Electron paramagnetic resonance (EPR) studies on hydrogenase-1 (HYD1) purified from a mutant strain (AP6) of Escherichia coli enhanced in HYD1.

Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1. EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals. At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared. The detection of a 4Fe-4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear. The 4Fe-4S complex intensity was slightly increased. The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.

Pseudomonas aeruginosa for the evaluation of swimming pool chlorination and algicides.

Concentrations of ammonia and the chlorine stabilizer, cyanuric acid, which could be expected in swimming pools decreased the rate of kill by chlorine of the potential pathogen, Pseudomonas aeruginosa. The effect of cyanuric acid increased as the concentration of chlorine decreased, a fact of significance from a public health view. Quaternary ammonium algcides had little effect on the kill rate of chlorine, but an organic mercury algicide had a synergistic effect with chlorine when the chlorine activity was stressed by the addition of ammonia or the use of 100 times the normal concentration of bacteria. The effect of natural waters, rain, beaches, and swimming pools on the kill rate by 0.5 mg of chlorine per liter indicated that a treatment time of 1 hr or more was required to kill 99.9% of 10(6)Pseudomonas cells per ml. The synergism of chlorine and the organic mercury algicide was also demonstrated with these waters and with sewage treatment plant effluents. The necessity of developing and using laboratory tests which simulate conditions in swimming pools with heavy loads of swimmers, as opposed to tests in chlorine demand-free conditions, is discussed. Samples taken from well-supervised swimming pools when the swimmer load had been especially high required treatment times of 1 to 3 hr to obtain 99.9% kills of the potential pathogen, P. aeruginosa, with 0.5 mg of chlorine per liter.

Factors influencing the effectiveness of swimming pool bactericides.

Techniques for culturing, harvesting, and testing bacteria to evaluate bactericidal chemicals for swimming pools are described. Concentrations of 25, 50, and 100 mg of the chlorine stabilizer cyanuric acid per liter increased the time required for a 99% kill of Streptococcus faecalis by 0.5 mg of chlorine per liter at pH 7.4 and 20 C from less than 0.25 min without cyanuric acid to 4, 6, and 12 min, respectively. The effect of concentrations of ammonia nitrogen in the range found in swimming pools on the rate of kill of 0.5 mg of chlorine per liter and of chlorine plus cyanuric acid was tested. At concentrations of ammonia nitrogen greater than 0.05 mg per liter, faster rates of kill of S. faecalis were obtained with 100 mg of cyanuric acid per liter plus 0.5 mg of chlorine per liter than with 0.5 mg of chlorine per liter alone. When water samples from four swimming pools with low ammonia levels were used as test media, 0.5 mg of added chlorine per liter killed 99.9% of the added S. faecalis in less than 2 min, but water from a pool with a large number of children required 60 to 180 min of treatment.