JOHN W. FOSTER* AND ELIZABETH A. HOLLEY. Department ofMicrobiology, Marshall University School ofMedicine, Huntington, West Virginia 25701.
Vol. 148, No. 1
JOURNAL OF BACTERIOLOGY, Oct. 1981, p. 394-396 0021-9193/81/100394-03$02.00/0
Genetic Mapping of the Salmonella typhimurium pncB Locus JOHN W. FOSTER* AND ELIZABETH A. HOLLEY Department of Microbiology, Marshall University School of Medicine, Huntington, West Virginia 25701 Received 20 April 1981/Accepted 16 June 1981
The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative topyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.
The genetic locus that codes for nicotinic acid phosphoribosyl transferase in Salmonella typhimurium is designated pncB. This is the only enzyme associated with pyridine nucleotide cycle metabolism which is controlled by end-product repression (5, 6). Earlier mapping studies roughly placed pncB near purB at 25 map units (4). However, attempts to demonstrate linkage of pncB with purB failed. To study the regulation of this gene, we decided to map pncB more accurately on the S. typhimurium chromosome. All bacterial strains used throughout this investigation were derivatives of S. typhimurium LT2 and are listed in Table 1. The generalized transducing phage used were P22 (3), HT105/1int (14), and P1 cmr clrlOO (10). The minimal E medium of Vogel and Bonner (15), minimal lactose basal medium used to test for carbohydrate utilization, and amino acid and vitamin supplements were described previously (4). The relative position of pncB was first obtained by constructing a nadB pncB pyrC strain for use in an interrupted-mating experiment which locatedpncB counterclockwise relative to pyrC. This was contrary to the published map position of pncB on the genetic map (4). Several F'ts plasmids, which carry chromosomal genes in the area of pyrD through purB, were subsequently used in genetic complementation experiInents to localize the area in whichpncB resides. Recipient cells were made recA by the method of Kleckner et al. (8) and Sanderson and Hartman (13). The cells to be made recA were transduced with HT phage propagated on strain TT521, with selection for Tetr. Six of the Tetr transductants were purified by single-colony isolation, grown overnight on Tet plates at 420C, and inoculated into 0.5 ml of L broth (9). After incubation for 5 to 6 h at 370C, each strain was tested for sensitivity to UV irradiation, recA cells being more sensitive to this treatment than recA+ cells. Transfer of temperature-sensitive F'
factors was accomplished by mixing 0.1 ml of logarithmically growing donor and recipient cells directly on selective medium at 300C. Several pncB and nadB pncB mutants were crossed with the various F'ts strains. Primary selection was made for the acquisition of the lac+ phenotype. Subsequent scoring was made either for sensitivity of the pncB recipients to the nicotinic acid analog 6-aminonicotinic acid (genotypepncB/F'tspncB+) or for the ability of a nadB pncB mutant to utilize nicotinic acid as a source of NAD. The results (Table 2) clearly show that pncB+ is carried by F'ts603. In each case examined, pncB+ was dominant to pncB. Control complementation experiments were also performed with purB, pyrD, and pyrC. All putative merodiploids were shown to lose, irreversibly, the acquired trait at 420C. These results show that pncB must be located between gal and pyrD. An additional complementation experiment was performed by introducing the Escherichia coli F' factor F'126 into a nadB pncB strain of S. typhimurium (JF116). This F' factor carries E. coli host markers extending from nadA to trp and should encompass the E. coli pncB locus if it resides at a location analogous to its Salmonella counterpart. Complementation was achieved with regard to the pyrC7 locus (Table 2), but no complementation was achieved with respect to pncB. This suggests that pncB in E. coli may map outside the region covered by F'126. The data presented in Table 2 also confirmed an aroA map position between gal and pyrD in S. typhimurium (11) but indicated that glt is actually located between put and purB, contrary to its published map position (13). Transduction experiments were subsequently performed to establish linkage of pncB with another known genetic marker in this region. Negative results (
