Evans, Katy
(2007)
The roles of surface ultrastructures in the predatory life cycle of Bdellovibrio bacteriovorus.
PhD thesis, University of Nottingham.
Abstract
Bdellovibrio bacteriovorus is a ubiquitous Gram-negative 8-proteobacterium that is predatory on other Gram negative bacteria. It's prey includes many pathogenic bacteria, including E. coli, Salmonella, Proteus and Pseudomonas species; as such, there is much interest in the study of Bdellovibrio with a view to its potential use as an alternative antimicrobial therapy.
Key to any future research into medical applications of this bacterium is an understanding of its predatory life cycle. Attack phase Bdellovibrio uses rapid flagellar motility through liquid environments to find its prey; upon collision with a suitable prey cell, it forms a strong attachment to the cell and generates a pore in the prey outer membrane. The predator then squeezes through this pore, loses its flagellum and establishes itself in the prey periplasm; the entry pore is then resealed, the prey rapidly killed with its peptidoglycan being modified to produce a rounded, osmotically stable structure termed the bdelloplast. Once safely within the bdelloplast, the Bdellovibrio secretes an arsenal of lytic enzymes into the prey cytoplasm which allow transport of the constituent monomers of DNA, RNA, protein et cetera from the prey back into the growing Bdellovibrio. The predator elongates as a spiral shaped filament within the periplasm of the prey and once the contents of the cytoplasm are exhausted, the filament septates into progeny Bdellovibrio which then re-synthesise flagella and lyse the remains of the prey cell to become free swimming attack phase predators.
In this thesis, the role of flagellar motility in the predatory lifestyle of Bdellovibrio was studied through individual insertional inactivation of each of the 6 genes found in the B. bacteriovorus HDIOO genome that encode the flagellar filament protein, FliC. Only one was found to be essential for filament formation yet, contrary to previous hypotheses, this non-motile mutant was still found to be predatory when applied to immobilised prey in a novel fluorescence assay. Thus, the role of Type IV pili in predation was investigated through both microscopic, mutational and transcriptional means. Abolition of the Type IV pilus fibre forming protein, PilA, resulted in a mutant that had no pilus fibres and was incapable of predation using the same assay as in the non-motile FliC mutant strain.
Throughout this work, experimental challenges have necessitated the continuing development of electron microscopic techniques for whole cell, flagella, pili and bdelloplast imaging and development of fluorescence methods for use in Bdellovibrio, such as novel use of GFP-encoding transposon mutagenesis and chemical dye-based assays.
Work presented here has demonstrated that pili, not flagella, are the bacterial nanomachines required for prey entry and that GFP is a viable tool for future study of this fascinating and potentially therapeutic predator.
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