Function and application of contractile injection systems and jumbophage RNA polymerases - Project Summary The proposal contains two subprojects, each with fundamental science and application components. The first subproject aims to build a quantitative description of the processes that take place when contractile injection systems – phage tails, tailocins, and the type VI secretion system – breach the envelope of a bacterial cell. This information will be used in the design and retargeting of R-type pyocins (tailocins) to medically important pathogens. The second subproject is focused on the RNA synthesis machinery of jumbophages (i.e. very large phages). We aim to characterize how the non-virion RNA polymerase (nvRNAP) encoded by jumbophage AR9 recognizes its promoters and how this enzyme can be used for in vitro transcription and RNA amplification. A contractile injection system, at a minimum, consists of an outer contractile sheath, inner rigid tube, and baseplate. The sheath and tube are fixed to each other at the baseplate-distal end. The initial, extended-sheath conformation is a high-energy, metastable state of the system. In phages and tailocins, the baseplate carries receptor-binding proteins, interaction of which with their cell surface ligands causes an avalanche of structural changes: the baseplate changes it conformation, the sheath contracts and drives the tube through the cell membrane. Recently, we have characterized the energetics of sheath contraction of the R-type pyocin using modeling, solution biophysics, and single molecule measurements. This proposal will identify and describe the mechanisms by which the baseplate is activated by receptor-binding proteins (tail fibers) and how the baseplate triggers sheath contraction. The pyocins are being explored as a tunable antimicrobial platform because they can be retargeted to a bacterial host of interest by altering their tail fibers. The quantitative description of pyocin baseplate activation will be used to define principles for the design of hybrid tail fibers. We have recently partially characterized how the AR9 nvRNAP recognizes its deoxyuridine-containing promoter element in the template strand of double-stranded DNA. Structurally, this enzyme resembles a trimmed down version of the bacterial multisubunit RNA polymerase. The enzyme contains no α or ω subunits while the slightly shortened β and β′ subunits are encoded by two separate AR9 genes each. The AR9-encoded promoter specificity subunit is distantly related to σ factors. Considering that other jumbophages use AR9-like RNA polymerases for transcription of their genomes that are nearly an order of magnitude larger than the T7 genome, the AR9 nvRNAP may represent a more powerful tool for in vitro transcription and RNA amplification than the current gold-standard T7 RNA polymerase. We will identify the strongest AR9 nvRNAP promoters and optimal reaction conditions to create an nvRNAP-driven in vitro transcription system. We will describe how the enzyme recognizes promoters of different strengths in atomic detail using X-ray crystallography, cryo-electron microscopy, and molecular dynamics simulations.
