Å). These structures show for the first time that T7 core proteins are able to form tubular structures. A special feature of these structures is that they seem to present partially folded domains, whose density is not observed in the reconstructions. Moreover, the formation of the gp15-gp16 complex allows the complete folding of gp15, suggesting that gp16 acts as a chaperone assisting the assembly of the translocation complex. The solved domain of gp16 also shows a transglycosylase canonical motif, which could be involved in the degradation of peptidoglycan layer of E. coli.
The study of the T7 core complex inside the capsid was performed using cryo- EM and a special data processing of this mismatched symmetry complex, which allowed us to obtain a core-tail complex model at 4.84 Å resolution.
Altogether, our results suggest that the interaction of gp15 and gp16 core proteins show a novel type of assisted folding and allowed us to propose an assembly process.
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RESUMEN
El transporte del genoma del ADN viral a través de membranas celulares es uno de los procesos más interesantes del ciclo vital de los bacteriófagos. En la mayoría de los virus bacterianos este proceso es llevado a cabo por la maquinaria de la cola, la cual comparte similitudes estructurales con los sistemas de secreción bacterianos. Sin embargo, en algunos virus que presentan cola, ésta es demasiado corta para atravesar la pared bacteriana. El bacteriófago T7, miembro de la familia Podoviridae, presenta una cola corta y no contráctil y, es un sistema interesante para el estudio de estos virus que cruzan la doble membrana que presenta E. coli (Gram-negativa). Con el fin de alargar el complejo de la cola, ha sido postulado que, durante la infección, un complejo interno de la cabeza del virus, el core, es translocado a través del canal de la cola y ensamblado como una estructura tubular en el periplasma bacteriano permitiendo la translocación del ADN viral.
En este trabajo, hemos clonado y purificado las proteínas del core gp15 y gp16.
Ambas proteínas fueron capaces de interaccionar in vitro y hemos podido resolver la estructura de dos componentes del complejo del core, gp15 aislada y en el complejo con gp16, por criomicroscopía electrónica a resolución cuasi-atómica (a 3,64 y 3,18 Å). Estas estructuras muestran por primera vez como proteínas del core de T7 son capaces de formar estructuras tubulares. Una característica especial de estas estructuras es que presentan dominios parcialmente plegados, cuya densidad no es observada en las reconstrucciones. Además, la formación del complejo gp15-gp16 permite el plegamiento completo de gp15, sugiriendo que la proteína gp16 actúa como chaperona, asistiendo al ensamblaje del complejo de translocación. El dominio resuelto de gp16 muestra además un motivo transglicosilasa canónico, el cual podría estar involucrado en la degradación del peptidoglicano de E. coli.
El estudio del complejo del core de T7 dentro de la cápsida fue llevado a cabo usando criomicroscopía electrónica y el procesamiento de datos para solucionar el problema en las diferencias de simetría de la estructura, el cual nos permitió obtener un modelo del complejo core-cola a 4,84 Å de resolución.
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En resumen, nuestros resultados sugieren que la interacción de gp15 y gp16 de las proteínas del core muestra un tipo de plegamiento asistido, lo cual nos permite proponer un modelo de ensamblaje de estas proteínas como estructuras tubulares en el periplasma durante el proceso de infección.
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1. INTRODUCTION
Cellular membranes can be considered as semipermeable barriers to ions and macromolecules. The mechanism of transport of proteins or nucleic acids across the membranes is a specialized process that has been developed along the evolution (Agarraberes & Dice, 2010). Bacteria have developed diverse mechanisms for the transport and exchange of substrates with other bacteria in order to react to modify their environment, as well as in several crucial processes such as pathogenicity, adaptation and survival (Costa et al., 2015). In this complex process a variety of macromolecular nanomachines are involved (Gerlach & Hensel, 2007; Holland, 2010).
The challenge of these macromolecular complexes is to be able to cross a bacterial wall of variable thickness composed by an outer membrane (OM), inner membrane (IM) and the peptidoglycan (PG) in the case of Gram-negative bacteria, or only a thick PG and OM in Gram-positive bacteria. The design of these macromolecular structures allows them to puncture the membranes and protect their translocation substrates from bacterial nucleases, proteases, and from the influence of periplasmic and cytoplasmic pH, altogether maintaining the cellular integrity and the electrochemical gradient of protons across the bacterial membrane. The best-characterized translocation machineries are the secretion systems from Gram-negative bacteria. These machineries exhibit similarities in structure, shape and function. While some of them expand from the IM to the OM and they are capable of assist in pore formation and transport of substrate through their central channel, others have been localized only in OM of Gram-negative bacteria (Costa et al., 2015). These complex molecular machines present structural similarities with the bacteriophage tails (Kanamaru, 2009; Leiman et al., 2009; Pell et al., 2009), that emerge to provide an optimized solution to cross through the bacterial cell wall for the transport of viral genome into bacteria. Tail complexes specifically recognize bacterial host cells, penetrate the cell envelope and deliver the nucleic acid into the cytoplasm (Bertin et al., 2011; Rossmann & Rao, 2012). The tailed phages, generally, show a common strategy to infect the host and they have strong structural homologies, although the protein machinery responsible for host adsorption has specific characteristics depending on each phage family (Nobrega et al., 2018). Some phages, as
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Podovirus, present a short non-contractile tail, which is not long enough to puncture the double membrane (Casjens, 2011), and others do not present tail machinery at all. These viruses have developed an alternative mechanism to cross the bacterial envelope using internal capsid proteins or membranes that are able to build tubular structures during infection, thus guiding the DNA translocation process through the bacteria wall (Guo et al., 2014; Peralta et al., 2013).
The subject of study of this thesis is T7 bacteriophage which belongs to the Podoviridae family. This virus, that infects E. coli Gram-negative bacteria, shows a tail that is not able to penetrate by itself the bacterial membrane. T7 presents a group of internal proteins named “the core” which are involved in DNA stabilization inside the capsid and ejection during infection (Agirrezabala et al., 2005; Hu et al., 2013). Although it has been hypothesized before that the core proteins were able to form a tubular structure expanding from the inner to outer membrane of the bacteria, this is the first time that is been possible to structurally show that these proteins are able to build tubular structures. In this introduction we will first give an overview of the different structure and function of bacterial and viral translocation systems to finish with a description of the T7 ejection mechanism and the machinery involved in this process, the core proteins.
1.1. Overview on bacterial macromolecular transport complexes and puncturing devices
Bacteria have a specific nanomachines whose function consists in translocating proteins, DNA and small molecules directly through large membrane-associated multiprotein complexes from the cytoplasm to the extracellular host. As it has been mentioned in the previous section, bacterial secretion systems from Gram-negative bacteria are the best characterized structures (Figure 1). In order to expand from the inner to the outer membrane the simplest version of these complexes, the type 1 and 2 secretion systems (T1SS and T2SS, Figure 1 a and b), are formed by: an inner and outer membrane components, which participates in substrate recognition and membrane anchoring; a channel component that protects the translocation substrate and
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cytoplasmic ATPase, as in T2SS (Figure 1 b), that provides the energy for the transport process (Nivaskumar & Francetic, 2014). The structures of type 3, 4 and 6 secretion systems (T3SS, T4SS and T6SS, Figure 1 c, d and e) are more complex as they also count with structures that connect with the host bacteria. While the T3SS and T4SS consists respectively of an extracellular needle and pilus, that mediates contact with the target cell (Cornelis, 2006; Cornelis, 2010; Alvarez-Martinez & Christie, 2009); the T6SS (Figure 1 e) is a specialized weapon with the ability of contract, puncture and inject substrates into multiple bacterial species and eukaryotic cells (Russell et al., 2014). T6SS is one of the most studied secretion systems and shows structural/function similarities with the molecular components of phage tails that will be described later in this work, such as E.
coli phage T4 (Davidson & Maxwell, 2018) or l phage (Pell et al., 2009).
Fig. 1. The secretion systems (types I-VI) in Gram-negative bacteria. The different protein components are shown schematically in colors. Adapted from Costa et al. 2015.
Other bacterial structures related to bacteriophage tail assemblies are bacteriocins. Bacteriocins, also termed pyocins, are compounds that are assembled and ejected to the extracellular medium by bacteria aiming to kill the competitor bacteria from the same or a related species. They are classified in F-type and R-type pyocins, being non-contractile or contractile devices respectively (Figure 2 a and b) (Nakayama et al., 2000). In order to accomplish their function, they bind to another bacterium, form a pore on the membrane and inject the effector proteins by a spiked tube, resulting in a polarization of the membrane and bacteria death (Lee et al., 2016).
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Fig. 2. The bacterial phage tail-like complexes. These structures are homologous and evolutionarily related to phage components. They share structures like: baseplate, tail tube and tail fibers. Figure adapted from Nobrega et al., 2018.
Bacteria have evolved a wide range of mechanisms to adapt themselves into the environment, just as the nanomachineries needed for these purposes. In the same way, in nature there are conserved structures for similar functions such as bacteriophages, which have developed sophisticated machineries to overcome the bacterial membranes or barriers and deliver their genomes into the bacterial interior (Basler et al., 2012;
Davidson & Maxwell 2018; Kanamaru, 2009; Leiman et al., 2009).
1.2 Bacteriophages
Bacteriophages or bacterial viruses, also known as phages, are virus that infect bacteria and are the most abundant entities on Earth. They were independently discovered in the early 1900s by two different scientists: Frederick Twort, a British pathologist, and Fèlix d’Herelle, a French-Canadian microbiologist (Salmond & Fineran, 2015). Their influence is very wide in ecosystems, by promoting exchange of genetic material between bacteria, pathogenicity by stimulating development of bacterial defence mechanisms against phage, and evolution, by selecting for genetic variation of bacteria (Gencay et al., 2019). Due to these bacteriophage-bacteria interactions, they have been proposed (and exploited) as useful tools in molecular biology almost since their discovery. Furthermore, the increase of antibiotic resistance in pathogens has recently provided a renewed interest in phage therapy, which is used as an agent in the treatment of human infections, as well as in agriculture, veterinary science, industry and
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food safety (Ackermann, 2009; Doss et al., 2017; Gencay et al., 2019; Salmond & Fineran, 2015).
1.2.1 Bacteriophage structure
Phage classification is currently done using both genetical and morphological information, as well as structure: at least 5500 phages have been examined by electron microscopy since the use of negative staining in 1959 (Ackermann, 2009). The genome of phage consists of double-stranded (ds) or single-stranded (ss) of DNA or RNA, and their sizes vary from very small and simple to highly compacted and complex (for example, MS2 phage genome which present 3.2 kb ssRNA or 500 kb dsDNA in Bacillus phage G). The morphological types of virions include those with a tail (96% of virions), polyhedral, filamentous or pleomorphic (which represent only the ~3-4% virions). There are also some phages, which have lipid or lipoprotein envelopes (Ackermann, 2009; Doss et al., 2017; Gencay et al., 2019; Salmond & Fineran, 2015).
The genome of the phages is located in a container (named capsid), which is built by multiple copies of one or a few proteins. The shape of this container can be filamentous or icosahedral, and this latter assembly is present in the majority of phages as it maximises the volume content using a minimum surface. Based on crystallographic arguments, 60 copies of a single protein are the minimum protein content to build an icosahedral capsid, where all the subunits have the same type of interactions assembled into 12 pentons. In order to increase the size of the container the virus modifies the interactions between the proteins and introduces different number of hexons. This process follows specific geometric rules described by the triangulation number (T), a parameter that determines the limited number of ways that 60 x T subunits might accommodate to an icosahedral lattice (Figure 3) (Caspar & Klug, 1962).
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Fig. 3. The triangulation number of icosahedral capsids. Spherical capsids assemble from 60 asymmetric units, arranged with icosahedral symmetry. To enable the creation of larger capsid shells, each asymmetric unit may comprise more than one capsid protein, arranged with quasi- equivalent packing. The permitted numbers of subunits in each asymmetric unit are given by the T-number series. Here icosahedra are represented with T-numbers of 1 (a), 3 (b), 4 (c) and 7 (d).
Every single capsid protein is colored according to its quasi-equivalent position. Figure reproduced from Baker & Bhella 2013.
Double-stranded DNA (dsDNA) tailed phages, called Caudovirales, belong to the most representative class of phages (Bertin et al., 2011; Doss et al., 2017; Salmond &
Fineran, 2015). They can infect a wide variety of hosts and are common in many different environments (Nobrega et al., 2018). These viruses are built by an icosahedral capsid with a tail protein complex, which is assembled in one special vertex of the capsid (Cuervo & Carrascosa, 2012). In these viruses one of the capsid pentamers is replaced by a protein named the portal or connector, which builds an entry channel to the viral capsid and serves as a docking point for the tail complex. The size of the capsid varies between 50 - 100 nm and the length of the tail depends on the phage family, from 300 Å in P22 to about 4550 Å in phage G (Aksyuk & Rossmann, 2011). According to the diverse morphology of the tail bacteriophages can be divided into three families (Figure 4): Myoviridae (25%), Siphoviridae (61%) and Podoviridae (14%). Myoviridae family (Figure 4 a) presents a long and contractile tail. Some examples are groups T4, Mu, SPO1, FH, φ92, φKZ (Ackermann, 2003; Fokine & Rossmann, 2014). Although, the best
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Myophage characterized is T4 (Hu et al., 2013; Taylor, N. M. et al., 2016). This contractile tail consists of an internal tube, an external sheath tube, a baseplate and a terminator complex. The structure responsible for recognition and attachment to the host cell is the receptor binding proteins (RBPs) in the baseplate. Siphoviridae, such as phage T5, l, p2, T1, L5, c2, ψM, HK97and SPP1 possess long, flexible, non-contractile tails without a sheath (Figure 4 b) (Ackermann, 2003; Fokine & Rossmann, 2014). Some Siphoviridae that infect Gram-positive bacteria present a baseplate-like structure like Myoviridae phage’s baseplate, nevertheless, Siphophages that infect Gram-negative bacteria have no baseplate (Nobrega et al., 2018). Inside the tail tube they present the tape measure protein (TMP) that determines the tube length. Podoviridae family, comprises viruses such as φ29 (Xiang et al., 2006), T7 (Cuervo et al., 2013), P22 (Lander et al., 2006), e15 (Jiang et al., 2006), P-SPP7 (Liu et al., 2010), N4 (Choi et al., 2008), K1E and K1-5 (Leiman et al., 2007), have a short non-contractile tail that consists of an upper tail adaptor protein that connects the tail with the connector protein (Figure 4 c). Also, the tail is surrounded by 6-12 trimeric tail fibers or tail spikes (Cuervo et al., 2013; González-García et al., 2015b).
Fig. 4. Caudovirales: double-stranded DNA (dsDNA) tailed phages. All of them present a capsid that enclose and protect the genome and assemble a tail. a. Myoviridae family, the only tailed phages with a contractile tail sheath. b. Siphoviridae family, with long and non-contractile tail.
Both Myoviridae and Siphoviridae families have a baseplate at the distal end of the tail to which receptor-binding proteins (RBPs), such as tail fibers and tail spikes, are attached. c. Podoviridae family presents a short and non-contractile tail without baseplate. Figure adapted from Nobrega et al. 2018.
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1.2.2 Bacteriophage life cycle and assembly
Bacteriophage life cycle can be lytic or lysogenic (Figure 5). The mechanism of viral infection will be detailed in further sections of this manuscript; briefly, the phage attachment to the host bacteria involves a strong interaction between the phage receptor binding protein (RBP) and one of the surface components (receptor). Then, the phage injects its genetic material into the bacteria and starts the viral replication process. The host provides the molecular machinery needed to replicate the phage genetic material and produce the progeny phage. The following strategy of replication depends on whether the phage is lytic (virulent virus) or lysogenic (temperate virus) (Figure 5). In the case of lytic life cycles of phages, they are able to replicate their genome and take about 1 hour per generation and produce 50-500 new viral progeny.
Lysogenic phages can replicate their genome in concert with the bacteria DNA, either in a free, plasmid-like state, or integrated into the bacterial chromosome, where it can reside quiescently for many host generations. These are called prophages and under stress conditions can leave of this state and produce more virions that are released from bacteria and involve the host death (Casjens, 2011; Doss et al., 2017; Roach & Donovan, 2015; Young, 2013).
Fig. 5. Phage life cycles: They can have lytic or lysogenic life cycles. To infect a host bacterium, a phage will first interact with receptors on the host cell, adsorb and then inject its genome. The
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subsequent replication strategy will depend on whether the phage is virulent (lytic) or temperate (lysogenic). Figure adapted from Salmond & Fineran 2015.
The assembly pathway comprises different sequentially orchestrated steps in conducing to the formation of the mature virus (Figure 6). In the case of dsDNA phages, it starts with the formation of the prohead (Aksyuk & Rossmann, 2011). In order to package the DNA, the connector protein in the prohead interacts with the large subunit of the terminase, an ATPase that provides the chemical energy for the DNA translocation. The viral DNA is specifically recognized by another protein (small subunit of the terminase) or a viral RNA (pRNA) (Aksyuk & Rossmann, 2011; Cuervo &
Carrascosa, 2012). The large terminase subunit has two main functions: providing the energy for the DNA translocation, and an integrated nuclease activity that cuts individual chromosomes usually from the DNA concatemer in order to assure the encapsidation of the whole genome. The DNA packaging is a process that needs the hydrolysis of ATP (one molecule of ATP for each 2.5 bp) (Casjens, 2011; Dauden et al., 2013). While the DNA is encapsidated the prohead changes its conformation in a process named maturation (Aksyuk & Rossmann, 2011). Once that the DNA is fully packaged the connector channel is closed by the tail proteins in order to form the fully mature particle.
Finally, the liberation of mature phages involves endolysin and holins phage proteins that are responsible for the degradation of bacterial peptidoglycan producing the lysis from within the bacteria.
Fig. 6. Scheme of the assembly process of tailed phages. There are several distinct steps of dsDNA tailed phage assembly: 1) Assembly of a prohead, or a spherical shell of capsid protein filled with scaffolding protein that contains a dodecameric connector. 2) Packaging of DNA using the energy of ATP. 3) Maturation of proheads into angular mature heads. 4) Attachment of the neck and tail proteins or a preassembled tail. Reproduced from Aksyuk & Rossmann 2011.
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1.2.3 Bacteriophage infection
The majority of phages (Caudovirales) use a specialized structure, the tail, for host recognition, cell wall penetration and genome injection into the host (Arnaud et al., 2017; Fokine & Rossmann, 2014; González-García et al., 2015a; Hu et al., 2013;
Letellier et al., 2004; Nobrega et al., 2018). Although the attachment to the cell surface could be achieved through many mechanisms depending on the tail structure and bacterial host, the injection of the genome through the Gram-positive or Gram-negative bacterium wall present several common steps: receptor recognition, degradation of wall sugars, puncturing of the bacteria membrane and delivery of the genetic material. The tail appears to provide the best solution to the transfer of macromolecules into bacteria (Davidson et al., 2012).
i. Host receptor recognition
The host receptor recognition by tailed phages is carried out by the interaction of receptor binding proteins (RBPs), named baseplates, fibers or spikes depending on the phage. These structures, at the tail distal end, interact with the receptors on the bacterial membrane surface. It is thought that during this first interaction a reversible attachment of the tail allows the correct tail orientation relative to the bacterial membrane and, afterwards, an irreversible interaction with the same or a different component triggers the tail channel opening conducing to the genome ejection (González-García, et al., 2015a; Taylor et al., 2016). Gram-negative and Gram-positive bacteria present differences in thickness, uniformity and lipid content of the cell wall in the receptors for adsorption (Bertin et al., 2011; Nobrega et al., 2018). Viruses can use lipids, sugars or proteins as receptors (Rossmann & Rao, 2012). In Gram-positive bacteria, the main component of the cell wall is peptidoglycan and teichoic acid.
Unfortunately, only a few receptors have been found in Gram-positive bacterial viruses in comparison with Gram-negative receptors viruses, due to complexity and density of the cell wall (Nobrega et al., 2018). In the case of Gram-negative bacteria viruses, the principal receptor is lipopolysaccharide (LPS), which presents smooth and rough varieties. Even though both possess core polysaccharides and lipid A, they own the
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presence (smooth) or absence (rough) of O-antigens. Phages can recognize the O- antigen with their tail fibers or tail spike proteins, and they can hydrolyze the O-antigen to puncture the cell wall with the tail (Nobrega et al., 2018).
ii. Degradation of peptidoglycan
The largest single molecule of the bacterial cell, the peptidoglycan, has to be degraded during infection. In bacteria, this molecule has also to be degraded in order to be renovated during the cell growth to maintain the integrity of the cell. Both, Gram- negative and positive bacteria, possess special peptidoglycan hydrolases for the insertion and recycling of new glycan in the bacterial wall. There are several classes of peptidoglycan hydrolases: lysozymes, lytic transglycosylases, N-acetylmuramyl-L- alanine amidases and endopeptidases. Lysozymes and lytic transglycosylases are similar enzymes; both break the glycosidic bond between NAM and NAG of peptidoglycan (Moak & Molineux, 2004).
A lysozyme-like enzymatic activity has also been found in phages, associated to the degradation of the peptidoglycan layer in the viral infection process. Indeed, external and internal polysaccharides of the bacteria are recognized and disrupted by RBPs (named baseplates, fibers or spikes depending on the phage), which possess enzymatic activities specific to these sugars. This enzymatic domain is found as a part of a large protein or associated with other structural components of the virion that limit its enzymatic activity (Molineux, 2001; Moak & Molineux, 2004). Their location within the virions is variable, but, they are usually located within the tail complex (Xiang et al., 2008). The best documented example of peptidoglycan hydrolases is found in T4 Myoviridae phage, which presents a complicated complex tail and a very efficient mechanism of infection. The protein uncharged to disrupt the peptidoglycan layer is the baseplate protein gp5 (Arisaka et al., 2003). In the lipid containing phages such as PRD1 have been shown to contain two lysine activities in P15 and P7 proteins, although neither of these proteins is essential for infectivity, P7 plays an accessory role in genome penetration of the infected cell (Moak & Molineux, 2004). In podophages there have been also described some examples as P22 gp9 tailspike trimer (Wang et al., 2019) or Phi29 gp13 found in the tail know (Xiang et al., 2008; Xu et al., 2016). The case of T7
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podophage is special as this enzymatic activity has not been located in the tail complex.
Indeed, as it will be described in later sections, it has been shown that the internal core protein gp16, known to be ejected from the virion before the phage genome, has a lytic transglycosylase motif (Moak & Molineux, 2000; Molineux, 2001).
iii. Examples of genome delivery
Myoviridae T4 presents one of the best-characterized infection processes. Once that the baseplate recognizes the bacterial receptor, the tube can interact with the bacterial membrane and change its conformation until the sheath contraction. The contraction moves the entire phage particle closer to the cell surface and drives the rigid internal tail tube through the bacterial cell envelope (Leiman & Shneider, 2012; Taylor et al., 2016). The final step of the infection is the translocation of DNA and proteins from the phage capsid into the host-cell cytoplasm. The tail is thought of as a passive conduit in this process because it does not appear to contain a source of energy necessary to make it an active participant of this translocation event (Leiman & Shneider, 2012).
Another example of membrane puncturing device is described in the small phi29 virus which must penetrate the thick external peptidoglycan layer and membrane of Gram-positive Bacillus subtilis and then. The insertion inside the membrane of a hydrophobic peptide belonging to the p9 know protein, allows the ejection of the genome through its stubby and non-contractile tail (Xu et al., 2016).
It has been postulated that during infection, some Podophages infecting Gram- negative bacteria, enlarge their short-tail during the infection process, and form a new tubular protein structure which allows the DNA ejection into the hosts (Cuervo et al., 2013; González-García et al., 2015a; González-García et al., 2015b; Hu et al., 2013;
Nobrega et al., 2018). In the case of the bacteriophage P22 it has been shown that two of the six tail spikes that hydrolyze the O antigen allow the virion become perpendicularly oriented and the needle penetrates the outer membrane (Steinbacher et al., 1997). Afterwards the internal head protein gp7 is ejected, assembled and extended from the tail into the cell surface. Then, the second internal head protein gp20 is also ejected and assembled into a tubular structure into the periplasm. A third internal head protein gp16 is needed to complete the channel into the cytoplasm (Wang et al.,
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2019). The hallmark example of Podovirus is T7, as it will be described later on in this manuscript, the mechanism of ejection of this virus postulated also to involve the ejection of internal core proteins that would form a tubular complex in the periplasmic space. However, the adsorption machinery of T7 is different from the one described in P22.
iv. Models for DNA transport: chemiosmotic and difference of pressure
The dsDNA (usually such as B-form) in mature phages has to be highly packaged in a tiny capsid. Depending on each species, a phage can have between 35 and 50 kb DNA packaged into a ~ 60 nm diameter of capsid (for instance, in T7 bacteriophage the capsid is 55 nm in diameter) (Panja & Molineux, 2010). This dsDNA is in a condensed state and, by single-molecule experiments its concentration has been estimated in up to a maximum at ~ 500 mg per ml (Chemla et al., 2005; Smith et al., 2001).DNA ejection of tailed phages into the host cell is a translocation mechanism in the opposite direction to that required for active DNA packaging, and this has led to the proposal of different theories on how these two different processes carry out DNA transport. Inamdar et al. in his work published in 2006, explain in a theoretical way the physical phenomena involved in DNA translocation in bacteriophages. They can be summarised in: a) the diffusion of the DNA along its length chain, b) driving forces due to stress on the DNA inside the viral capsid, c) resisting forces associated with osmotic pressure in the host, d) cell confinement effects that constrain the injected chain and, e) ratcheting and pulling forces associated with DNA-binding proteins in the host cell cytoplasm.
There are basically two different hypotheses about how DNA crossing trough bacterial membranes (Letellier et al., 2004). The first proposal explains how the packaging machinery could introduce DNA under sufficient pressure into the capsid during morphogenesis to allow spontaneous ejection of the genome during the infection process (Riemer & Bloomfield, 1978). However, the second one proposes that it is the electrochemical gradient of protons across the bacterial membrane, which drives the force for DNA transportation (Earnshaw & Casjens, 1980).
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However, the review by Molineux and Panja in 2013, proposed two new models for in vivo viral DNA ejection: the continuum mechanic model and the hydrodynamic model. The continuum mechanic model considers that the viral DNA inside the capsid is in a toroid form, and the ejection is facilitated by the internal ejection pressure and it can continue as long as this pressure is higher than the osmotic pressure of the outside environment (Inamdar et al., 2006). In the hydrodynamic model, during DNA ejection water diffuses across the capsid in order to neutralize the osmotic pressure (Lemay et al., 2013; Panja & Molineux, 2010) and when the capsid is full, the DNA will be pushed by hydrostatic pressure gradient forward the tail until the pressure will be equilibrated.
1.3 The T7 bacteriophage
T7 bacteriophage is a well-characterized member of Podoviridae family and the model of study of this work. It presents a lytic cycle and infects E. coli bacteria. The viral particle is composed by a 55 nm icosahedral capsid and a 23 nm short non-contractile tail (Figure 7 a, b, c).
1.3.1. T7 bacteriophage structure
The mature capsid of bacteriophage T7 possesses 415 copies of the major shell protein gp10 (with a triangulation number T=7). The tail machine is a macromolecular complex formed by four proteins: the connector (gp8), the adaptor (gp11), the nozzle (gp12) and the fibers (gp17) (Figure 7 d, f with more detail) (Cuervo et al., 2013;
González-García et al., 2015a, b). This complex shows a tubular shape 293 Å long and 175 Å wide, organized into two 12-fold rings (gp8 and gp11) and a 6-fold nozzle (gp12) (Cuervo et al., 2019). During assembly, the tail is essential to close the connector channel after packaging and during infection the tail fibers specifically recognize the bacterial receptors transmitting the opening signal for DNA delivery (Cuervo et al., 2013). The capsid and the tail are connected by the connector that sits at one of the vertexes of the capsid. This protein presents a central channel that constitutes the entry door to the viral capsid (Cuervo et al., 2019).
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The most conspicuous structure of T7 viral particle is the internal core, a cylindrical structure of ~290 Å height and ~170 Å width located on top of the connector.
This complex is composed by three proteins: gp14 (~ 20.8 kDa), gp15 (~84.2 kDa) and gp16 (~ 144 kDa) (Figure 7 d, e more detailed) (Agirrezabala et al., 2007; Bhardwaj et al., 2014; Leptihn et al., 2016). Agirrezabala et al. estimated the number of copies of these internal core proteins in the immature virion as: 12 copies of gp14, 8 copies of gp15 and 4 copies of gp16. Up to date there is no atomic structure of the core components, this is due to the complexity of the image processing workflow induced by the existence of different mismatched symmetries inside the virus particle.
Fig. 7. The T7 bacteriophage structure. a, b. A representative micrograph and 2D average image of T7 bacteriophage (scale bar 230 Å). c. A 3D model of T7 phage. T7 presents an icosahedral capsid with 55 nm long and a short non-contractile tail with 23 nm long. d. A cross section of the 3D model of T7 bacteriophage. It shows the concentric rings of DNA and the core complex inside the capsid and, the tail assembled in the connector or portal vertex. e. In more detail, the T7 core complex inside the capsid: gp16 protein in red, gp15 in yellow, gp14 in blue and the connector gp8 in green. Figure adapted from Agirrezabala et al. 2005. f. Tail proteins of T7: the connector gp8 in purple, the adaptor gp11 in green and the nozzle gp12 in orange. It can be appreciated on top of connector gp8, the conspicuous core. Figure represented from Cuervo et al. 2019.
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1.3.2 T7 assembly pathway
The formation of T7 procapsid involves the interaction of the capsid proteins gp10, the scaffold protein gp9, the connector protein gp8 and the core complex gp14- gp15-gp16. The internal core proteins are not required for the morphogenesis of the prohead, but they are essential to stabilize the viral genome. In order to package the DNA, the prohead interacts with the large subunit of the terminase (gp19), while DNA is bound into the small subunit of the terminase (gp18) (Agirrezabala et al., 2005; Dauden et al., 2013).
Concomitant with the packaging process occurs the procapsid expansion into the mature capsid (maturation) that involves drastic changes in the interaction surface between pentons and hexons of the gp10 capsid protein (Ionel et al., 2010; Guo et al., 2014). Relevant structural changes take place also during this maturation process within the core complex-connector, probably triggered by the internal pressure of the DNA that is fully occupying capsid volume (Agirrezabala et al., 2005). As a result of these movements, the crown connector domain moves closely to the core complex (Agirrezabala et al., 2005) and the core-connector complex is pushed towards the outside of the capsid by the packaged DNA, probably forcing the terminase detachment when the DNA is totally translocated into the capsid (Agirrezabala et al., 2005; Cuervo et al., 2019). During this process the connector gp8 closes the channel valve retaining the DNA before the assembly of the tail proteins (Figure 8) (Agirrezabala et al., 2007;
Dauden et al., 2013; Cuervo et al., 2019).
The movement of the connector outside the capsid also allows its interaction with the tail proteins, the tail adaptor (gp11) and tail nozzle (gp12), triggering the open conformation of the connector valve and allowing the movement of the DNA along the tail channel that will be retained by four closing gates of the nozzle protein in the mature virus (Figure 8) (Cuervo et al., 2013; Cuervo et al., 2019). Finally, the gp17 N-terminal domain of the fibers is attached to the interface between the adaptor and the nozzle to complete the T7 assembly pathway (Cuervo et al., 2013; González-García et al., 2015a).
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Fig. 8. Scheme of the T7 bacteriophage assembly pathway. The capsid is shown in pink, the connector (gp8) in purple, the core complex (gp14-gp15-gp16) in light blue, the terminase in gray, the adaptor (gp11) in green, the nozzle (gp12) in orange, the fibers in dark blue, and the DNA in black. When the connector is in the prohead, the terminase–connector interaction stabilizes the open conformation, allowing DNA packaging. When the terminase leaves the complex, the connector channel is closed, in order to prevent the DNA release from the capsid.
The interaction of the connector with the adaptor protein re-establishes the open conformation of the connector channel valve, permitting the DNA to slide along the tail channel up to the nozzle, ready for ejection. In the mature virus, the gates of the nozzle protein close, retaining the DNA in the tail channel. These gates are closed until the reorganization of the nozzle, which is triggered by the interaction of the tip and the fibers with the host membrane. Then, the gates are opened and the viral genome is ejected. Figure reproduced from Cuervo et al. 2019.
1.3.3 The T7 infection process and the core complex
Bacteriophage T7 starts Escherichia coli infection by the interaction of its tail fibers with the LPS on the cell surface (Figure 9, step 1) (González-García et al., 2015b;
Kemp et al., 2004; Kemp et al., 2005). Firstly, the phage is reversibly bound to a primary receptor that allows the correct tail orientation related to the bacterial surface (Figure 9, step 2). Secondly, an irreversible binding event takes place. Gonzalez-García et al.
showed that this irreversible interaction involved the rough LPS, at least in an in vitro ejection experiments (Figure 9, step 3). Binding to the host cell is termed adsorption, and it occurs through electrostatic interactions (Kemp et al., 2005). After that, the tail suffers a conformational change (Figure 9, step 4): the gates of the nozzle are opened allowing the completely opening of tail channel. Then, the internal core proteins gp14- gp15-gp16 are disassembled to be transferred through the tail channel in order to enlarge the short non-contractile tail (Figure 9, step 5).
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It has been described that Gram-negative bacteria (like Escherichia coli) have an inner and an outer membrane (IM and OM, respectively) 150 Å apart, separated by an intermediate peptidoglycan layer in the periplasmic space (Bhardwaj et al., 2014). T7 is an interesting system to study how podophages puncture the double membranes of Gram-negative bacteria, as the tail is shorter than the whole intermembrane space (Casjens & Molineux, 2012; Cuervo et al., 2013; González-García et al., 2015a; González- García et al., 2015b; Hu et al., 2013). The first hypothesis was proposed by Hu et al. in a study by cryo-electron tomography, where they described the presence of a tubular and a temporal structure during infection of T7. They hypothesized that the core complex formed by gp14, gp15 and gp16, after adsorption, could be disaggregated from their folded structure inside the mature virus, and then move through the opening channel completely unfolded in order to leave the capsid and to enlarge the short tail (Figure 9, step 5). This extended short tail could also protect the genome from the periplasmic endonuclease Endo I (Panja & Molineux, 2010).
Fig. 9. Model of T7 ejection process divided into five main steps: Step 1. The mature T7 virus is completely assembled and presents all the components: the capsid (dark blue), DNA (inside the capsid in dark blue), the core complex (orange), the connector (light green), the tail (dark green), the fibers (red), and the bacterial membrane composed by the lipopolysaccharide (LPS), the outer membrane (OM), the peptidoglycan layer (PG) and the inner membrane (IM). Step 2. The phage is bound reversibly to a primary receptor that allows the correct tail orientation related with the bacterial surface. Step 3. The irreversible interaction takes place through the LPS. Step 4.
Conformational changes in the tail and opening channel take place. Step 5. The folded structure
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of the core complex is disassembled, translocated through the opened channel and folded again in the periplasmic space to enlarge the short non-contractile tail of T7.
More specifically, according to the current hypothesis, gp14 protein should be translocated (partially) unfolded through the channel of the connector protein and tail complex. After its ejection, gp14 is refolded and forming a pore into the OM of E. coli, allowing the passage of gp15 and gp16 across the outer membrane (Leptihn et al., 2016).
The gp15-gp16 proteins should also be translocated through the channel in an unfolded conformation, and then they would oligomerize as a tubular structure and span into periplasm, IM and cytoplasm. It is still unknown which one of them would be the first to exit from the virion to the periplasmic space, however the transglycosylase enzymatic activity of the gp16 N-terminal domain suggests that it might interact with the peptidoglycan layer to degrade it and that this protein could exit first (Black &
Thomas, 2012; Moak & Molineux, 2000; Moak & Molineux, 2004). This lytic transglycosylase motif of gp16 is essential during infection to overcome the highly cross- linked peptidoglycan (Moak & Molineux, 2000; Moak & Molineux, 2004). The sequence analysis of gp16 has shown that the N-terminal of this protein has homology with the C- terminal sequence of the 70 kDa E. coli soluble lytic transglycosylase SltY (Moak &
Molineux, 2000). Indeed, in this work, Moak & Molineux showed that the glutamate 37 (E37) of T7 gp16 is similar to glutamate 478 of SltY of E. coli, and that the expected catalytic residue for transglycosylase activity of gp16 is not absolutely essential but it is important for the infection at low temperature because their latent periods are extended.
Furthermore, recently it has been shown that gp16 is able to bind to the lipids of IM and form a pore, while gp15 interacts with the DNA (Lupo et al., 2015; Leptihn et al., 2016). Besides, these two proteins, gp15 and gp16 have the property of fully recovering their structure after thermal unfolding (Leptihn et al., 2016).
When the DNA channel formed by gp14, gp15 and gp16 is complete, the translocation of the genome into the cytoplasm of the bacteria takes place. In addition to this function as an extensible tail, recent studies have demonstrated that gp15 and gp16 are actively involved in the transportation of approximately ~1kb of the 40kb T7 genome into the bacteria (Black & Thomas, 2012; Chang et al., 2010). After this first
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internalization of the DNA, further translocation of the DNA is coupled to transcription, first by E. coli and then by T7 RNA polymerases (Chang et al., 2010; Garcia & Molineux, 1995).
1.4 3D reconstruction of macromolecules using single particle cryo- electron microscopy (cryo-EM)
The development of structural analysis techniques has made possible the understanding of the biomolecular function and the mechanistic properties of proteins or protein complexes (Nogales & Scheres, 2015). Currently, several methods are used to determine the structure of a macromolecule such as x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (cryo-EM). Along decades, x-ray crystallography has been the main method to solve protein structures.
Indeed, up to the present day, most protein structures at atomic resolution have been solved by x-ray crystallography. Nevertheless, solving certain structures such as membrane proteins or large, flexible complexes have been difficult to tackle due to the difficulty to obtain suitable crystals. On the other hand, NMR measures distance- dependent interactions between atoms, and it has been mainly used to determine the structure of small proteins (40-50 kDa). Nevertheless, solving large protein structures is limited by its size constraints (Method of the year 2015.2016; Avramov et al., 2019). The limitations of these traditional techniques for structural studies have been a barrier, especially in their application to large complexes, membrane proteins, polymers and multiple conformational states in macromolecular assemblies (Nogales & Scheres, 2015;
Nogales, 2016).
Proteins can scatter electrons about ten thousand times more than x-rays and, and this property made possible to use electron microscopes using electromagnetic lenses. These electrons have to be under high vacuum and they are accelerated down the microscope column at 80-300 kV voltages. Modern electron microscopes have the potential to make images with atomic-level detail (Fernandez-Leiro & Scheres, 2016).
Electrons are one type of ionizing radiation, which is the general term given to radiation that is capable of removing the tightly bound, inner-shell electrons from the attractive field of the nucleus by transferring some of its energy to individual atoms in the