Somers, Hannah
(2016)
Characterising Bdellovibrio bacteriovorus HD100 peptidoglycan modification genes during distinct cell cycle events.
PhD thesis, University of Nottingham.
Abstract
Bdellovibrio bacteriovorus are predatory bacteria, which prey on other Gramnegative bacterial species. Bdellovibrio modifies the cell wall peptidoglycan of its prey at several stages of its predatory life cycle: during invasion and entry into the prey periplasm, during the rounding of the prey into a stabilised bdelloplast (in which Bdellovibrio grow, replicate and divide) and at the end of the cycle, at which point the resulting progeny lyse the bdelloplast before repeating the predation process.
Bdellovibrio also modify another source of cell wall peptidoglycan during these processes: their own. During the invasion of prey cells, Bdellovibrio adjust their shape to enter prey cells through the size-limited pore in the prey cell envelope. Following this, Bdellovibrio peptidoglycan must be modified during its filamentous growth, division and possibly during the construction and insertion of flagella prior to progeny release. The modification of two “sets” of peptidoglycan was significant in the considerations behind this project.
Pioneering work in the 1970s deduced that the combined actions of Bdellovibrio peptidoglycan-modifying enzymes, including an N-deacetylase, were involved in the prey invasion process. Further work by these researchers also proposed mechanisms of bdelloplast stabilisation following predatory invasion. Since then the Bdellovibrio genome has been sequenced, and transcriptomic data alongside bioinformatic investigation, has provided several potential gene targets which may encode such “invasion-enabling” enzymes. I aimed to investigate the roles of these candidate gene products and determine their role in these predatory processes. Other predicted peptidoglycan-active enzymes have been researched for their roles in either the predatory or non-predatory lifestyles of Bdellovibrio in order to begin to separate prey-active proteins from self-active enzymes.
Previous work observed that the prey bdelloplast peptidoglycan is Ndeacetylated at the N-acetyl-muramic acid (MurNAc) and N-acetyl-gluco samine (GlcNAc) residues within the glycan strands. It was initially hypothesised that these modifications would inhibit early acting peptidoglycanactive enzymes, however recent work from our laboratory, to which I contributed, showed that the combined deletion of two GlcNAc N-deacetylases inhibited degradation of the bdelloplast during the final lytic event, leaving prey bdelloplast “ghost” structures behind following predation [110]. A third Bdellovibrio-encoded N-deacetylase enzyme (Bd0993) was found to contribute to prey cell modification (a gene deletion mutant showed a significant reduction in invasion efficiency). Further analysis has implicated this gene product in the stabilisation of the rounded bdelloplast, beginning to shed light on the internal modifications employed by Bdellovibrio when inside the intraperiplasmic niche.
Also identified within transcriptomic analyses of early predation by Bdellovibrio were three lysozyme-encoding genes. RT-PCR analyses and fluorescent labelling of these gene products identified that these proteins act during different stages of the predatory cycle. Bd1411 was shown to modify selfpeptidoglycan during the invasion process, facilitating the predator entry into prey (∆bd1411 showed significantly reduced invasion efficiency), while its close homologue Bd1413 was shown to act later in the life cycle, associated with elongation of the growing Bdellovibrio filament. Bd0314 was shown to cleave the peptidoglycan wall of the bdelloplast at the end of the predatory cycle as the ∆bd0314 mutant left exhibited intact peptidoglycan structures behind at the end of the predatory process. Combinatorial deletions did not prevent predatory activity, therefore it has been concluded that other enzyme classes are also likely involved in these crucial life cycle stages to produce the Bdellovibrio predatory niche.
The lysozyme genes were implicated in the host-independent (HI) lifestyle as the fluorescently-labelled gene products were observed within rounded, axenically-grown cells. Further analysis has led to the development of a model of HI survival and the role of peptidoglycan-active enzymes in this lifestyle, a novel area of research. One other such protein was investigated for its role in this alternate growth mode. Bd1285 is a putative lytic transglycosylase that when deleted left the predatory life cycle unaffected, but prevented the growth of HI cells in liquid media, implicating this protein in successful growth.
Although there are more peptidoglycan-active enzymes likely to be involved in these varied processes, and control at the post-transcriptional level is still to be uncovered, this work has identified and characterised several interesting mechanisms that contribute to successful predation by Bdellovibrio in its unique niche.
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