The Expression and Diversity of Catalases in Isolates of Genus Comamonas in Response to the Oxidative Stress of a Polluted Environment

We have evaluated the role of monofunctional heme-containing catalase encoded by cat-1 gene from the soil bacterium Comamonas terrigena N3H in the response to various forms of oxidative stress. Our results indicate that this constitutively expressed catalase represents the major source for the defence of Comamonas terrigena cells against toxic peroxides but the cells can express also a second form of catalase that is bigger and its regulation is probably more complicated. The sequence analysis confirmed the presence of highly conserved catalase sequence motifs in two environmental strains of Comamonas terrigena but in those strains that were not exposed to oxidative stress, no such sequence motif could be detected. The results obtained underline the importance of catalase expression in the defence mechanism against oxidative stress in bacterial cells.     

Oxidative stress-induced expression of catalases in Comamonas terrigena

When grown under oxidative stress, catalatic as well as peroxidatic activity is increased in the Gram-negative bacteriumComamonas terrigena N3H. Two distinct hydroperoxidases were demonstrated by a specific staining. Based on their molar masses and their sensitivity toward 3-amino-1,2,4-triazole and high temperatures, they were identified as dimeric catalase-1 (Cat-1; 150 kDa), and as a tetrameric catalase-2 (Cat-2; 240 kDa) with enhanced peroxidatic activity, respectively. These two catalases differ in their expression during the bacterial growth; whereas the expression of the smaller enzyme (Cat-1) is induced by 0.5 mmol/L peroxides in the medium, and to a lesser degree by 25 mg/L Cd²⁺, Cat-2 (typical catalase) is almost specifically induced with cadmium ions.     

Enrichment, isolation and characterization of dialkyl sulfosuccinate degrading bacteriaComamonas terrigena N3H andComamonas terrigena N1C

Microorganisms derived from soil, sludge, sewage or river water contaminated by detergents or mineral oils were enriched and tested for the capability of primary biodegradation of dialkyl sulfosuccinates (DASS). Adapted mixed cultures, containing predominantly Gram-negative bacteria, degraded di-n-hexyl sulfosuccinate (DHSS) more readily than di-n-octyl sulfosuccinate (DOSS). Bacterial isolates obtained from enriched mixed cultures that show the highest rates of primary biodegradation and assimilation of DASS belong to the genusComamonas. Under nongrowth conditions, the DHSS primary biodegradation by these strains follows the 1st order reaction kinetics. The rate constants of primary degradation of DHSS by resting cells ofC. terrigena N3H were found to be 30 times higher than when catalyzed byC. terrigena N1C. The capacity for accelerated removal of surface-active properties of DHSS byC. terrigena N3H was retained within the range of pH 4–10 and at temperatures up to 55°C, while DHSS degradation byC. terrigena N1C was completely inhibited above 48°C. Temperature and pH limits for DHSS primary degradation by both strains were to a great extent wider than those for the viability of the cultures. Biodegradation capacity and metabolic stability ofC. terrigena N3H make this strain interesting from both scientific and technological point of view.     

Evidence that phenol phosphorylation to phenylphosphate is the first step in anaerobic phenol metabolism in a denitrifying Pseudomonas sp.

Anaerobic phenol degradation has been shown to proceed via carboxylation of phenol to 4-hydroxybenzoate. However, in vitro the carboxylating enzyme was inactive with phenol; only phenylphosphate (phosphoric acid monophenyl ester) was readily carboxylated. We demonstrate in a denitrifying Pseudomonas strain that phenylphosphate is the first detectable product formed from phenol in whole cells and that subsequent phenylphosphate consumption parallels 4-hydroxybenzoate formation. These kinetics are consistent with phosphorylation being the first step in anaerobic phenol degradation. Various cosubstrates failed so far to act as phosphoryl donor for net phosphorylation of phenol in cell extracts. Yet, cells anaerobically grown with phenol contained an enzyme that catalyzed an isotope exchange between [U-¹⁴C]phenol and phenylphosphate. This transphosphorylation activity was anaerobically induced by phenol but was stable under aerobic conditions and required Mn²⁺ and polyethylene glycol. Activity was optimal at pH 5.5 and half-maximal with 0.6 mM Mn²⁺, 0.2 mM phenylphosphate, and 1 mM phenol. It is proposed that the phenol exchange/transphosphorylation reaction is catalyzed as partial reaction by an inducible phenol phosphorylating enzyme. The isotope exchange demands that a phosphorylated enzyme was formed in the course of the reaction, which might be similar to the phosphotransferase system of sugar transport.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

 Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C₁–C₆) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring-fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ringcleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11–0.74 h^-1), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a 1-m³ bioreactor supplied with 1 g nitrogen l^-1 for removal of toluene was 57 g day^-1 and for removal of trichloroethylene was 3.4 g day^-1. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts. Received: 26 June 1995 / Received revision: 4 September 1995 / Accepted: 20 September 1995     

Effect of temperature and pH on phenol removal using purified Coprinus cinereus peroxidase

The removal of phenol by peroxidase-catalysed polymerization was examined using purified Coprinus cinereus peroxidase. The phenol removal efficiency increased with a decrease in the reaction temperature over the range of 0–70 °C, though only a trace of enzyme activity with 4-aminoantipyrine (4-AAP), phenol and hydrogen peroxide was found at 0 °C. The optimum pH value for phenol removal was 9.0, while the enzyme expressed maximum activity at pH 7.5 in the presence of 4-AAP, phenol and hydrogen peroxide. By measuring residual enzyme activity in the polymerizing reaction mixture, it was shown that enzyme inactivation by free radicals was more suppressed at 0 °C than at 40 °C and that the adsorption of the enzyme on the polymerized precipitate was more suppressed at pH 9.0 than that at pH 7.5.     

Lag period of 14CO2 evolution from dioctyl sulpho[2,3-14C]succinate in relation to adaptation of bacterium, Comamonas terrigena, to dialkyl esters of sulphosuccinate

Comamonas terrigena, strain N3H, which was isolated from soil polluted with crude oil products, degraded dioctyl sulphosuccinate, a synthetic commercial surfactant. The primary degradation of this compound, the cleavage of ester bonds between octyl groups and sulphosuccinate, lasted significantly shorter time than the subsequent breakdown of the sulphosuccinate moiety of dioctyl sulpho[2,3-¹⁴C]succinate. ¹⁴CO₂ evolution had a significant shorter lag period with cells in Tris/phosphate medium, without inorganic sulphate and adapted to surfactant, than unadapted cells. The acceleration of the primary degradation by adapted cells also suggest that some enzymes involved in surfactant degradation are inducible. The bacterium may be useful for bioremediation.     

Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters

Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l⁻¹ glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Degradation and decolorization of monosodium glutamate wastewater with <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

Degradation and decolorization of monosodium glutamate wastewater (MSGW) with Coriolus versicolor were firstly carried out. The effects of various operation parameters namely wastewater concentrations, pH, culture time and incidence of sterilization on maximum percentage of degradation and decolorization of wastewater were investigated. Studies of mycelium and enzyme for C. versicolor degradation and decolorization were estimated in this study. Ten percentage of wastewater concentration and pH = 5.0 were found to be the most suitable ones among the other experiments. The highest degradation and decolorization efficiency of wastewater was obtained at the fifth day of cultivation, which was displayed with more than 70% chemical oxygen demand removal, 83% total sugar removal and 55% color removal, respectively. Sterile operation had no remarkable effect on the degradation and decolorization efficiency for C. versicolor. Mycelium and the extra cellular fungal enzyme were both necessary for the degradation and decolorization of MSGW. C. versicolor possesses great potential and economic advantages in MSGW treatment.     

The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance,pathogenicity and asexual structure development in Phytophthora infestans

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil–borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.     

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0–11.0 and 25–40 °C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24 h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.     

Growth of <Emphasis Type="Italic">Trametes versicolor</Emphasis> on phenol

Trametes versicolor 1 was shown to grow on phenol as its sole carbon and energy source. The culture growth and degradation ability dependence on culture medium pH value was observed. The optimal pH value of a liquid Czapek salt medium was 6.5. The investigated strain utilized completely 0.5 g/l phenol in 6 days. The dynamics of the phenol degradation process was investigated. The process was characterized by specific growth rate μ_max 0.33 h⁻¹, metabolic coefficient k = 4.4, yield coefficient Y _x/s  = 0.23 and rate of degradation Q = 0.506 h⁻¹. The intracellular activities of phenol hydroxylase (0.333 U/mg protein) and cis,cis-muconate lactonizing enzyme (0.41 U/mg protein) were demonstrated for the first time in this fungus. In an attempt to estimate the occurrence of gene sequences in T. versicolor 1 related to phenol degradation pathway a dot blot analysis with total DNA isolated from this strain was performed. Two synthetic oligonucleotides were used as hybridizing probes. One of the probes was homologous to the 5′end of phyA gene coding for phenol hydroxylase in Trichosporon cutaneum ATCC 46490. The other probe was created on the basis of cis,cis-muconate lactonizing enzyme coding gene in T. cutaneum ATCC 58094. The results of these investigations showed that T. versicolor 1 may carry genes similar to those of Trichosporon cutaneum capable to degrade phenol.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading <Emphasis Type="Italic">Candida albicans</Emphasis> TL3

A eukaryotic catechol 1,2-dioxygenase (1,2-CTD) was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity. The 1,2-CTD was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration, and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and found to be a homodimer with a subunit molecular weight of 32,000. Each subunit contained one iron. The optimal temperature and pH were 25°C and 8.0, respectively. Substrate analysis showed that the purified enzyme was a type I catechol 1,2-dioxygenase. This is the first time that a 1,2-CTD from a eukaryote (Candida albicans) has been characterized. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF mass analyses provided information of amino acid sequences for BLAST analysis, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314. We conclude that the hypothetical protein is 1,2-CTD.     

TNT transformation products are affected by the growth conditions of Raoultella terrigena

High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [¹⁴C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.     

Isolation and Characterization of Phenol-Degrading Soil Bacterium Xanthobacter flavus

A soil bacterium isolated from a contaminated site degraded phenol when provided as the sole carbon and energy source in the medium. The bacterium was identified as Xanthobacter flavus MTCC 9130. This microbial strain was able to tolerate phenol up to 1000 mg L^?1 concentration. The lag phase increased with the increase in phenol concentration. The optimum growth temperature was 37°C. The organism efficiently utilized phenol and could degrade it completely within 120 h when initial concentration was less than 600 mg L^?1. Degradation of phenol was through ortho pathway, enzyme assay through cell-free extract exhibited the presence of catechol 1,2-dioxygenase. The specific activity was 0.146 μ mol min^?1 mg^?1 protein. However, higher concentrations of phenol in the medium had a negative effect on the growth of the bacterium. Hence this ability of Xanthobacter flavus can be effectively used for bioremediation studies of phenol-contaminated sites.