Intracellular 2-keto-3-deoxy-6-phosphogluconate is the signal for carbon catabolite repression of phenylacetic acid metabolism in Pseudomonas putida KT2440

Abstract

The growth pattern of Pseudomonas putida KT2440 in the presence of glucose and phenylacetic acid (PAA), where the sugar is used in preference to the aromatic compound, suggests that there is carbon catabolite repression (CCR) of PAA metabolism by glucose or gluconate. Furthermore, CCR is regulated at the transcriptional level. However, this CCR phenomenon does not occur in PAA-amended minimal medium containing fructose, pyruvate or succinate. We previously identified 2-keto-3-deoxy-6-phosphogluconate (KDPG) as an inducer of glucose metabolism, and this has led to this investigation into the role of KDPG as a signal compound for CCR. Two mutant strains, the edd mutant (non-KDPG producer) and the eda mutant (KDPG overproducer), grew in the presence of PAA but not in the presence of glucose. The edd mutant utilized PAA even in the presence of glucose, indicating that CCR had been abolished. This observation has additional support from the finding that there is high phenylacetyl-CoA ligase activity in the edd mutant, even in the presence of glucose+PAA, but not in wild-type cells under the same conditions. Unlike the edd mutant, the eda mutant did not grow in the presence of glucose+PAA. Interestingly, there was no uptake and/or metabolism of PAA in the eda mutant cells under the same conditions. Targeted disruption of PaaX, a repressor of the PAA operon, had no effect on CCR of PAA metabolism in the presence of glucose, suggesting that there is another transcriptional repression system associated with the KDPG signal. This is the first study to demonstrate that KDPG is the true CCR signal of PAA metabolism in P. putida KT2440. INTRODUCTIONIn Pseudomonas putida, the phenylacetic acid (PAA) catabolic gene clusters consist of 15 genes that are organized into five contiguous operons (del Peso-Santos et al., 2008; Di Gennaro et al., 2007; Jiménez et al., 2002;Luengo et al., 2001;Olivera et al., 1998). The PAA pathway ( Fig. 1) is a major route of a complex functional unit that catalyses the transformation of structurally related compounds such as styrene, tropic acid, ethylbenzene and 2-phenylethylalanine into phenylacetyl-CoA (PA-CoA), a common intermediate (Luengo et al., 2001). It has been reported that PA-CoA, and not PAA, is the true inducer of the PAA catabolic pathway and that the PaaX repressor controls PAA-catabolic gene expression (Ferrández et al., 2000;García et al., 2000). Binding of PaaX to its cognate DNA binding sites is abolished when the inducer, PA-CoA, interacts with the PaaX protein (Bartolomé-Martín et al., 2004; del Peso-Santos et al., 2006; Ferrández et al., 2000;García et al., 2000). Interestingly, along with the StyR/StyS system, PaaX also regulates the styrene degradation pathway by binding to the promoter region of the styrene upper pathway in Pseudomonas sp. strain Y2 (del PesoSantos et al., 2006). PaaX is also involved in the transcriptional regulation of the pac gene, which encodes penicillin G acylase in Escherichia coli W (Galán et al., 2004;Kim et al., 2004). PaaX is involved in the co...

Carbon catabolite repression inPseudomonas: optimizing metabolic versatility and interactions with the environment

“…In Pseudomonas, however, only fructose is transported into the cell through a PTS system , and fructose does not play an important role in CCR (Cases et al, 1999;Velázquez et al, 2004;Kim et al, 2009). In Pseudomonas, however, only fructose is transported into the cell through a PTS system , and fructose does not play an important role in CCR (Cases et al, 1999;Velázquez et al, 2004;Kim et al, 2009).…”

“…Moreover, phosphorylation of PtsO seems to be needed for glucose-mediated repression of Pu, because mutation of the conserved His residue at PtsO that presumably serves as a phosphoryl acceptor rendered the protein inactive. However, there are several indications that the levels of glucose metabolites in P. putida, and in particular those of 2-keto-3-deoxy-6-phosphogluconate (KDPG), are critical for glucose-mediated catabolite repression, and may directly or indirectly affect the phosphorylation of PtsN (Velázquez et al, 2004;Kim et al, 2009). In the presence of glucose, transfer of the phosphoryl group to PtsO would increase the levels of nonphosphorylated PtsN, the form that is believed to mediate Pu repression.…”

Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86

“…On the other hand, glucose suppresses the toluene metabolism through 2-keto-3-deoxy-6-phosphogluconate (KDPG), which acts as a signal molecule to enable the repression. KDPG has also been shown to act as a signal molecule in the repression of phenylacetic acid metabolism (Kim et al, 2009). Transcriptomic studies from glucose+toluene-grown cells of strain KT2440 demonstrate the downregulation of genes involved in the glucose transport (OprB channel, and components of the ABC transport system).…”

UV Mutagenesis of Aspergillus flavus for Enhanced Mannanase Synthesis and Catabolite Activation Studies

“…The cyclic AMP-CRP complex facilitates binding of RNA polymerase to the promoter site of catabolite sensitive operon which alleviates the repression state. The molecular machinery behind CCR varies widely across the species, with CCR being enforced and operable at different levels including transcriptional (Kimata et al, 1997), post-transcriptional (Gorke and Vogel, 2008), translational (Parker et al, 1997) and biochemical regulations (Kim et al, 2009) which has fascinated scientists for over half a century.…”