Transcriptional analysis of Bdellovibrio bacteriovorus responses to different prey and environments

Bdellovibrio bacteriovorus is a small, highly motile Gram-negative bacterium that preys upon other Gram-negative bacteria. It does this by burrowing through the outer layers of the prey cell and establishing itself in the periplasm, before consuming the prey contents and using these for growth as a filament. When the prey contents are depleted, this filament septates to produce new B. bacteriovorus progeny which burst out of the prey shell and go on to attack further prey. Its prey includes pathogens of plants, animals and humans, including multidrug-resistant pathogens which are emerging as a major health threat. Thus it has potential as a novel therapeutic to overcome the lack of new antibiotics. This prospect is particularly attractive as genetic resistance to B. bacteriovorus predation has not been demonstrated, rather a plastic resistance to a sub-population is seen, which when recovered remains as susceptible to predation as the parent population. There are many stages to this complex predatory lifecycle: swimming or gliding to search out potential prey, attachment to and detecting suitable prey, formation of, and entry into, a pore in the outer layers which is then re-sealed. Then killing and rounding of the prey is followed by staged degradation of the prey contents, then growth and division of the predator ultimately leading to new prey emerging. Further, Host Independent (HI) mutants are capable of growth in rich nutrients in the lab, with divergent morphologies observed. Some transcriptional studies have proved invaluable as a tool for studying some of these processes, but a complete lifecycle study is lacking. Here, we present a high-resolution transcriptional profile throughout the predation cycle giving insights to all of these various stages. For B. bacteriovorus to fulfil its promise as a novel antimicrobial agent, more needs to be understood about predation outwith the paradigm laboratory conditions. In order to address this, predation carried out on a multidrug-resistant clinical isolate of Serratia marcescens was subjected to transcriptional analysis, including predation by a B. bacteriovorus mutated by deletion of the global regulator DgcC, a strain incapable of HI growth. Cluster analysis provides an unbiased means of ordering data by calculating distances between datapoints in n-dimensions, allowing grouping of gene expression from different experiments. Here, I develop a pipeline for the analysis of B. bacteriovorus RNA-Seq data with cluster analyses to bring further insights to both unpublished work from our laboratory and by re-analysing datasets of published work by other groups. These analyses discover that groups of genes are tightly sequentially regulated throughout the predatory cycle, giving insight to their functions. They show that predation upon Serratia is significantly different from that of predation on E. coli, with different transcriptional profiles throughout the predation cycles. The response of B. bacteriovorus to exposure to pooled human serum seems to be one of protection from the antimicrobial elements in serum rather than metabolism of potential nutrients in the medium. The response to nutrient broth and non-prey Gram-positive Staphylococcus aureus surprisingly includes genes which are specific to Gram-negative prey modification, suggesting that the obligate predator B. bacteriovorus co-regulates predation and nutrient utilisation pathways. Many groups of genes are identified by analyses of mutant transcription as important in various regulatory pathways. Along with insights into the predation process and condition responses, the gene clusters generated in this project by novel re-analyses of data identify many targets for future projects to better understand the predation process and how B. bacteriovorus reacts in more clinically relevant conditions; a prerequisite for the fulfilment of its promise as a potential novel antimicrobial therapy