1_BACTERIOFAGOS.ppt

Preserial high-resolution electron microscope (1938) from Siemens and Halske. In 1939 first EM micrograph of a virus: TMV by Ruska et al.

“The properties of bacteriophages have frequently paved the way for major discoveries in biology and medicine.”

Microbe 1(4): 164-165, 2006

Researchers are approaching phage with renewed interest, seeking to use them in agriculture and also to treat human infections.

(ASM News, 71(10): 453-455, 2005).

MOLECULAR BIOLOGY, DAVID FREIFELDER

Structural components of the T4 particle. Features of the particle have been

resolved to about 3 nm. The positions of several head, tail, baseplate, and tail fiber proteins are indicated. MMBR 67(1): 86-156, 2003

MICROBIOLOGY, DAVIES-DULBECCO-EISEN-GINSBERG

Electron micrographs of bacteriophage T4. (A) Extended tail fibers recognize the bacterial envelope, and its prolate icosahedral head contains the 168,903-bp dsDNA genome. (B) The DNA genome is delivered into the host through the internal tail tube, which is visible protruding from the end of the contracted tail sheath.

Microbiology & Molecular Biology Reviews 67(1): 86-156, 2003

MICROBIOLOGY, DAVIES-DULBECCO-EISEN-GINSBERG

CICLO DE MULTIPLICACION

1. ADSORCION. (2-4 MINUTOS)

- ES ESPECIFICA (RECEPTORES EN LA SUPERFICIE DE LA BACTERIA).

- DESORGANIZACION DE LA MEMBRANA PLASMATICA-REVERSIBLE

SE BLOQUEA LA SUPERINFECCION POR FAGOS DEL MISMO TIPO.

- DISTURBIOS METABOLICOS.

- REARREGLO EN LA SINTESIS DE MACROMOLECULAS: SE INHIBE LA

SINTESIS DE DNA, RNA Y PROTEINAS DE LA BACTERIA.

2. PENETRACION

EXPERIMENTO DE HERSHEY Y CHASE

GENES VI, BENJAMIN LEWIN

EXPERIMENTO 1. 3 MINUTOS CENTRIFUGAR 90% 35S AGITAR SOBRENADANTE A 2000 RPM 10% 35S BACTERIA

E. coli + FAGO T4 ( 35S )

EXPERIMENTO 2. 3 MINUTOS CENTRIFUGAR ~0% 32P AGITAR SOBRENADANTE A 2000 RPM 100% 32P BACTERIA

E. coli + FAGO T4 ( 32P )

MOLECULAR BIOLOGY, DAVIS FREIFELDER

CELL 118(4): 419-429, 2004

Figure 5. Baseplate Conformational Switch Schematics(A and B) The phage is free in solution. The long tail fibers are extended and oscillate around their midpoint position. The movements of the fibers are indicated with black arrows. The proteins are labeled with their corresponding gene numbers and colored as in Figure 1A. Domains of gp7 and gp10 are labeled as in Figure 2A.(C and D) The long tail fibers attach to their surface receptors and adapt the “down” conformation. The fiber labeled “A” and its corresponding attachment protein gp9 interact with gp11 and with gp10, respectively. These interactions, labeled with orange stars, probably initiate the conformational switch of the baseplate. The black arrows indicate tentative domain movements and rotations, which have been derived from the comparison of the two terminal conformations. The fiber labeled “B” has advanced along the conformational switch pathway so that gp11 is now seen along its 3-fold axis and the short tail fiber is partially extended in preparation for binding to its receptor. The thick red arrows indicate the projected movements of the fibers and the baseplate.(E and F) The conformational switch is complete; the short tail fibers have bound their receptors and the sheath has contracted. The phage has initiated DNA transfer into the cell.

Comparison of the Baseplate in the Two Conformations(A and B) Structure of the periphery of the baseplate in the hexagonal and star conformations, respectively. Colors identify different proteins as in Figure 1A: gp7 (red), gp8 (blue), gp9 (green), gp10 (yellow), gp11 (cyan), and gp12 (magenta). Three baseplate proteins (gp8, gp9, and gp11), with the available complete crystal structures, are shown as Cα traces. The density of the short tail fibers in the star conformation is based on the crystal structure of the receptor binding, C-terminal fragment of gp12 Thomassen et al. 2003 and on the corresponding density from the hexagonal conformation of the baseplate. Directions of the long tail fibers are indicated with gray rods. The three domains of gp7 are labeled with letters A, B, and C. The four domains of gp10 are labeled with Roman numbers I through IV. The C-terminal domain of gp11 is labeled with a black hexagon or black star in the hexagonal or star conformations, respectively. The baseplate 6-fold axis is indicated by a black line.(C and D) Structure of the proteins surrounding the hub in the hexagonal and star conformations. The proteins are colored as follows: spring green, gp5; pink, gp19; sky blue, gp27; violet, putative gp48 or gp54; beige, gp6-gp25-gp53; orange, unidentified protein at the tip of gp5. A part of the tail tube is shown in both conformations for clarity.

http://www.seyet.com

T-Even Bacteriophage Infecting a Cell


T-Even Bacteriophage-Contracción

Cyanophage P-SSP7, a marine Podovirus

+ DNA - DNA

Este virus infecta a una cyanobacteria marina, Prochlorococcus marinus, que es responsable de gran parte de la fotosíntesis en los oceanos.

Catching a Glimpse of Infection's Opening ActLiu et al. (2010) provides a view of the structural changes in a marine podovirus, cyanophage P-SSP7, that accompany release of its double-stranded DNA genome. P-SSP7 and related phages infect one of the world's most abundant organisms, Prochlorococcus marinus, a marine photosynthetic cyanobacterium that collectively account for a sizeable proportion of ocean photosynthesis.

The authors use single-particle cryo-EM to analyze the virus structure both with and without packaged DNA and establish clear differences at the portal vertex, including alterations at the nozzle tip and in the position of the tail fibers. From this analysis it appears that the position of the tail fibers relative to an adapter protein determines whether the nozzle valve will be open to allow release of the genomic contents of the phage. The authors then examine by cryo-electron tomography P-SSP7 in the process of infecting Prochlorococcus cells and provide evidence that the tail fibers are positioned in an orientation similar to that observed in empty phage. This observation supports the view that a change in tail fiber position, likely upon interaction with a suitable host cell-binding site, precedes a series of conformational changes that trigger nozzle opening.

X. Liu et al. (2010). Nat. Struct. Mol. Biol. Published online June 13, 2010. 10.1038/nsmb.1823.

MOLECULAAR BIOLOGY, DAVID FREIFELDER

BACTERIOFAGO PRD1ASM NEWS 68 (7): 330-335, 2002

BACTERIOFAGO PRD1: INGRESO DEL DNA A LA BACTERIA GRAM-NEGATIVA

OM= OUTER MEMBRANE, PG= PEPTIDOGLYCAN, CM=CYTOPLASMIC MEMBRANE

ASM NEWS 68 (7): 330-335, 2002


3. SINTESIS DE MACROMOLECULAS VIRALES

- TRANSCRIPCION:

mRNA TEMPRANO: SINTESIS DE PROTEINAS PARA REPLICACION

mRNA TARDIO: SINTESIS DE PROTEINAS ESTRUCTURALES.

- TRADUCCION

- REPLICACION

4. MADURACION Y LIBERACION

MOLECULAR BIOLOGY, DAVID FREIFELDER

CELL 94(2): 147-150, 1998

Images of Epsilon 15, a virus that infects the bacterium Salmonella. Cross section of the viral particle's interior, obtained with an advanced magnifier called a cryoelectron microscope. The right-side is a computer graphic highlighting the salient features of the virus. Scientists have had difficulty resolving the internal features of viruses with nonsymmetric components such as Epsilon 15, but Jiang's team made improvements to the computer software used to process the electron microscopy images. (Graphic courtesy of Nature magazine/Jiang Laboratories.) Microbe 1(4): 164-165, 2006

Motor molecular: ATPasas de anillo que convierten energía química en energía mecánica para introducir 100 pares de bases/segundo. La presión interna maxima es de 60 atmosferas. (fuerza de una molecula de miosina 3-5 pNewtons. Fago 55pNewtons)

Bustamante y Col., NATURE 43: 748-752, 2001; 421: 423-427, 2003

CINETICA DE INGRESO DEL DNA

INTRODUCCION DEL DNA I

INTRODUCCION DEL DNA II


T-Even Bacteriophage Assembly


1. ADSORCION. (2-4 MINUTOS)

- ES ESPECIFICA (RECEPTORES EN LA SUPERFICIE DE LA BACTERIA).

- DESORGANIZACION DE LA MEMBRANA PLASMATICA-REVERSIBLE

POR INCORPORACION DE PROTEINAS DEL FAGO A LA MISMA. SE

BLOQUEA LA SUPERINFECCION POR FAGOS DEL MISMO TIPO.

- DISTURBIOS METABOLICOS.

- REARREGLO EN LA SINTESIS DE MACROMOLECULAS: SE INHIBE LA

SINTESIS DE DNA, RNA Y PROTEINAS DE LA BACTERIA.

2. PENETRACION

EXPERIMENTO DE HERSHEY Y CHASE

3. SINTESIS DE MACROMOLECULAS VIRALES

- TRANSCRIPCION:

mRNA TEMPRANO: SINTESIS DE PROTEINAS PARA REPLICACION

mRNA TARDIO: SINTESIS DE PROTEINAS ESTRUCTURALES.

- TRADUCCION

- REPLICACION

4. MADURACION Y LIBERACION

http://www.microbelibrary.org/ASMOnly/details.asp?id=1870&Lang=

T-Even Bacteriophage Infecting a Cell


Intezyme-ivect-micelle

1_BACTERIOFAGOS.ppt

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