Biomedical Engineering Reference
In-Depth Information
sequences upstream from the promoter (Morales and Lewis,
2006
). This system limits maximal
PDTC production to instances in which the siderophore accumulates in the immediate environ-
ment of the producing organisms and thus may avoid wasteful investment in the system when
the siderophore diffuses away or has a limited half-life.
The concentration threshold for activation of
pdt
gene expression is apparently quite low.
PDTC transcriptional activation could be reconstituted in a strain of
P. putida
(KT2440) lacking
pdt
genes. That organism could not be rescued from chelator-induced inhibition by PDTC and is
actually inhibited by PDTC (Leach and Lewis,
2006
; Sebat et al.,
2001
). Deletion of genes for the
outer membrane receptor (
pdt
K) or the inner membrane transporter (
pdt
E) of DSM 3601 did
not produce appreciable negative effects on the transcriptional response to exogenous PDTC,
even though the respective strains could no longer use PDTC for iron acquisition (Leach and
Lewis,
2006
). Together these data indicate that PDTC can enter cells through a non-specific
mechanism where it can then bind to and activate PdtC for transcriptional activation.
The functions of other genes found in
pdt
clusters are less clear either because they have
not been tested experimentally (
pdt
N,
pdt
P), because their inferred functions do not fit within
current models of biosynthesis or transport (
pdt
M), or because alignment searches in existing
databases have not yielded informative hits (
pdt
L). PdtN is a putative ABC transporter of the
major facilitator superfamily (MFS). It has similarity to efflux permeases such as AraJ, leading
to speculation that it may be involved in export of PDTC. A truncated
pdt
gene cluster that
lacked
pdt
N, O and P genes showed no CT transformation, whereas deletion of
pdt
O and
pdt
P
led to decreased but detectable CT transformation (Lewis et al.,
2000
). It was not determined
whether PDTC had accumulated intracellularly in the
pdt
N deletion-containing strain.
PdtP is a putative S-adenosylmethionine-dependent O-methyltransferase. Since deletion of
pdt
P (and
pdt
O) led to a decrease rather than complete stop in PDTC production, it can be
inferred as having an accessory function, perhaps enhancing gene expression or biosynthetic
activity/pathway efficiency. No O-methyltransferase activity is required in either of the
hypothesized PDTC biosynthetic pathways presented thus far (described above), but a potential
role in regulation via post-translational modification has been presented (Sepulveda-Torres
et al.,
2002
). To achieve increased PDTC production, the target of this modification could be a
biosynthetic enzyme or a transcriptional regulator.
It is not known whether
pdt
M is required for PDTC production; no
pdt
M mutants have
been tested. PdtM is a putative pyridoxal phosphate-containing enzyme with similarity to
several aminotransferases. Again, the suggested biochemical function (functional group trans-
fers involving pyridoxal phosphate) has not been incorporated in current hypotheses regarding
PDTC biosynthesis.
PdtL is perhaps the most enigmatic gene product of the
pdt
cluster. No tests of its necessity
for PDTC biosynthesis have been described to date. It is a protein of 87,538 daltons (Da) and
sequence alignment searches identified as homologs only hypothetical proteins (proteins found
through
in silico
translation of genome sequences but as yet not known to be expressed in the
respective organisms).
9.5 PDTC-MEDIATED CT TRANSFORMATION
PDTC is produced by aerobic or denitrifying
P. stutzeri
KC cells growing under iron-limited
conditions. The following sections summarize major factors affecting PDTC production and CT
transformation activity.
Search WWH ::
Custom Search