Tara Spires and R. Malcolm Brown, Jr.
Department of Botany, The University of Texas at Austin, Austin, Tx., 78713
A sample of T7 bacteriophage donated by Dr. Ian Molineux of the microbiology department at UT Austin was observed using the Philips 420 TEM for the purpose of imaging the double helix of DNA and observing the structure of a DNA associated protein. A bacteriophage was chosen to observe DNA because DNA is difficult to visualize alone since it does not effectively absorb negative stain. When a bacteriophage is carefully lysed, its DNA will remain attached to the capsid making it easier to find for observation.
Bacteriophages insert their genome into a host bacterium where it is translated into viral proteins that aid in replication of viral genetic material and in production of new viruses.1 T7 contains linear, double stranded DNA and it attacks Escherichia coli, a gram negative species of bacteria. The short tail of T7 which consists of several distinct proteins attaches the virus to cell receptor proteins and mediates the passage of DNA from the phage to the bacterium.2 Bayer found that T7 associates with receptors where the cell wall and membrane adhere to each other.3 Introduction of the viral genetic material into an E. coli cell proceeds from the genetic left end of the linear DNA strand.4 Dr. Molineux asserts that there is a protein ejected from the phage head into the cell that aids in making the bridge between the cell wall and the cell membrane of an E. coli cell to facilitate genome insertion. (unpublished data) We have imaged this protein or a complex of this protein with other ejected proteins and gained a rough idea of its structure using the Philips 420 TEM.
The sample of CsCl purified T7 bacteriophage with a concentration of 1013 phage/ml was dialyzed in distilled water overnight to promote gentle phage lysis with DNA still associated with its capsid. This dialyzed sample was then diluted 1000 fold and 5ml was placed on a formvar coated TEM grid for one minute. The grid was then negatively stained and observed in the TEM at 100kV or 120kV and magnifications ranging from 21000X to 210000X. Images were captured with the Gatan camera and processed in the IBAS imaging system.
An example of a normal T7 virus is seen in figure 1. The double helix of T7 DNA was visualized at 275000X magnification (see fig. 2). DNA was observed associated with a lysed capsid. The strands of DNA at the end of the segment in figure 2(right side of the photograph) appear to have separated. Figure 3 shows DNA at lower magnification associated with a viral capsid.
The protein associated with T7 DNA insertion into E. coli was visualized. As seen in figure 4, it has a claw shape. The dimensions of the protein (based upon calibrations of the IBAS monitor with the 3.35Å spacing in a graphite lattice) are approximately 50nm long and 38nm wide (at its widest point.)
One unexpected benefit of these observations was the visualization of capsomere subunits which make up the icosahedral viral capsids (see fig. 5).
DNA visualization was made possible by lysis of phage particles
in dH2O, however, the water could also possibly change the confirmation
of the DNA associated protein or the DNA itself (such as the separation
of strands seen in fig. 2.) The dimensions
observed for the protein complex ejected for the phage head (50nm
by 30nm) are larger than the width of the tail of T7 particles.
In the images we captured, the widest portion of the tails of
the viruses ranged from 9nm to 15nm. This size discrepancy indicates
that the tail must change to allow passage of the protein complex
or that the protein complex cannot be ejected in its final form.
One possible explanation for the process of ejection is that
the protein is not folded into its final conformation when it
moves out of the phage head but folds after it passes through
the tail. The shape of the protein complex could also have been
changed by the distilled water solution causing a slight distortion
of its size. More studies should be conducted using solutions
more natural to the phage and trying different methods of lysis;
however, this approach presents problems with TEM imaging because
of salts in solution that can crystallize on the grid impeding
ACKNOWLEDGEMENTS We would like to thank Dr. Ian Molineux for his generous donation of a T7 sample. This work is supported in part by Welch Foundation Grant F1217 to R.M.B. and the Office of Naval Research grant N00014-95-1-0933.
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