Classic techniques designed to understand bacterial gene regulation in vitro are still
used today to elucidate genetic mechanisms and pathways. However as much as we
can learn from in vitro studies of bacterial behaviour, it is impossible to fully recreate
the complex and multifaceted environment the bacterium faces in vivo. To this end
the development of techniques to allow us to study gene regulation in vivo is
important as these can then be applied to dissect bacterial pathogenesis and should
uncover novel therapeutic targets.
Recombinase in vivo expression technology (RIVET) provides a heritable irreversible
marker of gene activity in the host and has been applied recently to discover genes
important for bacterial virulence in the host. The aim of this project was to develop a
novel RIVET system using genes from the enterohaemorrhagic Escherichia coli
0157:H7 (EHEC)ftm operon and the beta-lactamase reporter gene (bid). This system
was named FR1VET (Jim recombinase in vivo expression technology).
The basis to FRIVET is a completely synthetic operon placed in single copy in the
EHEC chromosome at the Jim locus. The arabinose (ara) inducible promoter was
tested in the system initially to validate the system in vitro. The key aim of these in
vitro tests was to understand the working tolerances of the FRIVET operon and define
appropriate control points. Emphasis was placed on 'setting' the system and
controlling the levels of recombination achieved.
Construction of the FRIVET operon on allelic exchange vectors and subsequent
exchange into a Shiga-like toxin negative EHEC strain was successful. In vitro tests
using the ara promoter proved the system functioned as intended and was
experimentally stable. When EHEC promoters, in particular LEE5, were tested in
vitro, considerable difficulty was encountered in controlling levels of recombination
and setting the system to the off status. This is essential for any use of the system to
examine gene expression in vivo. Therefore the emphasis of future work must be on
defining appropriate measures for controlling in vitro activity of chosen promoters
when preparing constructs for in vivo challenges.
With appropriate modifications the FRIVET system has the potential to produce
valuable data about EHEC gene expression in the host and therefore to contribute to
our understanding of the complex regulation required to establish colonisation,
maintain infection and induce pathology in vivo.