Temperature-sensitive Hydrogenase and Hydrogenase Synthesis in a Psychrophilic Bacteriuam

MATERIALS AND METHODS UPADHYAY, J. (Washington State University, Pullman) AND J. L. STOKES. Temperature-sensitive hydrogenase and hydrogenase synthesis in a psychrophilic bacterium. J. Bacteriol. 86:992998. 1963.-Hydrogenase and its synthesis were more heat-sensitive in psychrophilic strain 82 than in mesophilic Escherichia coli. The enzyme -as not formed above 20 C by the psychrophile, whereas it was formed by E. coli and other mesophiles at 45 C. Aerobically grown cells of strain 82 do not contain hydrogenase but could be induced to form the enzyme by incubation with glucose and amino acids. Hydrogenase adaptation proceeded best at pH 8.0. The psychrophile hydrogenase was destroyed 50% by exposure to 60 C for 2 hr compared with 25%c destruction of mesophile hydrogenase under the same conditions. The psychrophile hydrogenase was most active at pH 9.0, and the mesophile hydrogenase was most active at pH 10.0 or higher. Evidence is accumulating that oxidative, fermentative, and lipolytic enzymes and enzymeforming systems in psychrophilic microorganisms are abnormally sensitive to heat (Upadhyay and Stokes, 1963). Additional evidence was supplied recently by Baxter and Gibbons (1962), who found that in a psychrophilic Candida species respiration of intact cells and activity of alcohol dehydrogenase were more sensitive to temperatures above 10 C than in a mesophilic species, C. lipolytica. During our investigations of a temperaturesensitive formic hydrogenlvase in a gram-negative, rod-shaped psychrophilic bacterium, strain 82 (Upadhyay and Stokes, 1963), we encountered a temperature-sensitive hydrogenase and hydrogenase-forming system in the same bacterium. Some of the properties of the enzyme and enzymeforming system are described in the present paper. The modified Brain Heart Infusion (BHI) medium (medium A as previously described; Upadhyay and Stokes, 1963) was used for maintenance of stock cultures and growth of strain 82. The cultures were grown either anaerobically in glass-stoppered bottles completely filled with medium A or aerobically on agar plates. Unless otherwise stated, the cultures were incubated for 24 hr at 20 C and for 12 to 14 hr at 30 C. For manometric experiments, the cells were centrifuged, washed, and suspended in either 0.05 M NaOHboric acid buffer (pH 9.0) or 0.05 M or 0.1 M K2HPO4-KH2PO4 buffer (pH 7.0 and 8.0) as indicated. The cell suspensions were usually adjusted to 450 Klett units (red filter). The Warburg vessels contained 2 ml of cell suspension, 0.3 ml of 0.05 M methylene blue, and 0.2 ml of 10% KOH in the center well to absorb CO2. The gas phase was hydrogen, and the bath temperature was 20 C. Hydrogenase was measured quantitatively by determining the rate of H2 consumption. Hydrogenase was determined also by H2 evolution with reduced methyl viologen (Peck and Gest, 1956). The results are expressed either as QH, values (the microliters of H2 consumed per hour per milligram of dry cells) or as the microliters of H2 Consumed or produced per hour per vessel. There was virtually no endogenous H2 consumption or production. Mesophilic strains of Escherichia coli, Proteus vulgaris, and Aerobacter aerogenes were used for comparative purposes. They were grown and treated in the same general way as strain 82. Additional details of technique will be presented later as needed.