Metabolic diversity in bacterial degradation of aromatic compounds

Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and recalcitrant. Microbes, however, have evolved the ability to utilize these highly reduced and recalcitrant compounds as a potential source of carbon and energy. Aerobic degradation of aromatics is initiated by oxidizing the aromatic ring, making them more susceptible to cleavage by ring-cleaving dioxygenases. A preponderance of aromatic degradation genes on plasmids, transposons, and integrative genetic elements (and their shuffling through horizontal gene transfer) have lead to the evolution of novel aromatic degradative pathways. This enables the microorganisms to utilize a multitude of aromatics via common routes of degradation leading to metabolic diversity. In this review, we emphasize the exquisiteness and relevance of bacterial degradation of aromatics, interlinked degradative pathways, genetic and metabolic regulation, carbon source preference, and biosurfactant production. We have also explored the avenue of metagenomics, which opens doors to a plethora of uncultured and uncharted microbial genetics and metabolism that can be used effectively for bioremediation.     

Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris.

The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic compounds for growth under anaerobic and aerobic conditions. Many phenolic, dihydroxylated, and methoxylated aromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001 in both the presence and absence of oxygen. Some compounds were metabolized under only aerobic or under only anaerobic conditions. Two other strains, CGC023 and CGD052, had similar anaerobic substrate utilization patterns, but CGD052 was able to use a slightly larger number of compounds for growth. These results show that R. palustris is far more versatile in terms of aromatic degradation than had been previously demonstrated. A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respect to anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobic breakdown of diverse aromatics. Another mutant (CGA047) was unable to grow anaerobically on either benzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown on more complex aromatic compounds. This indicates that R. palustris has two major anaerobic routes for aromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate.     

Biodegradation of halogenated organic compounds.

In this review we discuss the degradation of chlorinated hydrocarbons by microorganisms, emphasizing the physiological, biochemical, and genetic basis of the biodegradation of aliphatic, aromatic, and polycyclic compounds. Many environmentally important xenobiotics are halogenated, especially chlorinated. These compounds are manufactured and used as pesticides, plasticizers, paint and printing-ink components, adhesives, flame retardants, hydraulic and heat transfer fluids, refrigerants, solvents, additives for cutting oils, and textile auxiliaries. The hazardous chemicals enter the environment through production, commercial application, and waste. As a result of bioaccumulation in the food chain and groundwater contamination, they pose public health problems because many of them are toxic, mutagenic, or carcinogenic. Although synthetic chemicals are usually recalcitrant to biodegradation, microorganisms have evolved an extensive range of enzymes, pathways, and control mechanisms that are responsible for catabolism of a wide variety of such compounds. Thus, such biological degradation can be exploited to alleviate environmental pollution problems. The pathways by which a given compound is degraded are determined by the physical, chemical, and microbiological aspects of a particular environment. By understanding the genetic basis of catabolism of xenobiotics, it is possible to improve the efficacy of naturally occurring microorganisms or construct new microorganisms capable of degrading pollutants in soil and aquatic environments more efficiently. Recently a number of genes whose enzyme products have a broader substrate specificity for the degradation of aromatic compounds have been cloned and attempts have been made to construct gene cassettes or synthetic operons comprising these degradative genes. Such gene cassettes or operons can be transferred into suitable microbial hosts for extending and custom designing the pathways for rapid degradation of recalcitrant compounds. Recent developments in designing recombinant microorganisms and hybrid metabolic pathways are discussed.     

Plasmid determined degradation of sulfo-aromatic compounds by Pseudomonas sp. BS1304]

Utilized sulfo-aromatic compounds of Pseudomonas sp. BS1304 were isolated. It was shown that the first step of conversion is desulfonation with following meta-cleavage of the substituted aromatic ring. At least two steps are controlled by the plasmid pBS1004 (120 kb), belonging to the IncP-9 incompatibility group. The degradative plasmid marked by Tn5-lac may be mobilized to P. putida, P. aeruginosa, P. mendocina, where a degradative phenotype is expressed.     

水体沉积物中微生物厌氧呼吸耦合多环芳烃降解的研究进展

多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是一种具有致癌、致畸、致突变的持久性有机污染物。本文在分析国内外主要水体沉积物中PAHs污染状况的基础上,综述了近几年有关厌氧水体沉积物中微生物以硝酸盐、Fe(III)以及硫酸盐为电子受体进行呼吸耦合PAHs降解的研究概况。此外,还总结了基于微生物的PAHs降解基因组、蛋白质组、代谢组以及菌群水平上互作网络的研究进展,以期为进一步加速PAHs污染水体沉积物原位生物修复提供科学理论参考。     

Genetic systems of biodegradation: organization and regulation of expression

The review discusses the current state of genetic analysis of degradative processes. Attention is mainly given to the structural and functional organization of the plasmid systems of degradation of organic compounds in gram-negative bacteria. The data available on the regulation mechanisms of D plasmids' metabolic operons, based on the most studied models of xyl and nah operons, are critically analyzed. The problems of evolution of plasmid D systems are considered conceptually as well as the principles of the experimental strategy of developing new metabolic pathways under laboratory conditions. The prospects of constructing the strains capable of efficient degradation of xenobiotics are considered in brief.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Limited Roles for Salicylate and Phthalate in Bacterial PAH Bioremediation

We have isolated and identified 12 previously unreported strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria. Three of these isolates belong to the genus Burkholderia, with the remainder comprised of two Pseudomonas species, and seven strains from the genus Sphingomonas. These isolates were examined for their ability to utilize a variety of three- to five-ring PAHs as sole sources of carbon and energy. All were also checked for the ability to grow on salicylic acid and phthalic acid, two compounds that are key intermediates in almost all published PAH degradative pathways. Only 3 of the 12 strains were able to grow on both of these monoaromatic acids, while three others (all Sphingomonas) could not grow on either. The implications of these findings on the universal applicability of known PAH catabolic pathways, and on possible induction of PAH degradation by pathway intermediates, are discussed.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Biomimetic oxidation of lignin model compounds by simple inorganic complexes

Copper peroxydisulfate has been shown to mimic "ligninases" in the oxidative degradation of Dihydroanisoin, Veratrylglycerol-beta-guaiacyl ether and veratryl alcohol. A unified mechanism leads to predictable degradative pathways. These are initiated by single-electron oxidation of aromatic substrates to aryl cation radicals as common intermediates to both the enzymic and biomimetic reactions. Our preliminary results show that simple complexes can facilitate the oxidative degradation of lignin model compounds.     

The ins and outs of ring-cleaving dioxygenases

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

Degradation of aromatic amino acids by Rhodococcus erythropolis

A Gram-positive Rhodococcus erythropolis strain S1 was shown to assimilate aromatic amino acids such as L-phenylalanine, L-tyrosine, L-tryptophan, D-phenylalanine, D-tyrosine and D-tryptophan, which were utilized not only as the sole carbon source but also as a suitable nitrogen source. The highest growth on these aromatic amino acids occurred at a temperature of 30°C. L-Phenylalanine, L-tyrosine and L-tryptophan degradative pathways would appear to be independent, and to be induced alternatively. The strain S1 also showed the ability to assimilate peptides which consisted of only L-phenylalanine and L-tyrosine.     

Epoxy coenzyme a thioester pathways for degradation of aromatic compounds

Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.     

Engineering of a psychrophilic bacterium for the bioremediation of aromatic compounds

Microbial degradation of aromatic hydrocarbons has been studied with the aim of developing applications for the removal of toxic compounds. Efforts have been directed toward the genetic manipulation of mesophilic bacteria to improve their ability to degrade pollutants, even though many pollution problems occur in sea waters and in effluents of industrial processes which are characterized by low temperatures. From these considerations the idea of engineering a psychrophilic microorganism for the oxidation of aromatic compounds was developed.In a previous paper it was demonstrated that the recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 (PhTAC/tou) expressing a toluene-o-xylene monooxygenase (ToMO) is able to convert several aromatic compounds into corresponding catechols. In our work we improved the metabolic capability of PhTAC/tou cells by combining action of recombinant ToMO enzyme with that of the endogenous P. haloplanktis TAC125 laccase-like protein. This strategy allowed conferring new and specific degradative capabilities to a bacterium isolated from an unpolluted environment; indeed engineered PhTAC/tou cells are able to grow on aromatic compounds as sole carbon and energy sources. Our approach demonstrates the possibility to use the engineered psychrophilic bacterium for the bioremediation of chemically contaminated marine environments and/or cold effluents.     

The Ins and Outs of Ring-Cleaving Dioxygenases

ABSTRACT

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.