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Catabolism of Phenylacetic Acid in Escherichia coli

CHARACTERIZATION OF A NEW AEROBIC HYBRID PATHWAY*
Open AccessPublished:October 02, 1998DOI:https://doi.org/10.1074/jbc.273.40.25974
      The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced. It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA. The 14 paa genes are organized in three transcription units:paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing thepaaX regulatory gene. The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA). The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation. The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid β-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates. Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified. ThePa promoter is negatively controlled by thepaaX gene product. As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative. The aerobic catabolism of PA in E. coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria.
      PA
      phenylacetic acid
      bp
      base pair(s)
      FNR
      ferredoxin-NADP+ reductase
      2-HPA
      2-hydroxyphenylacetate
      HPLC
      high performance liquid chromatography
      kb
      kilobase pair(s)
      ORF
      open reading frame
      PA-CoA
      phenylacetyl-coenzyme A
      PCR
      polymerase chain reaction.
      Escherichia coli living in the animal gut encounters aromatic compounds such as phenylacetic acid (PA),1 phenylpropionic acid, and their hydroxylated derivatives, as a result of the action of intestinal microflora on plant constituents, the amino acids phenylalanine and tyrosine, fatty acids with a terminal phenyl substituent, and some of their metabolites (
      • Ferrández A.
      • Garcı́a J.L.
      • Dı́az E.
      ,
      • Burlingame R.
      • Chapman P.J.
      ,
      • Mohamed M.
      • Fuchs G.
      ). The aerobic catabolism of these aromatic compounds by E. coli could occur close to the epithelial cells in the guts of warm-blooded animals, as well as in soil, sediment, and water once E. coli is excreted from its intestinal residence (
      • Savage D.C.
      ). The ability of E. coli to mineralize 3- and 4-hydroxyphenylacetic acids (
      • Cooper R.A.
      • Skinner M.A.
      ), 3-phenylpropionic, 3-(3-hydroxyphenylpropionic), and 3-hydroxycinnamic acids (
      • Burlingame R.
      • Chapman P.J.
      ,
      • Burlingame R.P.
      • Wyman L.
      • Chapman P.J.
      ), and phenylacetic acid (
      • Burlingame R.
      • Chapman P.J.
      ,
      • Cooper R.A.
      • Jones D.C.N.
      • Parrott S.
      ) has been reported previously. Recently, the molecular characterization of these catabolic pathways, with the only exception of that for PA degradation, has been carried out (
      • Ferrández A.
      • Garcı́a J.L.
      • Dı́az E.
      ,
      • Prieto M.A.
      • Dı́az E.
      • Garcı́a J.L.
      ,
      • Stringfellow J.M.
      • Turpin B.
      • Cooper R.A.
      ,
      • Spence E.L.
      • Kawamukai M.
      • Sanvoisin J.
      • Braven H.
      • Bugg T.D.H.
      ,
      • Dı́az E.
      • Ferrández A.
      • Garcı́a J.L.
      ), demonstrating that E. coliis endowed with its own set of genes and enzymes for the catabolism of aromatic compounds, and that they are similar to those of other microorganisms more relevant in the environment such as bacteria of the genus Pseudomonas.
      Although PA is a common source of carbon and energy for a wide variety of microorganisms, the bacterial catabolism of this natural aromatic compound is still poorly understood (
      • Miñambres B.
      • Martı́nez-Blanco H.
      • Olivera E.R.
      • Garcı́a B.
      • Dı́ez B.
      • Barredo J.L.
      • Moreno M.A.
      • Schleissner C.
      • Salto F.
      • Luengo J.M.
      ,
      • Vitovski S.
      ). Earlier reports suggested that aerobic PA catabolism implicated the typical initial attack by hydroxylation of the aromatic ring with the formation of the corresponding 2,5- or 3,4-dihydroxyphenylacetate as intermediates (
      • Vitovski S.
      ). However, much of this evidence was circumstantial, and none of the typical aerobic routes that could explain PA degradation were responsible of this catabolism in different PA-degrading bacteria (
      • Vitovski S.
      ,
      • Olivera E.R.
      • Reglero A.
      • Martı́nez-Blanco H.
      • Fernández-Medarde A.
      • Moreno M.A.
      • Luengo J.M.
      ). According to these data, it has been recently shown thatPseudomonas putida U mineralizes PA aerobically through a novel catabolic pathway, which does not follow the conventional routes for the aerobic catabolism of aromatic compounds and whose first step is the activation of PA to phenylacetyl-coenzyme A (PA-CoA) by the action of a PA-CoA ligase (
      • Miñambres B.
      • Martı́nez-Blanco H.
      • Olivera E.R.
      • Garcı́a B.
      • Dı́ez B.
      • Barredo J.L.
      • Moreno M.A.
      • Schleissner C.
      • Salto F.
      • Luengo J.M.
      ,
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ). In this sense, the participation of a PA-CoA ligase in the aerobic catabolism of PA has been also inferred from its specific induction during growth on PA of different bacterial strains (
      • Vitovski S.
      ,
      • Velasco A.
      • Alonso S.
      • Garcı́a J.L.
      • Perera J.
      • Dı́az E.
      ).
      Here we present the cloning, genetic characterization, mechanism of regulation, and a partial biochemical characterization of the PA biodegradation pathway from E. coli W. This work reveals that the PA degradation in E. coli follows an unusual route for the aerobic catabolism of aromatic compounds, which involves CoA derivatives. With the molecular characterization of thepaa-encoded pathway, all aromatic catabolic routes so far reported in E. coli are now described at the molecular level.

      DISCUSSION

      In this report, we describe the molecular characterization of the PA catabolic pathway of E. coli. Previous work had shown that, whereas E. coli K-12 and E. coli W were able to grow on PA as the sole carbon source, this catabolic ability was lacking in E. coli C (
      • Burlingame R.
      • Chapman P.J.
      ). The molecular analysis presented here confirm the previous observations, indicating that a 33.3-kb DNA fragment that appears to contain the paa genes responsible of the PA catabolism in E. coli W is lacking in E. coli C as well as in the mutant strain E. coliW14. However, we have shown here that the ability of E. coliK-12 to grow on PA was strain-dependent, with point mutations or small gene rearrangements being the most probable reason for the PA phenotype of some K-12 laboratory strains such as DH5α, HB101, and DH1.
      The paa genes from E. coli W were located in a chromosomal 15.4-kb DNA fragment cloned in plasmid pAAD, and they mapped at the right end of the mao region (Fig. 1 A), which is involved in the transformation of 2-phenylethylamine into PA (
      • Ferrández A.
      • Prieto M.A.
      • Garcı́a J.L.
      • Dı́az E.
      ,
      • Yamashita M.
      • Azakami H.
      • Yokoro N.
      • Roh J.H.
      • Suzuki H.
      • Kumagai H.
      • Murooka Y.
      ,
      • Hanlon S.P.
      • Hill T.K.
      • Flavell M.A.
      • Stringfellow J.M.
      • Cooper R.A.
      ). As the equivalentmao genes in E. coli K-12 have been mapped at min 31.0 on the chromosome (
      • Sugino H.
      • Sasaki M.
      • Azakami H.
      • Yamashita M.
      • Murooka Y.
      ), and two PA mutants of E. coli K-12 had been located in this chromosomal region (
      • Cooper R.A.
      • Jones D.C.N.
      • Parrott S.
      ), a similar location of the paa genes in the chromosome of E. coli W can be suggested.
      The nucleotide sequence of the paa cluster revealed the presence of 14 ORFs, paaZpaaABCDEFGHIJKpaaXY (Figs. 1 and 2), that corresponded with those of unknown function whose Protein Identification Database accession numbers are g1787653–g1787664, g1787666, and g1787667, respectively, and that have been recently sequenced in E. coli K-12 (accession numbers AE000236,AE000237, D90777, and D90778) (
      • Blattner F.R.
      • Plunkett III G.
      • Bloch C.A.
      • Perna N.T.
      • Burland V.
      • Riley M.
      • Collado-Vides J.
      • Glasner J.D.
      • Rode C.K.
      • Mayhew G.F.
      • Gregor J.
      • Davis N.W.
      • Kirkpatrick H.A.
      • Goeden M.A.
      • Rose D.J.
      • Mau B.
      • Shao Y.
      ). Although the left end of thepaa cluster was near to the maoA gene both in E. coli W and K-12, the right end of the paacluster was different in the two strains. Thus, although thepaaY stop codon was found 231 bp upstream of the ATG start codon of the ydbC gene in E. coli W (Fig. 2), a 9.2-kb sequence encoding a long ORF (ydbA) disrupted by two insertion sequences (IS2 and IS30) was found between paaY (Protein Identification Database accession number g1787667) and ydbC in E. coli K-12 (
      • Kröger M.
      • Wahl R.
      ). The presence of insertion sequences near the paa cluster and the location of this cluster in a nonessential region of the chromosome (
      • Henson J.M.
      • Kopp B.
      • Kuempel P.L.
      ) provide some clues on the possible mechanisms of gene mobilization of a catabolic cassette that would enhance bacterial adaptability, and could explain the heterogeneity observed among different E. coli strains respect to their ability to mineralize PA. It is also noteworthy that the mao genes for the metabolism of 2-phenylethylamine, an aromatic amine whose degradation gives rise to PA, lie adjacent to the paa cluster responsible for the further catabolism of PA. This association between genes belonging to the same catabolon (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ), i.e. genes involved in convergent degradative routes, could be considered as an important evolutionary and adaptive advantage. Another example of such association within a PA catabolon has been recently described in the pathway for styrene degradation in Pseudomonas sp. Y2, where the stygenes responsible of the oxidation of styrene to PA are in tight association with the genes involved in PA degradation (
      • Velasco A.
      • Alonso S.
      • Garcı́a J.L.
      • Perera J.
      • Dı́az E.
      ).
      A. Velasco, S. Alonso, J. Perera, J. L. Garcı́a, and E. Dı́az, unpublished data.
      The genetic arrangement of the paa cluster and the mutagenesis of pAAD with transposon Tn1000 revealed that the 14 paa genes are organized in three transcriptional units, two of them, paaZ and paaABCDEFGHIJK, essential for the catabolism of PA, and a third one, paaXY, that contains the paaX regulatory gene. An overall sequence comparison analysis of the paa gene products showed that they were homologous to the recently described pha genes responsible of the catabolism of PA in P. putida U (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ) (Fig. 8 B). Here, we have presented experimental evidence that the paaK gene product is the PA-CoA ligase of E. coli W (Fig. 3 B), an activity that had been detected in this strain when it was grown in PA-containing medium (
      • Vitovski S.
      ). Analysis of the primary structure of PaaK (Fig. 2) revealed that residues103SSGTTGKPTV112 match the AMP-binding site consensus sequenceT(SG)-S(G)-G-(ST)-T(SE)-G(S)-X-P(M)-K-G(LAF) in acyl-adenylate-forming enzymes (residues that predominate at that position are underlined, with alternates given in parentheses;X represents a hypervariable position) (
      • Chang K.-H.
      • Xiang H.
      • Dunaway-Mariano D.
      ). It is worth noting that the Lys residue of this signature motif is substituted by Thr in all phenylacetyl-CoA ligases so far sequenced, i.e.PaaK, PhaE (
      • Miñambres B.
      • Martı́nez-Blanco H.
      • Olivera E.R.
      • Garcı́a B.
      • Dı́ez B.
      • Barredo J.L.
      • Moreno M.A.
      • Schleissner C.
      • Salto F.
      • Luengo J.M.
      ) and PaaK_Y2 (
      • Velasco A.
      • Alonso S.
      • Garcı́a J.L.
      • Perera J.
      • Dı́az E.
      ), an observation that supports recent studies showing that this residue does not assume a major role in ATP binding (
      • Chang K.-H.
      • Xiang H.
      • Dunaway-Mariano D.
      ). The sequences236DIYGLSE242 and302YRTRD306 (underlined are the stringently conserved residues) in PaaK also match the conserved motifs II and III that may contribute to the substrates binding sites in acyl-adenylate-forming enzymes (
      • Chang K.-H.
      • Xiang H.
      • Dunaway-Mariano D.
      ).
      Figure thumbnail gr8
      Figure 8Comparison of the paa cluster of E. coli W with the pha cluster of P. putida U. A, comparison of the genetic organization of the paa and pha clusters.Blocks with similar shading orhatching indicate homologous regions encoding potential functional units in both gene clusters. The location and size of the intergenic regions, are also indicated. Bent arrowsrepresent the promoters. B, percentages of amino acid sequence identity between the analogous paa and pha gene products. Note that genes phaJK do not have counterparts in the paa cluster, and that genespaaB and paaI have not been described in thepha cluster.
      The detection of radiolabeled PA-CoA inside E. coli W14 (pAAD::Tn1000 derivative 3) cells, indicates that disruption of the paaZ gene causes a blockade of the PA catabolic pathway leading to the accumulation of this CoA derivative, and confirms the physiological role of PaaK in the catabolism of PA in this microorganism. Assuming that the paaK gene product catalyzes the first enzymatic step of the PA catabolic pathway, the polar effects caused by the Tn1000 insertions within the potential paa catabolic operon containing thepaaK gene at its 3′-end, can explain why pathway intermediates did not accumulate in E. coli W14 cells expressing the corresponding pAAD::Tn1000derivatives. The degradation of PA in P. putida U also appears to require PA-CoA as the first intermediate of the pathway (
      • Miñambres B.
      • Martı́nez-Blanco H.
      • Olivera E.R.
      • Garcı́a B.
      • Dı́ez B.
      • Barredo J.L.
      • Moreno M.A.
      • Schleissner C.
      • Salto F.
      • Luengo J.M.
      ), and a similar situation could be inferred in other bacteria that are able to use aerobically PA as the sole carbon source (
      • Vitovski S.
      ,
      • Velasco A.
      • Alonso S.
      • Garcı́a J.L.
      • Perera J.
      • Dı́az E.
      ). The aerobic catabolism of aromatic compounds via their initial activation to CoA derivatives constitutes an unusual strategy that resembles anaerobic degradation mechanisms (
      • Egland P.G.
      • Pelletier D.A.
      • Dispensa M.
      • Gibson J.
      • Harwood C.S.
      ), and could be a widespread way of PA catabolism in bacteria. The participation CoA ligases in the initial step of the aerobic catabolism of 2-aminobenzoate (
      • Altenschmidt U.
      • Fuchs G.
      ) and benzoate (
      • Altenschmidt U.
      • Oswal B.
      • Steiner E.
      • Herrmann H.
      • Fuchs G.
      ) in Azoarcus evansii KB740 (formerlyPseudomonas sp. KB740), ferulate in P. putida(
      • Zenk M.H.
      • Ulbrich B.
      • Busse J.
      • Stöckigt J.
      ) and Pseudomonas fluorescens (
      • Gasson M.J.
      • Kitamura Y.
      • McLauchlan W.R.
      • Narbad A.
      • Parr A.J.
      • Parsons E.L.H.
      • Payne J.
      • Rhodes M.J.C.
      • Walton N.J.
      ), and 2-furoic acid in P. putida Fu1 (
      • Koenig K.
      • Andreesen J.R.
      ) has been also reported, and the existence of a CoA ligase has been suggested for the aerobic catabolism of salicylate in Rhodococcus sp. strain B4 (
      • Grund E.
      • Denecke B.
      • Eichenlaub R.
      ) and thiophen-2-carboxylate (
      • Cripps R.E.
      ). Moreover, some dehalogenation mechanisms of aromatic compounds also involve CoA thioester formation in aerobiosis (
      • Dunaway-Mariano D.
      • Babbitt P.C.
      ). Although the rationale for utilizing such hybrid pathways, i.e. aerobic catabolic pathways endowed with typical features of an anaerobic catabolism, is not known, it has been suggested that they could represent a strategy of facultative microorganisms to cope with the fluctuations of oxygen supply (
      • Niemetz R.
      • Altenschmidt U.
      • Brucker S.
      • Fuchs G.
      ). In this sense, the existence of a hybrid pathway for the catabolism of PA in E. coli could reflect the facultative anaerobe character of this bacterium.
      All or some of the paaABCDE genes appear to be responsible of the second enzymatic step in the catabolism of PA in E. coli. Thus, the expression of paaK and paaABCDE genes in E. coli W14 caused the consumption of PA and the accumulation of 2-HPA in the culture medium. However, 2-HPA appears not to be a true intermediate in the PA catabolic pathway as it does not support growth of E. coli W and is not consumed even when E. coli W cells are growing also in the presence of PA. Interestingly, a similar lack of growth on 2-HPA and accumulation of this compound after adding PA to some cultures of PA mutant strains from E. coliK-12 (
      • Cooper R.A.
      • Jones D.C.N.
      • Parrott S.
      ) and P. putida U (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ), has been also observed. Although the possibility that exogenous 2-HPA does not enter the cells cannot be ruled out, the fact that 2-HPA formation requires the simultaneous expression of the paaK and paaABCDEgenes strongly suggests that 2-HPA is not a true intermediate in PA degradation but derives from the accumulation of a hydroxylated PA-CoA intermediate that cannot be further degraded. The excretion to the culture medium of a hydroxylated aromatic compound as a dead-end product derived from the intracellular accumulation of a hydroxylated CoA derivative has been also reported in the hybrid pathway for the catabolism of 2-aminobenzoate (
      • Altenschmidt U.
      • Fuchs G.
      ), and could be a general strategy of the cells to prevent the possible metabolic risk of depletion of the intracellular pool of CoA (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ,
      • Chohnan S.
      • Furukawa H.
      • Fujio T.
      • Nishihara H.
      • Takamura Y.
      ).
      The second catabolic step in PA degradation in E. coli seems to be, therefore, the hydroxylation of PA-CoA. Although we could not detect a hydroxylated CoA derivative in E. coli W14 (pAAD::Tn1000 derivative 3) cells, intracellular accumulation of 2-HPA-CoA has been observed during the catabolism of PA by a PA- P. putida U mutant strain (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ). Sequence comparison analyses of the paaABCDE gene products revealed that the PaaE protein (356-amino acid length) showed significant similarity with the class IA-like reductases (Table I). These enzymes are members of the ferredoxin-NADP+ reductase (FNR) family and they contain a FNR-like domain consisting of a FMN(FAD)- and a NAD(P)-binding region (
      • Correl C.C.
      • Batie C.J.
      • Ballou D.P.
      • Ludwig M.L.
      ). The residues55RCYS58 in PaaE fit the RXYS consensus motif for binding of the isoalloxazine ring of the flavin cofactor, and residues 121GSGITP126 and216CGPAAM221 match the GXG(X)2–3P and CG(X)3–4M sequences for the binding of the NAD(P) ribose and NAD(P)-pyrophosphate-nicotinamide moieties of the nicotinamide cofactor, respectively (
      • Rosche B.
      • Tshisuaka B.
      • Hauer B.
      • Lingens F.
      • Fetzner S.
      ). At the C terminus of the FNR-like domain, residues 299–337 in PaaE correspond to the CX 4CXXCX 24–34C conserved motif of the plant-type ferredoxin [2Fe-2S] binding domain (
      • Rosche B.
      • Tshisuaka B.
      • Hauer B.
      • Lingens F.
      • Fetzner S.
      ). Other members of the extended FNR family are the reductase components of the methane, alkene, phenol, and toluene diiron monooxygenases (
      • Small F.J.
      • Ensign S.A.
      ,
      • Pikus J.D.
      • Studts J.M.
      • Achim C.
      • Kauffmann K.E.
      • Münck E.
      • Steffan R.J.
      • McClay K.
      • Fox B.G.
      ,
      • Johnson G.R.
      • Olsen R.H.
      ,
      • Powlowski J.
      • Sealy J.
      • Shingler V.
      • Cadieux E.
      ,
      • Lipscomb J.D.
      ), a group of bacterial hydrocarbon oxidation enzymes that comprises an evolutionarily related protein family (
      • Pikus J.D.
      • Studts J.M.
      • Achim C.
      • Kauffmann K.E.
      • Münck E.
      • Steffan R.J.
      • McClay K.
      • Fox B.G.
      ). These soluble multicomponent monooxygenases contain, in addition to the reductase component, a heteromultimeric (αβγ) oxygenase component, a low molecular weight activator protein (
      • Johnson G.R.
      • Olsen R.H.
      ,
      • Powlowski J.
      • Sealy J.
      • Shingler V.
      • Cadieux E.
      ,
      • Lipscomb J.D.
      ), and, in some cases, a Rieske-type ferredoxin (
      • Small F.J.
      • Ensign S.A.
      ,
      • Pikus J.D.
      • Studts J.M.
      • Achim C.
      • Kauffmann K.E.
      • Münck E.
      • Steffan R.J.
      • McClay K.
      • Fox B.G.
      ). Interestingly, the primary structure of the PaaA protein (309-amino acid length) shows the two repeats of residues EX 2H separated by approximately 100 amino acids (positions 155–158 and 249–252) that characterize the dinuclear iron binding-site of the large (α) oxygenase subunit of the methane, phenol, and toluene diiron monooxygenases (
      • Pikus J.D.
      • Studts J.M.
      • Achim C.
      • Kauffmann K.E.
      • Münck E.
      • Steffan R.J.
      • McClay K.
      • Fox B.G.
      ). Moreover, the amino acid sequence of PaaB (95-amino acid length) shows the strictly conserved residues found in the low molecular weight dissociable activator protein that is required for optimal turnover of the oxygenase component in multicomponent diiron monooxygenases (
      • Qian H.
      • Edlund U.
      • Powlowski J.
      • Shingler V.
      • Sethson I.
      ). Therefore, these sequence comparison analyses suggest that genes paaABCDE may encode the five subunits of a diiron multicomponent oxygenase, with PaaB being the effector protein and PaaE the reductase that mediates electron transfer between NAD(P)H and the PaaACD oxygenase component. It is worth noting that the paaE gene product can constitute the first example of a reductase subunit from a multicomponent oxygenase that shows a reversed domain order, i.e. a FNR-like N-terminal domain and a plant-type ferredoxin C-terminal domain, which supports the previous hypothesis that class IA-like reductases may have been recruited for a variety of aromatic ring oxidation reactions (
      • Nakatsu C.H.
      • Straus N.A.
      • Wyndham R.C.
      ). Moreover, the putative PaaABCDE oxygenase, and its counterpart encoded by thephaFGHI operon of P. putida U (Fig. 8 B), may represent the first reported multicomponent oxygenase acting on a CoA-activated aromatic acid.
      The paaZ gene product appears to be responsible of the third enzymatic step of the PA catabolic pathway. The putative PaaZ protein (681-amino acid length) presents an N-terminal region (residues 1–527) whose primary structure shows similarity with that of aldehyde dehydrogenases (Table I). In this sense, the PaaZ residues229FTGSAATG236 and291GQKCTAIR298, respectively, match the consensus NAD(P)+-binding site and the active site motif spanning the catalytic cysteine (underlined) of aldehyde dehydrogenases (
      • Ferrández A.
      • Prieto M.A.
      • Garcı́a J.L.
      • Dı́az E.
      ,
      • Liu Z.-J.
      • Sun Y.-J.
      • Rose J.
      • Chung Y.-J.
      • Hsiao C.-D.
      • Chang W.-R.
      • Kuo I.
      • Perozich J.
      • Lindahl R.
      • Hempel J.
      • Wang B.-C.
      ). Moreover, the sequence254MEADSLN260 in PaaZ encompasses the potential catalytic glutamic acid residue (italicized) of aldehyde dehydrogenases (
      • Ferrández A.
      • Prieto M.A.
      • Garcı́a J.L.
      • Dı́az E.
      ,
      • Liu Z.-J.
      • Sun Y.-J.
      • Rose J.
      • Chung Y.-J.
      • Hsiao C.-D.
      • Chang W.-R.
      • Kuo I.
      • Perozich J.
      • Lindahl R.
      • Hempel J.
      • Wang B.-C.
      ). The amino acid sequence of the C-terminal region of PaaZ shows similarity to that of the maoC and nodN gene products of unknown function (Table I). As has been suggested for the analogous PhaL protein of P. putida U (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ) (Fig. 8 B), the paaZ gene product in E. coli might catalyze the aromatic ring cleavage of the hydroxylated CoA derivative formed during PA degradation. Nevertheless, the formation by PaaZ of a non-aromatic CoA cyclic intermediate, similar to that described as the product of the reaction catalyzed by the aminobenzoyl-CoA monooxygenase-reductase during the aerobic catabolism of 2-aminobenzoate (
      • Langkau B.
      • Ghisla S.
      ), cannot be ruled out.
      The paaF, paaG, paaH, and paaJ gene products show significant sequence similarities to fatty acid β-oxidation enzymes (Table I), and therefore can tentatively constitute a β-oxidation-like pathway involved in the successive oxidation reactions of the non-aromatic CoA intermediate. Interestingly, a β-oxidation-like mechanism is another typical feature of the anaerobic catabolism of aromatic compounds (
      • Egland P.G.
      • Pelletier D.A.
      • Dispensa M.
      • Gibson J.
      • Harwood C.S.
      ). The primary structure of the putative PaaF (255-amino acid length) and PaaG (262-amino acid length) proteins shows similarity with that of members of the enoyl-CoA hydratase/isomerase superfamily (
      • Dunaway-Mariano D.
      • Babbitt P.C.
      ,
      • Müller-Newen G.
      • Janssen U.
      • Stoffel W.
      ) (Table I). The paaH gene encodes a protein (475-amino acid length) that shares the signature sequence motives of 3-hydroxyacyl-CoA dehydrogenases (
      • He X.-Y.
      • Deng H.
      • Yang S.-Y.
      ) (Table I), thus suggesting that it could attack the product of the reaction catalyzed by the PaaF and PaaG enzymes. Although the paaI gene product (140-amino acid length) did not show a high level of sequence similarity with other proteins in the data bases, the paaJ gene product (401-amino acid length) presented a significant sequence similarity with the PcaF and CatF β-ketoadipyl-CoA thiolases (Table I), residues 90 and 386 in PaaJ being the putative catalytic cysteines. As PcaF and CatF catalyze the last step in the ortho-cleavage pathway for the aerobic degradation of protocatechuate and catechol, respectively (
      • Harwood C.S.
      • Parales R.E.
      ), it is tempting to speculate that PaaJ and its analogous PhaD protein in P. putida U (Fig. 8) are also responsible for the last enzymatic step in PA degradation.
      In the paa cluster, we have identified three promoters,Pz, Pa, and Px, which drive the expression of genes paaZ, paaABCDEFGHIJK, and paaXY, respectively (Figs. 6 and 8 A). The expresion of thepaa-encoded catabolic pathway is inducible, and it has been shown that the Pa promoter is negatively controlled by thepaaX gene product (Table III and IV). The PaaX protein (316-amino acid length) contains a stretch of 25 residues at amino acids 39–64 that shares similarity with the helix-turn-helix motif predicted to be important for DNA recognition and binding in other transcriptional repressors such as GntR (
      • Fujita Y.
      • Miwa Y.
      ) and FadR (
      • DiRusso C.C.
      • Heimert T.L.
      • Metzger A.K.
      ). The GntR and FadR binding sites within the respective promoters contain a region of dyad symmetry, which is located very close to the transcription initiation sites (
      • Fujita Y.
      • Miwa Y.
      ,
      • DiRusso C.C.
      • Heimert T.L.
      • Metzger A.K.
      ,
      • Black P.N.
      • DiRusso C.C.
      ). Interestingly, a region of dyad symmetry can also be found centered near the transcription initiation sites in thePa promoter (Fig. 2). As the repression caused by PaaX was only alleviated by PA in the presence of the PaaK protein (Table IV), PA-CoA appears to be the true inducer of the paa-encoded pathway. In this sense, gel retardation assays have confirmed PA-CoA as the effector molecule.
      A. Ferrández, J. L. Garcı́a, and E. Dı́az, manuscript in preparation.
      Therefore, PaaX constitutes the first reported transcription factor regulated by CoA derivatives that controls the catabolism of aromatic compounds. It is worth noting that the FadR transcriptional repressor, which is regulated by acyl-CoA compounds and shows local similarity to PaaX, is also controlling the expression of genes involved in β-oxidation mechanisms (
      • Black P.N.
      • DiRusso C.C.
      ).
      Overlapping the 3′-end of paaX, we have found the putative ATG translation initiation codon of the paaY gene. A palindromic sequence (ΔG value of −15.7 kcal/mol) followed by a (T)7 tract is located 42 bp downstream of the TAA stop codon of paaY (Fig. 2), and may act as a ρ-independent transcription terminator of the putativepaaXY operon. Although the primary structure of thepaaY gene product (196-amino acid length) and its analogous PhaM protein from P. putida U (Fig. 8 B) show several repeats of the hexapeptide (LIV)GX 4motif that characterizes the members of the bacterial transferases family, e.g. the CaiE protein from the carnitine operon of E. coli and the Fbp ferripyochelin-binding protein of P. aeruginosa (Table I), the physiological role of these proteins in PA catabolism is still unknown.
      Comparative studies of the whole structure and organization of thepaa and pha clusters from E. coli and P. putida U, respectively (Fig. 8 A), revealed interesting functional and evolutionary data. Thus, although thepha genes appear to be cotranscribed in four discrete DNA segments or modules encoding the six different functional units for the catabolism of PA, i.e. the β-oxidation and activation (phaABCDE), hydroxylation (phaFGHI), transport and dearomatization (phaJKL), and regulation (phaMN) units, the paa cluster showed the transcriptional coupling of the hydroxylation-β-oxidation-activation functional units into the single operon paaABCDEFGHIJK (Fig. 8 A). As there is good evidence that operons coding for the catabolism of aromatic compounds are assembled in a stepwise manner from existing catabolic genes (
      • van der Meer J.R.
      • de Vos W.M.
      • Harayama S.
      • Zehnder A.J.B.
      ), it is tempting to speculate that the paa cluster from E. coli arose by the fusion of some gene blocks that are contiguous but separately regulated in thepha cluster of P. putida U, and therefore it could be considered as a further step in the evolution toward a single regulon of a common ancestral gene cluster involved in PA catabolism. Moreover, the differences in gene order within some of the DNA modules, and the relative locations of these modules in the paa and pha clusters, suggest that various DNA rearrangements have occurred during their evolution. As the G+C content of thepaa (52.5%) and pha (63.5%) genes averaged a value close to the mean G+C content of E. coli (51.5%) and P. putida (60%) genomic DNA (
      • Nakamura Y.
      • Gojobori T.
      • Ikemura T.
      ), it could be thought that these two set of genes have been imprisoned within each host over a long period of evolution. Especially remarkable is the observation that the phaJ and phaK genes of P. putidaU, encoding a permease and a specific-channel-forming protein for the uptake of PA, respectively (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ), are absent in the paacluster from E. coli W (Fig. 8 A). Interestingly, the phaJ gene product shows significant amino acid sequence identity (62.1%) with the product of the yjcG gene that is located at min 92.2 of the E. coli K-12 chromosome (
      • Kröger M.
      • Wahl R.
      ). Whether a permease, such as the putative YjcG protein, and a channel-forming protein are required for the catabolism of PA in E. coli is still an open question.
      The identification and genetic characterization of the hybridpaa-encoded pathway complete our knowledge on the pathways so far described for the aerobic catabolism of aromatic compounds in E. coli. Although, in some Pseudomonas and Acinetobacter species, a supraoperonic clustering of the aromatic catabolic genes has been observed in a limited region of the chromosome, the aromatic catabolic clusters are dispersed throughout the genome in E. coli, with cluster mhp(3-(3-hydroxyphenyl)propionate and 3-hydroxycinnamate) at min 8 (
      • Ferrández A.
      • Garcı́a J.L.
      • Dı́az E.
      ,
      • Spence E.L.
      • Kawamukai M.
      • Sanvoisin J.
      • Braven H.
      • Bugg T.D.H.
      ), paa at min 31, hca (3-phenylpropionate) at min 57.5 (
      • Dı́az E.
      • Ferrández A.
      • Garcı́a J.L.
      ), and hpa (3- or 4-hydroxyphenylacetate) at min 98 (
      • Prieto M.A.
      • Dı́az E.
      • Garcı́a J.L.
      ). These data also indicate that E. coli is not an “empty box” for the catabolism of aromatic compounds; on the contrary, it is endowed with typical aerobic degradation routes as well as with a novel hybrid pathway, which are considered among the most ubiquitous aromatic compound catabolic systems and therefore are thought to be closer to the central catabolism than those involved in the degradation of xenobiotic compounds (
      • Barnes M.R.
      • Duetz W.A.
      • Williams P.A.
      ).
      The results presented in this work provide a framework for additional studies to determine the role and properties of the enzymes involved in PA catabolism through a hybrid aerobic pathway that is likely to be a widespread route for the metabolism of this aromatic compound. In this sense, the cloned paa genes should be useful as probes to identify homologous genes from distinct groups of bacteria. Moreover, we anticipate that the unique features of the aerobicpaa-encoded pathway will reveal novel catabolic activities that can be of great biotechnological interest to improve some microorganisms for the degradation of PA-related aromatic environmental pollutants (e.g., styrene), and for the synthesis of pathway intermediates that can be useful for the production of new or modified antibiotics and plastics (
      • Olivera E.R.
      • Miñambres B.
      • Garcı́a B.
      • Muñiz C.
      • Moreno M.A.
      • Ferrández A.
      • Dı́az E.
      • Garcı́a J.L.
      • Luengo J.M.
      ).

      ACKNOWLEDGEMENTS

      We thank M. K. B. Berlyn for strains MG1063 and MG1655, S. Jaenecke for plasmids pSJ3 and pSJ19Not, A. Prieto and J. Varela for assistance with the gas chromatography-mass spectrometry and N-terminal amino acid sequence analyses, respectively, and A. Dı́az and G. Porras for their help with the sequencing. The excellent technical assistance of E. Cano, M. Carrasco, and F. Morante is gratefully acknowledged. We are indebted to K. N. Timmis and D. Pieper for facilitating the short visit of A. Ferrández to their laboratory.

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