Mol Gen Genet (1986) 205:507-514 MGG © Springer-Verlag1986 The pyridine nucleotide cycle of Salmonella typhimurium: Genetic characterization of the pncXA operon Jean M. Hill-Chappell, Michael P. Spector, and John W. Foster Marshall University School of Medicine Huntington, WV 25705, USA Summary. A series of Mudl and TnlO insertions were iden- tified in the pncA chromosome region of Salmonella typhi- murium which is responsible for the production of nicotin- amide deamidase. Both pneA (resulting in no nicotinamide deamidase activity) and pncX (resulting in lowered nicotin- amide deamidase activity) insertions were constructed. In addition, mutants which could utilize nicotinamide as a sole source of nitrogen were isolated. These mutants, designated pncH, hyperproduce nicotinamide deamidase. Genetic stu- dies utilizing pncX-lacZ and pncA- lacZ operon fusions indicate that pncX::TnlO insertions reduce transcription ofpncA --lac while pncH mutations increase the expression of both pncA-lacZ and pncX-lacZ. The gene order was determined as purB-pncA-pncX-gdh with transcription of both pncA and pncX occurring in the counterclockwise direction. Merodiploid studies suggest a model whereby pncX and pncA form an operon with the major promoter occurring upstream from pncX. A second, weaker promoter for pneA must be situated between pncX and pncA. The pncH mutations appear to occur in the pncX promoter (pncXp) increasing promoter activity. Key words: Pyridine nucleotide cycle NAD metabolism - Salmonella typhimurium Genetics Introduction The biosynthesis and metabolism of nicotinamide adenine dinucleotide (NAD) in Salmonella typhimurium includes a de novo and several recycling pathways termed pyridine nucleotide cycles (PNC, Foster and Moat 1980). Two loci involved in PNC metabolism have been identified based in part upon the aquisition of analog resistance to 6-amino nicotinic acid (6ANA) and/or 6-amino nicotinamide (6ANAm). They are pncA (nicotinamide deamidase, 6ANAm%ANA ~) and pncB (nicotinic acid phosphoribosyl- transferase [NAPRTase], 6ANAmr6ANAr). Earlier reports from this laboratory indicated that pncA was constitutively expressed while pncB was found to exhibit repression fol- lowing growth on high concentrations of nicotinic acid (NA), a precursor for NAD (Foster et al. 1979b). The re- pression ofpncB is presumably due to changes in intracellu- lar NAD concentrations. Mapping studies have placed pncA at 27 units on the S. typhimurium linkage map (Foster etal. 1979a; Sanderson and Roth 1983). A comparison Offprint requests to. J.W. Foster with the linkage map of Escherichia coli places pncA within the major inversion region residing near one end of that inversion (Foster et al. 1979a; Rosenfeld et al. 1982; Riley and Anilionis 1978; Bachmann 1983). Another locus, originally described by Hughes et al. (1983), has been designatedpncX. In contrast to pncA muta- tions which lack all demonstrable nicotinamide deamidase activity, pncX mutations produce a reduced level of this enzyme. The mutations, all isolated as TnlO insertions, mapped in the vicinity of pncA. Hughes et al. (1983) sug- gested that the pncX phenotype may either represent TnlO insertions into pncA with some transcription ofpncA occur- ring off an internal TnlO promotor or, alternatively, that the insertions may inactivate a positive regulatory gene for pncA. We describe in this communication the isolation of pncA::Mud and pncX::Mud insertions using the Mu cts dl (Ap Lac) and the Mu cts dl-8 (Hughes and Roth, 1983) vectors. These fusions were used to examine the expression of pncA and pncX. TnlO insertions into pncA and pncX were utilized to examine the effect each mutant locus has upon the expression of the other. In addition, TnlO inser- tions near pncA were used to construct pncX deletions, in- versions and point mutations. Additional mutants, desig- nated pncH, were isolated as hyperproducers of nicotin- amide deamidase and the effect that pncH mutations have on the expression of pncA and pncX was examined. The data indicate that pncX and pncA form an operon with a major promoter, pncXp, which allows transcription of both pncX and pncA. A minor promoter occurs between pncX and pncA that will result in a lowered expression of pncA. Materials and methods Bacterial strains and culture conditions. All strains used in this study were derivatives of Salmonella typhimurium LT-2 and are listed in Table 1. The minimal medium of Vogel and Bonner (1956) and complete LB medium were used and supplemented as described earlier (Foster et al. 1979a). MOPS medium was as described by Neidhardt et al. (1974) MOPS medium lacking a nitrogen source is referred to as MOPS-N. Chemicals and reagents. All chemicals used were of analyti- cal quality. Nicotinic acid (NA) and nicotinamide (NAm) were both used as pyridine sources at a concentration of