Substrates and pathways preference of two novel species of Rhodococcus for aromatic compounds degradation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that are hazardous to human health. It has been demonstrated that members of the Mycobacterium genus are among the most effective degraders of PAHs, but few studies have focused on the degradation of PAH mixtures. In this study, single and mixed PAH metabolism was investigated in four phylogenetically distinct Mycobacterium species with respect to (i) parent compound degradation, (ii) bacterial growth, (iii) catabolic gene expression, and (iv) metabolite production. Synergistic and antagonistic effects on four model PAH compounds (benzo[a]pyrene, pyrene, fluoranthene, and phenanthrene) characterized degradation of mixtures in a strain- and mixture-dependent manner. The mixture of pyrene and phenanthrene, in particular, resulted in antagonized degradation by three out of four bacterial species, and further studies were narrowed to investigate the degradation of this mixture. Antagonistic effects persisted over time and were correlated with reduced bacterial growth. Antagonized degradation of PAH was not caused by preferential degradation of secondary PAHs, nor were mixture compounds or concentrations toxic to cells growing on sugars. Reverse transcription-PCR (RT-PCR) studies of the characterized catabolic pathway of phenanthrene showed that in one organism, antagonism of mixture degradation was associated with downregulated gene expression. Metabolite profiling revealed that antagonism in mixture degradation was associated with the shunting of substrate through alternative pathways not used during the degradation of single PAHs. The results of this study demonstrate metabolic differences between single and mixed PAH degradation with consequences for risk assessment and bioremediation of PAH-contaminated sites. Mycobacterium species are promising organisms for environmental bioremediation because of their ubiquitous presence in soils and their ability to catabolize aromatic compounds. PAHs can be degraded effectively as single compounds, but mixed substrates often are subject to degradative inhibition, which may explain the persistence of these pollutants in soils. Single and mixed PAH degradation by diverse Mycobacterium species was compared, with associated bacterial growth, gene expression, and metabolite production. The results demonstrate that antagonism characterized degradation in a strain- and mixture-dependent manner. One strain that was versatile in its pathway use of single chemicals also efficiently degraded the mixture, whereas antagonism in other the strains was associated with altered metabolic profiles, indicating unusual pathway use. The impacts of this work on risk assessment and bioremediation modeling studies indicate the need to account for mixture-generated intermediates and to recognize mixture degradation as a property distinct from that of PAH substrate range.

Study of the regulation of the syntheses of enzymes of the catechol and protocatechuate pathways in Pseudomonas putida has shown that two groups of enzymes are subject to coordinate control. cis,cis-Muconate-lactonizing enzyme and muconolactone isomerase, which are uniquely associated with the catechol pathway, constitute the first coordinate block of enzymes. The synthesis of these enzymes, as well as that of catechol oxygenase (which is regulated independently), seems to be induced by cis,cis-muconate. The second coordinate block of enzymes comprises β-carboxy-cis,cis-muconate-lactonizing enzyme and γ-carboxymuconolactone decarboxylase, which are uniquely associated with the protocatechuate pathway, and β-ketoadipate enol-lactone hydrolase, which is functional in both the protocatechuate and the catechol pathways. This group of enzymes seems to be induced by β-ketoadipate or β-ketoadipyl coenzyme A. Moraxella lwoffii, which degrades protocatechuate and catechol through identical step-reactions, regulates the synthesis of the enzymes mediating these conversions by a different mechanism.

Complete metabolic study by dibutyl phthalate degrading Pseudomonas sp. DNB-S1.

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.

Metabolism of veratric acid and other aromatic compounds has been studied in two strains of Pycnoporus cinnabarinus. In non-agitated cultures which contained cellulose as an additional carbon source, veratric acid was demeth(ox)ylated to vanillic acid which accumulated in the medium. Under these conditions, 14CO2 evolution from [4-O14CH3]-veratric acid preceded that from [3-O14CH3]-veratric acid in the case of both strains. 14CO2 evolution was markedly accelerated and increased when 100% oxygen was employed instead of air. Oxygen had not so strong effect on the decarboxylation of 14COOH-labelled vanillic and p-hydroxybenzoic acid but it did increase decarboxylation of 14COOH-labelled veratric acid, indicating the effect of oxygen on the preceding demeth(ox)ylation. There were indications, for example rapid demethylation of veratric acid in early stages of growth when apparent phenol oxidase (laccase) activity was zero, for an existence of a separate demethylase enzyme. However, the participation of phenol oxidases in demeth(ox)ylation cannot be ruled out. Degradation pattern of vanillic acid was basically similar in P. cinnabarinus compared to Sporotrichum pulverulentum (Phanerochaete chrysosporium). Also the effect of carbon source was similar: cellulose as a carbon source enhanced degradation of vanillic acid through methoxyhydroquinone whereas in glucose medium, vanillic acid was reduced to the respective aldehyde and alcohol.

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

Finding novel chemoreceptors that specifically sense and trigger chemotaxis toward polycyclic aromatic hydrocarbons in Novosphingobium pentaromativorans US6-1

BackgroundThe aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved.ResultsUsing whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds.ConclusionsThis study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories.

Extracts of benzoate-grown Pseudomonas putida have high levels of β-carboxy-cis,cis-muconate-lactonizing enzyme and γ-carboxymuconolactone decarboxylase, even though these enzymes are uniquely associated with the protocate-chuate pathway. Since these activities might result from nonspecific catalysis by the enzymes which catalyze the analogous reactions in the catechol pathway, cis,cis-muconate-lactonizing enzyme and muconolactone isomerase were purified extensively and their specificities were examined. Crystalline preparations had no detectable activity on the carboxylated substrate analogues of the parallel convergent pathway. The enzymes of the catechol pathway do, however, share some physical characteristics with the enzymes that catalyze the analogous reactions in the protocatechuate pathway.

Two new intermediates were identified in the protocatechuate pathway of Pseudomonas putida. The first of these, γ-carboxymuconolactone (γ-carboxy-γ-carboxymethyl-Δα-butenolide), is the product of the enzymic lactonization of β-carboxy-cis, cis-muconate. Enzymic decarboxylation of γ-carboxymuconolactone gives rise to β-ketoadipate enollactone (γ-carboxymethyl-Δβ-butenolide), the second newly discovered intermediate in the protocatechuate pathway. β-Ketoadipate enol-lactone, which was isolated and physically characterized, is also an intermediate in the catechol pathway; the catechol and protocatechuate pathways converge at this point. β-Ketoadipate enol-lactone is hydrolyzed to β-ketoadipate by an enzyme which is essential for utilization of either catechol or protocatechuate. Studies with Moraxella lwoffii showed that this organism also degrades protocatechuate and catechol by the pathways characteristic of P. putida. Elucidation of the bacterial pathway for the dissimilation of protocatechuate revealed that the three step-reactions responsible for the conversion of this compound to β-ketoadipate enol-lactone are analogous with the step-reactions responsible for the conversion of catechol to β-ketoadipate enol-lactone.

Earthworms can expedite di-(2-ethylhexyl) phthalate (DEHP) degradation in soils, but limited information is available on the key DEHP-degradation pathways and related genes during the vermicomposting process. In this study, DEHP degradation, degradation-related genes and bacterial communities were investigated by metagenomic analysis. DEHP degradation efficiency was significantly and 65.69% higher in vermicomposting treatment than natural soils. Earthworm supplement remarkably increased the contents of humic acid, humus and fulvic acid in soils. Both humic acid and earthworm gut positively stimulated soil microbes potentially responsible for DEHP degradation. Betaprotebacteria, Acidobacteria, Variovorax, Hydrogenophaga, Limnobacter, Ramlibacter, Pseudomonas, Acinetobacter, Paracoccus and Achromobacter significantly contributed to DEHP degradation pathways. From functional gene analysis, there were remarkable differences in dominant DEHP degradation pathways between soils (catechol pathway), earthworm cast (protocatechuate pathway), and earthworm gut (protocatechuate and catechol pathways). Our findings proposed two possible mechanisms of earthworms in accelerating DEHP degradation, stimulating the activities of indigenous degraders to augment the catechol pathway in soils and providing an extra protocatechuate pathway in earthworm gut. This study, for the first time, offers new insights into the impacts of vermicomposting on DEHP degradation genes and pathways, providing valuable scientific evidence for improving DEHP bioremediation in contaminated agricultural soils.

Polycyclic aromatic hydrocarbon (PAHs) are common soil contaminants of concern due to their toxicity toward plants, animals and microorganisms. The use of indigenous or added microbes (bioaugmentation) is commonly used for bioremediation of PAHs. In this work, the biodegradation rates and changes in the bacterial community structure were evaluated. The enrichment culture was useful for unambiguously identifying members of the soil bacterial community associated with PAH degradation and yielded a low diversity community. No significant difference in the rate of PAH degradation was observed between the microcosm receiving only PAHs or PAHs and bioaugmentation. Moreover, identical matches to the bioaugmentation inoculum were only observed at the initial stages of PAH degradation on day 8. After 22 days of incubation, the substantial degradation of all PAHs had occurred in both microcosms and the PAH contaminated soil had statistically significant increases in Alphaproteobacteria. There were also increases in Betaproteobacteria. In contrast, the PAH contaminated and bioaugmented soil was not enriched in PAH degrading Proteobacteria genera and, instead, an increase from 1.6% to 8% of the population occurred in the phylum Bacteroidetes class Flavobacteria, with Flavobacterium being the only identified genus. In addition, the newly discovered genus Ohtaekwangia increased from 0% to 3.2% of the total clones. These results indicate that the same soil microbial community can give rise to different PAH degrading consortia that are equally effective in PAH degradation efficiency. Moreover, these results suggest that the lack of efficacy of bioaugmentation in soils can be attributed to a lack of persistence of the introduced microbes, yet nonetheless may alter the microbial community that arises in response to PAH contamination in unexpected ways.

To investigate the effects of physiological properties on polycyclic aromatic compound (PAH) degradation, the surface tension and emulsification activities, and cell surface hydrophobicity of five PAH-degrading yeast isolates were compared to Saccharomyces cerevisiae from cultures grown with glucose, hexadecane, or naphthalene as carbon sources. The cell surface hydrophobicity values for the five yeast strains were significantly higher than for S. cerevisiae for all culture conditions, although these were highest with hexadecane and naphthalene. Strains with higher hydrophobicity showed higher rates of naphthalene and phenanthrene degradation, indicating that increased cell hydrophobicity might be an important strategy in PAH degradation for the five strains. Emulsification activities increased for all five yeast strains with naphthalene culturing, although no relationship existed between emulsification activity and PAH degradation rate. Surface tensions were not markedly reduced with naphthalene culturing.

The catabolism of aromatic compounds in Corynebacterium glutamicum was investigated by genome data mining and by experimental analysis. Results indicated that C. glutamicum assimilated different aromatic compounds such as phenol, p-cresol, benzoate, 4-hydroxybenzoate, vanillate, vanillin, resorcinol, 3,5-dihydroxytoluene and 2,4-dihydroxybenzoate. Genome data indicated, and enzyme assays confirmed; the existence of multiple ring-cleavage pathways for the catabolism of central aromatic intermediates; the protocatechuate and catechol branches of the β-ketoadipate pathway, two similar hydroxyquinol pathways and the gentisate pathway. Two putative hydroxyquinol 1,2-dioxygenase genes (ncg11113 and ncg12951) were cloned and functionally identified in Escherichia coli. The genes encoding enzymes for the conversion of phenol, benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate and vanillate in the central β-ketoadipate pathways were mapped on the chromosome of C. glutamicum. A unique 30-kb (approximately 1% of the entire genome) catabolic island that channels the degradation of various aromatic compounds was mapped to position 2525-2555 kb of the genome. The global analysis and characterization of aromatic degradation pathways provided new insights into the metabolic ability of C. glutamicum in addition to its well-known ability to produce various amino acids and vitamins.

Aromatic compound degradation by iron reducing bacteria isolated from irrigated tropical paddy soils