Vol. 138, No. 1

JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 264-267 0021-9193/79/04-0264/04$02.00/0

Mapping hisS, the Structural Gene for Histidyl-Transfer Ribonucleic Acid Synthetase, in Escherichia coli JACK PARKER* AND SCOTT E. FISHMAN Southern Illinois University, Carbondale, Illinois 62901 Microbiology, of Department Received for publication 1 February 1979

The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-( guaA, guaB)-hisS-glyA.

Although a great deal is known about the genetics of the his operon in enteric bacteria and about the regulation of the histidine biosynthetic enzymes (5, 14, 17), very little is known about hisS and its product, histidyl-tRNA synthetase (HisRS). In Salmonella typhimurium several hisS mutants have been isolated (4, 9, 10), and the hisS locus has been placed at 55.6 U on the 100-U map of S. typhimurium, in a region bounded by purC at 53.6 U and glyA and purG at 56.5 and 56.7 U, respectively (13). This region contains other markers, including strB, a pleiotropic mutation which leads to altered regulation of HisRS (17). In E. coli, a hisS mutant has been isolated (8) and the mutation's map position has been reported to be in a position analogous to hisS in S. typhimurium (8; A. Bock, G. Nass, and F. C. Neidhardt, unpublished data). This approximate map location was later confirmed by Wyche et al., (17) who found that S. typhimurium strains carrying the E. coli plasmid F'142, which covers the entire region, had an increased level of HisRS activity, as would be expected from gene dosage. Although, in general, the genomes of E. coli and S. typhimurium are quite similar, there are regions where there are considerable distortions and rearrangements. Unfortunately one such region seems to be that which contains the hisS gene. By using the same size map units, the region is longer in S. typhimurium than in E. coli, and the orders of several of the loci are reported to be different in the two organisms (2, 11). We set about making a more detailed map of this region in E. coli to facilitate genetic and regulatory studies on HisRS. The report that hisS is in approximately the same region in the two organisms and that it is contained on the E. coli plasmid F'142 would indicate that it should be cotransducible with some markers in the area from dapE to glyA. We began by doing P1mediated generalized transduction, using the hisS mutation reported by Nass (8), and other

markers in this region (2). All strains used are reported in Table 1. P1 transductions were performed as described by Lennox (6). Tranaductants were selected on M9 minimal medium agar plates (1) supplemented with 0.4% D-glucose, thiamine at 10 itg/ml, the appropriate amino acids at 50 ,ug/ml, and the appropriate bases at 20 ,ug/ml. The upp mutation was screened by resistance to 100 ,ug of 6azauracil per ml. Strains containing hisS210 require histidine at 37°C or above when growing on plates, and crude extracts of them contain less than 25% of the normal level of HisRS activity at 37°C under our assay conditions (S. E. Fishman, unpublished data). Data from a number of different P1 transductions are given in Table 2. The cotransduction frequencies from these crosses were used to determine map distances by the equation of Wu (16) and used to construct the map given in Fig. 1, using dapE at 52.6 min as a reference point. An unambiguous order for most of the loci in this region was obtained. The data were not sufficient to order upp and purG with respect to outside markers or to order guaA and guaB. (No further attempt was made to order guaA and guaB.) The order dapE-upp-purG was derived from cross A in Table 3. Reciprocal crosses with dapE as a donor were not performed, since strains carrying the dapE9 allele grow very poorly on minimal medium even with high amounts of added diaminopimelic acid (J. Parker, unpublished data). The order dapE-purG-gua-hisS, which can be determined by two factor crosses, differs from the published order for the first three markers (2). To confirm this order, two different types of experiments were done. The order dapE-purGhisS was first confirmed by a three-factor cross, cross B (Table 3). The order purG-gua-hisS was then confirmed by crosses of strains containing these mutations with a series of specialized transducing lambda bacteriophage which all car-

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VOL. 138, 1979

NOTES

265

TABLE 1. Bacterial strains Strain

Genotype

Source

hisS210pheSI F. C. Neidhardt Hfr, Hayes type, thi-I reIAl dapE9 AA. L. Taylor via CGSC Hfr, Hayes type, thi-1 reIAl glyA6 AA. L. Taylor via CGSC Hfr, Hayes type, thi-1 reUA guaA21 AA. L. Taylor via CGSC F-, thi-1 purFI argHl hisGI glyA6 rpsL8 9 or A. L. Taylor via CGSC 14 malAU (Ar) mtt-2 xyl-7 lacY) or lacZ4 supE44? H677 F-, thi-1 his-68 tyrA2 trp-45purC50 malAl (Xr) P. G. deHann via CGSC rpsL125 lacYl gal-6 xyl-7 mtl-2 tonA2 tsx-70 A- supE44 H712 As H677 except guaB22 and purC+ P. G. deHaan via CGSC PCO631 P. G. deHaan via CGSC Hfr, Reeves 4 type carB671 metBl purG48 argF58 relAI SA53 deoA22 udp-l upp-l metBI argF58 relAl R. Pritchard via CGSC JK15 dapE glyA argH rpsL; other markers not AT97xAT2681 checked JK16 hisS glyA argH rpsL; other markers not JK15XP1(NP2106) checked JK24 purC glyA argH rpsL; other markers not JK15xP1(H677) checked malA' (A) derivative of H712 JK67 JK119 dapE purG argH rpsL JK15xPl(PCO631) a CGSC, E. coli Genetic Stock Center, Yale University, New Haven, Conn. B. J. Bachmann, Curator. NP2106 AT978 AT2457 AT2465 AT2681

TABLE 2. Cotransduction frequenciesa % Cotransduction of unselected marker

Selected marker

dapE

purC+ purG+

purC 86 (69/80)b -

guaA+

-

upp 20 (7/35)

purG 20 (27/134)c

-

-

-

85 (68/80)h

-

d

hisS glyA O (0/80))b 7.4 (12/162)d e 4 (1/22)f 23 (5/22)f 60 (15/25)81 (42/52)' -

72 (13/18)' guaB 0 (0/200)k 18 (45/253)e, kglyA+ a The percent cotransduction for various markers was determined for a number of crosses, using strains and P1 vir grown on strains given in Table 1. The data presented are compiled from 10 crosses. Values in parentheses indicate the actual colony counts in each case. -, No such cross was done.

b

c

d

JK15xP1(H677). JK119xP1(NP2106).

JK119xPl(SA53).

eJK15xPl(NP2106). f JK24xP1(NP2106).

' PCO631xP1(NP2106). h AT2465XP1(SA53).

'AT2465xP1(NP2106).

i JK67xP1(PCO631). k

JK16xP1(H677).

ried guaB and guaA and are derived from AcI857S7 (S. E. Fishman, K. R. Kerchief, and J. Parker, manuscript in preparation). The transductions were performed by the method of Signer (12). Since the transducing phage are temperature-inducible and the hisS phenotype is only apparent at high temperatures, transductions with AguaBs selecting for hisS+ were done at 42°C, where only recombinants would survive. The data from these crosses are also summarized

in Fig. 1. Since AdguaB2 carries hisS but not purG and AdguaB3 and 5 carry purG but not hisS, the gene order must be purG-gua-hisS. No attempt was made to determine the location of xseA, the gene for exonuclease VII, but the P1 transduction data of Chase and Richardson (3) would make it likely that it is carried by some of the AdguaB-transducing phages. The reason for the difference between the order dapE-purG-gua and that found by Stou-

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J. BACTERIOL.

NOTES TABLE 3. Transductants from three factor crosses

Cross

Recipient

A

JK119 (dapE purG)

B

JK119 (dapE purG)

Selected

Unselected markers

No. of col

SA53 (upp)

dapE+

purG+ upp+

NP2106 (hisS)

dapE+

0 5 28 2 16 6 77 0

Donor

purG+ upp purG upp+ purG upp

purG+ hisS+ purG+ hisS

purG hisS+ purG hisS

toz

Ina

-

~

dopE purC

-53

uipp

g purG gvuoA/0 hisS

-54

gIyA

. 55 FIG. 1. A genetic map of the E. coli chromosome in the hisS region. This map is based on the 100-min E. coli map, using dapE at 52.6 min as a reference point. Most map distances were calculated by the equation of Wu (16) from the contransduction frequencies of loci with dapE as given in Table 2. The position of glyA is based upon the additive map distances from dapE to hisS and hisS to glyA and is close to that given by Bachmann et al. (2). The guaA/ B loci were placed according to their average cotransduction frequencies with hisS, upp, and purG. The lines to the left indicate the extent of the E. coli chromosome carried by the different lambda transducing bacteriophage, none of which was screened for upp.

thamer et al. (13) is unclear but is probably a result of the use by those authors of two-factor conjugation experiments and the closeness of the markers. The gene order we obtained is the same as that given for S. typhimurium, except that the positions of purG and purI appear to be reversed in E. coli. The effect of the strB+ allele of Salmonella on HisRS synthesis can be mimicked by using the E. coli plasmid F'142 (17). This would indi-

cate that a gene involved in HisRS regulation also maps in this region in E. coli. If the order of the genes in the two organisms is identical one would expect this strB+-like gene of E. coli to be located between gua and hisS. This work was supported by NIGMS grant #lROlGM25855-01 and a special SIU research grant to J.P. Our sincere thanks to F. C. Neidhardt and B. Bachmann for strains.

LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virus resistant mutants of Escherichia coli strain B. Proc. Natl. Acad. Sci. U.S.A. 32:120-126. 2. Bachmann, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 3. Chase, J. W., and C. C. Richardson. 1977. Escherichia coli mutants deficient in exonuclease VII. J. Bacteriol. 129:934-947. 4. DeLorenzo, F., D. S. Straus, and B. N. Ames. 1972. Histidine regulation in SabnoneUa typhimurium X. Kinetic studies of mutant histidyl transfer ribonucleic acid synthetases. J. Biol. Chem. 247:2302-2307. 5. Hartman, P. E., Z. Hartman, R. C. Stahl, and B. N. Ames. 1971. Clasification and mapping of spontaneous and induced mutations in the histidine operon of Salmonella. Adv. Genet. 16:1-34. 6. Lennox, E. S. 1955. Transduction of linked genetic characters of the host bacteriophage P1. Virology 1:190201. 7. Low, K. B. 1972. Escherichia coli K-12 F-prime factors, old and new. Bacteriol. Rev. 36:587-607. 8. Nass, G. 1967. Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase. Mol. Gen. Genet. 100:216224. 9. Roth, J. R., and B. N. Ames. 1966. Hiatidine regulatory mutants in SalmoneUa typhimrrium. II. Histidine regulatory mutants having altered histidyl-tRNA synthetase. J. Mol. Biol. 22:325-334. 10. Roth, J. R., D. N. Ant6n, and P. E. Hartman. 1966. Histidine regulatory mutants SalmoneUa typhinuriun. I. Isolation and general properties. J. Mol. Biol. 22:305323. 11. Sanderson, K. E., and P. E. Hartman. 1978. Linkage map of SalmoneUa typhiurium, edition V. Microbiol. Rev. 42:471-519. 12. Signer, E. R. 1966. Interaction of prophages at the atts0 site with the chromosome of Escherichia coli. J. Mol. Biol. 15:243-255. 13. Stouthamer, A. H., P. G. deHann, and H. J. J. Nijkamp. 1965. Mapping of purine markers in Escherichia coli K 12. Genet. Res. 6:442-453.

VOL. 138, 1979 14. Umbarger, H. E. 1978. Amino acid biosynthesis and its regulation. Annu. Rev. Biochem. 47:533-606. 15. Winkler, M. E., D. J. Roth, and P. E. Hartman. 1978. Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon. J. Bacteriol. 133:830-843.

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16. Wu, T. T. 1966. A model for three-point analysis of random general transduction. Genetics 54:405-410. 17. Wyche, J. H., B. Ely, T. A. Cebula, M. C. Snead, and P. E. Hartman. 1974. Histidyl-transfer ribonucleic acid synthetase in positive control of the histidine operon in Salmonella typhimurium. J. Bacteriol. 117:708-716.