NAD (NAD ϩ ) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD ϩ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD ϩ de novo synthesis pathway. Herein, we report the importance of the NAD ϩ salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD ϩ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (K m of 17 M, k cat of 50 s Ϫ1 , k cat /K m of 3.0 ϫ 10 3 s Ϫ1 · mM Ϫ1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD ϩ salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD ϩ and NADH.IMPORTANCE NAD ϩ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD ϩ decomposition is, in general, more rapid. This study emphasizes that NAD ϩ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.KEYWORDS NAD ϩ , Thermus thermophilus, nicotinamidase, salvage synthesis N AD (NAD ϩ ) is an essential molecule for cellular metabolism. It serves as an electron donor/acceptor for many redox reactions in cellular metabolism. It also serves as a precursor for NADP ϩ and a substrate for both bacterial DNA ligases and ADP ribosyl transferases (1, 2). Due to its importance as a cofactor for many oxidoreductases, NAD ϩ is also an essential compound in biotechnology applications (3); however, NAD ϩ is known to be chemically unstable, especially at high temperatures, where it nonenzymatically decomposes to ADP-ribose and nicotinamide (4,5).Cells need to maintain a certain concentration of NAD ϩ for metabolism, and different organisms possess different pathways for synthesizing NAD ϩ , such as the de novo biosynthesis pathway and the salvage pathway (6). In the salvage pathway,

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