Pyridine nucleotide cycle of Salmonella typhimurium: isolation and characterization of pncA, pncB, and pncC mutants and utilization of exogenous nicotinamide adenine dinucleotide

Abstract

Mutants of Salmonella typhimurium LT-2 deficient in nicotinamidase activity (pncA) or nicotinic acid phosphoribosyltransferase activity (pncB) were isolated as resistant to analogs of nicotinic acid and nicotinamide. Information obtained from interrupted mating experiments placed the pncA gene at 27 units and the pncB gene at 25 units on the S. typhimurium LT-2 linkage map. A major difference in the location of the pncA gene was found between the S. typhimurium and Escherichia coli linkage maps. The pncA gene is located in a region in which there is a major inversion of the gene order in S. typhimurium as compared to that in E. coli. Growth experiments using double mutants blocked in the de novo pathway to nicotinamide adenine dinucleotide (NAD) (nadc) and in the pyridine nucleotide cycle (pnc) at either the pncA or pncB locus, or both, have provided evidence for the existence of an alternate recycling pathway in this organism. Mutants lacking this alternate cycle, pncC, have been isolated and mapped via cotransduction at 0 units. Utilization of exogenous NAD was examined through J. S. GOt& SA797 purC proA46 ilv-K. E. Sandersonb 405 rha-461 str' M-10 fla-56 fim HU180 metP760gal J. S. Gots trpA49 trpA49 K. E. Sanderson Hfr SA534 137 cwc serA13 rfa-3058

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities

“…The addition of cold NAm to the tipA49 reaction mixture prevented most of the NAD pyrophosphatase activity. Whole cells of S. typhimnurium were shown previously to contain NAD pyrophosphatase activity (7). The enzyme, however, has not been demonstrated in vitro.…”

“…Sal-moneUa typhinurium was shown to posses at least two PNCs: a four-membered cycle referred to as PNC IV, and a six-membered cycle referred to as PNC VI ( Fig. 1) (7,9,18). Although some of the enzymes in these cycles have not previously been demonstrated in vitro, both have been shown to recycle NAD in vivo, with PNC IV being the predominant cycle (6).…”

“…The present investigation was initiated to demonstrate the presence or absence of nicotinamide mononucleotide (NMN) glycohydrolase, NAD pyrophosphatase, and NAD glycohydrolase in cell-free extracts of S. typhimurium. NMN glycohydrolase and NAD pyrophosphatase, although suggested in S. typhimurium by several studies (7,9,18), have not been assayed previously in vitro. The results presented show that both of these enzymes exist and reveal the presence of two NAD glycohydrolase activities.…”

“…tipA49 was kindly provided by K. E. Sanderson, University of Calgary, Calgary, Alberta, Canada. JF34 is an nadAl mutant of t*pA49 whose isolation and characterization were described elsewhere (7). The cultural conditions used for growth were described previously (18).…”

“…Nicotinic acid (NA), NAm, NMN, nicotinic acid mononucleotide (NAMN), desamido-NAD, NAD, NADH, NADP, and thionicotinamide adenine dinucleotide were purchased from Sigma Chemical Co., St. Louis, Mo. and were checked for purity by using chromatographic techniques described previously (7,17 zymatic degradation with snake venom phosphodiesterase I as described previously (18). All other chemicals were of analytical quality.…”