Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon

Abstract

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a beta-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

Figure 1

Biochemical organization of the PhAc-CoA catabolon. PHPhAs, poly(3-hydroxyphenylalkanoic acid)s; TA, tropic acid; TCA, tricarboxylic acid cycle. Box indicates the catabolon core.

Figure 2

(A) PCR amplification of the DNA fragments included between a sequence of Tn5 (5′ → 3′ ACTTGTGTATAAGAGTCAG) and different oligonucleotides present in the genome of P. putida U separated by 400–600 bp. Mw, size markers; C, control without oligonucleotides. (B) Analysis of the P₁, P₂, and P₃ promoters. The existence of a functional promoter implies the expression of the lacZ gene present in the plasmid pRS551. Blue color is generated by the hydrolysis of 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal). The upper three patches are E. coli MC4100 transformed with the parental promoter-probe plasmid; the lower three patches are E. coli MC4100 transformed with the recombinant plasmid carrying the indicated promoter.

Figure 3

Gene disruption by homologous recombination. P₁, P. putida promoter belonging to the pha catabolic pathway; Km^R, kanamycin-resistance gene; P_Km^R, kanamycin-resistance promoter; T_Km^R kanamycin-resistance terminator.

Figure 4

Genetic map of the pha catabolic pathway and hypothetical function of the different proteins. A putative intermediate is shown in brackets.

Figure 5

Bacterial growth (OD₅₄₀) of P. putida U (▾, ▿) or mutants in which the genes encoding: PhAc-CoAL (phaE) (•, ○) or the enoyl-CoA hydratase I (phaA) had been disrupted (▪, □) were cultured in MM containing 5 mM PhAc (▾, •, ▪) or in MM containing 5 mM PhH (▿, ○, □). Disruption of phaA, phaB, phaC, or phaD genes led to similar results.

Figure 6

(A) Multiple alignment of the amino acid sequence of PhaJ, the proline permease from Salmonella typhimurium (PUTP-SALTY) and a sodium/solute symport protein from E. coli (YJCG-ECOLI). (B) Putative transmembrane regions of PhaJ. (C) Pairwise alignment of the amino acid sequence of PhaK from P. putida U and the D2 protein from Pseudomonas aeruginosa (OprD). ∗ indicates that a position in the alignment is perfectly conserved; ., that it is well conserved.

Figure 7

Analysis of the PhAc transport system when P. putida U and various mutants were cultured in MM containing glucose (5 mM), and once the sugar had been exhausted 1 mM PhAc (inducer) was added to the bacterial population. Shown is [1-¹⁴C]PhAc uptake by P. putida U (▾) and by mutants in which the genes encoding permease (▪), the specific channel (▴), or the repressor protein (•) have been disrupted. Arrow indicates addition of PhAc.