Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defence compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defence compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach

ABSTRACT The release of synthetic chemical pollutants in the environment is posing serious health risks. Enzymes, including oxygenases, play a crucial role in xenobiotic degradation. In the present study, we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants. Fosmid clones possessing dioxygenase activity were further sequenced, and their genes were identified using bioinformatics tools. Two positive fosmid clones, SD3 and RW1, suggested the presence of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC-SD3) and catechol 2,3-dioxygenase (C23O-RW1), respectively. Recombinant versions of these enzymes were purified to examine their pollutant-degrading abilities. The crystal structure of BphC-SD3 was determined at 2.6-Å resolution, revealing a two-domain architecture, i.e., N-terminal and C-terminal domains, with the sequential arrangement of βαβββ in each domain, characteristic of Fe-dependent class II type I extradiol dioxygenases. The structure also reveals the presence of conserved amino acids lining the catalytic pocket and Fe3+ metal ion in the large funnel-shaped active site in the C-terminal domain. Further studies suggest that Fe3+ bound in the BphC-SD3 active site probably imparts aerobic stability. We further demonstrate the potential application of BphC-SD3 in biosensing of catecholic compounds. The halotolerant and oxygen-resistant properties of these enzymes reported in this study make them potential candidates for bioremediation and biosensing applications. IMPORTANCE The disposal and degradation of xenobiotic compounds have been serious issues due to their recalcitrant properties. Microbial oxygenases are the fundamental enzymes involved in biodegradation that oxidize the substrate by transferring oxygen from molecular oxygen. Among oxygenases, catechol dioxygenases are more versatile in biodegradation and are well studied among the bacterial world. The use of catechol dioxygenases in the field is currently not practical due to their aerobically unstable nature. The significance of our research lies in the discovery of aerobically stable and halotolerant catechol dioxygenases that are efficient in degrading the targeted environmental pollutants and, hence, could be used as cost-effective alternatives for the treatment of hypersaline industrial effluents. Moreover, the structural determination of novel catechol dioxygenases would greatly enhance our knowledge of the function of these enzymes and facilitate directed evolution to further enhance or engineer desired properties.

Identification of catechol dioxygenases of acinetobacter species, with improved ability to degrade crude oil

Background: Various microbes involved in alkanes degradation or reducing organics oil products have been identified among. The goal of this work was to screen the soil bacteria with potential ability to degrade alkanes and characterized using a combination of genetic and physiological methods.Methods: The microbial species of soil sample was isolated and the loss of hydrocarbon was calculated periodically by gravimetric analysis. Isolated bacterial strain with potential ability to degrade the engine oil in Baghdad city was further tested for hydrocarbon-degrading abilities using 2, 6-DCPIP. Colonies of bacteria were counted using serial dilution methods of the soil sample. One of the highly bioactive degrading strains ISN53 was selected and a different fraction of crude extract were analyzed for their hydrocarbon degrading activity. The purified active fraction characterised in LC/MS and SDS PAGE. Presence of chlorocatechol dioxygenases was checked by PCR and expression was analyzed in RT PCR. The envelope of ISn21 isolate was checked for any changes under different conditions using microbial adhesion to the hydrocarbon test (MATH). Results: Serratia marcesens (Sm53) and Acinetobacter venetianus (ISn21) were isolated and ISn21 showed maximum growth on the third day and up to 72% degradation on the 7th day of incubation. LC-MS/MS spectrometry analysis identified the bioactive enzyme catechol dioxygenases in crude extract. The purified catechol dioxygenases enzyme had 35 kDa weight which showed the maximum activity between 48 to 72 hour of incubation. The expression of its gene was 2 fold more than control the maximum expression was observed at 60 to 72 hours of incubation. ISn21 was extremely hydrophobic both in MMV and in LB medium.Conclusion: Using a naturally available resources would be better with efficiency at degrading diesel. Use of like microorganisms during bioremediation operation might supply worthy advances in this important biotechnological/ biological domain.

Identification of catechol dioxygenases of acinetobacter species, with improved ability to degrade crude oil

Background: Various microbes involved in alkanes degradation or reducing organics oil products have been identified among. The goal of this work was to screen the soil bacteria with potential ability to degrade alkanes and characterized using a combination of genetic and physiological methods.Methods: The microbial species of soil sample was isolated and the loss of hydrocarbon was calculated periodically by gravimetric analysis. Isolated bacterial strain with potential ability to degrade the engine oil in Baghdad city was further tested for hydrocarbon-degrading abilities using 2, 6-DCPIP. Colonies of bacteria were counted using serial dilution methods of the soil sample. One of the highly bioactive degrading strains ISN53 was selected and a different fraction of crude extract were analyzed for their hydrocarbon degrading activity. The purified active fraction characterised in LC/MS and SDS PAGE. Presence of chlorocatechol dioxygenases was checked by PCR and expression was analyzed in RT PCR. The envelope of ISn21 isolate was checked for any changes under different conditions using microbial adhesion to the hydrocarbon test (MATH). Results: Serratia marcesens (Sm53) and Acinetobacter venetianus (ISn21) were isolated and ISn21 showed maximum growth on the third day and up to 72% degradation on the 7th day of incubation. LC-MS/MS spectrometry analysis identified the bioactive enzyme catechol dioxygenases in crude extract. The purified catechol dioxygenases enzyme had 35 kDa weight which showed the maximum activity between 48 to 72 hour of incubation. The expression of its gene was 2 fold more than control the maximum expression was observed at 60 to 72 hours of incubation. ISn21 was extremely hydrophobic both in MMV and in LB medium.Conclusion: Using a naturally available resources would be better with efficiency at degrading diesel. Use of like microorganisms during bioremediation operation might supply worthy advances in this important biotechnological/ biological domain.