JOURNAL OF BACTERiOLOGY, Nov. 1970, p. 658-667Copyright a 1970 American Society for Microbiology

Characterization of Pyridoxine Auxotrophs ofEscherichia coli: Serine and PdxF Mutants

WALTER B. DEMPSEY AND HAJIME ITOHMicrobial Genetics Unit, Veterans Administration Hospital, Dallas, Texas 75216, and

Department ofBiochemistry, University of Texas Southwestern Medical School,Dallas, Texas 75235

Received for publication 30 July 1970

At least six phenotypically distinct classes of mutants of Escherichia coli whichrequire serine or pyridoxine or both can be isolated. Three of the six classes lack 3-phosphoserine-2-oxoglutarate aminotransferase. One of these classes contains WG5,a mutant previously characterized as containing the pdxFS allele. The aminotrans-ferase isolated from this mutant has been compared to that isolated from wild-typeE. coli and found to have apparently normal affinity for pyridoxal 5'-phosphate, butreduced affinity for pyridoxamine 5'-phosphate.

The sequence of reactions used in the biosyn-thesis of pyridoxal 5'-phosphate by organismswhich do not require an external source of pyri-doxine has not been fully elucidated. [Pyridoxineis used here to mean the family of six compounds:pyridoxol, pyridoxal, pyridoxamine, and theirrespective 5'-phosphate esters (2). Pyridoxolspecifically refers to 2-methyl-3-hydroxy-4, 5-bis(hydroxymethyl) pyridine. A pyridoxinelessorganism is one which cannot synthesize one ormore of the biologically essential compounds fastenough to maintain normal growth rates. Suchmutants then require one of the six compoundsfor normal growth but do not necessarily respondto all of them.] It has been possible, however, todivide pyridoxineless mutants of Escherichiacoli into five groups by transduction (4), and tosubdivide two of the five groups by cross-feedingand other nutritional tests (8). One group ofpyridoxineless mutants was further characterizedas lacking pyridoxol 5'-phosphate oxidase (3).This report describes the enzyme defect in a

second pyridoxineless mutant, namely one con-taining the pdxF5 allele (4). The enzyme defectsin other groups of pyridoxineless mutants remainunknown.The sequence of reactions used in the biosyn-

thesis of serine was fully elucidated by Pizer (13)and Umbarger et al. (17). They showed that thebiosynthesis of serine in both E. coli and Salmo-nella typhimurium involves the oxidation of3-phosphoglycerate to a 3-phosphohydroxypyru-vate, the transamination of 3-phosphohydroxy-pyruvate to 3-phosphoserine, and finally thehydrolysis of 3-phosphoserine to serine. Thefirst reaction is catalyzed by 3-phosphoglycerate

dehydrogenase, and E. coli mutants lacking thisactivity have serA genotype (17). The second reac-tion is catalyzed by 3-phosphoserine-2-oxoglu-tarate aminotransferase (POA) and mutantslacking this activity have serC genotype (6). Thethird reaction is catalyzed by 3-phosphoserinephosphatase, and mutants lacking this activityhave serB genotype (17).Mutants with serA and serB genotype are well-

known, but mutants with serC genotype were onlyrecently found and described (6). These lattermutants surprisingly require both pyridoxol andserine for growth, whereas mutants carrying eitherserA or serB genotype require only serine. Theresulting possibility that 3-phosphoserine not onlyis a precursor of serine but also is a precursor ofpyridoxine has been discussed (7).The purpose of this present work is to show

that mutants bearing the pdxF allele constitute a

special class of serC mutants, namely ones whichappear to be expressed in a gene product, POA,that has an altered affinity for pyridoxamine5'-phosphate.

MATERIALS AND METHODSStrains. WG1, wild-type E. coli B, is the parent

organism for all bacterial mutants herein described.It was originally obtained from A. L. Koch. WG5(formerly B-B6-5), WG3, WG25, WG1027, and ourstrain of Plbt phage were described previously (4, 8).Strain WG1145 (serC 57) has also been described (6).Strains B-166 and 22-99 were obtained from J. G.Morris.

Media. Glucose minimal medium was as describedpreviously (4). In all cases glucose was added onlyafter separate autoclaving. MgSO4 was likewiseseparately autoclaved. Mannitol minimal medium

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contained separately autoclaved mannitol at 0.2%final concentration instead of glucose. ML agar hasbeen described (4). DS medium had the followingcomposition: potassium phosphate, 0.15 M, pH 7.8;0.6% glucose, 15 mm (NH4)2SO4, 0.4 mm MgSO4, and0.01 mM FeSO4.

Isolation and characterization of mutants. Thegeneral procedure used to isolate mutants was thatdescribed for pyridoxine mutants (5). For each experi-ment all colonies which could grow at 42 C on glucoseminimal medium supplemented with 300 mg of L-serine per liter and 0.1 mg of pyridoxal per liter wererestreaked on identical medium. After overnightgrowth at 42 C, the streaked plates were used asmaster plates to test, by replica plating, the ability ofthe cells to grow on glucose minimal medium con-taining (per liter): 0.1 mg of pyridoxal, 300 mg ofL-serine, and 300 mg of glycine, both singly and incombinations. Only one isolated colony of eachnutritional type was then selected for two furthersequential single-colony isolations on ML agar; theremainder were discarded.

Growth requirements. The pyridoxol requirement at37 C was determined as previously described forpyridoxine mutants (8). The anaerobic serine require-ment was determined at 37 C in an identical wayexcept that L-serine in concentrations from 0 to 300mg/liter replaced pyridoxol. The aerobic serine re-quirement at 37 C was tested in vigorously shakencultures. Each culture contained 50 ml of glucoseminimal medium plus various amounts of L-serine in a250-ml Erlenmeyer flask. Each flask was inoculatedwith 107 saline-washed cells which had been freshlygrown in glucose minimal medium containing 300mg of L-serine per liter. After 22 hr, the absorbancyat 650 nm was determined, and samples from eachflask or tube, diluted to contain approximately 100 to200 cells, were plated onto ML agar. After growththese cells were replica plated to glucose minimal agarwith and without supplements to test for revertants.Only data from samples with less than 1% revertantswere used. For WG1 145, the requirements were deter-mined anaerobically only, with 70 ng of pyridoxol/mlin all tubes having varying serine concentrations and280 jug of serine/ml in all tubes having varyingpyridoxol concentrations. In all cases, pyridoxol wasused as the free base. After 22 hr of incubation at37 C, cell mass was determined by turbidimetry asdescribed earlier (2).

Synthesis of pyridoxine during serine starvation.Immediately before use, each mutant was restreakedand its nutritional properties were rechecked byreplica plating. Each strain was than grown overnightwith vigorous shaking at 39 C in 50 ml of glucoseminimal medium containing 500 mg of L-serine and0.1 mg of pyridoxol per liter. For B166 the mediumalso contained glycolaldehyde at 120 mg/liter. Thecells were centrifuged for 10 min at 5,000 X g and25 C, washed once in saline, and used in entirety toinoculate 1 liter of fresh identical medium at 39 C.The cultures were grown with vigorous shaking to adensity of approximately 0.6 mg (dry weight)/ml,centrifuged for 15 min at 4,000 X g and 35 C, andwashed once with saline. They were then put in 1 liter

PYRIDOXINE 659

of glucose minimal medium at 39 C, and vigorousshaking was resumed for 3 hr. Duplicate samples of 5ml each were removed at the time of inoculation andevery 30 min thereafter. The turbidity of these samplesat 650 nm was first measured to determine cell mass,after which they were mixed with 5 ml of 0.11 NH2SO4 and refrigerated until all samples were taken.After 3 hr of growth, the cells were harvested, washedwith saline, and stored at -70 C. After hydrolysis ofthe acidified samples at 121 C for 5 hr, the total pyri-doxine content of the samples was determined bybioassay as described previously (2). Samples fromWG1241 starved for 60 min or longer turned deepyellow upon hydrolysis and precipitated a brownmaterial. This material was removed by centrifuga-tion before assay.Enzyme assays. For assays which compared enzyme

activities between strains, 3-phosphoglycerate dehy-drogenase was assayed by the method of Umbargeret al. (17); POA and 3-phosphoserine phosphatasewere assayed by the method of Pizer (13). For all ofthese assays, the frozen cells were thawed, disruptedby ultrasonic oscillation, and carried through theammonium sulfate step of the partial purificationdescribed by Pizer (13).The assay of POA during purification of this en-

zyme from WG5, as well as comparative studies be-tween the mutant enzyme and the wild-type enzyme,was made by following reduced nicotinamide adeninedinucleotide (NADH) oxidation in the followingsystem (Itoh and Dempsey, submitted for publication).In a cuvette of 1-cm light path were put: 5 ,imoles ofO-phospho-L-serine, 2 jAmoles of a-ketoglutarate,0.05 jAmole of pyridoxal 5'-phosphate, 0.125 mg ofNADH, 200 ,gmoles of potassium phosphate (pH 7.5),5 ,umoles of NaF, 1 jumole of dithiothreitol, 0.5 jAmole ofethylenediaminetetraacetic acid, 6 to 10 units of3-phosphoglycerate dehydrogenase [purified fromchicken liver through the phosphocellulose step bythe method of Sallach (15)] aminotransferase, andwater to 1 ml final volume. Reaction was started bythe addition of a-ketoglutarate, and decrease inabsorbancy at 340 nm at 25 C was followed. A molarabsorbancy index of 6,200 for NADH was used toconvert the observations to micromoles of NADHoxidized. One unit of enzyme was the amount whichoxidized 1 ;umole ofNADH per min at 28 C. ApparentMichaelis constants were determined with enzymewhich had been dialyzed against glutamate.

Synthesis of pyridoxine by WG5 during growth onL-serine. One liter of glucose minimal medium con-taining 300 mg of L-serine per liter was inoculatedwith a saline-washed suspension of WG5 which hadbeen grown overnight in 50 ml of the same medium.The culture was shaken vigorously at 37 C until thecell density reached 0.5 mg/ml (dry weight). The cellswere harvested by centrifugation at 35 C for 20 minat 4,000 X g, washed once with saline, and suspendedagain in saline. Identical medium lacking only L-serinewas then inoculated with 60% of these cells. Shakingat 37 C was resumed, and samples were withdrawnfor total pyridoxine and cell mass determinations asdescribed above.

Miscellaneous. Transductions with Plbt phage

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were as described previously (4). Protein was deter-mined by the method of Lowry et al. (9). Pyridoxolhydrochloride was a gift of Merck & Co., Rahway,N.J. All concentrations of this and other pyridoxinecompounds refer to the free base. All other chemicalswere obtained from commercial sources.

RESULTS

WG5 (formerly B-B6-5), a pyridoxineless strainof E. coli B carrying the pdxFS allele, was isolatedseveral years ago after ultraviolet irradiation ofwild-type E. coli B (8). Although this strain wasfirst distinguished from other pyridoxinelessorganisms by its behavior in cross-feeding tests(8) and more recently by its behavior in trans-ductional analyses (4), it had been little studiedbecause it reverted readily to prototrophy.As reported earlier (Dempsey, Bacteriol Proc.,

p. 131, 1969), WG5 grows slowly at room tem-perature in unsupplemented glucose minimalmedium and is stimulated to fuller growth atroom temperature by some metabolites relatedto the aspartate family of amino acids and in-hibited by others. These effects are not seen incultures incubated at 42 C.

In an effort to explain these effects, we exam-ined still other compounds and found thatL-serine could not only stimulate the rate ofgrowth but could in fact fully replace the pyri-doxine requirement of WG5 in simple media at37 and 42 C. The same survey revealed that WG5did not respond with full growth to any otheramino acid, including glycine. The serine effectwas sufficiently dramatic that an explanation forit was sought. This report presents the results ofour investigations of this phenomenon.Four other pyridoxineless strains isolated after

ultraviolet mutagenesis were previously reportedto be closely linked to WG5 by P1 transduction(4). These strains, WG4, WG143, WG144, andWG148, also grew fully in glucose minimal me-dium supplemented with either pyridoxol orL-serine. The sixth strain described in the originalreport was lost.The amount of serine required for full growth

of WG5 in glucose minimal medium withoutpyridoxine was next determined. In still cultures,WG5 required approximately 0.3 g of L-serineper g (dry weight) of cells, an amount similar tothat of serineless mutants (14). In cultures shakenvigorously at 37 to 39 C, a similar amount ofserine was required to maintain a doubling timeof approximately 60 min, but we also found thatWG5 would grow slowly but fully in a mediumcontaining as little as 10 mg of L-serine per liter.During growth in glucose minimal medium

containing serine, WG5 synthesized normalamounts of pyridoxine. For example, the data in

Fig. 1 show that, when the initial L-serine con-centration was 300 mg/liter, the pyridoxine con-tent of the culture was approximately 450 nmolesof pyridoxine/mg of dry cell weight, a value ap-proximately equal to that of wild-type E. coli (2).Figure 1 also shows that WG5 was unable tocontinue synthesis of pyridoxine at the normalrate after serine was removed from the medium.

Since WG5 was originally classified as a pyri-doxineless mutant, the finding that serine couldreplace pyridoxine led us to reexamine thisoriginal classification. The first evidence that theclassification was correct was nutritional. WG5grew to the same extent in glucose minimal me-dium supplemented with 0.4 ,uM pyridoxol aswild-type E. coli B (WG1) did in unsupplementedmedium. This amount of pyridoxol (i) is equiva-lent to the total pyridoxine which wild-type cellsmake under the same conditions (2), and (ii) isan amount that will prevent further de novo syn-thesis of pyridoxine (2). The actual nutritionalrequirement of WG5 was next determined to be50 ,g of pyridoxol per g of dry cells (Fig. 2) or0.3 nmole of pyridoxol per mg of dry cells. Thisamount was equivalent to that previously foundfor other pyridoxineless auxotrophs (8).The second evidence that WG5 was a true

pyridoxineless mutant came from direct demon-stration (presented below) that extracts of WG5possess altered activity of an enzyme recentlyshown to be essential for pyridoxine biosynthesis(7), namely POA. The third evidence is shown inFig. 1 and 4, namely, that WG5 synthesizedpyridoxine at greatly reduced rates after starva-tion for either serine or serine and pyridoxine.WG5 now appeared to bear some resemblance

to the three pyridoxineless mutants described in

1.0

0.8

0.6

0.4

0.2

WG5 without -.-with serine serine

1 2 3 4 5 6 7 8

TIME ( in hours)FIG. 1. Cell mass and pyridoxine changes of WGS

during growth on serine and during starvation forserine. Symbols: 0, milligrams of dry cells/milliliter;

l, nanomoles of total pyridoxine/milliliter; 0, nano-

moles of total pyridoxine/milligram of dry cells.

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some detail several years ago by M(Woods (11) as having an unexplainedship with serine and glycolaldehyde.gested that a potentially profitable waythe lesion in WG5 would be to isolate ctants showing growth responses to ;

pyridoxol, or both, and to compare therties and the properties of the two muteand 22-99 of Morris and Woods whichavailable and still unexplained (J. G.personal communication, 1969) with the rof WG5. Accordingly, we isolated newand found that, among 94 independentthat grew at 42 C in minimal medium cpyridoxol and serine but not in unsuppmedium, at least six different nutritional iwere distinguishable. These differentresponses allowed the mutants to be divthe six classes shown in Table 1.

Class a mutants included organiseither serA or serB genotype, and class Iincluded organisms with pdx genotype.these types of mutants had been previolacterized (13, 17). Class c mutants conta

FIG. 2. Anaepyridoxol.

10 20 30 40

pyridoxol (pg /liter)robic growth response of

Drris and tants with a single mutation causing a doublerelation- requirement for pyridoxine and serine (6). Therhis sug- growth requirements of this type of mutant areto define depicted in Fig. 3. These mutants have been)ther mu- shown to lack POA (6) and, accordingly, haveserine or received the designation serC mutants.ieir prop- Class d mutants had a phenotype identical toants B166 that of WG5. Class e mutants had not been de-were still scribed before. The remaining class f could haveMorris, included one or more of the previously unchar-

?roperties acterized "serine" mutants that would not usex mutants glycine (16), but none of the mutants in this classt mutants grew in glucose minimal medium which containedontaining glycylglycine (16) or p-aminobenzoic acid (1) in-Alemented stead of serine.responses Mutants representing classes d, e, and f to-growth gether with WG5, and WG1 (wild type) and the

tided into B166 mutant of Morris and Woods were studiedfurther in two ways. First, to find out whether

;ms with any were true pyridoxineless auxotrophs, their5 mutants ability to synthesize pyridoxine during starvationBoth of for required nutrients was determined. Second,

asly char- to determine whether any were of serA, serB, orlined mu- serC genotype, their content of each of the three

enzyme activities known in serine biosynthesiswas determined.The rate of pyridoxine biosynthesis was deter-

mined by measuring the cell mass and the totalpyridoxine content (2) in samples taken every 30min from cultures which were being starved ofserine and pyridoxine. All cultures were firstgrown in an identical glucose medium supple-mented with serine and pyridoxol and then trans-ferred to unsupplemented glucose medium toinitiate starvation. [It had previously been shownthat growth in media supplemented with pyri-doxol prior to starvation for an amino acid had

50 no detectable effect on the rate of pyridoxinebiosynthesis seen during amino acid starvation(2). In other words, there was no apparent repres-

WG5 to sion of pyridoxine biosynthesis.] Figure 4 showsthe data obtained from these measurements. The

TABLE 1. Classes of mutants that require serine or pyridoxine, or both

Growth in glucose minimal medium supplemented withClass Phenotypes Potyesris No. of strains- _ _ -___-_______

designation included in class Prototype strains isolated Serine Glcine Pyridoxol Pyridoxol p(300 Mg/ (30mg/ (70 pg/ and Py sridoxoliter) liter liter) glycine and erine

a SerA, SerB WG1143, 39 + + _ + +WG1100

b PdxA,B,C,D, WG3,WG25, 21 _ _ + + +E,G,H,J,K WG1027

c SerC WG1145 20 _ _ _ + +d PdxF WG1245,WG5 3 + _ + + +e Unknown WG1228 3 + + + + +f Unknown WG1241 7 + _ _ _ +

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2E.

0

00

0

100 200 300 0.01 0D2 0.03 0.04

L-Serine (mg/ier) Pyridoxol (mgAiter)FiG. 3. Anaerobic growth response ofa SerC mutant to pyridoxol and to serine in media containing excess ofthe

unvaried nutrient.

1 2 3 1 2 3 1 2 3

TIME (in hours)FIG. 4. Cell mass and pyridoxine changes during starvation ofmutants for serine and pyridoxine. Symbols: *,

milligrams of dry cells/milliliter; El, nanomoles of total pyridoxine/milliliter; 0, nanomoles of total pyridoxinelmilligram ofdry cells.

data showed that (i) the growth rate of each ofthe mutants after starvation for serine and pyri-doxine had begun was less than that of WG1(wild-type E. coli); (ii) none of the mutantsreached the same final density as WG1; (iii)WG5, WG1228, and B166 appeared to stoppyridoxine biosynthesis after starvation was initi-ated and therefore could be classed as pyridoxine-less organisms; (iv) WG1241 synthesized pyri-doxine at 2.6 x 10-10 mole per hr per mg, a ratetwice that usually seen (2, 7). Similar studies withclass a and class c mutants as defined in Table 1were reported previously (7).

After 3 hr of starvation for serine and pyridox-ine, the cells were collected by centrifugation,washed with saline, and stored at -70 C untilall strains had been grown in similar manner.Assays for each of the known enzymes of serinebiosynthesis were then performed on a portionof these cells by the methods described above.The data from these assays are shown in Table 2together with the data from similar assays madeon 22-99 [the other mutant of Morris and Woods(11)] and WG3, a pyridoxineless mutant. Thedata in Table 2 showed that WG5, WG1228,B166, and 22-99 all could be classified as serC

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TABLE 2. Specific activities of serine biosyntheticenzymes in several mutants

Class designation Strain no.

UnclassifiedbdefdUnclassifiedUnclassified

WG1WG3WG5WG1228WG1241WG1245B-16622-99

Activitya

3-Phos-phogly-cerate

dehydro-genase

0.0250.010.0150.0300.0110.0190.0120.002

3-Phospho-serine-oxoglu-tarate-

aminotrans-ferase

0.230.22

<0.004<0.0040.310.4

<0.004<0.004

3-Phos-phoserinephos-phatase

0.030.020.020.020.020.02

<0.010.02

aExpressed as micromoles per minute per milli-gram of protein.

mutants since they all lacked the same enzymeactivity, namely, 3-phosphoserine-2-oxoglutarateaminotransferase. The other strains tested con-tained apparently normal amounts of each of theserine biosynthetic enzymes.To determine whether the defect in strain WG5

was genetically linked to that in WG1145, a mu-tant strain known to lack POA as the result of asingle mutation (6), WG1145 was transduced toSer+ with Plbt phage grown on WG5. Transduc-tion mixtures were plated on glucose minimalmedium containing pyridoxol. A total of 184transductants were isolated and purified on thesame medium. This medium would be expectedto support growth of transductants with PdxFphenotype but would not support growth ofWG1145 which requires both pyridoxine andserine for growth. All of the 184 transductantsgrew on glucose medium supplemented only withpyridoxol as well as on glucose medium supple-mented only with L-serine, but not on minimalmedium. This showed that this nutritional char-acteristic of WG5 had been transferred toWG1145. This 100% genetic linkage was inter-preted to mean that the pdxF5 gene from strainWG5 had been incorporated during each repairof the genetic defect in strain WG1145, and,together with the enzyme assays, provided evi-dence that the pdxFS gene ofWG5 and the serC57gene of WG1145 were alleles. It would not havebeen possible to detect full transductants inreciprocal crosses.

If pdxFS and serC57 were alleles, the nutri-tional differences between the strains had to beexplained in terms of different kinds of behaviorof the alleles or the product derived from thealleles. One explanation would be that the productof the serC57 allele in WG1145 was totally inac-

tive, whereas the product of the pdxF5 allele mayhave had only a decreased affinity for an activatoror coenzyme. This hypothesis was tested by com-paring the ability of pyridoxal 5'-phosphate(PALP) to activate POA in extracts preparedfrom WG5, WG1145, and other mutants. Table 3shows that the extracts of WG5 had POA activitywhen pyridoxal 5'-phosphate was present duringthe assay. No other extract was activated byPALP.The restoration of POA activity to WG5 ex-

tracts by PALP could have arisen either becausethe partial purification required before assay (13)had removed PALP from the enzyme, or becausethe PALP-free enzyme was the principal form ofPOA in the starting material. If PALP restoredactivity to the POA made by WG5 only becausethe POA had had its PALP removed by the

TABLE 3. Effect of pyridoxal 5'-phosphate on3-phosphoserine-oxoglutarate aminotrans-

ferase activity

Strain no.

(A) WG1WG3WG5WG25WG1027WG1 145WG1228WG1228WG1241B-16622-99

(B) WG5 with-out PALPadditiond

WG5 with10-4 MPALPe

(C) WG5fWG5fWG5SWG5S

Microgramsof proteinper assay

70707970705015030070300300

7530075

300

Specific activity ofaminotransferasea

Assayed Assayedwithout PALP with PALPb

0.230.22

<0.0040.240.27

<0.004<0.0040.0013c0.31

<0.004<0.004

0.10

0.13

<0.004<0.004<0.004<0.004

0.250.230.250.260.25

<0.004<0.0040.004c0.38

<0.004<0.004

0.15

0.18

0.28No data0.25

No data

a Expressed as micromoles per minute per milli-gram of protein.

b PALP, pyridoxal 5'-phosphate.c Calculated from extent of reaction after 2 hr.d WG5 disrupted in buffer containing 104 M

PALP, purified without further PALP addition.6 WGS disrupted in buffer containing 104 M

PALP, purified with 104 M PALP in all solutions.f Freshly grown with serine, not starved.g Freshly grown with pyridoxol, not starved.

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purification procedure (a procedure which hadlittle effect on wild-type enzyme), then it shouldhave been possible to add PALP to freshly brokencells, perform the partial purification withoutfurther PALP addition, and end up with inactiveenzyme. In this case the mutation in WG5 couldbe classified as one which decreased the affinitybetween pyridoxal 5'-phosphate and the POAmade by WG5. If, on the other hand, PALP re-stored activity because the POA made by WG5was present orginally without any attachedPALP,then the mutation in WG5 was not necessarilyone which affected the dissociation constant be-tween POA and PALP. In this case, PALP addedonce to broken cells before the partial purificationwould not be removed by the purification, and thepartially purified enzyme would be active withoutfurther PALP addition.This test was then performed. In these experi-

ments, cells of WG5 which had been starved forserine and pyridoxine for 3 hr were disrupted inbuffer which was 10' M in PALP. The extractswere then divided into two equal portions, andthe partial purification which included centrifuga-tion, streptomycin treatment, ammonium sulfateprecipitation, and solution in tris(hydroxy-methyl)aminomethane buffer was continued inthe normal way on one-half. The second half wastaken through each of the purification steps also,but all solutions used in the purification were10-4 M in PALP. Assay for POA was made onboth halves both in the presence of 10- MPALPand in its absence. The results, as shown in Table3 (B) suggested that WG5 enzyme did lose someactivity during the purification, but also sug-gested that the conversion of active POA to inac-tive POA by loss of PALP during purificationwas not the sole source of inactive POA.The finding that PALP was not easily removed

from the aminotransferase by the partial purifica-tion indicated that the 3 hr of starvation of theculture for pyridoxine and serine before assaymight also be unable to effect total conversion ofthe aminotransferase to the PALP-free state, andsuggested that this enzyme could possibly be en-tirely in this state before starvation was begun.To test this hypothesis, we grew WG5 in two

kinds ofminimal medium, one supplemented onlywith 300 mg of L-serine per liter and the othersupplemented only with 100 gg of pyridoxol perliter. The cells were harvested by centrifugation inlate exponential phase, washed once with saline,frozen immediately at -70 C, and then assayedwith and without pyridoxal 5'-phosphate. Theresults (C, Table 3) showed that the aminotrans-ferase was isolated largely in the PALP-free state

and ruled out extended starvation for pyridoxineand serine as the cause of PALP loss.

Finally, three pyridoxineless mutants, known tocarry pdx alleles unlinked by Pl transductioneither to each other or to pdxF5 (4), were testedto see whether loss of POA activity is charac-teristic of all pyridoxine starvations. The mu-

tants, WG3, WG25, and WG1027, were firstgrown in minimal medium with pyridoxol andthen starved for 3 hr for pyridoxine. Figure 5

shows that their growth during pyridoxine starva-tion was the same as that which had been foundfor WG5. [This type of growth is characteristic ofvitamin mutants during starvation (18).] Afterstarvation, the mutants were harvested and as-sayed as described in the preceding experimentsand each was found (A, Table 3) to have POAof normal specific activity which could not besignificantly enhanced by addition of pyridoxal5'-phosphate. These data showed that normalPOA is not inactivated by pyridoxine starvationof pyridoxine auxotrophs. These findings led usto the conclusion that the form of POA presentin WG5 was not normal POA which had beeninactivated by loss of PALP. Therefore, PALPdid not have its effect on WG5 extracts solely byactivating normal PALP-free enzyme.To clarify the nature of the mutation in the

POA of WG5, the enzyme was purified by themethod of Itoh and Dempsey (submitted forpublication) from 60 g (wet weight) of WG5which had been grown at 30 C without aerationin DS medium containing 100 Mug of pyridoxol perliter.The properties of the enzyme from WG5 were

then compared to the properties of the enzymefrom WGI grown in the same medium withoutpyridoxol. Table 4 shows that, after severalidentical steps of purification, the specific activityof the mutant enzyme is less than that of wildtype. It also shows that purified enzyme from bothorganisms lost a different proportion of activityafter simultaneous exhaustive dialysis in thesame beaker against the same solution of 0.1 M

1.2

0.8

0.4

1 2 3 1 2 3 1 2 3TIME (in hours)

FuG. 5. Cell mass and pyridoxine changes duringstarvation of pyridoxineless mutants for pyridoxine.Symbols are identical with those of Fig. 4.

,WG3 #w WG25 WGO27

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TABLE 4. Comparison of the specific activity of3-phosphoserine-2-oxoglutarate aminotransferase

purifiedfrom WGS with that from WGI

SpecificPurification step protein Totlactivityu

WGS WG1

mgCrude extract. 15,000Protamine sulfate..... 14,400Ammonium sulfate. .. 7,980 46.7 0.0060.027DEAEb-cellulose...... 1,130 45.5 0.04 0.18Hydroxylapatite ...... 67.2 31.6 0.47 2.2Biogel P-100.......... 16.9 10.3 0.61 3.7

a Expressed as micromoles per minute per milli-gram of protein. Dialysis against potassiumglutamate (pH 6.0) reduced activity of WG5 to 0.027and activity ofWG1 to 1.12 when assay was performedwithout pyridoxal S'-phosphate. These activities were0.73 and 2.77, respectively, with pyridoxal 5'-phosphate.

b Diethylaminoethyl.

potassium glutamate. In the latter case, the mu-tant enzyme was almost inactive without pyri-doxal 5'-phosphate addition, but the wild-typeenzyme retained 40% of its activity.When the apparent Michaelis constants of

these two pyridoxine compounds were deter-mined by plotting reciprocal velocity againstreciprocal coenzyme concentration, we foundthat the value for PALP was 1.6 X 10-6 M forboth WG5 POA and WG1 POA, but that thevalue for pyridoxamine 5'-phosphate was 4.4 x10-5 for WG5 POA and 1.6 X 10-5 M for WGIPOA. It appeared from these data that the WG5enzyme would tend to permit dissociation ofpyridoxamine 5'-phosphate more readily thanthe wild-type enzyme would.One puzzling finding of Morris (10) was still

unexplained. He had shown that B166 starvedfor glycolaldehyde, serine, and pyridoxine failedto synthesize pyridoxine (we confirm theseresults in Fig. 4). He also showed that that, givenglycolaldehyde, B166 synthesized pyridoxine.Accordingly, we tested glycolaldehyde for itsability to replace the pyridoxine requirement ofWG1145 and WG5. The findings were that glu-cose minimal medium containing glycolaldehydein concentrations of 20 mg/liter allowed somemoderate but in no case full growth of WG5,but WG1145 did not grow at all in this medium ineither the presence or absence of serine.

Since we show (Table 2) that both the B166and the 22-99 mutants of Morris and Woods (11)appear to be double mutants, we feel that theorganisms may have been treated with heavy

doses of mutagens before isolation and thus thespecific glycolaldehyde effect of Morris (10) maybe due to the presence of a third mutation. Clearlymore data are needed to resolve this question.

DISCUSSIONOur findings show that at least six classes of

mutants that are distinguishable by their varyingresponses to supplementation with serine andpyridoxine can be isolated from E. coli. Sur-prisingly, members of three of the six classes(WG1145, WG5, WG1228) appear to lack thesame enzyme, namely, 3-phosphoserine-2-oxo-glutarate aminotransferase.The data presented here, however, were col-

lected primarily to allow description of the geneticdefect in a member of that class of mutantslabeled (d) in Table 1, i.e., those that requireeither pyridoxine or serine for growth. StrainWG5, which bears the pdxFS allele, is the proto-type of this class.Table 3 indicates that POA activity is absent

from extracts of WG5, both when the mutant isfreshly grown in minimal medium supplementedwith either serine or pyridoxol and when themutant is starved of pyridoxol and serine for 3 hr.Table 3 also shows that the activity of POA canbe restored by the addition of pyridoxal 5'-phos-phate to extracts of WG5 and that, once restoredby PALP addition, the activity is not easily lostagain. Table 4 shows that (i) POA purified fromWG5 has a lower specific activity than the WG1enzyme at the same stage of purification, and (ii)dialysis against 0.1 M glutamate at pH 6 convertsthe WG5 enzyme to inactive enzyme, whereasidentical dialysis of the WG1 enzyme inactivatesonly a portion of this enzyme. Finally, the affini-ties of both wild-type and WG5 enzyme for pyri-doxal 5'-phosphate were found to be equal, butthe affinity of the mutant enzyme for pyridox-amine 5'-phosphate was only one-fourth that ofwild type.The data presented here show that the pdxF5

gene is probably allelic with the serC57 genebecause (i) strains containing these alleles,such as WG5 (pdxFS), WG1145 (serC57), havealtered POA activity, and (ii) the gene causingthe nutritional phenotype of WG5 (namely,Pdx or Ser) is 100% linked to the gene causingthe nutritional phenotype of WG1145 (namely,Pdx + Ser).The data presented here also show that the

wild-type gene that is allelic to pdxFS or serC57is required for the biosynthesis of pyridoxine.The evidence for this comes from the discoverythat SerC mutants such as WG1145 require both

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DEMPSEY AND ITOH

serine and pyridoxol for growth (Fig. 3), and thatPdxF mutants such as WG5 make reducedamounts of pyridoxine during starvation forserine and pyridoxine (Fig. 1, 4). It was shownpreviously that mutants containing the serC57allele cannot synthesize pyridoxine (7). Thisfinding might mean that 3-phosphoserine is arequired, common precursor of both serine andpyridoxine (scheme A, Fig. 6) or that, althoughPOA is an enzyme common to the biosynthesis ofboth serine and pyridoxine, it catalyzes theformation of two different compounds of whichone is an as yet undescribed compound requiredfor pyridoxine biosynthesis and the other is 3-phosphoserine (scheme B, Fig. 6). Biosyntheticschemes analogous to both of those above arewell known in E. coli.The total findings raise several questions. (i)

How does pyridoxol supply all the growth re-quirements of WG5? (ii) How does serine supplyall the growth requirements of WG5? (iii) Whydoes POA from WG5 require PALP for activity?

Pyridoxol may permit growth of WG5 byallowing POA to function in vivo. In this pro-posal, the externally supplied pyridoxol is takenup, converted to pyridoxal 5'-phosphate which isthen used both to activate the POA and to con-vert any other PALP-requiring enzyme to itsactive form. The cell can now biosynthesize itsown serine.

Serine may permit the biosynthesis of pyri-doxine and growth of WG5 in scheme A, Fig. 6,by repressing or inhibiting 3-phosphoserinephosphatase enough to allow a small accumula-tion of 3-phosphoserine. Serine (Scheme B), byrepressing or inhibiting 3-phosphoglyceratedehydrogenase but not POA, may reduce thecompetition between pyridoxine and serine pre-cursors for POA and thus allow more pyridoxinesynthesis.POA from WG5 is isolated in an inactive form

possibly because conditions of growth or isola-tion expose the enzyme to sufficiently high con-centrations of the appropriate amino acids toleave the enzyme predominantly in the pyridox-amine 5'-phosphate form which then dissociates.This explanation is suggested from the findingthat pyridoxamine 5'-phosphate dissociates morereadily from the mutant enzyme than it does fromwild-type enzyme.As a corollary to these findings, the data of

Table 2 identify the B166 strain of Morris andWoods (11) as a serB, serC double mutant.Their mutant 22-99 is identified as a serA, serCdouble mutant. The enzymatic defects in thesestrains had not been previously identified (J. G.Morris, personal communication, 1969). Thedata of Table 2 leave unresolved the nature ofthe mutation in WG1241 and in WG1245.

A|3-phampoe lRNE

3-PHOPHO- -phosphoserine phoShItose3-PHOSPHO- pphohycerote *2 oxw 3H OGLYCERATE dehydoqenose omintrnslse SERINE

PYRUVATE -. -? , PYRIDOXNE|

B3-PHO0SPH40-

&.WPHO9O- 23 phosphoycerte HYRO- 3-POSPHO- 3- phosphoserine SERINEGLYCERATE dehydrogenose S ERINE phosphotose

PYRUVATE 3- phosphoserine

2-osoglutkate

ommoronsweosePYRIOXKINE

COMPOUNDY - ,. ?* PYRIOKINEPRECURSOR

FIG. 6. Two possible schemes to show role of3-phosphoserine aminotransferase in pyridoxine biosynthesis.

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ACKNOWLEDGMENTS

I want to thank J. G. Morris for his kindness in providingstrains B166 and 22-99. The assistance of A. C. Kern, K. Sims,and G. Learmont is gratefully acknowledged.

This investigation was supported by a general research supportgrant from the Public Health Service to the University of TexasSouthwestern Medical School.

LITERATURE CITED

1. Davis, B. D. 1951. Aromatic biosynthesis. III. Role of p-

aminobenzoic acid in the formation of vitamin B12. J.Bacteriol. 62:221-230.

2. Dempsey, W. B. 1965. Control of pyridoxine biosynthesis inEscherichia coli. J. Bacteriol. 90:431-437.

3. Dempsey, W. B. 1966. Synthesis of pyridoxine by a pyridoxalauxotroph of Escherichia coli. J. Bacteriol. 92:333-337.

4. Dempsey, W. B. 1969. Characterization of pyridoxine auxo-

trophs of Escherichia coli: P1 transduction. J. Bacteriol.97:1403-1410.

5. Dempsey, W. B. 1969. Characterization of pyridoxine a,ixo-trophs of Escherichia coli: chromosomal position of linkagegroup I. J. Bacteriol. 100:295-300.

6. Dempsey, W. B. 1969. 3-Phosphoserine transaminase mutantsof Escherichia coli B. J. Bacteriol. 100:1114-1115.

7. Dempsey, W. B. 1969. Evidence that 3-phosphoserine may bea precursor of vitamin B6 in Escherichia coli. Biochem. Bio-phys. Res. Commun. 37:89-93.

8. Dempsey, W. B., and P. F. Pachler. 1966. Isolation and char-

acterization of pyridoxine auxotrophs of Escherichia coli.J. Bacteriol. 91:642-645.

9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Rand-all. 1951. Protein measurement with the Folin phenol re-

agent. J. Biol. Chem. 193:265-275.10. Morris, J. G. 1959. The synthesis of vitamin B6 by some mu-

tant strains of Escherichia coli. J. Gen. Microbiol. 20:597-604.

11. Morris, J. G., and D. D. Woods. 1959. Interrelationships ofserine, glycine and vitamin B6 in the growth of mutants ofEscherichia coli. J. Gen. Microbiol. 20:576-596.

12. Newman, E. B., and B. Magasanik. 1963. The relation ofserine-glycine metabolism to the formation of single-carbonunits. Biochim. Biophys. Acta 78:437-448.

13. Pizer, I. 1963. The pathway and control of serine biosynthesisin Escherichia coli. J. Biol. Chem. 238:3934-3944.

14. Pizer, L. I., and M. L. Potochny. 1964. Nutritional and regula-tory aspects of serine metabolism in Escherichia coli. J.Bacteriol. 88:611-619.

15. Sallach, H. J. 1966. D-3-Phosphoglycerate dehydrogenase,p. 216-220. W. A. Wood (ed.), Methods in enzymology,vol. 9. Academic Press Inc., New York.

16. Simmonds, S. and D. A. Miller. 1957. Metabolism of glycineand serine in Escherichia coli. J. Bacteriol. 74:775-783.

17. Umbarger, H. E., M. A. Umbarger, and P. M. L. Siu. 1963.Biosynthesis of serine in Escherichia coli and SalmonellaTyphimwurium. J. Bacteriol. 85:1431-1439.

18. Wilson, A. C., and A. B. Pardee. 1962. Regulation of flavinsynthesis by Escherichia coli. J. Gen. Microbiol. 28:283-303

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