JOURNAL OF BACTERIOLOGY, Nov. 1973, p. 656-662 Vol. 116, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Deletion Mapping and Orientation of the Histidine Operon of Escherichia coli on a Transducing Bacteriophage ALESSANDRA AVITABILE, CARMELO B. BRUNI, MARIA S. CARLOMAGNO-CERILLO, MARYLIN MEYERS, GERMANA VIGLIAR, AND FRANCESCO BLASI Centro di Endocrinologia ed Oncologia Sperimentale del CNR, IIIstituto di Patologia Generale, 2a Facolt& di Medicina, Via Sergio Pansini 5, 80131, Naples, Italy, and Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland 20014 Received for publication 31 July 1973 The defective prophage 080icI857dhis has been mapped through both marker rescue and deletion analysis. Deletions have been isolated which put residual his genes close to trp genes. Analysis of these deletions shows that the histidine operon on the prophage is oriented clockwise as on the bacterial chromosome, thus opposite to the orientation of the trp operon. The presence of the his promoter-operator region is inferred by the ability of the prophage-carrying strain to derepress sequentially under conditions in which the histidine concen- tration is limiting. In addition to his, the gnd gene is also present on the prophage and is located between his and trp operons. The bacterial genes are inserted in the right arm of the prophage and substitute for all of the late function genes, except for the first three. These data indicate that the "sense" strand for transcription of the his operon in vivo must be the "R" strand. A specialized transducing phage carrying the strain, and 480h is a host-range mutant which can entire Escherichia coli K-12 histidine operon, grow on tonB hosts (10). The symbol 480iX indicates a O8OiXc1857dhis (480dhis) was recently isolated hybrid phage (480 and lambda) which carries the is immunity region of lambda (19) with a temperature- in our laboratory (2). The transducing phage sensitive repressor mutation (c ,857). Phage 480 defective (2), which is not surprising since the amber lysates were obtained from R. E. Wolf, Jr., and his operon is made up of about 11,000 nucleo- had been prepared and titrated on a supD strain (CA tide pairs (4). In view of the use of 080dhis in 5013). Transduction was carried out as described by studies of the regulation of the expression of the Adams (1). his operon conducted in vitro, we have carried Solid and liquid media. The media employed have out a more detailed genetic and physiological been described previously (2). study of 480dhis lysogens with the aim of Enzymatic assays. The cells from a 200-ml culture answering the following questions: (i) is the his were harvested at an optical density of 0.650 at 650 nm, centrifuged at 4 C, washed with 0.05 M tris(hy- operon of 080dhis under histidine control? (ii) droxymethyl)aminomethane (Tris) buffer, pH 7.8, does 480dhis carry bacterial genes other than suspended in 5 ml of the same buffer, and passed his? (iii) which region of the phage genome has through a French pressure cell at 12,000 psi. The been replaced by the bacterial genes? and (iv) extract was centrifuged for 45 min at 30,000 x g at what is the direction of transcription of the his 4 C, passed through a column (1 by 7 cm) of Sephadex operon on the prophage genome, and thus which G-50 coarse (washed and eluted with 0.05 M Tris one of the phage strands acts as a template in buffer, pH 7.8), and assayed for protein and for the in vivo transcription of the his operon? enzymatic activities. Protein concentration was esti- mated by the method of Lowry et al. (13), with bovine serum albumine as standard. The activity of gluco- MATERIALS AND METHODS nate-6-phosphate dehydrogenase (EC 1.1.1.44) was Bacterial strains. The strains used in this study assayed by measuring the increase in reduced nicotin- are described in Table 1. Genotype and phenotype amide adenine dinucleotide phosphate (NADPH) symbols follow the nomenclature of Demerec et al. absorption at 340 nm dependent on 6-phosphogluco- (6). nate and cell extract (7). Assays of the histidine Phage strains. The phage strains used were 080 enzymes and definition of enzymatic units have been and several of its mutants. Phage 480v is a virulent described by Martin et al. (15). 656 VOL. 116, 1973 ORIENTATION OF his OPERON ON 080dhis 657 TABLE 1. Bacterial strains Strain J Genotypea | Source and/or reference Escherichia coli K-12 SB1801 his gnd deletion, rha2A, mal, lambdar P. Hartman (2) FB53 SB1801 lysogen for 080iA and 4680iAdhis (2) FB73 SB1801 lysogen for 480i' dhis (2) FB78 his FIE71 gnd tonB trp Derived from FB73; FB82 his (H)AFIE72 gnd tonB trp spontaneously re- FB88 his GDCBHAFIE73 gnd tonB sistant to 480v FB89 his GDCBHAFIE74 gnd tonB trp and colicin V, B. DF710 F- edd gnd tyrA pyrD thi str D. G. Fraenkel (16) RW84 F- pgi edd eda (gnd his deletion) str R. E. Wolf, Jr. CA5013 supD R. E. Wolf, Jr. Salmonella typhimurium LT2 TR48 F' hisDa2378/hisBE612 trpA8 purE801 J. C. Loper (8) SC419 F' hisG5961/hisOF644 trpl30str J. C. Loper (8) SC7 F' hisB1205/hisOF644 trpl3O str J. C. Loper (8) SC83 F' hisA5925/his0F644 trpl3O str J. C. Loper (8) SC41 F' hisF5971/hisOF644 trpl30 str J. C. Loper (8) a Genetic symbols for E. coli according to Taylor and Trotter (21) and for S. typhimurium according to Sanderson (17). Isolation of deletions of 80dhis prophage. A RESULTS 5-ml culture of FB73 in Z broth was centrifuged, and the cells were resuspended in 0.1 ml of 080v lysate Presence of gnd on 480dhis. The episome (1012 plaque-forming units [PFU]/ml) and 0.4 ml of (F'... 402 his+) which has been integrated at colicin lysate (10). This bacterial suspension was tonB as a step preliminary to the isolation of incubated at 34 C for 15 min, and samples were 480dhis (2) carries the structural gene for glu- spread on Z plates. After 24 to 48 h at 34 C, survivors conate-6-phosphate dehydrogenase (gnd) (R. E. were purified by streaking on Z plates. The survivors Wolf, Jr., personal communication). were tested by replica-plating for resistance to 480v To assess whether the gnd gene is also present and colicin, for sensitivity to 80h, and for auxotro- phy. Auxotrophy was tested for requirement of histi- on 080dhis, strain RW 84 was transduced to dine and tryptophan, and for the ability to grow on His+ with a lysate from strain FB53 (double histidinol. Histidinol is the immediate precursor of lysogen for 080 and 480dhis). His+ transduc- histidine and its utilization requires a functionally tants were checked for ability to grow on gluco- active histidinol dehydrogenase (EC 1.1.1.23; product nate as sole carbon source. The recipient RW84 of the second structural gene, the hisD gene [4]). (his, gnd, edd) cannot grow on gluconate as sole Marker rescue with 080 amber mutants. FB 73 carbon source, since it lacks both gluconate- was grown at 34 C in minimal citrate medium (22) 6-phosphate dehydrogenase (gnd) and Entner- supplemented with 0.2% glucose and induced at 41 C Doudoroff gluconate-6-phosphate dehydrase for 15 min. A loopful of induced FB73 was streaked on (EC 1.1.1.43; edd). Either one of these two a lawn of SB1801, and lysates of the different 080 enzymes is necessary for the utilization of amber mutants (106 to 8 x 108 PFU/ml on strain CA5013) were cross-streaked. The presence of an area gluconate (16; R. E. Wolf, Jr., personal commu- of lysis at the confluence of the streaks was taken as nication). Transductants were selected on mini- an indication of recombination between 480dhis and mal plates supplemented with glucose, purified, 480 amber mutants. and then tested for growth on minimal plates Pattern of derepression of the his operon. Strain supplemented with 0.5% gluconate. All of eight FB73 grown at 34 C in minimal citrate (22) with 0.2% transductants tested (from two individual ly- glucose was derepressed by the addition of thiazolo- sates) were able to grow on gluconate as sole D,L-alanine (final concentration, 1 mM). Samples carbon source. were withdrawn at various times before and after The presence of gnd in the genome of 080dhis derepression, and the specific activity of several of the was confirmed by enzymatic assays. Gluconate- histidine enzymes was determined (15). The scheme of the experiment closely followed that described by 6-phosphate dehydrogenase activity is absent in Berberich, Venetianer, and Goldberger (3). a strain (SB1801) containing a total deletion of The enzyme levels of strain FB82 were measured on his and gnd genes, but can be detected in the cells cultured in minimal citrate (22) with 0.2% same strain after lysogenization with k80dhis. glucose, supplemented with tryptophan (30 ug/ml) In fact, strain FB73 (SB1801, singly lysogenic and histidine (20 jg/ml) or histidinol (150 ig/ml). for 480dhis) contains roughly wild-type levels of 658 AVITABILE ET AL. J. BACTERIOL. gluconate-6-phosphate dehydrogenase, whereas ture-sensitive (showing that they still contain the parental strain, SB1801, has no detectable the temperature-sensitive lambda repressor enzyme activity. The results of these experi- (c,857)), tonB (because of the selection used), ments are presented in Table 2. This activity is and His- (a property which has been scored missing in all three classes of his mutants (see for). Furthermore, classes I and III are Hol- below). (unable to use histidinol instead of histidine), Derepression of the histidine operon car- whereas class II mutants are Hol+.