The species of Comamonas testosteroni is the most common human pathogen of the genus, which can be associated with acute appendicitis, infections of the bloodstream, the peritoneal cavity, cerebrospinal fluid, inflammatory bowel disease, and in general, bacteremia. According to the literature, Comamonas testosteroni has destructive activity to a wide range of toxic chemical compounds, including chlorobenzenes. The specified strains were isolated from the soil of the organochlorine waste landfill, where hexachlorobenzene (HCB) was predominant. These strains were expected to be capable of degrading HCB. Microbiological (bacterial enrichment and cultivating, bacterial biomass obtaining), molecular biology, biochemical (enzymatic activities, malondialdehyde measuring, peroxidation lipid products measuring), and statistical methods were carried out in this research. The reaction of both strains (UCM B-400 and UCM B-401) to the hexachlorobenzene presence differed in the content of diene and triene conjugates and malondialdehyde, as well as different catalase and peroxidase activity levels. In terms of primary peroxidation products, diene conjugates were lower, except conditions with 20 mg/L HCB, where these were higher up to two times, than the pure control. Malondialdehyde in strain B-400 cells decreased up to five times, in B-401, but increased up to two times, compared to the pure control. Schiff bases in strain B-400 cells were 2-3 times lower than the pure control. However, in B-401 cells Schiff bases under higher HCB dose were in the same level with the pure control. Catalase activity was 1.5 times higher in all experimental variants, compared to the pure control (in the strain B-401 cells), but in the B-400 strain, cells were 2 times lower, compared to the pure control. The response of the two strains to hexachlorobenzene was similar only in peroxidase activity terms, which was slightly higher compared to the pure control. The physiological response of Comamonas testosteroni strains to hexachlorobenzene has a typical strain reaction. The physiological response level of these strains to hexachlorobenzene confirms its tolerance, and indirectly, the ability to destroy the specified toxic compound.
- MeSH
- Antioxidants MeSH
- Chlorobenzenes MeSH
- Comamonas testosteroni * MeSH
- Hexachlorobenzene * MeSH
- Catalase MeSH
- Humans MeSH
- Lipids MeSH
- Malondialdehyde MeSH
- Lipid Peroxidation MeSH
- Soil MeSH
- Schiff Bases MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
OBJECTIVE: Comamonas testosteroni Pb50 is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This bacterial strain is capable of utilizing phenol as the sole carbon and energy source. Although examples are known in which the C. testosteroni utilizes phenol for growth or metabolism, much less information are known on the nature of the phenol-oxidizing enzymes in this microorganism. Therefore, the occurrence and cellular location of phenol hydroxylase (EC 1.14.13.7), the enzyme participating in the first step of phenol degradation, catalyzing its hydroxylation to catechol in a bacterial Comamonas testosteroni Pb50 strain grown in the presence of phenol as a sole carbon and energy source are the aims of this study. METHODS: Combination of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300 was used for isolation of phenol hydroxylase detectable in the medium in which C. testosteroni was cultivated. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephacryl S-300 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (phenol consumption and/or catechol formation). RESULTS: Whereas low activity of phenol hydroxylase was detected in cytosol isolated from C. testosteroni, more than 16-fold higher activity of this enzyme was found in the medium in which C. testosteroni was cultivated. The presence of phenol hydroxylase extracellular activity suggests that this microorganism may secrete the enzyme into the extracellular medium. Using the procedure consisting of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300, the enzyme was isolated from the medium to homogeneity. The formation of catechol mediated by purified phenol hydroxylase is strictly dependent on the presence of NADPH, which indicates that this enzyme is the NADPH-dependent phenol hydroxylase. The enzyme is a homotetramer having a molecular mass of 240 000, consisting of four subunits having a molecular mass of 60 000. The optimum pH of the enzyme for the phenol oxidation is pH 7.6. CONCLUSION: The results are the first report showing isolation and partial characterization of extracellular NADPH-dependent phenol hydroxylase of a bacterial C. testosteroni Pb50 strain capable of oxidizing phenol to catechol. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by bacteria.
- MeSH
- Time Factors MeSH
- Comamonas testosteroni enzymology genetics MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Extracellular Space enzymology genetics MeSH
- Phenol metabolism MeSH
- Catalysis MeSH
- Catechols metabolism MeSH
- Cloning, Molecular MeSH
- Hydrogen-Ion Concentration MeSH
- NADP metabolism MeSH
- Oxidation-Reduction MeSH
- Mixed Function Oxygenases genetics isolation & purification metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Phthalic acid isomers are the monomers of phthalate molecules, also known as phthalic acid esters, widely employed in the plastics industry. This study aims to investigate the biodegradation of phthalic acid (PA) and terephthalic acid (TPA) by five industry-borne Comamonas testosteroni strains: 3APTOL, 3ABBK, 2B, 3A1, and C8. To assess the ability of C. testosteroni strains to biodegrade phthalic acid isomers in fermentation media, an analytical method was employed, consisting of high-performance liquid chromatography (HPLC) analyses. Subsequently, molecular screening of the genomic and plasmid DNA was conducted to identify the degradative genes responsible for the breakdown of these chemicals. The genes of interest, including ophA2, tphA2, tphA3, pmdA, and pmdB, were screened by real-time PCR. The five C. testosteroni strains effectively degraded 100% of 100 mg/L PA (p = 0.033) and TPA (p = 0.0114). Molecular analyses indicated that all C. testosteroni strains contained the pertinent genes at different levels within their genomes and plasmids, as reflected in the threshold cycle (Ct) values. Additionally, DNA temperature of melting (Tm) analyses uncovered minor differences between groups of genes in genomic and plasmid DNA. C. testosteroni strains could be excellent candidates for the removal of phthalic acid isomers from environmental systems.
