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Mapping of Nrda and Nrdb in Escherichia Coli K-12 JAMES A

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Mapping of Nrda and Nrdb in Escherichia Coli K-12 JAMES A JOURNAL OF BACTERIOLOGY, Dec. 1976, p. 810-814 Vol. 128, No. 3 Copyright © 1976 American Society for Microbiology Printed in U.S.A. Mapping of nrdA and nrdB in Escherichia coli K-12 JAMES A. FUCHS* AND H. OLLE KARLSTROM' Medicinska Nobelinstitutet, Biokemiska Avdelningen, Karolinska Institutet, Stockholm, Sweden, and Department ofBiochemistry, University of Minnesota, College ofBiological Sciences, St. Paul, Minnesota 55108* Received for publication 15 July 1976 The structural genes coding for the Bi and B2 subunits of the enzyme ribonucleoside diphosphate reductase, nrdA (formerly designated dnaF) and nrdB, respectively, have been mapped in Escherichia coli. They are located at approximately 48 min. The gene order in this region of the E. coli chromosome was found to be purF glpT nrdB nrdA nalA cdd dcd his. In Escherichia coli, the enzyme ribonucleo- thioredoxin reductase obtained from E. coli B were side diphosphate reductase (RDP reductase) available in our laboratory. Davis minimal medium (E.C. 1.17.4.1) is responsible for the conversion (6) and L broth (15) were used. of ribonucleotides to deoxyribonucleotides. It Bacterial strains. All ofthe bacterial strains used thus in this study were derivatives ofE. coli K-12 and are catalyzes the first reaction in a pathway described in Table 1. specific for the synthesis of deoxyribonucleic Selection and testing of phenotypes. The follow- acid precursors (4). RDP reductase contains two ing conditions were used to detect the phenotypes subunits designated Bi and B2. Each of the caused by the indicated mutations: subunits is composed of two polypeptide chains nrdA (formerly dnaF [20]): Failure of cells to (4). A mutant containing a structurally altered grow at 40°C on either enriched or minimal plates Bi subunit (8) and a mutant containing a struc- supplemented with 20 Ag of thymidine per ml. turally altered B2 subunit (7, 9) have been nrdB: Inhibition of growth by hydroxyurea (0.25, characterized. The mutations present in these 0.50, or 1.0 mg/ml) in minimal media in the pres- strains have been designated nrdA and ence of 20 gg of thymidine per ml. Sensitivity of nrdB, both nrdB and nrdB+ is increased at a lower tem- respectively. The mutated Bi subunit was perature (30°C) and in thymine auxotrophs. shown to be extremely thermolabile in vitro (8), nalA: Resistance to 40 ,ug of nalidixic acid per ml. and the mutated B2 subunit was found to have cdd in thyA+ strains: Inhibition of growth by 5- a greatly decreased activity, which could be fluorodeoxycytidine (0.2 /,g/ml). partially restored by the addition of high con- cdd in thyA strains: Growth with 5-methyl de- centrations of sodium acetate (7). The nrdA oxycytidine as thymine source. mutation prevents the growth of strains har- cdd in pyr strains: Growth with deoxycytidine boring it at temperatures above 40°C even in (20 ,ug/ml) as pyrimidine source. enriched media. The nrdB mutant was isolated cdd in thyA pyr strains: Growth with 5-methyl deoxycytidine and deoxycytidine as thymine and from a specially constructed strain as a deoxy- pyrimidine sources, respectively. uridine auxotroph (7), but the mutation was gipT: Inability to utilize 0.4% a-glycerol phos- found to be more easily recognizable by the phate as a carbon source. Isolation ofgipT mutants increased sensitivity to hydroxyurea that it was accomplished by planting a culture on minimal conferred to strains harboring it. We have used plates containing 0.4% glycerol as a carbon source these properties to map the nrdA and nrdB and 0.2 mg of phosphonomycin per ml. Resistant genes. In the course of these studies we also colonies were purified and screened for those unable located the approximate map position of two to use 0.4% a-glycerol phosphate as a carbon source additional deoxyribonucleotide-metabolizing (13). genes, cdd and dcd, the genes dcd (formerly paxA [16]): Sensitivity to 100 pAg of coding for deoxy- 5-bromodeoxyuridine per ml at 42°C. cytidine (cytidine) deaminase and deoxycyti- Conjugation. Matings between many different dine triphosphate deaminase, respectively. Hfr strains and a single female were carried out as follows. A total of 25 Hfr strains were grown over- MATERIALS AND METHODS night in 0.1 ml of L broth in separate compartments Materials. [3H]cytidine diphosphate was obtained of an autoclavable nylon microculture container from Schwarz/Mann. Hydroxyurea was a gift from (E.L.E.S.A., 20129 Milan, Italy). The cultures were E. R. Squibb and Sons. Purified thioredoxin and diluted 10-fold and grown for 2 h. A 0.3-ml sample of each Hfr was transferred to a new block and com- ' Present address: University Institute of Microbiology, bined with 0.3 ml of the exponentially growing fe- Copenhagen, Denmark. male. At various times samples from each mating 810 VOL. 128, 1976 RDP REDUCTASE-DEFICIENT MUTANTS OF E. COLI 811 TABLE 1. E. coli strains used Strain Sex of chromosome Source of Genotype derivation/reference KL16 Hfr AB2572 Hfr CGSC4280 F'129 argG metB leu his recA mtl xyl malA gal lacY or lacZ str Barbara Bachman (KLF29/JC1553) LD195 F- his argG metB leu lacY or klcZ malA xyl mtl gal str tpp cdd dcd nrdB 7, 9 (P1) E101 F- thr leu thi thyA dra drm pup tonA lacY supE nrdA J. Gross (20) K-43 F- thi tonB trp his met cys str gal lac tonA tsx (A) G. Bertani KK391 F- thi met cys str gal lac tonA tsx nrdB cdd See text KK395 F- thr leu thi thyA dra drm pup tonA lacY supE nrdA nalA From E101 KK408 F- thi met cys str gal lac tonA tsx nrdB cdd thyA From KK391 KK419 F- met drm thyA nalA glpT Constructed in our lab KK420 F- thi met cys str gal lac tonA tsx nrdB cdd thyA glpT From KK395 KK424 F- thr leu thi thyA dra drm pup tonA lacY supE nrdA nalA glpT From KK395 KK342 F'129 his metB leu lacY or lacZ nalA str xyl dcd cdd tpp nrdB (P1) See text KK343 F'129 his metB leu lacY or kzcZ naIA str xyl dcd cdd tpp nrdB (P1) See text HD1038 F- his metB leu argG lacY or lacZ malA str xyl mtl dcd gal 17 JC411 F- his metB leu argG lacY or kacZ malA str xyl gal mtl 17 LD181 F- his metB leu lacY or lacZ malA gal str xyl mtl dcd cdd tpp 7 CGSC4249 F'103 argG metB his trp leu mtl xyl malA gal lacY or lacZ str Barbara Bachman (KLF3/JC1153) LD188 F- his metB his trp leu lacY malA str xyl mtl dcd cdd tpp pyrE thyA 6 were transferred to selective agar plates by inverted recombinants from noninterrupted matings nails held by a Plexiglas template. were analyzed for unselected markers. Since Interrupted and long-term matings were per- the recipient contains cdd (mutation in the formed as described by Curtiss et al. (5). P1 transduction. P1 transductions were con- gene coding for cytidine [deoxycytidine] deami- ducted essentially as described by Lennox (12) ex- nase) which had previously been located clock- cept that phage P1 vir-1 was used. Map locations are wise from his (0. Karlstrom, unpublished given according to the 100-min map (1). data, and in Salmonella [2]), cdd+ was ana- Enzyme assays. RDP reductase assays were con- lyzed as well. Table 2 indicates that the selec- ducted as previously described (4). tion of nrd+ resulted in a low frequency of Protein estimation. Protein concentrations in inheritance of unselected markers. Selection of crude extracts were estimated from the absorbance his+ was accompanied by a much higher fre- at 280 and 260 nm (1-cm light path) by the following quency of cdd+ and nrd+. Furthermore, cdd+ equation: protein concentration (milligrams per and nrd+ usually appeared together. This milliliter) = 1.55 A280 - 0.76 A26 (19). would indicate a gene order ofnrdB cdd his. In this experiment, unselected markers were un- RESULTS AND DISCUSSION derrepresented due to the restriction exerted by Mapping ofnrdB by conjugation. A series of phage P1, which is present as a prophage in Hfr strains was crossed to the nrdB mutant LD195. LD195, and after 30 min samples were spotted Before the nrdB mutation could be trans- on plates containing streptomycin (500 ug/ml) ferred via P1 transduction, it was necessary to and hydroxyurea (1 mg/ml). Hfr KL16, which transfer it to a nonlysogenic strain. F'129, transfers counterclockwise with an origin at which includes the region ofthe E. coli chromo- approximately 60.5 min, gave numerous recom- some from 44 to 50 min including the nrd+ binants, whereas Hfr AB2572, which also genes, from strain CGSC428 was transferred to transfers counterclockwise but with an origin of strain LD195 by selecting for his+ to obtain transfer of approximately 45.5 min, gave no strain KK342. An nrdB cdd homozygote KK343 recombinants. This indicates that nrdB lies be- was obtained from strain KK342 by selecting tween 45.5 and 60.5 min. An interrupted mat- for resistance to 0.1 ,ug of 5-fluorodeoxycytidine ing utilizing strains KL16 and LD195 was car- per ml. Since difficulties were experienced in ried out, selecting for either hydroxyurea and transferring the nrdB mutation to the chromo- streptomycin resistance or histidine prototro- some ofanother strain via episome transfer and phy and streptomycin resistance. The results homozygotization, strain KK343 was subjected indicated that hydroxyurea resistance is trans- to ultraviolet light and crossed to strain K-43 in ferred 2.7 to 5.0 min before his, and thus nrdB an Hfr-type mating selecting for trp+ recombi- is located between 47 and 49 min.
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