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A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, alpha-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by alpha-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. alpha-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to alpha-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of intial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was perferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells respect to molecular weight (61,700) and reaction mechanism, but may be regulated differently.

Aims:  Legionella bacteria ubiquitously colonize natural freshwater and are responsible for legionellosis in humans. Several cases of legionellosis have been associated in particular with the use of whirlpool spas. The objective of this study was to verify whether real‐time PCR is applicable for the quantification of Legionella spp. in spa water. Methods and Results:  The study compared concentrations obtained by real‐time PCR vs that obtained by conventional culture for 101 spa water samples. For the culture method, Legionella spp. were detected and quantified in 14 of 101 samples with measured concentrations ranging from 250 to 3·5 × 105 CFU l−1. With the real‐time PCR method, Legionella spp. were detected and quantified in 42 of 101 samples with concentrations ranging from 1000 to 6·1 × 107 GU l−1. Results revealed a significant but weak correlation (r2 = 0·1867) between the two methods. The positive predictive value (35%) of the PCR method compared to conventional culture herein was low. In contrast, the negative predictive value was excellent, reaching 93%. Conclusions:  Real‐time PCR could be used as a screening tool to rapidly ascertain the absence of Legionella spp. in spa water. However, a positive result involves the need to resort to conventional culture. Significance and Impact of the Study:  Data of this study highlighted the pros and cons of quantification of Legionella spp. in spa water with real‐time PCR using a commercial quantitative PCR kit in a routine laboratory, when compared to conventional culture.

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