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

6.3 Regulatory Networks Controlling Plant-Induced Genesj121
expressed to rescue the egf mutant strain in an IVETscreen; an example of this is the
rspL activator, which has been shown to be expressed in the plant rhizosphere, but at
relatively low levels [4]. Thirdly, the activators are transiently expressed in the plant
environment and expression is not for a sufficient period of time to rescue the strain.
Transient gene expression has been demonstrated in other systems [22].
Two plant-inducible genes (wssE and cueA) had more than three regulators controlling
their expression compared with the other gene systems for which only one or two
regulators were identified. In SBW25, cueA encodes a copper transporting P1-type
ATPase [31]. To date, the majority of bacterial genes for copper homeostasis that have
been characterized encode P1-type ATPases, and their expression is induced by high
levels of extracellular copper [43]. Notably, the copper-exporting ATPase in Gramnegative
bacteria, such as E. coli and P. putida, is regulated by a MerR-type activator
(CueR) [44], whereas in the Gram-positive bacterium Enterococcus hirae, it is regulated
by a repressor protein CopY and a copper chaperone CopZ [45]. SPyVET analyses
identified CueR, a putative MerR-type activator, as a regulator of cueA. This was
consistent with the previous work on P. putida that cueA is activated by CueR in a
copper-responsive manner [46]. Additionally, SPyVET analysis suggested that CueA
activated the expression of cueA and that CopZ negatively regulated cueA. It was also
found that CueR and CopZ activated a plant-inducible permease locus of unknown
function in SBW25 that was originally identified by IVET. These data suggest a role for
the permease locus in copper homeostasis, which is currently under investigation.
The second plant-induced gene controlled by multiple regulatory inputs in
SBW25 is wssE. The wssE gene is part of an operon (wssABCDEFGHIJ) that encodes
a membrane-bound cellulose synthase complex. The production of cellulose is
important for bacterial fitness in the sugar beet rhizosphere and particularly the
phyllosphere [2]. The function of cellulose in plant colonization is unknown, but like
many secreted extracellular polysaccharides (EPS), it is central to the formation of
biofilms formed by SBW25. A total of seven regulators have been identified in
controlling expression of wssE, which include repressors (AwsX, WspF, AlgZ
(AmrZ) and FleQ) and activators (WspR, AwsR and AlgR). AlgR and AlgZ (AmrZ)
have previously been implicated in controlling expression of another EPS, alginate
[47]. In P. aeruginosa, AlgZ (AmrZ) is a transcriptional activator that acts on alginate
genes, but in SBW25, it acts as a repressor, in this case of wss expression. Although
SBW25 carries an intact alginate operon, the production of alginate has not been
identified either in vivo or in biofilms. This indicates that cellulose is probably the
more important EPS for plant colonization and that the regulators have been recruited
to either control expression of both EPS gene systems or just the wss system,
with alginate gene regulation becoming redundant.
FleQ is the master regulator of the flagellum biosynthesis genes [48] and this is the
first time the regulator has been implicated in controlling the expression of EPS
genes. However, the inverse relationship between EPS and flagellum gene expression
is well documented [49]. Removing the bulk of the IS transposon from the fleQ
mutant via Cre–loxP recombination [41] and subjecting the resulting unmarked fleQ
mutant to another round of transposon mutagenesis led to the discovery that FleQ
repressed the activity of AlgR. Since FleQ is an RpoN-interacting enhancer binding