BIOAUGMENTATION WITH PSEUDOMONAS STUTZERI KC FOR CARBON TETRACHLORIDE REMEDIATION - Bioaugmentation for Groundwater Remediation - page 257

even higher. A stability constant of that magnitude suggests that Cu(II) augmentation would
not normally be necessary for effective in situ remediation.
In addition to Cu(II) and Fe(III), PDTC forms complexes with a variety of elements,
including naturally occurring and synthetic transition metals, actinides, and lanthanides
(Cortese et al., 2002 ; Neu et al., 2000 , 2001 ). In several cases, the PDTC:metal complexes of
toxic elements were less inhibitory than the metals alone (Cortese et al., 2002 ). This finding, and
the reactivity of PDTC with other toxic elements suggests a possible function of PDTC in
detoxification (Zawadzka et al., 2009 ). Whether this type of selection has influenced evolution
of the synthesis and use of PDTC is unknown.

9.4 GENETIC REQUIREMENTS FOR PDTC PRODUCTION
By characterizing genetic requirements of a particular biochemical system, information can
be provided for use in genetic 'improvement' or optimization above naturally-evolved levels of
expression. Also, insight may be gained into the biosynthetic mechanisms and pathways, which
can allow greater predictive capability with regard to which nutrients and/or cofactors may
become limiting during sustained operation of a bioprocess. Genes necessary for CT transfor-
mation were first identified independently by two groups, using either a spontaneously-arisen
mutant (Lewis et al., 2000 ), or transposon-derived mutants (Sepulveda-Torres et al., 1999 ).
Deoxyribonucleic acid (DNA) capable of complementing the CT transformation/PDTC
production phenotype of those mutants was obtained and sequenced, allowing further delinea-
tion of a gene cluster sufficient to confer PDTC production upon non-producing pseudomo-
nads (Lewis et al., 2000 ).
That gene cluster was denoted the pdt gene cluster and the alignments of the amino acid
sequences of proteins encoded within the pdt gene cluster with annotated proteins allowed
assignment of putative biosynthetic, transport (uptake and export) and regulatory functions to
individual genes (Lewis et al., 2000 ; Sepulveda-Torres et al., 2002 ). The sequence of a second
pdt gene cluster (that of P. putida DSM 3601) has since been obtained (Genbank accession
AY319946) and shown to be capable of conferring PDTC production upon other pseudomonads
(T. A. Lewis, Montana State University, unpublished). In addition, genes from different
organisms required for synthesis of a second thiocarboxylic acid-containing siderophore,
thioquinolobactin, have been described ( qbs genes; Matthijs et al., 2004 ).
The alignment of the two pdt gene clusters is shown in Figure 9.4 . The minimum set of
genes sufficient for PDTC production has not yet been established, and it is possible that some
genes within the clusters are superfluous or have modulating effects as opposed to providing
an absolutely required function. However, comparisons of pdt gene clusters can contribute to
our understanding of minimal requirements for conferring a selective benefit, assuming that
sufficient time has elapsed during their evolution to allow deletion of random, intervening

P. stutzeri KC

CDE FGH I J K L M N O P

P. putida
DSM 3601

KCPEL M F HI J ' N

1 kb

Figure 9.4. Gene clusters encoding PDTC biosynthesis and utilization functions in P. stutzeri KC
and P. putida DSM 3601. Sequences used for open reading frame identification are GenBank
accession #s AF196567and AY319946.