Tomography of type IV secretion system complexes (360G-Wellcome-093353_Z_10_Z)

4.1 Visualisation of available T4SS subcomplexes by ET. Constructs encoding core- and mega-complexes of the pKM101 T4SS plasmid will be transformed into BL21 DE3 and B/r E. coli strains for inducible expression. Samples of induced BL21 DE3 cells will be high-pressure frozen, freezesubstituted into organic solvents and embedded in a plastic resin. Different freeze-substitution protocols will be tested, so as to determine the optimal temperature steps, identify the most suitable fixatives, stains and resin, together with their concentrations, giving rise to the smallest loss of ultrastructure and the best contrast, as assessed by TEM. Room temperature ET will be performed on ultrathin sections of thus prepared resin blocks. The same approach will initially be applied to every new T4SS system or its overexpressed portion. Although the stains and fixatives used in this strategy would almost certainly lead to some structural artefacts, they also greatly improve contrast and are hence desirable for the initial detection of T4SS complexes. This will facilitate the subsequent artefact-free visualisation of T4SS assemblies embedded in vitreous ice, when the strict low electron dose regime severely constraints the signal-tonoise ratio and contrast. Simultaneously, all constructs will also be transformed into B/r H266 cells which when grown in minimal media do not exceed ~0.6 ?m in diameter and are hence suitable for whole-cell CET (24). Tomograms will be recorded at cryogenic temperatures at 300 keV. Low-pass and other types of filtering, together with various denoising methods will also be used. The challenging task of cryo-sectioning will further be attempted on any complexes successfully observed either in plastic-sections or in whole cells (25). 4.2 Approaches to localization of T4SS complexes in tomograms Due to a relatively small size of currently overexpressable and stable T4SS complexes and the possibly low level of constitutive T4SS expression (26), their unambiguous detection under electron microscope will be assisted by using different electron-dense high-contrast labels. Both post- and pre-embedding immunogold labelling will be used to tag an extracellular Flag-tag introduced between ?-helices 2 and 3 of VirB10 in VirB10-containing complexes (9). In this technique, the antigen-specific primary antibody is detected with a suitable secondary antibody conjugated to nano-size gold particles of defined diameter, which can be easily observed under electron microscope. Additionally, a recently developed iron-loaded ferritin-label will be translationally fused to individual T4SS subcomplex subunits and the resulting construct expressed in fur gene knockout B/r H266 E. coli strain, hence providing 3-dimensional information, as opposed to immunogold technique where labelling is limited to section surfaces (24). In addition to electron-dense labelling, determination of positions of T4SSs or of their smaller complexes in the bacterial membrane will be attempted by tracking of extracellular conjugative pili, which are constitutively expressed by exponentially growing E. coli harbouring derepressed T4SS operons. One example of such system is the R1-16 plasmid (27), which will be studied by ET in BL21 DE3, TOP10 or thin B/r cells. If required, discrimination of T4SS pili from bacterial flagella will be achieved through bacteriophage-display approach (28). R1-16 is a deletion mutant of plasmid R1-19, which itself is a derepressed high conjugation frequency mutant of plasmid R1, a close relative of the extensively studied, archetypal F-system (27). 4.3 Cloning of inducible T4SS subcomplexes Despite the possible localization of constitutively expressed T4SSs being restricted to the double-membrane, trace levels of their expression renders them unlikely to be observed in an exposure area of a tomogram. This problem becomes particularly profound for averaging of reconstituted individual sub-volumes, where large numbers are required for better structural characterization. This limitation imposes a need for efficient inducible overexpression of functional T4SS machines. Achievement of this goal would also prove indispensible for numerous functional studies on Type IV secretion. During the proposed PhD research, work will therefore focus also on restriction-free and other approaches to clone the functional T4SS operon of the self-transmissible R388 system (29) downstream of inducible promoters. Overexpression of A. tumefaciens T4SS genes with acetosyringone for ET studies will also be investigated and assessed by the presence of T4SS pili under TEM. In the event of successful detection and purification of new T4SS subcomplexes through pull-down experiments, single particle EM studies will be undertaken. Successful visualization of a wild-type T4SS would open a wide range of possibilities to study its functional and associated structural dynamics.