Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

120j 6 Molecular Mechanisms Underpinning Plant Colonization
Figure 6.4 The SPyVET principle. (a) IVET
strains will not grow in vitro in the absence of
exogenous EGF owing to lack of P ES::egf expression
(see Figure 6.2), but regulatory
mutants (suppressors) that result in PES expression
restore prototrophy enabling growth
in the absence of exogenous EGF (Giddens,
Jackson, Moon and Zhang, unpublished).
(b) Two transposons were used to mutate
IVET strains and disrupt expression of environment-specific
regulators. (i) miniTn5 was
used to solely disrupt repressors and (ii) IS-O-
Km was used to disrupt repressors and induce
environment-independent expression of
neighboring activators by virtue of a constitutively
expressed nptII promoter near one
end of the transposon.
disruption of a repressor gene enable expression of the plant-inducible gene linked
to the dapB::lacZ reporter, thus allowing expression of dapB and growth. Similarly,
IS-O-Km/hah nptII promoter activity enabled expression of positive regulators that
activate the plant-inducible gene fusion. By using arbitrary-primed PCR [41] and
sequencing, it was possible to identify the insertion points of the transposons, which
were then mapped to the SBW25 genome sequence.
Approximately 2–3 million mutants of each plant-inducible gene fusion were
screened and a total of 16 regulators were identified for eight plant-inducible genes
(Giddens, Jackson, Moon and Zhang, unpublished). Most of the activators identified
were not isolated by IVET. This implies one of several possibilities. First, these
activators are constitutively active and are themselves regulated post-transcriptionally.
These would be recovered from an IVET screen through plants, but eliminated
as housekeeping-type gene fusions that are also active in vitro. For example, one
gene, algR, was identified as a positive regulator of the plant-inducible wss gene
cluster, and also identified as a positive activator of hydrogen peroxide resistance
[42]. This trait is expressed in vitro, which suggests the regulator must be active
both in vitro and in vivo. A second possibility is that the activators are too weakly