Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.     

The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.     

A two-component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate in Pseudomonas putida

The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, l-Phe, and l-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).     

Aerobic metabolism of phenylacetic acids in Azoarcus evansii

The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-(14)C]PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus. A chromosomal region from A. evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.     

Benzoate-dependent induction from the OP2 operator-promoter region of the TOL plasmid pWWO in the absence of known plasmid regulatory genes.

Expression of the lower catabolic pathway of the TOL plasmid pWWO requires an aromatic acid inducer and the product of the xylS regulatory gene. Pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (OP2 [or Pm]), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known TOL plasmid regulatory genes. Induction was not seen in transformed Escherichia coli cells or in a P. putida mutant lacking chromosomally encoded benzoate catabolic functions.     

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.     

Physical map of the aromatic amine and m-toluate catabolic plasmid pTDN1 in Pseudomonas putida: location of a unique meta-cleavage pathway

A restriction endonuclease map was derived for the aromatic amine and m-toluate catabolic plasmid pTDN1 present in Pseudomonas putida UCC22, a derivative of P. putida mt-2. The plasmid is 79 +/- 1 kbp in size and can be divided into a restriction-site-deficient region of 51 +/- 1 kbp and a restriction-site-profuse region of 28 kbp which begins and ends with directly repeating sequences of at least 2 kbp in length. A mutant plasmid isolated after growth of the host on benzoate had lost the restriction-profuse region by a straightforward recombinational loss retaining one copy of the direct repeat. Analysis of clones, deletion and Tn5 insertion mutants strongly suggested that the meta-cleavage pathway of pTDN1 was situated in the region readily deleted. The catechol 2,3-dioxygenase (C23O) gene of pTDN1 showed no hybridization or restriction homology to previously described C23O genes of TOL plasmids pWW0 and pWW15. In addition, there was little homology between intact pTDN1, pWW0 and pWW15, suggesting the presence of a unique meta-cleavage pathway. We also demonstrated that pTDN1 did not originate from P. putida mt-2 chromosome.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacterium Cupriavidus necator JMP134

Cupriavidus necator JMP134 is a model for chloroaromatics biodegradation, capable of mineralizing 2,4-D, halobenzoates, chlorophenols and nitrophenols, among other aromatic compounds. We performed the metabolic reconstruction of aromatics degradation, linking the catabolic abilities predicted in silico from the complete genome sequence with the range of compounds that support growth of this bacterium. Of the 140 aromatic compounds tested, 60 serve as a sole carbon and energy source for this strain, strongly correlating with those catabolic abilities predicted from genomic data. Almost all the main ring-cleavage pathways for aromatic compounds are found in C. necator : the β-ketoadipate pathway, with its catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage branches; the (methyl)catechol meta ring-cleavage pathway; the gentisate pathway; the homogentisate pathway; the 2,3-dihydroxyphenylpropionate pathway; the (chloro)hydroxyquinol pathway; the (amino)hydroquinone pathway; the phenylacetyl-CoA pathway; the 2-aminobenzoyl-CoA pathway; the benzoyl-CoA pathway and the 3-hydroxyanthranilate pathway. A broad spectrum of peripheral reactions channel substituted aromatics into these ring cleavage pathways. Gene redundancy seems to play a significant role in the catabolic potential of this bacterium. The literature on the biochemistry and genetics of aromatic compounds degradation is reviewed based on the genomic data. The findings on aromatic compounds biodegradation in C. necator reviewed here can easily be extrapolated to other environmentally relevant bacteria, whose genomes also possess a significant proportion of catabolic genes.     

Complete nucleotide sequence and evolutionary significance of a chromosomally encoded naphthalene-degradation lower pathway from Pseudomonas stutzeri AN10

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced the entire naphthalene-degradation lower pathway of P. stutzeri AN10, this being, together with the upper-pathway reported previously (Bosch R. et al., 1999a. Gene 236, 149-157) the first complete DNA sequence for an entire naphthalene-catabolic pathway. Eleven open reading frames were identified. The nahGTHINLOMKJ genes encode enzymes for the metabolism of salicylate to pyruvate and acetyl-CoA, and nahR encodes the NahR regulatory protein. Our findings suggest that catabolic modules were recruited through transposition events and recombination among tnpA-like genes, and subsequent rearrangements and deletions of non-essential DNA fragments allowed the formation of the actual catabolic pathway. Our results also suggest that the genes encoding the xylene/toluene-degradation enzymes of P. putida mt-2 (pWW0) have coexisted with the nah genes of the P. stutzeri AN10 ancestral genome. This could allow the selection, via recombination events among homologous genes, for a combination of genes enabling the metabolism of a given aromatic compound in the ancestral host strain. Such events accelerate the evolution of modern catabolic pathways and provide new genetic material to the environment, ultimately resulting in improved, natural, bioremediation potential.     

Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB.

The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned. We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns. The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.     

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.     

p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon.

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.     

NahY, a Catabolic Plasmid-Encoded Receptor Required for Chemotaxis of Pseudomonas putida to the Aromatic Hydrocarbon Naphthalene

Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.     

Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales

The relevance of the β-proteobacterial Burkholderiales order in the degradation of a vast array of aromatic compounds, including several priority pollutants, has been largely assumed. In this review, the presence and organization of genes encoding oxygenases involved in aromatics biodegradation in 80 Burkholderiales genomes is analysed. This genomic analysis underscores the impressive catabolic potential of this bacterial lineage, comprising nearly all of the central ring-cleavage pathways reported so far in bacteria and most of the peripheral pathways involved in channelling of a broad diversity of aromatic compounds. The more widespread pathways in Burkholderiales include protocatechuate ortho ring-cleavage, catechol ortho ring-cleavage, homogentisate ring-cleavage and phenylacetyl-CoA ring-cleavage pathways found in at least 60% of genomes analysed. In general, a genus-specific pattern of positional ordering of biodegradative genes is observed in the catabolic clusters of these pathways indicating recent events in its evolutionary history. In addition, a significant bias towards secondary chromosomes, now termed chromids, is observed in the distribution of catabolic genes across multipartite genomes, which is consistent with a genus-specific character. Strains isolated from environmental sources such as soil, rhizosphere, sediment or sludge show a higher content of catabolic genes in their genomes compared with strains isolated from human, animal or plant hosts, but no significant difference is found among Alcaligenaceae, Burkholderiaceae and Comamonadaceae families, indicating that habitat is more of a determinant than phylogenetic origin in shaping aromatic catabolic versatility.     

Synergistic Allostery,a Sophisticated Regulatory Network for the Control of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis

The shikimate pathway, responsible for aromatic amino acid biosynthesis, is required for the growth of Mycobacterium tuberculosis and is a potential drug target. The first reaction is catalyzed by 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Feedback regulation of DAH7PS activity by aromatic amino acids controls shikimate pathway flux. Whereas Mycobacterium tuberculosis DAH7PS (MtuDAH7PS) is not inhibited by the addition of Phe, Tyr, or Trp alone, combinations cause significant loss of enzyme activity. In the presence of 200 μm Phe, only 2.4 μm Trp is required to reduce enzymic activity to 50%. Reaction kinetics were analyzed in the presence of inhibitory concentrations of Trp/Phe or Trp/Tyr. In the absence of inhibitors, the enzyme follows Michaelis-Menten kinetics with respect to substrate erythrose 4-phosphate (E4P), whereas the addition of inhibitor combinations caused significant homotropic cooperativity with respect to E4P, with Hill coefficients of 3.3 (Trp/Phe) and 2.8 (Trp/Tyr). Structures of MtuDAH7PS/Trp/Phe, MtuDAH7PS/Trp, and MtuDAH7PS/Phe complexes were determined. The MtuDAH7PS/Trp/Phe homotetramer binds four Trp and six Phe molecules. Binding sites for both aromatic amino acids are formed by accessory elements to the core DAH7PS (β/α)₈ barrel that are unique to the type II DAH7PS family and contribute to the tight dimer and tetramer interfaces. A comparison of the liganded and unliganded MtuDAH7PS structures reveals changes in the interface areas associated with inhibitor binding and a small displacement of the E4P binding loop. These studies uncover a previously unrecognized mode of control for the branched pathways of aromatic amino acid biosynthesis involving synergistic inhibition by specific pairs of pathway end products.