Production of catalases by Comamonas spp. and resistance to oxidative stress.

Bacterial isolates Comamonas terrigena N3H (from soil contaminated with crude oil) and C. testosteroni (isolated from the sludge of a wastewater treatment plant), exhibit much higher total catalase activity than the same species from laboratory collection cultures. Electrophoretic resolution of catalases revealed only one corresponding band in cell-free extracts of both C. testosteroni cultures. Isolates of C. terrigena N3H exhibited catalase-1 and catalase-2 activity, whereas in the collection culture C. terrigena ATCC 8461 only catalase-1 was detected. The environmental isolates exhibited much higher resistance to exogenous H2O2 (20, 40 mmol/L) than collection cultures, mainly in the middle and late exponential growth phases. The stepwise H2O2-adapted culture of C. terrigena N3H, which was more resistant to oxidative stress than the original isolate, exhibited an increase of catalase and peroxidase activity represented by catalase-1. Pretreatment of cells with 0.5 mmol/L H2O2 followed by an application of the oxidative agent in toxic concentrations (up to 40 mmol/L) increased the rate of cell survival in the original isolate, but not in the H2O2-adapted variant. The protection of bacteria caused by such pretreatment corresponded with stimulation of catalase activity in pretreated culture.

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 bacterium Comamonas 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.

Potential application of catalase-peroxidase from Comamonas terrigena N3H in the biodegradation of phenolic compounds.

Comamonas terrigena N3H is a gram-negative rod-shaped bacterium that was isolated from contaminated soil in Slovakia. This bacterium showed remarkable biodegradation properties. We investigated the expression and functioning of two catalase isozymes in this bacterium. The typical catalase could be induced by cadmium ions, whereas the catalase-peroxidase enzyme was constitutively expressed. Since C. terrigena lacks the key enzyme for complete degradation of phenols (phenolhydroxylase), we analysed the possible removal of phenol by the two catalases of this bacterium. Addition of phenol to the culture medium led to increased expression of the catalase-peroxidase. Applying oxidative stress prior to phenol administration markedly induced the expression of the typical catalase, irrespective of the nature of the added agent. Thus, the rate of phenol degradation is rather reduced under these conditions, while growth of the cells is not impaired. We concluded that phenol peroxidation in C. terrigena can be largely attributed to the action of a catalase-peroxidase. The potential application of this enzyme in the removal of phenol from the environment is discussed.

Protein expression in the stressed Vibrio strains.

In a conjunction process using Escherichia coli SM10 (pLOF) KmR APR as donor and Vibrio S141 SmR as recipient, several mutants were constructed: Vibrio PH 101, V. PH 106, and V. PH 109 with lowered ability to synthesize poly-beta-hydroxybutyrate. The survival and metabolic activities of parent and mutant strains were estimated when they were subjected to stress conditions (starvation of carbon and energy sources and/or cadmium treatment). Using two-dimensional electrophoresis, the synthesis of stress proteins was demonstrated. Vibrio cultures consecutively exposed to CdCl2 and then to starvation or vice versa responded similarly metabolically. These results show increased proteosynthetic activity of the stressed Vibrio cells, indicating that the primary cadmium treatment induced the expression and synthesis of the protective proteins, enabling the cells to cope with the secondary stress.

Polynucleotide phosphorylase from Streptomyces aureofaciens: purification and properties.

1. Polynucleotide phosphorylase from a chlortetracycline-producing strain of Streptomyces aureofaciens was isolated by Polymin P fractionation. Using chromatography on DEAE-cellulose and Sephadex G-150 the enzyme, which appears homogeneous in gel chromatography and sedimentation analysis, was purified 2000-fole giving a final yield of 15%. 2. The sedimentation coefficient (s-o 20, w) of the native enzyme in 0.2 M NaCl is 9.15 S and its molecular weight is 210 000 plus or minus 15 000. Molecular weight estimated by sodium dodecylsulfate gel electrophoresis was about 100 000. 3. We have determined the optimal conditions for nucleoside 5'-diphosphate polymerization, their phosphate exchange and phosphorolysis of polyribonucleotides catalysed by polynucleotide phosphorylase from S. aureofaciens. 4. Chlortetracycline is a competitive inhibitor of S. aureofaciens polynucleotide phosphorylase. 5. Polynucleotide phosphorylase is activated in the polymerization reaction by ionic strength (K+, Na+, NH4+) while polyribonucleotide phosphorolysis is activated only by NH4+.