Biomedical Engineering Reference
In-Depth Information
DNA (Lawrence and Roth,
1996
). Comparisons that include the
qbs
gene cluster also may help
elucidate the genes required for the biosynthesis of thiocarboxylate groups on siderophore
molecules.
The two
pdt
clusters show strong conservation in terms of gene content, with significant
differences in terms of synteny (the co-location of genes along the chromosome). Two genes
are not conserved among the
pdt
clusters: (1)
pdt
D, encoding a putative methyltransferase, is
found only in the strain KC cluster; and (2)
pdt
O, a putative acylCoA dehydrogenase (ACAD),
also has no clear homolog in the DSM 3601
pdt
cluster. PdtO' of the DSM 3601 cluster is also a
putative ACAD but with only approximately 28% sequence identity to PdtO, whereas the other
apparent orthologs show between 46% and 79% identity. This suggests that PdtO and PdtO'
encode a common function (presumably biosynthetic) for PDTC-producing organisms, but may
have been independently 'recruited' into their respective gene clusters.
The similarity of PDTC to dipicolinic acid (DPA) led Hildebrand et al. (
1986
) to assume its
intermediacy in the PDTC biosynthetic pathway. In this view, PDTC biosynthesis could be
envisioned as a branch off the lysine and diaminopimelate pathway of bacteria (and plants)
which includes 4-hydroxytetrahyrodipicolinate (Blickling et al.,
1997
). Conversion into DPA
could be accomplished by a dehydratase, and a flavin adenine dinucleotide (FAD)-type dehy-
drogenase, potentially encoded by
pdt
I. Apparent confirmation of DPA as a precursor of PDTC
was obtained from experiments that showed incorporation of a deuterium label from DPA into
PDTC (Hildebrand et al.,
1986
). Mass spectrometry of other labeled compounds identified
sulfenic acids, and led the authors to propose a pathway including an acylsulfenic acid
intermediate, hydrolysis of the acylsulfenic acid, and reduction to thiocarboxylic acid.
A monooxygenase would seem critical to such a pathway (for oxygen addition to a thioester)
but is not found among the
pdt
genes. The sequence data have instead suggested a different
route, related to the well-characterized production of thiocarboxylic acids at the C-termini of
certain proteins (e.g., MoeD, a small subunit of
E. coli
molybdopterin synthase or ThiS [Begley
et al.,
1999
; Godert et al.,
2007
; Leimk¨hler and Rajagopalan,
2001
; Taylor et al.,
1998
]).
Homologs of
pdt
F, G, H, and J are found in the
qbs
gene cluster (Matthijs et al.,
2004
).
The pathway suggested by the putative enzymatic activities includes adenylate activation of
a carboxylic acid precursor by PdtJ, followed by sulfur transfer from cysteine. A cysteine
desulfurylase (PdtF) would pass the sulfur atom from the cysteine via a cysteine persulfide
modification. This would be transferred to a sulfur transferase enzyme (PdtGH) to form a
C-terminal thiocarboxylic acid. How a protein thiocarboxylate is used to form a small-molecule
thiocarboxylate like PDTC or quinolobactin is not clear. Resolution of the actual PDTC
biosynthetic pathway awaits biochemical characterization of the relevant enzymatic activities
encoded in
pdt
clusters.
Expression of
pdt
genes is under transcriptional regulation involving the ferric uptake
regulator (
fur
; Sepulveda-Torres et al.,
2002
) and the product of the
pdt
C gene (Morales and
Lewis,
2006
). This regulation is ostensibly quite similar to that seen for the siderophore
pyochelin, characterized in
P. aeruginosa
(Michel et al.,
2005
).
fur
regulation explains the
original observation of iron regulation of CT transformation activity; the
fur
repressor binds
intracellular ferrous iron when supplies are adequate and forms a DNA-binding complex that
blocks transcription downstream of its cognate recognition elements ('
fur
box' sequences).
fur
box elements have been identified upstream of
pdt
F in both
pdt
gene clusters, and upstream of
pdt
K and within the
pdt
C/P intergenic region of DSM 3601 (Sepulveda-Torres et al.,
2002
);
Gen Bank AF196567, AF149851, AY319946). The PdtC protein is an AraC-type transcriptional
regulator homologous to PchR, a regulator of pyochelin biosynthetic and transport genes
dependent upon pyochelin for DNA-binding activity (Michel et al.,
2005
). Maximal expression
from the
pdt
F promoter requires the
pdt
C gene and the siderophore (PDTC) itself, as well as
Search WWH ::
Custom Search