Metabolism of Benzoate and the Methylbenzoates by Pseudomonas putida (arvilla) mt-2: Evidence for the Existence of a TOL Plasmid

Mutant strains of Pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of m- or p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin C. The mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase and 4-hydroxy-2-oxovalerate aldolase; unlike the wild type they utilize benzoate by the ortho pathway. Evidence is presented that these mutants have lost a plasmid coding for the enzymes of the meta pathway, which may be transmitted back to them or into other P. putida strains. Preliminary results from these mutants and from a mutant defective in the regulation of the plasmid-carried pathway suggest that the wild type contains two benzoate oxidase systems, one on the plasmid which is nonspecific in both its catalysis and its induction and one on the chromosome which is more specific to benzoate as substrate and is specifically induced by benzoate.     

Anaerobic degradation of protocatechuate (3,4-dihydroxybenzoate) by Thauera aromatica strain AR-1

The denitrifying bacterium Thauera aromatica strain AR-1 grows anaerobically with protocatechuate (3,4-dihydroxybenzoate (DHB)) as sole energy and carbon source. This bacterium harbors two distinct pathways for degradation of aromatic compounds, the benzoyl-coenzyme A (CoA) pathway for benzoate degradation and the hydroxyhydroquinone (HHQ) pathway for degradation of 3,5-DHB. In order to elucidate whether protocatechuate is degraded via the benzoyl-CoA or the HHQ pathway, induction experiments were carried out. Dense suspensions of cells grown on protocatechuate or benzoate readily degraded benzoate and protocatechuate but not 3,5-DHB. Dense suspensions of 3,5-DHB-grown cells degraded 3,4- and 3,5-DHB at similar rates, but benzoate was not degraded. 3,5-DHB hydroxylating activity was found only in cells grown with this substrate. HHQ dehydrogenase activity was found in extracts of cells grown with 3,5-DHB and at a low rate also in protocatechuate-grown cells, but not in extracts of cells grown with benzoate. Activities of protocatechuyl-CoA synthetase and protocatechuyl-CoA reductase leading to 3-hydroxybenzoyl-CoA were found in extracts of cells grown with protocatechuate. There was no repression of the HHQ pathway by the presence of protocatechuate, unlike by degradation of benzoate. We conclude that protocatechuate is not degraded via the HHQ pathway because there was no evidence of a hydroxylation reaction involved in this process. Instead, our results strongly suggest that protocatechuate is degraded via a pathway which connects to the benzoyl-CoA route of degradation.     

Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways

In the denitrifying member of the beta-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V(max)/K(m), the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.     

Degradation of phenol under meso- and thermophilic, anaerobic conditions

Based on the results of preliminary studies on phenol degradation under mesophilic conditions with a mixed methanogenic culture, we proposed a degradation pathway in which phenol is fermented to acetate: Part of the phenol is reductively transformed to benzoate while the rest is oxidised, forming acetate as end product. According to our calculations, this should result in three moles of phenol being converted to two moles of benzoate and three moles of acetate (3 phenol + 2 CO2 + 3 H2O --> 3 acetate + 2 benzoate): To assess the validity of our hypothesis concerning the metabolic pathway, we studied the transformation of phenol under mesophilic and thermophilic conditions in relation to the availability of hydrogen. Hence, methanogenic meso- and thermophilic cultures amended with phenol were run with or without an added over-pressure of hydrogen under methanogenic and non-methanogenic conditions. Bromoethanesulfonic acid (BES) was used to inhibit methanogenic activity. In the mesophilic treatments amended with only BES, about 70% of the carbon in the products found was benzoate. During the course of phenol transformation in these BES-amended cultures, the formation pattern of the degradation products changed: Initially nearly 90% of the carbon from phenol degradation was recovered as benzoate, whereas later in the incubation, in addition to benzoate formation, the aromatic nucleus degraded completely to acetate. Thus, the initial reduction of phenol to benzoate resulted in a lowering of H2 levels, giving rise to conditions allowing the degradation of phenol to acetate as the end product. Product formation in bottles amended with BES and phenol occurred in accordance with the hypothesised pathway; however, the overall results indicate that the degradation of phenol in this system is more complex. During phenol transformation under thermophilic conditions, no benzoate was observed and no phenol was transformed in the BES-amended cultures. This suggests that the sensitivity of phenol transformation to an elevated partial pressure of H2 is higher under thermophilic conditions than under mesophilic ones. The lack of benzoate formation could have been due to a high turnover of benzoate or to a difference in the phenol degradation pathway between the thermophilic and mesophilic cultures.     

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.     

2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris

A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD(+); no activity was detected with NADP(+) as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K(m) values of 10 and 200 microM for 2-hydroxychc-CoA and NAD(+), respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

Regulation of benzoate-CoA ligase in Rhodopseudomonas palustris

Abstract: The first step in the anaerobic pathway of benzoate degradation by Rhodopseudomonas palustris is catalyzed by benzoate-coenzyme A ligase. To study factors influencing the synthesis of this enzyme, a polyclonal antiserum was prepared and in immunoblot assays. Benzoate-CoA ligase was synthesized when cells were grown with benzoate, as well as with hydroxyl- and methyl-substituted benzoates. Partially reduced alicyclic compounds proposed to be intermediates in the benzoate pathway also induced benzoate-CoA ligase. Ligase synthesis was repressed by oxygen. The diversity of inducers is consistent with the observation that benzoate is a central intermediate in the degradation of a variety of aromatic acids with more complex structures.     

Anaerobic Degradation of the Benzene Nucleus by a Facultatively Anaerobic Microorganism

A bacterium was isolated by elective culture with p-hydroxybenzoate as substrate and nitrate as electron acceptor. It grew either aerobically or anaerobically, by nitrate respiration, on a range of aromatic compounds. The organism was identified as a pseudomonad and was given the trivial name Pseudomonas PN-1. Benzoate and p-hydroxybenzoate were metabolized aerobically via protocatechuate, followed by meta cleavage catalyzed by protocatechuic acid-4,5-oxygenase, to yield alpha-hydroxy-gamma-carboxymuconic semialdehyde. Pseudomonas PN-1 grew rapidly on p-hydroxybenzoate under strictly anaerobic conditions, provided nitrate was present, even though protocatechuic acid-4,5-oxygenase was repressed. Suspensions of cells grown anaerobically on p-hydroxybenzoate oxidized benzoate with nitrate and produced 4 to 5 mumoles of CO(2) per mumole of benzoate added; these cells did not oxidize benzoate aerobically. The patterns of the oxidation of aromatic substrates with oxygen or nitrate by cells grown aerobically or anaerobically on different aromatic compounds indicated that benzoate rather than protocatechuate was a key intermediate in the early stages of anaerobic metabolism. It was concluded that the pathway for the anaerobic breakdown of the aromatic ring is different and quite distinct from the aerobic pathway. Mechanisms for the anaerobic degradation of the benzene nucleus by Pseudomonas PN-1 are discussed.     

Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates.

The TOL plasmid-specified meta-cleavage pathway for the oxidative catabolism of benzoate and toluates branches at the ring cleavage products of catechols and reconverges later at 2-oxopent-4-enoate or its corresponding substituted derivatives. The hydrolytic branch of the pathway involves the direct formation of 2-oxopent-4-enoate or its derivatives, whereas the oxalocrotonate branch involves three enzymatic steps effected by a dehydrogenase, an isomerase, and a decarboxylase, which produce the same compounds. Evidence is presented which shows that benzoate and p-toluate can, under certain circumstances, be catabolized by the hydrolytic branch. However, in a fully functional pathway, only m-toluate is dissimilated via this branch, and benzoate and p-toluate are catabolized almost exclusively by the oxalocrotonate branch. The biochemical basis of this selectivity was found to reside in the high affinity of the dehydrogenase for ring fission products derived from benzoate and p-toluate and its inability to attack the ring fission product derived from m-toluate. Although isomerization of 4-oxalocrotonate occurs spontaneously in vitro, enzymatic isomerization was found to be essential for effective functioning of this branch of the pathway in vivo.     

New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp.

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds compounds completely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via benzoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aerobically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-hydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoate was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments with whole cells seemingly suggested a sequential induction of enzymes of a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Cells grown aerobically with benzoate contained a benzoate-CoA ligase (AMP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3-hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. Cells grown aerobically with 3-hydroxybenzoate contained a similarly active CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (AMP forming). Extracts from cells grown aerobically with benzoate catalyzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidation of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzyme, benzoyl-CoA 3-monooxygenase, was specifically induced during aerobic growth with benzoate and converted [U-14C]benzoyl-CoA stoichiometrically to [14C]3-hydroxybenzoyl-CoA.     

Regulation of terephthalate catabolism in Rhodococcus rubropertinctus

The regulation of terephthalate catabolism was studied in Rhodococcus rubropertinctus which decomposed this synthetic monomer. The pathway (a) of terephthalate (TP) catabolism is as follows: TP----benzoate----4-hydroxybenzoate----protocatechuate----pyrocatechol-- --cycle ortho-cleavage. The following results were obtained when studying why two other catabolic pathways were realized if benzoate and 4-hydroxybenzoate were taken as a sole carbon source, namely, (b) benzoate----pyrocatechol----cycle cleavage and (c) 4-hydroxybenzoate----protocatechuate----cycle cleavage. TP seemed to cause the divergence of pathways (a) and (b) by repressing the system of benzoate oxidation to pyrocatechol. In pathway (c), benzoate repressed the synthesis of enzymes which catalysed protocatechuate oxidation. Pathway (b) was switched over to (a) when the strain was grown in a medium containing TP and benzoate at a benzoate concentration above 5 mM. Here, the concentration of benzoate (first exogenous and later formed from TP) played a key role. R. rubropertinctus growth in a medium with TP and glucose had diauxic characteristics.     

The metabolism of aromatic compounds by Rhodopseudomonas palustris. A new, reductive, method of aromatic ring metabolism

1. Rhodopseudomonas palustris grows both aerobically and photosynthetically on aromatic acids. p-Hydroxybenzoate and protocatechuate are able to support aerobic growth; these compounds are metabolized by the protocatechuate 4,5-oxygenase pathway. 2. The photoassimilation of benzoate and hydroxybenzoates and the effects of air and darkness on the photoassimilation of benzoate are described. 3. Evidence in conflict with the pathway previously proposed for the photometabolism of benzoate is discussed. 4. The photometabolism of benzoate is accomplished by a novel reductive pathway involving its reduction to cyclohex-1-ene-1-carboxylate, followed by hydration to 2-hydroxycyclohexanecarboxylate and after dehydrogenation to 2-oxocyclohexanecarboxylate further hydration results in ring-fission and the production of pimelate. 5. Attempts were made to prepare cell-free extracts capable of dissimilating benzoate.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Loss of the toluene-xylene catabolic genes of TOL plasmid pWW0 during growth of Pseudomonas putida on benzoate is due to a selective growth advantage of 'cured' segregants

During growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative ('cured') strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWW0. Experiments with two plasmids identical to pWW0 but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the 'benzoate curing' occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate. Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWW0174, a derivative of pWW0 which has deleted the 39 kbp region but carries Tn401. Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWW0, was not lost from PaW701 during growth on benzoate. Competition between wild-type PaW1 and PaW174, which has the 'cured' phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously 'cured' derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10(-2) and 10(-3). We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the 'cured' segregants grow faster than the wild-type using the chromosomally determined beta-ketoadipate pathway.     

Biosynthetic Pathway for Veratryl Alcohol in the Ligninolytic Fungus Phanerochaete chrysosporium

Veratryl alcohol (VA) is a secondary metabolite of white-rot fungi that produce the ligninolytic enzyme lignin peroxidase. VA stabilizes lignin peroxidase, promotes the ability of this enzyme to oxidize a variety of physiological substrates, and is accordingly thought to play a significant role in fungal ligninolysis. Pulse-labeling and isotope-trapping experiments have now clarified the pathway for VA biosynthesis in the white-rot basidiomycete Phanerochaete chrysosporium. The pulse-labeling data, obtained with ¹⁴C-labeled phenylalanine, cinnamic acid, benzoic acid, and benzaldehyde, showed that radiocarbon labeling followed a reproducible sequence: it peaked first in cinnamate, then in benzoate and benzaldehyde, and finally in VA. Phenylalanine, cinnamate, benzoate, and benzaldehyde were all efficient precursors of VA in vivo. The isotope-trapping experiments showed that exogenous, unlabeled benzoate and benzaldehyde were effective traps of phenylalanine-derived ¹⁴C. These results support a pathway in which VA biosynthesis proceeds as follows: phenylalanine → cinnamate → benzoate and/or benzaldehyde → VA.