Catabolism of aromatics in Pseudomonas putida U

Abstract

Phenylacetic acid (PhAcOH) and 4‐hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4‐hydroxyphenylacetic acid‐3‐hydroxylase; and those blocked in the gene encoding homoprotocatechuate‐2,3‐dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc‐CoA ligase protein; and those lacking this enzyme activity.

Aerobic catabolism of phenylacetic acid in Pseudomonas putida U: biochemical characterization of a specific phenylacetic acid transport system and formal demonstration that phenylacetyl-coenzyme A is a catabolic intermediate

“…The existence of a different pathway involved in the specific degradation of PA is supported by two additional pieces of evidence: (i) phenylacetyl-CoA ligase (PCL) was not induced when P. putida U was cultured in a defined medium containing either 4-hydroxy-PA or many other phenylacetyl derivatives as the sole carbon source (26), and (ii) PA and 4-hydroxy-PA are catabolized by two unrelated pathways (32). These facts, together with the description of this enzyme (PCL) in other bacteria which also catabolized PA aerobically (35) as well as the identification of PA-CoA inside the bacteria (see below), lend a new perspective to this kind of research.…”

The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of l -Phenylalanine, l -Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

“…putida U and the mutant strains were cultured in MM containing Phe, Tyr, or 3-OH-PhAc (5 mM) as a source of intermediates and 4-OH-PhAc (5 mM) for support of bacterial growth. Moreover, 4-OH-PhAc, which requires a specific catabolic route, is not degraded through the homogentisate pathway (48), and it is not a substrate of 4-OH-PhPyr dioxygenase. When required, the cultures were centrifuged (5,000 ϫ g) and filtered through Millipore filters (pore size, 0.45 m) to eliminate bacteria.…”

“…To this end, we studied the catabolism of Phe and Tyr in Pseudomonas putida, a ␥-proteobacterium with great metabolic versatility that can use these amino acids as carbon and energy sources and that is considered a model system for environmental studies (35,49,50,75). By using two strains of P. putida that show different catabolic abilities with some natural aromatic compounds, P. putida U, a strain that is able to grow with 4-hydroxyphenylacetate (4-OHPhAc) or 3-hydroxyphenylacetate (3-OH-PhAc) as the sole carbon source (48), and P. putida KT2440, a strain whose complete genome is known (47) and that is unable to use of E. coli cells was carried out by using the RbCl method or by electroporation (Gene Pulser; Bio-Rad) (14). Oligonucleotides were synthesized with an Oligo-1000 M nucleotide synthesizer (Beckman Instruments).…”

Metabolism and Preferential Utilization of Phenylacetic acid and 4-Hydroxyphenylacetic Acid in Pseudomonas putida CSV86

“…A bacterial strain capable of utilizing PA and 4-HPA via different metabolic routes has been demonstrated earlier in P. putida U and Azoarcus evansii [5,14].…”

“…Homoprotocatechuate pathway involves initial hydroxylation of 4-HPA at C3 position to yield 3,4-DHPA, which is subsequently ring-cleaved to yield semialdehyde intermediate. This yellow colored intermediate ultimately leads to the formation of succinic and pyruvic acid (Figure 1, route c) [14,[24][25][26][27].…”