thik and thil loci of escherichia · thik andthil loci ofe. coli 709 shown in fig. 1, together with...

Vol. 151, No. 2JOURNAL OF BACTERIOLOGY, Aug. 1982, P. 708-7170021-9193/82/080708-10$02.00/0

thiK and thiL Loci of Escherichia coliNARIKO IMAMURA AND HIDEO NAKAYAMA*

Department ofFood and Nutrition, Yamaguichi Women's University, Yamaguchi, Yamaguchi-Ken 753,Japan

Received 1 December 1981/Accepted 5 April 1982

Mutants of Escherichia coli K-12 auxotrophic for thiamine phosphates wereproduced in stepwise fashion from the polyauxotrophic F- strain JC1552, viaintermediate production of thiamine auxotrophs that had lost the enzymaticactivity of either phosphomethylpyrimidine kinase or thiamine phosphate pyro-phosphorylase. They include two types: one responds to thiamine monophos-phate or thiamine pyrophosphate, and the other responds to thiamine pyrophos-phate only; the former lacks thiamine kinase activity, and the latter lacks thiaminemonophosphate kinase activity, in addition to the enzymatic defects caused by thefirst mutations. We found two new genes, for which we propose the designationsthiK and thiL, which govern the activities of thiamine kinase and thiaminemonophosphate kinase, respectively. By conjugation and P1 transduction, thethiK locus was mapped at about 25 min, between pyrC and purB and close tofabD. The relative order of thiK with respect to nearby genes was tentativelyestablished as pyrC-ptsG-fabD-thiK-purB. In the case of thiL, the locus wassituated at about 9 min, between tsx and acrA and probably 0.2 min clockwisefrom the former.

This paper continues the genetic analyses ofNakayama and Hayashi of a series of enzymescatalyzing the biosynthesis of thiamine pyro-phosphate (thiamine-PP) from the pyrimidineand the thiazole moieties of thiamine in Esche-richia coli (16-18). Mutants of E. coli auxotro-phic for thiamine have been described andmapped at three loci. The thiA and thiC genesparticipate in certain steps up to the biosynthesisof 4-methyl-5-3-hydroxyethylthiazole (hydroxy-ethylthiazole) and 2-methyl-4-amino-5-hydroxy-methylpyrimidine (hydroxymethylpyrimidine),respectively (7, 22). Strains carrying mutationsin the former gene respond to either thiamine orhydroxyethylthiazole, and those mutated in thelatter respond to thiamine or hydroxymethylpy-rimidine. The thiB gene was reported as proba-bly a structural gene for thiamine phosphatepyrophosphorylase (6), and the mutant of thistype manifested a phenotype of auxotrophy forthe intact form of thiamine. Next, experimentswith several kinds of pyrithiamine-resistant mu-tants have led to the assumption that they arealtered in the regulatory mechanism for thiaminebiosynthesis, and the thiO locus has been char-acterized and mapped as a probable operatorsequence for thiA, thiB, and thiC (7). Fromavailable data, which indicate that the four thigenes are situated very close together on thechromosome and that the synthesis of thiaminein this species is repressible and derepressibleby the intracellular thiamine concentration (5),those genes operating in the biosynthesis of

thiamine-PP, a coenzyme form of thiamine, havebeen expected to constitute the thi operon.Subsequently, a fifth gene, thiD, which affectsphosphomethylpyrimidine kinase activity; hasbeen identified and mapped at about 46 min onthe chromosome of this species (4). These stud-ies have shown that not all of the genes operat-ing in the biosynthesis of thiamine-PP are local-ized in one region, as a thi cluster.Nakayama and Hayashi have described two

types of mutants, which include a thiaminemonophosphokinase (thiamine kinase)-deficientmutant and a thiamine monophosphate (thia-mine-P) kinase-deficient mutant of E. coli strainW (16). Both mutants were isolated as doublyblocked mutants from a parent strain that lackedthe enzymatic activity of phosphomethylpyri-dine kinase, which catalyzes the reaction fromhydroxymethylpyrimidine-P to hydroxymethyl-pyrimidine-PP. Evidence was obtained withthese mutants which indicates that in E. coli thefree form of thiamine is not involved in de novosynthesis of thiamine-PP, but that thiamine-P,an exclusive product formed by condensation ofhydroxymethylpyrimidine-PP and hydroxyeth-ylthiazole-P, is directly phosphorylated to formthiamine-PP. When, however, the free form ofthiamine is supplied from the outside, it is takenup into the cells and then converted to thiamine-PP via intermediate formation of thiamine-P(17). Reactions and intermediates involved inthe biosynthesis of thiamine-PP from hydroxy-methylpyrimidine and hydroxyethylthiazole are

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thiK AND thiL LOCI OF E. COLI 709

shown in Fig. 1, together with mutant strainsthat lack the various enzyme activities. Genesspecifying the enzymes catalyzing the stepwisephosphorylation of thiamine have never beencharacterized. This paper describes new genes,for which we propose the designations thiK andthiL; thiK and thiL affect thiamine kinase andthiamine-P kinase, respectively. Our resultsshow that the loci are situated at two differentchromosomal sites. These results, together withprevious data (4), indicate that the loci of thegenes specifying the enzymes in the biosynthesisof thiamine-PP, rather than being clustered, arespread along the chromosome in at least fourgroups.

MATERIALS AND METHODS

Organisms. The bacterial strains used in the geneticwork are all derivatives ofE. coli K-12. The genotypesand sources of these strains are given in Table 1. Thechromosomal locations of several pertinent genes inthis species and the positions of the points of origin ofthe Hfr strains used are shown by Low (11).

Media. Minimal medium 56 as modified by Low (11)was used as a basal medium for the growth experi-

Hydroxymethylpyrimi

ments, including the selection for the desired recombi-nants and transductants. Since all of the mutant strainsauxotrophic for thiamine were derived from the poly-auxotrophic F- strain JC1552, this medium was sup-plemented with 25 Ftg of leucine, 25 ,ug of tryptophan,25 Ftg of histidine, 25 Fig of methionine, and 50 p.g ofarginine per ml. When strains were carrying thiD,thiK, or thiL mutations, the medium was additionallysupplemented with thiamine, thiamine-P, or thiamine-PP, respectively, at a final concentration of 20 nM. Forthe selection of Lac' or Gal' recombinants, glucose-free lactose or galactose was substituted for glucose inthe medium. Luria broth was used in the transductionexperiments, and Luria broth without glucose was alsoused in conjugation experiments. Since Luria brothdoes not contain sufficient thiamine-P and thiamine-PP, they were supplied when required.

Chemicals. Thiamine, thiamine-P, and thiamine-PPwere purchased from Sigma Chemical Co., St. Louis,Mo. Hydroxymethylpyrimidine and hydroxyethylthia-zole were gifts from Takeda Chemical Industries,Osaka, Japan. Hydroxymethylpyrimidine-P and hy-droxymethylpyrimidine-PP were prepared from 2-methyl4amino-5-bromomethylpyrimidine dihydro-bromide according to the method devised by Lewinand Brown (9). Hydroxyethylthiazole-P was pre-pared by phosphorylation of hydroxyethylthiazole bythe method of Miyagawa (13).idine Hydroxyethylthiazole

Hydroxymethyl-pyrimidine kinase

Hydroxymethylpyrimidine-P

Phosphomethyl-| pyrimidine kinase

_Hyroxyethyl-thiazole kinase

NI500, NI510, NI520NI512~~Hydroxymethylpyrimidine-PP Hydroxyethylthiazole-P

hiamine phosphatepyrophosphorylaseNI400, NI420

Thiamine I Thiamine-P

aliri- nkinasea|eNI510, NI512 i inemonophosph

NI520, NI512, NI420

Thiamine-PP

Ins ide

Cell membrane

FIG. 1. Biosynthetic pathway of thiamine-PP from hydroxymethylpyrimidine and hydroxyethylthiazole viaintermediate formation of thiamine-P in E. coli. Mutant strains missing enzymes are also listed.

Outside

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710 IMAMURA AND NAKAYAMA

TABLE 1. Bacterial strainsStrain Sex Relevant genotype' Source

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacYlrpsLi04 (tsx_i)b

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacYlrpsLi04 thiDI

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacY]rpsLi04 thiBI

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacYlrpsL104 thiDI thiKI

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacYlrpsL104 thiDI thiLI

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacYlrpsL104 thiDI thiKI thiL3

F- argG6 metBI his-i leu-6 trp-31 gal-6 lacYlrpsL104 thiBI thiL2

HfrHfr thi-iHfrHfr thi-iF- argG6 his-i leu-6 trp-31 rspL104 thiDI nalA

F- pyrC46 purB51 thi-i rpsL125F- thiDI pyrC46 purB51 thi-i rpsLi25 nalA

F- purB51 thi-iF- purB51 hemA30 thi-iF+ ptsG21 thi-lF- fabD89 thi-iHfr thi-i pyrC46F- argG6 metBI his-i trp-31 pyrC46 rpsL104

thiDI thiKIF- argG6 metBI his-i leu-6 trp-31 lacYI rpsL104

thiDI thiLIHfrHfrHfr proC29Hfr phoR17F- tsx-33 thi-i rpsL31F- tsx-29F- thi-i rpsL31 tsx-33 phoR17

Hfr phoA4F- acrAI

A. J. Clark, via CGSCC

Isolated from JC1552 by mutagenesis

Isolated from JC1552 by mutagenesis

Isolated from NI500 by mutagenesis

Isolated from N1500 by mutagenesis



K. B. Low, via CGSCK. B. Low, via CGSCK. B. Low, via CGSCW. Hayes, via CGSCmet+ recombinant from conjugation ofNI500 with AB259; then nalA was intro-duced

J. Beckwith, via CGSCnalA thiD recombinant from conjugation ofX7014a with NI50OMN

P. G. de Haan, via CGSCA. Stouthamer, via CGSCB. Magasanik, via CGSCS. Silbert et al., CGSCW. Maas, via CGSCleu+ pyrC recombinant from conjugation ofN1510 with MA1008

gal' recombinant from conjugation ofN1520 with KL208

K. B. Low, via CGSCP. Broda, via CGSCR. Curtiss, via CGSCA. Garen, via CGSCE. A. Adelberg, via CGSCA. L. Taylor, via CGSCphoR recombinant from conjugation ofAB1157 with C5

A. Garen, via CGSCH. Nakamura, via CGSC

a Genetic symbols are as given by Bachmann and Low (1).b Since JC1552 used in the present report did not show any difference in sensitivity to phage T6 compared with

strains that do not carry the tsx mutation, and since the strain became resistant to T6 when tsx was introducedfrom strains carrying this mutation, JC1552 and its derivatives were used as T6 sensitive.

c CGSC, Coli Genetic Stock Center, Department of Human Genetics, Yale University School of Medicine,New Haven, Conn.

Genetic procedures. Mating and strain constructionswere performed by procedures essentially the same asthose of Miller (12). When Thi+ recombinants wereselected from a mating mixture that contained eitherthiamine or its phosphate esters, the mixture waswashed twice by centrifugation at 6,000 x g for 10 minwith 10 volumes of saline before its dilution andplating. This prevented background growth of mutantscarrying the thi mutations in selective medium withoutthiamine. Phage P1 kc-mediated transductions wereperformed according to the directions of Lennox (8)and Taylor and Trotter (23).Assay of enzyme activities. Five enzymes are in-

volved in the biosynthesis of thiamine-PP from hy-droxymethylpyrimidine and hydroxyethylthiazole(Fig. 1). Another enzyme, thiamine kinase, catalyzesthe reaction from thiamine to thiamine-P. Our experi-ence has revealed that in E. coli the enzymatic activi-ties of the overall reaction of thiamine-PP biosynthesisfrom the pyrimidine and the thiazole moieties cannotbe assayed with cell extracts, because the activity ofthiamine phosphate pyrophosphorylase is inhibitedalmost completely by the addition of ATP at theconcentration required for the phosphorylating stepsof the remaining four reactions. The activities of theindividual enzymes were assayed with crude extracts

JC1552

N1500

N1400

N1510

N1520

N1512

N1420

KL983KL99KL208AB259NI50OMN

X7014aX7014aEN

PC0254S730LA12L48MA1008NI51OLP

N1520G

KL226Broda8X342C5AB1157AT1380AB1157PR

S26N43

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in the following reaction systems: for the assay ofhydroxymethylpyrimidine kinase (EC 2.7.1.49), 0.1 mlof 0.1 mM hydroxymethylpyrimidine-0.1 ml of 0.1 MATP; for phosphomethylpyrimidine kinase (EC2.7.4.7), 0.1 ml of 0.1 mM hydroxymethylpyrimidine-P-0.1 ml of 0.1 M ATP; for hydroxyethylthiazolekinase (EC 2.7.1.50), 0.1 ml of 0.1 mM hydroxyeth-ylthiazole-0.1 ml of 0.1 M ATP; for thiamine phos-phate pyrophosphorylase (EC 2.5.1.3), 0.1 ml of 0.1mM hydroxymethylpyrimidine-PP-0.1 ml of 0.1 mMhydroxyethylthiazole-P; and for thiamine kinase (EC2.7.1.89), 0.1 ml of 0.1 mM thiamine-0.1 ml of 0.1 MATP. In addition, each of the reaction mixtures con-tained 0.1 ml of 1 M potassium phosphate buffer (pH7.2), 0.1 ml of 0.1 M MgC92, and cell extracts (3.0 mgof protein) in a final volume of 1.0 ml. For thiamine-Pkinase (EC 2.7.4.16), the reaction mixture contained0.2 ml of 0.1 mM thiamine-P, 0.1 ml of 0.1 M ATP, 0.1ml of 1 M Tris-hydrochloride buffer (pH 7.5), 0.1 ml of0.1 M MgCl2, 0.27 ml of 2.5 M KCI, and cell extracts(4.0 mg of protein) in a final volume of 2.0 ml. After thereaction mixtures had been incubated at 37°C for 30min, both hydroxymethylpyrimidine-P and hydroxy-methylpyrimidine-PP formed in the hydroxymethyl-pyrimidine kinase reaction, hydroxymethylpyrimi-dine-PP formed in the phosphomethylpyrimidinekinase reaction mixture, hydroxyethylthiazole-Pformed in the hydroxyethylthiazole kinase reactionmixture, thiamine-P formed in the thiamine phosphatepyrophosphorylase and thiamine kinase reaction mix-tures, and thiamine-PP formed in the thiamine-P ki-nase reaction mixture were estimated by microbiologi-cal assay with paper bioautography, using threedifferent kinds of E. coli mutants auxotrophic forthiamine. Mutant 70-23 was used to detect thiamine,thiamine-P, and thiamine-PP. Mutants 70-17 and 26-43were used to detect the phosphate esters of hydroxy-methylpyrimidine and hydroxyethylthiazole, respec-tively. Detailed assay conditions with these mutantswere described previously (17).

RESULTSBiochemical characterization of newly isolated

mutants. The parental strain, JC1552, showedfull growth in minimal medium supplementedwith the five amino acids required by this strain.Under these conditions, the strain did not re-spond to any extent to the thiamine compounds,including hydroxymethylpyrimidine and hy-droxyethylthiazole. Mutants auxotrophic for thi-amine responded only to thiamine intermediatesthat were located beyond the genetic blocks orcompounds that the mutant cells were able toconvert into the intermediates. Since hydroxy-methylpyrimidine-PP cannot permeate throughthe cell membrane, mutants blocked at the stepof hydroxymethylpyrimidine-P phosphorylationto hydroxymethylpyrimidine-PP required thia-mine in the intact form. Mutant strains auxotro-phic for thiamine or its phosphate esters wereproduced by treating JC1552 with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) by the follow-ing procedures. JC1552 was first treated withNTG, and mutants N1500 and NI400, auxotro-

phic for the intact molecule of thiamine, wereisolated. Both of the mutants were further muta-genized, and resulting mutants that could growon thiamine-PP, but not on thiamine, were se-lected. Mutants NI510 and N1520 were derivedfrom NI500, and NI420 was derived from N1400.Mutants NI510 and N1520 differed only ingrowth response to thiamine-P: the former couldgrow on thiamine-P, whereas the latter couldnot. NI512 was produced from NI510 as a mu-tant that had lost its ability to grow on thiamine-P but that still responded to thiamine-PP. Ifdouble or triple mutations were induced bystepwise mutagenesis, multiple lesions wouldhave occurred successively from the earlier tothe later steps in the reaction sequences in-volved in the biosynthesis of thiamine-PP. Mu-tants with such multiple blocks should respondonly to the compounds that are located beyondthe last biochemical block. To clarify the rela-tionships between the sites and number of bio-chemical lesions of mutants and their growthresponses to the thiamine compounds, the activ-ities of six enzymes involved in the biosynthesisof thiamine-PP were assayed. Five enzymes,hydroxymethylpyrimidine kinase, phospho-methylpyrimidine kinase, hydroxyethylthiazolekinase, thiamine phosphate pyrophosphorylase,and thiamine-P kinase, are involved in thiamine-PP biosynthesis from hydroxymethylpyrimidineand hydroxyethylthiazole. In addition, anotherenzyme, thiamine kinase, operates in the uti-lization of the free form of thiamine suppliedfrom the outside. Mutants NI500 and NI400,produced from JC1552 by the first mutagenesis,were lacking in the activities of phosphomethyl-pyrimidine kinase and thiamine phosphate pyro-phosphorylase, respectively, whereas theactivities of the other enzymes involved in thia-mine-PP biosynthesis were comparable to thoseobserved in JC1552 (Table 2). The experimentsalso revealed that the second and third mutagen-esis resulted in corresponding mutations in thesemutants that caused double and triple lesions atthe enzymatic steps in thiamine-PP biosynthe-sis. In mutant N1420, for example, thiamine-Pkinase was lacking, in addition to the originalblock, thiamine phosphate pyrophosphorylasedeficiency. Mutants NI510 and NI520, both in-duced from a strain (NI500) that lacked phos-phomethylpyrimidine kinase activity, lackedthiamine kinase and thiamine-P kinase, respec-tively, in addition to the initial block. Further, inNI512, phosphomethylpyrimidine kinase, thia-mine kinase, and thiamine-P kinase were allinactivated by the three successive steps ofmutagenesis. These results support our assump-tion that mutant strains blocked at later steps inthe reaction sequence for the synthesis of thia-mine-PP can be produced by stepwise mutagen-

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TABLE 2. Activities of enzymes involved in the biosynthesis of thiamine-PP from hydroxymethylpyrimidineand hydroxyethylthiazole in mutant strains of E. colia

Enzyme activity (nmol of product/mg of protein per h)

Strain Thiainne b Hydroxymethyl- Phosphomethyl- Hydroxyethyl- Thiaminerequirement pyrimidine pyrimidine thiazole phosphate Thiamine Thiamine-Pkinase kinase kinase pyrophosphorylase kinase kinase

JC1552 None 1.59 0.47 0.45 2.02 1.15 0.11NI500 Thiamine 1.40 0 0.44 1.62 1.01 0.15N1400 Thiamine 1.74 0.62 0.58 0 0.80 0.22N1510 Thiamine-P 2.57 0 0.52 0.49 0 0.10NI520 Thiamine-PP 1.38 0 0.46 0.77 1.11 0N1512 Thiamine-PP 1.63 0 0.48 0.32 0 0N1420 Thiamine-PP 1.82 0.55 0.60 0 0.31 0

a Cells grown on a limiting amount of thiamine or its phosphate esters were harvested and washed with 0.02 Mcold potassium phosphate buffer (pH 7.2) containing 1 mM MgCl2. The pellet was suspended in the same bufferplus 1 mM MgCl2 and 1 mM 3-mercaptoethanol. For the assay of thiamine-P kinase, 0.01 M Tris-hydrochloridebuffer (pH 7.5) was used instead of the phosphate buffer. The cells in the suspension were disrupted by sonicoscillation, and the supernatant fraction obtained by centrifugation was used as crude extract. The activity ofeach enzyme is expressed as product(s) formed by crude extracts prepared from mutant organisms. Thereactions and assay systems are described in the text.

b The mutants that can grow on thiamine also respond to thiamine-P and thiamine-PP, and the mutant that cangrow on thiamine-P also responds to thiamine-PP.

esis. Among these mutants strains, mainlyNI510 and N1520 were used in the genetic analy-sis of thiamine-PP biosynthesis.

Location of thiK by conjugation and transduc-tion. In conjugational mapping, the location ofthe thiK gene on the chromosome was estimatedby determining the gradient of transmission ofthe wild-type allele for thiK from several Hfrstrains to NI510 (thiD thiK), a mutant auxotro-phic for thiamine-P. In these matings the num-bers of Leu+, Trp+, His', Arg+, and Met'recombinants were compared with those of thiDthiK+ recombinants, which can grow on thia-mine but not on thiamine-free medium. Since thethiD locus has been mapped on the chromosomeat about 46 min, the selection of thiD thiK +recombinants could be accomplished by choos-ing an appropriate Hfr strain or by adjusting themating time so that thiK+ could be transferredearly, but thiD+ could not. The results obtainedwith Hfr strains KL99 and KL208 indicated thatthe thiK mutation is located in the chromosomalregion between the point of origin of KL99(about 23 min) and the trp locus, presumablyvery close to the former (data not shown). Thiswas confirmed by an interrupted mating ofKL208 with NI510, which indicated that thewild-type allele corresponding to the thiK locustransferred at 3.7 min after trp (data not shown).As discussed previously, a mutant carrying

the thiK mutation has lost its ability to catalyzethe reaction from thiamine to thiamine-P, but itcannot manifest a thiamine-P requirement whenit is capable of forming thiamine-P through thede novo synthetic pathway via the phosphateesters of hydroxymethylpyrimidine and hy-droxyethylthiazole. When a medium containing

thiamine is used to select for thiK+ transduc-tants derived from a recipient strain carryingmutations in both thiD and thiK, one can expectthat two types of transductants, thiD+ thiK andthiD thiK+, will appear at about a 1:1 ratio,because these two genes are approximately 20min apart on the chromosome and becausephage P1-mediated transducing fragments aregenerally assumed to be cut at random from thedonor chromosome. Our observation that ap-proximately half of the transductants grown onthiamine medium required thiamine and that theremaining half could grow on thiamine-free me-dium was entirely consistent with the assump-tion described above. Transductants which hadlost the thiamine-P requirement but which stillrequired thiamine were subsequently analyzedto determine the cotransduction frequencies ofdonor unselected marker with the wild-type al-lele for thiK in a recipient strain carrying muta-tions in both thiD and thiK. When we wished touse thiK as an unselected marker, we had to usea recipient strain whose de novo synthetic path-way of thiamine-PP had already been blockedsomewhere before the formation of thiamine-P.For example, the thi-l mutation, which is verywidespread among the existing mutant strains ofE. coli K-12, could be used for this purpose,because this mutation is responsible for a bio-chemical block of hydroxyethylthiazole synthe-sis and, hence, manifests a requirement foreither thiamine or hydroxyethylthiazole. How-ever, in the case of X7014a, which carried thethi-l mutation already, the thiD mutation wasincorporated from N150OMN by conjugationbefore use, because the strain did not grow wellon minimal medium with uracil and adenine

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unless nutrient broth at a final concentration of10 pI/ml was also added. Nutrient broth presum-ably contains a substance which substitutes forhydroxyethylthiazole and which might havecaused a high background growth of thi-l trans-ductants in selective media without added thia-mine.We tested for cotransduction between thiK

and several markers in the region from 23 to 25min, and the data obtained are presented inTable 3. Among these nearby genes we foundthat the thiK mutation was cotransducible withboth pyrC and purB; the latter two genes havebeen placed on the chromosome in positionsbetween which cotransduction could occur onlyat a low frequency (21). The thiK mutation instrain N1510 was cotransduced with pyrC+when X7014aEN was used as the recipientstrain, whereas in a reciprocal cross, the trans-ductants that appeared on the selective platewere very few in number and cotransductioncould not be demonstrated. When crosses be-tween Pl grown on strain LA12 or L48 andstrain NI51OLP, which carried mutations in bothpyrC and thiK, were attempted, cotransductionof donor wild-type alleles corresponding to thetwo genes was observed at anticipated frequen-cies when transductants were selected forPyrC+, whereas a decreased frequency wasobserved in a cross between L48 and NI51OLPwhen Thi+ (thiD thiK+) was selected. As forlinkage with purB, no appreciable cotransduc-tion between thiK and purB was observed whenX7014a was used as the donor and Thi+ wasselected. The same situation was seen in S730when thiK+ was used as the selected marker.However, contrasting results were observedwith strain PC0254, in which cotransductionoccurred when either of the two markers wasused as the one selected. We concluded that thethiK locus lies between the pyrC and purB loci,although there were unexplained difficulties inthe cotransduction of thiK with the two markers,especially when Thi+ was selected.Cotransduction experiments were subse-

quently conducted to determine the position ofthe thiK locus relative to the loci of ptsG andfabD. Both ptsG and fabD were shown to co-transduce with thiK at high frequencies (53 and65%, respectively), suggesting that the thiK lo-cus is close to those loci. Semple and Silberthave reported, in their extensive studies on thefabD locus, that the locus is situated at 24 min,between pyrC and purB and close to ptsG, andthat their order could be pyrC, ptsG, fabD, andpurB (20). They also noted that cotransductionfrequencies between ptsG and pyrC were mark-edly reduced in all crosses involvingfabD donorstrains. From their studies, together with thefact that ptsG can be used only as an unselected

marker, we assumed that the ordering of ptsG,fabD, and thiK by three-factor transductionalcrosses with a strain carrying both ptsG andfabD might be difficult, even if such a straincould be constructed. We constructed a strain,NI51OLP, which carried mutations in the thiD,thiK, and pyrC loci in order to determine therelative positions ofpyrC, ptsG, and thiK and ofpyrC, fabD, and thiK, using three-factor trans-ductional crosses. In considering the set ofgenes pyrC, ptsG, and thiK, the order could beeither pyrC-ptsG-thiK (A) or pyrC-thiK-ptsG(A'). Of the pyrC+ transductants, 83% receivedneither ptsG nor thiK+ (Table 3, cross 15). Wealso found that pyrC cotransduced at frequen-cies of 17% with ptsG and 15% with thiK. Of thepyrC ptsG transductants, 14 of 16 inheritedthiK+ simultaneously, whereas the pyrC thiKtransductants, 14 in all, received ptsG as well. Itappeared, then, that gene order A was morelikely than gene order A', though the number oftransductants analyzed was not large and,hence, a definite conclusion should be avoided.In the case of another set of genes, pyrC, fabD,and thiK, their order could have been pyrC-fabD-thiK (B) or pyrC-thiK-fabD (B'). Three-factor transductional crosses between P1 donorstrain L48 fabD and recipient strain NI51OLPwere performed (Table 3, crosses 16 through18). It was noted that 81% of transductantsselected for the inheritance of pyrC+ receivedneitherfabD nor thiK+. When, however, thiK+transductants were selected in the same cross,56% received fabD but not pyrC+, and 40%received neitherfabD nor pyrC+. As for recom-binants selected for the inheritance of pyrC andthiK+ simultaneously, coinheritance of fabDincreased to 87%. Only 1 to 2% of the recombi-nants were pyrC+fabD+ thiK+ (Table 3, crosses16 and 17). Assuming that the least frequentrecombinant class arose from quadruple ex-changes rather than from double exchanges, thegene order must have been B. Therefore, iffabDis to the right of ptsG, as placed by Semple andSilbert, the relevant gene order tentatively as-signed was pyrC-ptsG-fabD-thiK-purB (Fig. 2).

Location of thiL by conjuption and transduc-tion. We produced two types of mutants carry-ing mutations in the thiL locus, constructingdoubly blocked mutants from strains that lackedthe enzymatic activity of either phosphomethyl-pyrimidine kinase or thiamine phosphate pyro-phosphorylase. Thus, strain NI520 carried boththiD and thiL mutations, and strain N1420 car-ried thiB and thiL mutations. To map the thiLlocus, strain NI520 was used mainly becauserecombinants produced from the strain could beselected in the manner similar to that previouslyused in strain NI510. Preliminary conjugationalmapping of thiL with four Hfr strains, KL208,

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TABLE 3. Transductional mapping of the thiK locus with respect to the nearby genesStrain Marker

Crossno. Donor

lb NIS10 thiK

2C X7014a purB

3 X7014a pyrC

4 NIS10 thiK

5 PC0254 purB

6d LA12 ptsG

7' NI510 thiK

8f L48 fabD

9 S730 purB

10 S730 hemA

11 LA12 ptsG

12 LA12 ptsG

13 L48 fabD

14 L48 fabD

15 LA12 ptsG

Recipient

X7014aEN pyrC

NIS10 thiK

NI510 thiK

PC0254 purB

NIS10 thiK

NI510 thiK

L48 fabD

NI510 thiK

NI510 thiK

NI510 thiK

X7014a pyrC

PC0254 purB

X7014a pyrC

PC0254 purB

NI51OLP thiK pyrCg

16 L48 fabD NIS1OLP thiK pyrC

17 L48 fabD NIS1OLP thiK pyrC

18 L48 fabD NI51OLP thiK pyrC

Selected Unselected

pyrC+ thiK

thiK+ purB

thiK+ pyrC

purB+ thiK

thiK+ purB

thiK+ ptsG

fabD+ thiK

thiK+ fabD

thiK+ purB

thiK+ hemA

pyrC+ ptsG

purB+ ptsG

pyrC+ fabD

purB+ fabD

pyrC+ ptsG+ thiKpyrC+ ptsG thiK+pyrC+ ptsG thiKpyrC+ ptsG+ thiK+

pyrC+ fabD+ thiKpyrC+ fabD thiK+pyrC+ fabD thiKpyrC+ fabD+ thiK+

thiK+ pyrC fabDthiK+ pyrC fabD+thiK+ pyrC+ fabDthiK+ pyrC+ fabD+

pyrC+ thiK+ fabDpyrC+ thiK+ fabD+

No. of colonies withdonor unselectedmarker/total no. ofcolonies selecteda

7/47, 5/34, 4/27

0/29

0/26

44/143, 16/64, 9/52

4/36

6/11, 22/42

108/165

36/55

0/45

0/90

12/96, 20/191, 7/104

30/113, 49/156, 8/40

11/103

10/55

77/9314/932/930/93

104/12813/1288/1283/128

114/20482/2047/2041/204

14/162/16

Cotransductionfrequency (%o)

15

27

11

53

65

65

<2

<1

10

28

11

18

83152

<1

811062

564031

8812

a Each fraction shows the result obtained from an independent experiment which included a different cultureof the recipient and a different phage stock.

b When thiK was used as the donor unselected marker, recipient strains had to have the de novo syntheticpathway of thiamine-PP already blocked somewhere before the formation of thiamine-P. The mutation thi-l wassuitable for this purpose except in the case of X7014a. X7014aEN is a strain in which thiD was incorporated byconjugation of X7014a with NISOOMN.

C Since NIS10 carries both thiD and thiK mutations and the two loci are approximately 20 min apart on thechromosome, half of the transductants derived from the cross of it with the wild-type alleles for both thiD andthiK should have been thiD+ thiK, and the other half should have been thiD thiK+ when selected on mediumcontaining thiamine. The thiD+ thiK transductants could grow in the absence of thiamine, whereas the thiDthiK+ transductants could not grow unless thiamine was added to the medium. Therefore, the latter type oftransductants were scored and the presence of unselected marker was tested.

d ptsG transductants could be distinguished from ptsG+ transductants in two different ways: transductantscarrying ptsG could not grow on the minimal medium containing 250 mg of glucose per liter, whereas ptsG+transductants could, and on MacConkey agar base indicator plates containing glucose (2), ptsG transductantsformed white colonies with a red central portion, whereas ptsG+ transductants produced dark red colonies.

e For the selection offabD+ transductants, the plates were incubated at 42°C.f WhenfabD was used as the donor unselected marker, colonies grown on the plates at 30°C were tested for

their ability to grow on the same medium at 42°C. Transductants that could grow at 30°C, but not at 42°C, werejudged as those carrying the fabD mutation.

8 NI51OLP (thiD thiK pyrC) was constructed by conjugation of NIS10 with MA1008.714

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pyrC

23.0 (23.3)

ptsG fabD thiK purBI I I I

24.0 (24.4)(24.7)

10

25.0 (25.2)

11,7-ll, 27-3* ,2

18-11 -

I

--

-15,4* 1

28 I

26.0

hemA

(26.4)

L 1 1FIG. 2. Genetic map of E. coli near the thiK gene as determined by transduction. Reference loci are placed as

on the map of Bachmann and Low (1). Numbers are cotransduction percentages obtained from crosses 1 through14 of Table 3. The order of ptsG, fabD, and thiK is based upon the results obtained from the three-factortransductional crosses (15 through 18) of Table 3. Abnormal low frequencies observed in crosses when thiK wasthe selected marker are indicated by asterisks.

KL226, AB259, and Broda8, using leu, lac, trp,and his as reference markers in their gradient oftransmission, placed the site of the thiL locus atabout 9 min on the chromosome. Conjugationalmapping of thiL with another strain, N1420,revealed that the mutation lay in the same posi-tion as determined with NI520 (data not shown).Based on the results of conjugational map-

ping, cotransduction between the thiL locus andseveral neighboring genes situated clockwisefrom lac was measured. The experiment wasfirst conducted to determine the relative positionof thiL to the loci lac and proC with the three-factor crosses shown in crosses 1 and 2 of Table4. In a cross between P1 grown on strain NI52OGand strain x342, carrying proC, 70% of theproC+ recombinants received neither lac northiL and 23% received thiL but not lkc. In thereciprocal cross, 73% of thiL+ recombinantsreceived neither lac+ nor proC and 25% of themreceived proC but not lac+. In these crossesthiL was cotransduced with proC at a frequencyof 27% and with lac at a frequency of 3%,whereas between thiL and lac the cotransduc-tion frequency was 8%. Therefore, thiL ap-peared to lie on the opposite side of proC fromlac, and the distance between thiL and proCappeared to be shorter than that between lac andproC. Next, a cotransductional cross was per-formed to examine the linkage ofphoR and tsxwith thiL. The three-factor cross (Table 4, cross3), involving phoR as the Pl donor and both lacand thiL as the markers in the recipient, showedthat 53% of the thiL+ recombinants receivedphoR but not lac, and 46% of them receivedneither phoR nor lac. In addition, the linkagebetween thiL and phoR was 54%, whereas thatbetween thiL and lac was only 2%. These resultssupport the assumption that thiL lies clockwisefrom phoR, away from lac. Since thiL cotrans-duced with phoR at a much higher frequency

than with proC, it became necessary to examinethe cotransduction frequency between thiL andtsx, a locus which has been placed clockwisefrom phoR, presumably closer to thiL. Trans-duction with two different Pl donor strains car-rying tsx showed that thiL cotransduced with tsxat frequencies of 69 to 75%, suggesting that thiLwas located within 0.2 min from tsx. Judgingfrom their high cotransduction frequencies, wehad to reexamine the map position of thiL rela-tive to tsx with the help of a three-factor crossincluding phoR. Strain AB1157PR, carryingboth phoR and tsx markers, was constructed forthis purpose by the conjugation of AB1157 withC5, and the strain was used as the donor. Of thethiL+ recombinants, 56% received both phoRand tsx simultaneously, 20% of them receivedtsx but not phoR, and 23% had neither tsx norphoR (Table 4, cross 6). Moreover, it should benoted that among 139 recombinants that hadreceived thiL together with phoR, 137 receivedtsx as well. These results clearly indicated thatthe gene order must have been phoR-tsx-thiL.To confirm the gene order, the linkage of thiLwith the additional markers phoA and acrA wasexamined. thiL was cotransducible with phoA ata frequency of about 26% and with acrA at afrequency of 3% (Table 4, crosses 7 and 8).These values were entirely consistent with theposition of thiL relative to the neighboring genesmentioned above (Fig. 3).

DISCUSSION

We describe in this paper two newly foundgenes, thiK and thiL, governing the activities ofthiamine kinase and thiamine-P kinase, respec-tively. thiK lies near 24 min and thiL lies at 9.4min on the chromosome. Mutants that can growon thiamine phosphates but not on the free formof thiamine were used in the investigation. They

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TABLE 4. Transductional mapping of the thiL locus

Strain Marker No. of coloniesCross with donor unselected Cotransductionno. Donor Recipient Selected Unselected marker/total no. of frequency (%)

colonies selected

ia N1520G lacY thiL X342 proC proC+ lac+ thiL+ 319/461 69lac+ thiL 104/461 23lac thiL+ 35/461 7lac thiL 3/461 1

2b X342 proC N1520G lacY thiL thiL+ lac proC+ 131/181 72lac proC 45/181 25lac+ proC 3/181 2lac+ proC+ 2/181 1

3c CS phoR NI52OG lacY thiL thiL+ phoR lac 119/224 53phoR lac+ 3/224 1phoR+ lac 102/224 46phoR+ lac+ 0/224 0

4d AB1157 tsx N1520G lacY thiL thiL+ tsx 145/193 75

5 AT1380 tsx N1520G lacY thiL thiL+ tsx 70/102 69

6 AB1157PR phoR tsx NI52OG lacY thiL thiL+ phoR tsx 137/245 56phoR tsx+ 2/245 1phoR+ tsx 49/245 20phoR+ tsx+ 57/245 23

7c S26 phoA NI52OG lacY thiL thiL+ phoA 48/196 25

8' N43 acrA NI52OG lacY thiL thiL+ acrA 5/193 3a Since strains that have received the thiL mutation from N1520 (thiD thiL) required thiamine-PP for growth,

thiL transductants could be determined by scoring the colonies that grew on thiamine-PP but not on thiamine.b Selection of thiL+ was done with thiamine, since thiD thiL+ transductants were produced.c Scoring of transductants carrying a phoR or phoA mutation was performed essentially as described by

Echols et al. (3). thiL+ colonies grown on tryptone-glucose medium containing thiamine and either an excess or alimiting amount of Pi were sprayed with a-naphthylphosphate and tetrazotized o-dianisidine. phoR coloniesturned light brown on plates with excess Pi, whereas phoA colonies remained white on both types of plates (15).

d Resistance or sensitivity of thiL+ transductants to phage T6 was determined by cross-streaking a loopful ofthiL+ transductants on a nutrient agar plate on which T6 lysate had been streaked.

' thiL+ transductants capable ofgrowing on the peptone-glucose-yeast extract medium of Nakamura et al. (14)containing 6 ng of acriflavine per ml werejudged acr+, and those unable to grow on the medium werejudged acr.

lac phoA proC phoR tsx thiLI . .a. --I- -t I

(7.8) 8.0 (8.7)(8.8)9.0 (9.2)

L1 33,2 - 3-FIG. 3. Genetic map ofE. coli near the th

determined by transduction. Reference locias on the map of Bachmann and Low (1). Nicotransduction percentages obtained from tments in Table 4.

acrA included two types: one requires thiamine-PP,I'-- and the other requires thiamine-P or thiamine-

10.0 (10.2) PP. Mutants showing such growth phenotypeshave never been obtained from a parental orga-nism prototrophic for thiamine, whereas theycan readily be produced by further mutation of amutant organism auxotrophic for thiamine. Thisindicates that the requirements for either thia-mine-P or thiamine-PP are manifested as theresult of additional mutations arising in a cellwhere de novo synthesis of thiamine-PP has

WiL gene as been blocked by the first mutation.are placed In the course of mapping experiments, weimbers are examined the cotransduction frequencies of thiKthe experi- with various markers in the 23- to 25-min region.

The results showed that thiK lies between pyrC

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and purB, although anomalously low frequen-cies of cotransduction for pyrC and purB withthiK were observed when thiK+ was selected forin strains X7014a and S730. The cotransductionfrequencies of pyrC with ptsG, fabD, and thiKpresented in crosses 1, 11, and 13 of Table 3appear not to coincide with the gene order ptsG-fabD-thiK in Fig. 2. However, from the resultsobtained with three-factor transductional cross-es between pyrC, ptsG, and thiK and betweenpyrC, fabD, and thiK (Table 3), thiK is mappedto the right of both ptsG and fabD, with thesethree genes being situated very close to oneanother. The fabD locus has been mapped atabout 24 min, between pyrC and purB and closeto ptsG, and their order was tentatively estab-lished as pyrC, ptsG,fabD, and purB (20). If thisis the case, the order of the relevant genes couldbe pyrC-ptsG-fabD-thiK-purB. In the case of thethiL locus, cotransduction frequencies of thiLwith several markers in the 8- to 10-min regionclearly show that the locus is situated betweentsx and acrA, and probably 0.2 min from theformer. However, there have been several re-ports which showed that the loci of nuvA andsrnA are situated in this chromosomal regionnear the tsx locus (10, 19, 24). From the frequen-cies of cotransduction between these two mark-ers and lac, proC, and txs, they are placed at 0.3and 0.5 min clockwise from tsx in the currentlinkage map of E. coli K-12 (1). Comparison ofthe cotransduction frequencies of thiL withthose of several nearby markers and those ofnuvA or srnA with lac, proC, and tsx presentedin these reports suggest that thiL would lie closeto nuvA, but further experiments are required todetermine the orientation of thiL, nuvA, andsrnA.

LITERATURE CITED

1. Bachmann, B. J., and K. B. Low. 1980. Linkage map ofEscherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56.

2. Curtis, S. J., and W. Epstein. 1975. Phosphorylation ofD-glucose in Escherichia coli mutants defective in glu-cosephosphotransferase, mannosephosphotransferase,and glucosekinase. J. Bacteriol. 122:1189-1199.

3. Echols, H., A. Garen, S. Garen, and A. Torriani. 1961.Genetic control of repression of alkaline phosphatase inEscherichia coli. J. Mol. Biol. 3:425-438.

4. Imamura, N., and H. Nakayama. 1981. thiD locus ofEscherichia coli. Experientia 37:1265-1266.

5. Kawasakil, T., A. Iwashima, and Y. Nose. 1969. Regulation

of thiamine biosynthesis in Escherichia coli. J. Biochem.(Tokyo) 65:407-416.

6. Kawmsl, T., T. Nakata, and Y. Nose. 1968. Geneticmapping with a thiamine-requiring auxotroph of Esche-richia coli K-12 defective in thiamine phosphate pyro-phosphorylase. J. Bacteriol. 95:1483-1485.

7. Kawasaki, T., and Y. Nose. 1969. Thiamine regulatorymutants in Escherichia coli. J. Biochem. (Tokyo) 65:417-425.

8. Lennox, E. S. 1955. Transduction of linked genetic charac-ters of the host by bacteriophage P1. Virology 1:190-206.

9. Lewin, L. M., and G. M. Brown. 1963. The biosynthesis ofthiamine. IV. Inhibition by vitamin B6 compounds. Arch.Biochem. Biophys. 101:197-203.

10. L_psett, M. N. 1978. Enzymes producing 4-thiouridine inEscherichia coli tRNA: approximate chromosomal loca-tions of the genes and enzyme activities in a 4-thiouridine-deficient mutant. J. Bacteriol. 135:993-997.

11. Low, K. B. 1973. Rapid mapping of conditional andauxotrophic mutations in Escherichia coli K-12. J. Bacte-riol. 113:798-812.

12. Mller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

13. Miyagwa, K. 1960. Studies on thiamine synthesis byyeast. III. Detection of active-pyrimidine and active-thiazole by means of bioautography. Vitamins 20:255-259.

14. Nakamura, H., T. Tojo, and J. Greenberg. 1975. Interac-tion of the expression of two membrane genes, acrA andpisA, in Escherichia coli K-12. J. Bacteriol. 122:874-879.

15. Nakata, A., G. R. Peterson, E. L. Brooks, and F. G.Rothman. 1971. Location and orientation of the phoAlocus on the Escherichia coli K-12 linkage map. J. Bacte-riol. 107:683-689.

16. Nakayama, H., and R. Hayashi. 1972. Biosynthesis ofthiamine pyrophosphate in Escherichia coli. J. Bacteriol.109:936-939.

17. Nakayama, H., and R. Hayashi. 1972. Biosynthetic path-way of thiamine pyrophosphate: a special reference to thethiamine monophosphate-requiring mutant and the thia-mine pyrophosphate-requiring mutant of Escherichia coli.J. Bacteriol. 112:1118-1126.

18. Nakayama, H., and R. Hayahi. 1974. Inhibition of thia-mine pyrophosphate utilization by thiamine or its mono-phosphate in Escherichia coli. J. Bacteriol. 118:32-40.

19. Ohlhl, Y. 1974. Genetic analysis of an Escherichia colimutant with a lesion in stable RNA turnover. Genetics76:185-194.

20. Semple, K. S., and D. F. Silbert. 1975. Mapping of thefabD locus for fatty acid biosynthesis in Escherichia coli.J. Bacteriol. 121:1036-1046.

21. Singer, E. R., J. R. Beckwith, and S. Brenner. 1965.Mapping of suppressor loci in Escherichia coli. J. Mol.Biol. 14:153-166.

22. Stouthame, A. H., P. G. de Haan, and H. J. J. Nlkamp.1965. Mapping of purine markers in Escherichia coli K12.Genet. Res. 6:442-453.

23. Taylor, A. L., and C. D. Trotter. 1967. Revised linkagemap of Escherichia coli. Bacteriol. Revs. 31:332-353.

24. Tbomas, G., and A. Favre. 1977. Localication genetiqued'une mutation qui rend la croissance de E. coli K12insensible & l'illumination & 365 nm. C. R. Acad. Sci. Ser.D 284:2285-2288.

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thik and thil loci of escherichia · thik andthil loci ofe. coli 709 shown in fig. 1, together with...

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