Figure 3 Bacterial growth of A1501 cultured in minimal medium containing 4 mM benzoate (black triangle), 8 mM benzoate (clear triangle),
0.4 mM 4-hydroxybenzoate (black dot) or 0.8 mM 4-hydroxybenzoate www.selleckchem.com/products/blasticidin-s-hcl.html (clear dot). High-performance liquid chromatography (HPLC) was used to measure the concentrations of catechol and muconate in the Tozasertib mw culture supernatants of the wild type A1501 and pcaD mutant A1603 grown on benzoate as the sole carbon source (Figure 4; see Additional file 1). During the initial phase of benzoate catabolism by A1501, small amounts of catechol (~30 μM) and cis, cis-muconate (~500 nM) were detected. After 24 h, benzoate was completely removed from the culture supernatants, and no metabolites could be detected (see Additional file 1). The inability of the pcaD mutant A1603 to grow on benzoate was further confirmed by HPLC analysis of culture supernatants. After 48 h, the concentration of benzoate remained almost unchanged in the culture supernatant of the mutant, while accumulation of catechol and cis, cis-muconate
was detected by Palbociclib supplier HPLC (Figure 4). As shown in Figure 1B, inactivation of PcaD completely blocked the conversion of β-ketoadipate enol-lactone to β-ketoadipate, resulting in accumulation Aldehyde dehydrogenase of the intermediates catechol and cis, cis-muconate derived from benzoate. These results provide experimental evidence that the two branches of the β-ketoadipate pathway converge at β-ketoadipate enol-lactone and that the products of pcaDIJF complete the conversion of the latter to TCA cycle intermediates in P. stutzeri A1501,
as documented in other Pseudomonas strains . Figure 4 Conversion of benzoate (BEN) to catechol (CAT) and cis, cis -muconate (CCM) by the pcaD mutant A1603. Cells were grown for 48 h in minimal medium supplemented with 4 mM benzoate. The elution profile of compounds separated by HPLC is shown. Accumulations of the intermediates catechol and cis, cis-muconate are indicated by red vertical arrows. As mentioned above, A1501 can grow well on benzoate, but not on 4-hydroxybenzoate, as the sole carbon and energy source. Therefore, we focused on the genetic organization of the A1501 ben-cat region. As shown in Figure 5A, nine ben and cat genes are in the same transcriptional orientation and the lengths of the intergenic regions vary.