Identifying the assembly pathway of cyanophage inside the marine bacterium using electron cryo-tomography.

Advances in electron cryo-tomography open up a new avenue to visualize the 3-D internal structure of a single bacterium before and after its infection by bacteriophages in its native environment, without using chemical fixatives, fluorescent dyes or negative stains. Such direct observation reveals the presence of assembly intermediates of the bacteriophage and thus allows us to map out the maturation pathway of the bacteriophage inside its host. For every bacterium, there are associated bacterial viruses. The virus has dual effects on its bacterial host: destroying the bacteria through virus multiplication inside them leading to cell lysis, and enriching the gene pool of its host by incorporating host genes in its genome and transferring them among hosts. The processes of virus multiplication and lateral gene transfer have been critical areas of research in bacteriology and phage biology since the beginning of the molecular biology era. Through mutagenesis of the bacteriophage combined with biochemical analysis of the bacteria during the course of phage morphogenesis, it has been possible to dissect the pathway of the maturation process of many enteric bacteriophages within their hosts. However, such a discovery process requires tedious and laborious work in making many mutants, analyzing the protein profiles, and examining the cell structures by conventional electron microscopy of plastic embedded cell sections. In electron cryo-tomography, the native structure of the bacteria is preserved in its physiological conditions for electron microscopic examination. Images of a frozen hydrated bacterium are recorded one at a time at different tilts, similar to a medical CT scan. These images can then be compiled to form a 3-D density volume where each voxel has a density value corresponding to the mass density of the biological contents of the bacterium. We applied this approach to study a Synechococcus cyanobacterium infected by Syn5 bacteriophage. Synechococcus WH8109 cell is a marine photosynthetic bacterium distributed throughout the world’s oceans. Marine bacteria play a critical role in the balance of the biosphere by absorbing carbon dioxide with the help of sunlight to generate biomass. It has been estimated that over 30% of carbon dioxide in the atmosphere are fixed by the marine bacteria through photosynthesis. Yet, very little is known about the pathogenesis of marine bacteria under the attack of cyanophages. The Synechococcus WH8109 has a thickness of ~0.7 µm, which is thin enough to allow the 200 keV electrons to penetrate the cell in its entirety. The electron microscope used in our study was equipped with a special type of electron optics, in which the scattered electrons undergo a 90 degree phase shift. This is equivalent to the well-known light optics called Zernike phase contrast microscopy. The implementation of such optics in an electron microscope is relatively straightforward and involves placing a phase plate at the objective lens’ focal plane. The phase plate is made of a thin 20-25 nm carbon film with a hole (~1 µm diameter) in the middle of the carbon film. The electron images recorded this way yield a contrast enhancement of a factor of 3-5 relative to conventional electron optics. Though the physics principle is simple, the practice of this methodology to record high quality images is technically demanding because the phase plate is readily subject to deterioration under electron irradiation. Nevertheless, we were able to generate ~100 tilt series of Synechococcus