Physiological and molecular genetic evaluation of the dechlorination agent, pyridine‐2,6‐bis(monothiocarboxylic acid) (PDTC) as a secondary siderophore of Pseudomonas

Abstract

The bacterial metabolite and transition metal chelator pyridine-2,6-dithiocarboxylic acid (PDTC), promotes a novel and effective means of dechlorination of the toxic and carcinogenic pollutant, carbon tetrachloride. Pyridine-2,6-dithiocarboxylic acid has been presumed to act as a siderophore in the Pseudomonas strains known to produce it. To explore further the physiological function of PDTC production, we have examined its regulation, the phenotype of PDTC-negative (pdt) mutants, and envelope proteins associated with PDTC in P. putida strain DSM 3601. Aspects of the regulation of PDTC production and outer membrane protein composition were consistent with siderophore function. Pyridine-2,6-dithiocarboxylic acid production was coordinated with production of the well-characterized siderophore pyoverdine; exogenously added pyoverdine led to decreased PDTC production, and added PDTC led to decreased pyoverdine production. Positive regulation of a chromosomal pdtI-xylE transcriptional fusion, and of a 66 kDa outer membrane protein (IROMP), was seen in response to exogenous PDTC. Tests with transition metal chelators indicated that PDTC could provide a benefit under conditions of metal limitation; the loss of PDTC biosynthetic capacity caused by a pdtI transposon insertion resulted in increased sensitivity to 1,10-phenanthroline, a chelator that has high affinity for a range of divalent transition metals (e.g. Fe(2+), Cu(2+), Zn(2+)). Exogenously added PDTC could also suppress a phenotype of pyoverdine-negative (Pvd-) mutants, that of sensitivity to EDDHA, a chelator with higher affinity and specificity for Fe(3+). Measurement of 59Fe incorporation showed uptake from 59Fe:PDTC by DSM 3601 grown in low-iron medium, but not by cells grown in high iron medium, or by the pdtI mutant, which did not show expression of the 66 kDa envelope protein. These data verified a siderophore function for PDTC, and have implicated it in the uptake of transition metals in addition to iron.

Biology of Pseudomonas stutzeri

“…It is highly likely that this is a secondary siderophore in this strain (200). P. stutzeri KC can degrade carbon tetrachloride (CT) (trade names, Freon 10 and Halon 104) to carbon dioxide, chloride ions, and other nonvolatile compounds, such as formate.…”

“…Strain KC secretes the molecule PDTC (pyridine-2,6-bisthiocarboxylate), which has a siderophore function. PDTC has been implicated in the uptake of other transition metals in addition to iron (200). It is believed to be an iron chelator that is fortuitously involved in CT transformation.…”

“…A pdt locus corresponding to the PDTC biosynthetic pathway in the strain KC genome has been mapped and cloned as a 25,746-bp insert in the pT31 cosmid (199,200,315). The pdt operon was sequenced, and a low-density PDTC microarray consisting of 17 PCR-amplified ORFs was printed on chemically modified glass substrates.…”

Role for Ferredoxin:NAD(P)H Oxidoreductase (FprA) in Sulfate Assimilation and Siderophore Biosynthesis in Pseudomonads

“…Using a pyoverdine-deficient genetic background, a transposon insertion library was screened for strains with altered PDTC production by exploiting the iron-containing dye chrome azurol S (CAS) (12,27). That screening procedure yielded only a single pdt insertion (pdtI) that has been described elsewhere (24). In the present study, we describe other insertions that led to the complete loss of or to a reduction in PDTC production.…”

“…1 predicts PDTC production to be limited by factors affecting intracellular cysteine levels, as has been seen for pyochelin and thioquinolobactin (16,17,24). The biochemistry and genetics of cysteine biosynthesis has been extensively studied in Gram-negative bacteria in which it constitutes a major regulon (25).…”