APPLIED AND ENVIRONMENTAL MICROBIOLoGY, Nov. 1977, p. 465472Copyright © 1977 American Society for Microbiology

Vol.34, No.5Printed in U.S.A.

L-Histidine Production by Histidase-Less Regulatory Mutantsof Serratia marcescens Constructed by Transduction

MASAHIKO KISUMI,* NORIYUKI NAKANISHI, TSUTOMU TAKAGI, AND ICHIRO CHIBATA

Research Laboratory ofApplied Biochemistry, Tanabe Seiyaku Company, Ltd., Yodogawa-ku,Osaka, Japan

Received for publication 11 April 1977

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited thegrowth of Serratia marcescens. These inhibitory effects were counteracted byL-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitorand TRA acts as both a false feedback inhibitor and a repressor. Mutantsresistant to each analog were isolated from a histidase-less mutant, because thewild-type strain possesses a potent histidase activity. 2MH-resistant mutantshad a feedback-insensitive phosphoribosyltransferase, but they produced onlysmall amounts of L-histidine. TRA-resistant mutants were divided into two typesaccording to their histidine productivity. A mutant of one type produced about8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levelsof histidine biosynthesis. Moreover, this mutant had a partially feedback-insen-sitive phosphoribosyltransferase. A mutant of the second type produced only asmall amount of L-histidine and had only derepressed enzyme levels. Accordingly,strains possessing the genetic alterations in both 2MH- and TRA-resistant mu-tants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The repre-sentative strain HT-2604 produced about 17 mg of L-histidine per ml.

The production of histidine has been carriedout mainly by extraction from protein hydroly-sates. For the fermentative production of histi-dine, a two-step method, i.e., fermentative pro-duction of L-histidinol using a histidine auxo-troph of Brevibacterium flavum and its micro-bial conversion into L-histidine, has been re-ported (20). Recently a direct fermentation wasdescribed, using histidine analog-resistant mu-tants of Corynebacterium glutamicum (2, 3) andB. flavum (H. Kamijo, 0. Mihara, and K. Ku-bota, Abstr. Annu. Meet. Agric. Chem. Soc. Ja-pan, Tokyo, p. 112, 1973).

In bacteria, histidine is synthesized from aden-osine 5'-triphosphate and 5-phosphoribosyl-1-pyrophosphate by nine histidine biosyntheticenzymes. This biosynthesis has been shown tobe controlled by both feedback inhibition ofphosphoribosyltransferase, the first enzyme inthe biosynthetic pathway, and repression of allhistidine biosynthetic enzymes (6, 12). On theother hand, histidine has been known to bedegraded by histidine decarboxylase or histidasein many bacteria (4). Therefore, the intracellularhistidine pool seems to be regulated by feedbackcontrol mechanisms and/or a degrading system,in addition to histidine permeation.We have been studying the fermentative pro-

duction of branched-chain amino acids by var-ious regulatory mutants of Serratia marcescens(14-18). These studies have convinced us thatwe would be able to make S. marcescens pro-duce any natural amino acid if feedback controlscould be released and degrading activities couldbe removed.A preliminary observation revealed that S.

marcescens has a potent histidine-degrading se-ries of enzymes, including histidase and urocan-ase. Therefore, it was presumed that not onlyhistidine but also urocanic acid, which is thefirst intermediate ofthe histidine-catabolic path-way and is useful as a sun-screening agent, wouldbe produced by mutants of S. marcescens.Matsumoto et al. (24, 25) described a gener-

alized transducing phage, PS20, ofS. marcescensSr4l. We expected that the application of thisgeneralized transduction would facilitate theconstruction of various amino acid producers ofS. marcescens Sr4l. Another strain of S. mar-cescens, HY, in which generalized transductionwas reported previously (5, 32), was not suitablefor our purpose because of its poor growth.

In this report, we describe the isolation ofhistidine regulatory mutants of S. marcescensSr4l and the construction of histidine producers,using a transductional procedure. The construc-

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tion of a urocanic acid producer of S. marcescenswill be described in a separate paper (submittedfor publication).

MATERIALS AND METHODS

Bacterial strains. S. marcescens Sr4l (24) and itsderivatives were used. Salmonella typhimuriumSB2802 (hisG46 his01242), which is defective in phos-phoribosyltransferase and contains the his01242 con-

stitutive mutation, was a gift from B. N. Ames.Chemicals. L-Histidine hydrochloride, urocanic

acid,,and L-histidinol phosphate, all A grade, were

products of Calbiochem. L-Histidinol hydrochloride,5-phosphoribosyl-1-pyrophosphate sodium salt, and1,2,4-triazole-3-D,L-alanine (TRA) were purchasedfrom Sigma Chemical Co. 3-Amino-1,2,4-triazole(AMT) was purchased from Tokyo Chemical Industry.2-Methyl-D,L-histidine (2MH) dihydrochloride was

chemically synthesized by us (26, 28). All other chem-icals were reagent grade.

Media. The complete medium was Difco nutrientbroth. Soft agar used for the phage study was supple-mented with 1 mM CaCl2 2H20. Minimal mediumwas that of Davis and Mingioli (10), modified byomitting the citrate and increasing the glucose to0.5%.Growth experiments. Cultures were grown in test

tubes (13 by 103 mm) containing 2 ml of minimalmedium. Supplements were added as indicated. Cul-tures in late-log or early-stationary phase in minimalmedium were diluted with fresh medium. Incubationwas carried out at 30°C with shaking (300 strokes/min,2-cm orbit). Growth was measured turbidimetricallyat 660 nm with a Hitachi electric photometer (typeEPO-B). An optical density of 0.10 corresponded to160,ug of dry cells per ml.

Isolation of mutants. The isolation of histidase-less mutants from S. marcescens Sr41 was carried outby mutagenesis with N-methyl-N'-nitro-N-nitroso-guanidine followed by selection of small colonies on

sodium succinate-L-histidine minimal medium platessupplemented with a small amount (0.0005%) of am-monium sulfate (27). Presumptive histidase-less mu-

tants were tested for the absence of histidase activity.The isolation of mutants resistant to 2MH or TRA

from histidase-less mutant Hd-16 was performed byusing the following procedure. A heavy inoculum (108cells) of strain Hd-16, which had been treated with N-methyl-N'-nitro-N-nitrosoguanidine, was plated out onminimal medium containing 1 mM 2MH or TRA. Inthe case of 2MH, sorbitol was used as the carbonsource in place of glucose. After 3 to 4 days of incu-bation at 30°C, large colonies appeared on each ofthe plates. These colonies were picked and screenedfor halo-producing activity by a cross-feeding test witha histidine auxotroph of S. marcescens. Several halo-producing colonies were purified and used for furtherstudy.Phage and transduction. Phage PS20, which is

a generalized transducing phage of S. marcescens Sr41,was kindly supplied by H. Matsumoto (24). Phagelysates were prepared according to the method ofMatsumoto et al. (24). Transduction was performedas follows: 1 ml of culture broth of the recipient strain

grown on nutrient broth (about 2 x 108 cells/ml) wasmixed with 3 ml of phage lysate containing about 1010plaque-forming units/ml, and the mixture was keptat 30°C for 30 min to allow for phage adsorption.After centrifugation, the cells were suspended in theoriginal volume of saline. When the resistance of TRAwas a selected marker, post-transductional cultivationwas necessary. Therefore, 0.2 ml of the cell suspensionwas inoculated on a nutrient agar slant and cultivatedfor 18 h at 30°C. The culture was harvested, washedwith saline, and resuspended in saline to a concentra-tion of 5 x 108 cells/ml. One-tenth milliliter of thesample was spread on the selective plate containingminimal medium supplemented with 1 mM TRA and5 mM AMT. The addition of AMT suppressed theappearance of spontaneous mutants with weak TRAresistance. After 3 to 4 days of incubation at 30°C,the large colonies that appeared were picked as thetransductants.Enzyme assays. The bacteria were cultured in

500-ml flasks containing 150 ml of minimal mediumat 30°C, with reciprocal shaking (140 strokes/min, 8-cm stroke). The cells grown exponentially were har-vested by centrifugation and washed twice with 0.05M tris(hydroxymethyl)aminomethane-hydrochloridebuffer (pH 7.5). The cells were suspended in the samebuffer and disrupted with a sonic oscillator (Kubotamodel 200M, 9 kHz) for 5 min at <50C. The sonicallytreated preparation was centrifuged at 27,000 x g for30 min at 0°C. The supernatant fluid was immediatelyused as cell-free extract for the enzyme assays, exceptfor the phosphoribosyltransferase assay, in which thedialyzed cell-free extract was used.The assays of histidine biosynthetic enzymes were

based on the methods described by Martin et al. (23).Phosphoribosyltransferase activity was assayed essen-tially according to the "coupled G-70 assay," which isdependent on a change in absorption at 290 nm whenthe enzyme is added to a solution of adenosine 5'-triphosphate and 5-phosphoribosyl-1-pyrophosphate.An extract of S. typhimurium SB2802 was added tothe assay mixture to pull the reaction to BBM III, N-(5'-phospho-D-1'-ribulosylformimino)-5-amino-1-(5"-phosphoribosyl)-4-imidazolecarboxamide, which hasan extinction 2.5 times higher than that of phospho-ribosyl-adenosine 5'-triphosphate. The histidinolphosphate phosphatase assay was based on the deter-mination of inorganic phosphate released. Histidinoldehydrogenase was assayed by the coupled-dye sys-tem. Specific activities of these histidine biosyntheticenzymes were calculated as described by Martin etal. (23) and are expressed in units per milligram ofprotein. Protein concentration was measured by themethod of Lowry et al. (21).

Cell-free extracts for the histidase assay wereprepared as described above, except that tris(hydrox-ymethyl)aminomethane-hydrochloride buffer was re-placed by 0.05 M potassium phosphate buffer (pH7.4). Histidase was assayed according to the methodof Magasanik et al. (22). The specific activity is ex-pressed in nanomoles of product formed per minuteper milligram of protein.

Fermentation experiments. The medium usedfor the production of L-histidine contained 10% glu-cose, 2% urea, 0.1% K2HPO4, 0.02% MgSO4 7H20,

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0.7% corn steep liquor, and 3% CaCO3. Glucose was

autoclaved separately and added aseptically to theremaining components of the medium. A loopful ofcells (grown overnight on nutrient agar slants) was

inoculated into 15 ml of the medium in 500-ml flasks.Incubation was carried out at 300C with reciprocalshaking (140 strokes/min, 8-cm stroke).Methods of analysis.Samples of the fermentation

broth were chromatographed on Toyo no. 53 paper

(solvent system, 1-butanol-acetic acid-water [4:1:1]),and the identification of L-histidine was carried outby using Pauly's reagent (1) and ninhydrin. The quan-

titative determination of L-histidine was performedby bioassay with Leuconostoc mesenteroides P-60.Glucose and total sugar were estimated by the meth-ods of Somogyi (31) and Dubois et al. (11), respec-

tively. Growth was estimated by measuring the opticaldensity at 660 nm of fermentation broth diluted with0.1 N HCl to dissolve CaCO3 in the broth and was

expressed as dry cell weight calculated from a standardcurve.

RESULTSEffects of 2MH and TRA on growth. To

select suitable analogs for the isolation of regu-

latory mutants, several structural analogs of L-histidine were examined for their inhibitory ef-fects on the growth of S. marcescens Sr4l. As aresult, 2MH and TRA were selected. A lowconcentration of 2MH (0.05 mM with respectto the L-isomer) inhibited the growth slightly,and complete inhibition was not observed even

at 1 mM (Fig. 1). On the other hand, growthinhibition by TRA was very strong, and com-

plete inhibition was observed at 1 mM (withrespect to the L-isomer).The inhibitory effect of 1 mM 2MH was com-

pletely prevented by 0.5 mM L-histidine, butthe effect of 1 mM TRA was only partiallyprevented by this concentration of L-histidine(Fig. 2). A concentration of L-histidline 20 timeshigher was necessary for complete reversal ofthe inhibition.

0.8-

'A 0. S0

0.3

o

Culture time (hr)FIG. 1. Growth inhibition of S. marcescens Sr4l

by 2MH (A) and TRA (B). Molar concentrations ofthe analogs are expressed with respect to the L-iso-mer.

0.8O-

,, 0.5c

X 0.3

C 0.

3.o a

A L-Histidineadded0.5mM0. I mM

0.05mm

None

2 3 4 5

B L-Histidineadded

0.5

mM

0.1

None

I- I0 1 2 3 4 5

Culture time (hr)FIG. 2. Effect of L-histidine on the growth inhibi-

tion of S. marcescens Sr41 by 2MH (A) and TRA (B).Symbols: (0) medium in the presence of a 1 mMconcentration of each analog; (0) medium in theabsence of both analogs.

Enhancement of 2MH-mediated growthinhibition. Since growth inhibition by 2MHwas rather weak, the selection of resistant mu-tants seemed difficult. Therefore, a means forenhancing growth inhibition was required. Cal-houn and Jensen (9) reported that the decreaseof carbon flow to the biosynthesis of aromaticamino acids by a change of carbon sources en-

hanced the inhibitory effects of the amino acidanalogs on the growth of Pseudomonas aerugi-nosa. Based on their observation, we searchedfor an effective carbon source which would en-

hance growth inhibition by 2MH. By changingthe carbon source from glucose to sorbitol,growth inhibition by 2MH was markedly en-hanced (Table 1). Thus, it was possible to use

2MH as an analog for the selection of resistantmutants. This specific enhancement of growthinhibition by sorbitol may be attributed to lowlevels of intraceliular precursors of L-histidinebiosynthesis, due to the alteration of carbonflow, in the cells grown with sorbitol.

Effect of histidine analogs on phosphori-bosyltransferase activity and on the for-mation of histidine biosynthetic enzymes.To ascertain the main cause ofgrowth inhibitionby 2MH and TRA, the sensitivity of phospho-ribosyltransferase to these analogs was exam-ined. In this experiment, a cell-free extract pre-pared from derepressed cells of strain Sr4l was

used, because the enzyme level in the wild-typestrain was very low. Derepression was effectedby the addition of AMT, which has been knownto be an inhibitor of imidazole glycerol phos-phate dehydratase, the enzyme catalyzing step7 of the histidine pathway (13). The levels ofphosphoribosyltransferase of strain Sr4l grownin the presence ofAMT increased about fivefoldover that of the wild type. Phosphoribosyltrans-ferase of strain Sr4l was inhibited by a low

VOL. 34, 1977 467

A 2MH B TRAadded addedNone p None0.05 0"00mm 5)0m00.5mm

0.5mmIrmM

III ~~~~~~~~~~~~I0 12 34 5 012 34 5

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TABLE 1. Effect of carbon sources on growth inhibition of S. marcescens Sr41 by 2MHGrowth (OD)" at:

Carbon sourcea Supplementb16h 24h 40h 48h

Glucose None 0.56 0.73 0.68 0.652MH 0 0.03 0.71 0.732MH + L-histidine 0.55 0.72 0.68 0.65

Maltose None 0.43 0.68 0.67 0.672MH 0.02 0.04 0.33 0.452MH + L-histidine 0.44 0.70 0.69 0.69

Mannose None 0.26 0.48 0.61 0.702MH 0.01 0.02 0.23 0.342MH + L-histidine 0.29 0.60 0.71 0.71

Sorbitol None 0.23 0.73 0.70 0.662MH 0 0 0.01 0.032MH + L-histidine 0.22 0.70 0.67 0.66

Glycerol None 0.41 0.72 0.70 0.682MH 0 0.03 0.74 0.772MH + L-histidine 0.42 0.70 0.69 0.68

a The carbon source of minimal medium was changed as indicated and added at a concentration of 0.5%.bL-Histidine and 2MH were added at concentrations of 0.1 and 1 mM (with respect to the L-isomer),

respectively.c The inoculum was approximately 2 x 106 cells/ml of a 5-h culture of strain Sr4l grown on glucose-salts

minimal medium, harvested, and diluted with saline. OD, Optical density.

0.1 0.5 5 10L-Histidine or its analogue

added (mM)FIG. 3. Effect of L-histidine and its analogs on the

activity of phosphoribosyltransferase of S. marces-

cens Sr4l. Molar concentrations of histidine analogsare expressed with respect to the L-isomer.

concentration of L-histidine (Fig. 3). The activityof the enzyme was reduced by 50% in the pres-ence of 0.1 mM L-histidine. 2MH and TRA alsoinhibited the enzyme, and 50% inhibition wasobserved at 2 mM 2MH or 5 mM TRA. If theseanalogs act as only false feedback inhibitors, theaddition of each analog would cause a reductionin the intracellular level of L-histidine and wouldprobably lead to derepression of histidine bio-synthetic enzymes. To test this possibility, theeffect of both analogs on the levels of histidinebiosynthetic enzymes, histidinol phosphatephosphatase and histidinol dehydrogenase, wasexamined. The addition of 2MH to minimal

medium increased the enzyme levels, but theaddition of TRA decreased them, as did theaddition of L-histidine (Table 2).

TABLE 2. Effect of histidine analogs on theformation of histidine biosynthetic enzymes in S.

marcescens Sr4lSp act"

Growth condi- Addition to mini-tion mal mediuma Phospha- Dehydro-

tase genase

Normal None 7.8 1.1L-Histidine 5.4 <0.5TRA 5.5 <0.52MH 12.7 2.0

Derepressed None 31.3 7.0with AM1t L-Histidine 15.8 3.5

TRA 18.6 4.42MH 35.4 8.4

a L-Histidine or its analog was added at 1 mM with respectto the L-isomer when the culture had reached a cell densityat 660 nm of approximately 0.3, and the cultivation was carriedout for 2 h.

I Phosphatase, histidinol phosphate phosphatase; dehydro-genase, histidinol dehydrogenase.

10 mM AMT and 1 mM adenine were added.

Since the action of TRA was not clear, anadditional experiment was performed withAMT-mediated, derepressed cells. In this case,TRA clearly repressed the enzymes, as did L-histidine. These results show that growth inhi-bition of S. marcescens by 2MH is due mainlyto false feedback inhibition. Furthermore, theresults also indicate that strong growth inhibi-tion by TRA is due to both false feedback inhi-bition and repression.Degradation of L-histidine and isolation

of a histidase-less mutant. In addition to therelease from regulatory mechanisms of biosyn-thesis, the lack of histidine-degrading activitywas considered to be indispensable in an L-his-

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tidine producer. Therefore, L-histidine degrada-tion by strain Sr4l was examined. Strain Sr4lutilized L-histidine as the sole source of carbonand/or nitrogen for growth (Table 3). The strainrapidly degraded L-histidine added to the fer-mentative medium (Table 4), and the accumu-

lation of urocanic acid and L-glutamic acid was

observed at an early stage of the cultivation.Moreover, histidase activity was detected in thecell-free extract (Table 5). These results indicatethat histidase is involved in L-histidine degra-dation. Accordingly, the removal of histidaseactivity was considered to be necessary for theconstruction of an L-histidine producer.

In a histidase-less mutant, strain Hd-16, his-tidase activity was not detected in spite of theaddition of either L-histidine or urocanic acid(Table 5). This strain could not utilize L-histi-dine as a source of carbon and/or nitrogen (Ta-ble 3) and did not degrade the amino acid inthe fermentative medium (Table 4). Apparently,strain Hd-16 completely lacked histidine-de-grading activity, and it seemed to be a suitableparent strain for isolating histidine regulatorymutants.Isolation and properties of mutants re-

sistant to 2MH or TRA. Mutants resistant to2MH or TRA were isolated from strain Hd-16,and they were tested for the ability to produceL-histidine. 2MH-resistant mutants representedby strain HdMHr581 produced only smallamounts of L-histidine (Table 6). On the otherhand, TRA-resistant mutants were divided intotwo types according to their histidine productiv-ity. Strains HdTr23 and HdTr51 produced about8 mg of L-histidine per ml, but strain HdTrl42produced only a small amount of the amino acid.To determine whether there were alterations

in the regulation of histidine biosynthesis inthese mutants, the levels of three enzymes in-volved in the pathway were examined (Table6). Strain HdMHr581 had the same enzyme

levels as strains Sr4l and Hd-16. On the otherhand, TRA-resistant mutant strains HdTr23,HdTr5l, and HdTrl42 had considerably in-creased enzyme levels. Also, the sensitivity ofphosphoribosyltransferase to L-histidine in thesemutants was examined. Since the 2MH-resistantmutant HdMHr581 possessed a low level of theenzyme, its cell-free extract was prepared fromAMT-mediated, derepressed cells. The enzymein strain HdMHr581 was insensitive to 10 mML-histidine. In strains HdTr23 and HdTr5l, theenzyme was not inhibited by 1 mM L-histidinebut was inhibited weakly by 10 mM. On theother hand, in strain HdTr142 the enzyme wasinhibited strongly by 1 mM L-histidine, as inthe wild type.

TABLE 4. Degradation of L-histidine infermentative medium by strains of S. marcescens

Residual L-histidineStanGrowth (mg/mi, dry wt) (Mg/ml)a

24h 48h 72h 24h 48h 72h

Sr4l 16.8 21.3 17.8 1.6 0.2 0Hd-16 16.0 22.4 18.0 9.6 9.4 9.6

a L-Histidine was added at a concentration of 10 mg/ml.

TABLE 5. Histidine-degrading enzyme in strains ofS. marcescens

Strain Addition to minimal me- Histidase spdiuma act

Sr41 None NDbL-Histidine 366Urocanic acid 338

Hd-16 None NDL-Histidine NDUrocanic acid ND

a The carbon source of minimal medium waschanged to 1.0% sodium succinate to avoid cataboliterepression caused by glucose (8; Kisumi et al., unpub-lished observation). L-Histidine or urocanic acid wasadded at a concentration of 0.2%.'ND, Not detectable.

TABLE 3. Utilization of L-histidine as a source of carbon or nitrogen by strains of S. marcescens

Growth conditiona Growth (OD) b

StrainCarbon source Nitrogen source 16 h' 20 h 24 h

Sr41 Glucose (NH4)2SO4 0.53 0.63 0.61Glucose L-Histidine 0.59 0.76 0.78L-Histidine (NH4)2SO4 0.08 0.18 0.43L-Histidine L-Histidine 0.15 0.33 0.67

Hd-16 Glucose (NH4)2SO4 0.48 0.64 0.68Glucose L-Histidine 0 0 0L-Histidine (NH4)2SO4 0 0 0L-Histidine L-Histidine 0 0 0

a Carbon and nitrogen sources of minimal medium were changed as indicated and added at a concentrationof 0.2%.

b The inoculum was approximately 2 x 106 cells/ml. OD, Optical density.c Length of incubation.

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TABLE 6. Accumulation of L-histidine and lack offeedback controls in mutants resistant to 2MH or TRAL-Histidine accumu- S Inhibition of transferase (%) by

lated (mg/ml) p acta L-histidinebStrain

48 h 72 h Transferase Phospha- Dehydro- 0.1 mM 1 mM 10 mMtase genase

Sr41 0 0 2.0 7.8 1.1 50 92 100Hd-16 0 0 2.0 7.3 0.9 52 95 100HdMHr581 0.5 0.8 <1.0 8.2 1.6 0 0 0HdTr23 5.2 7.8 27.2 56.6 12.1 0 0 23HdTr51 5.8 7.6 26.4 57.8 11.1 0 0 17HdTrl42 0.9 1.3 29.2 67.3 12.4 45 88 94a Transferase, Phosphoribosyltransferase.b In strains Sr4l, Hd-16, and HdMHr581, the inhibition was determined with AMT-mediated, derepressed

cells.

The results described above suggest that a

mutation to 2MH resistance eliminates feedbackinhibition and a mutation of TRA resistanceeliminates feedback inhibition and/orrepression. Moreover, the results indicate thatthe defect in both feedback inhibition andrepression is essential for L-histidine productionby S. marcescens.Transductional crosses between analog-

resistant mutants and properties of thetransductants. To obtain strains with higherhistidine productivities than that of strainHdTr23 or HdTr51, a large number of TRA-resistant mutants were isolated. However, noimproved strains could be obtained. Therefore,we attempted to construct strains possessinggenetic alterations in both the 2MH- and TRA-resistant mutants, using PS20-mediated trans-duction.

Since TRA-resistant mutants showed a strongcross-resistance to 2MH, a 2MH-resistant mu-tant served as the recipient and phage lysatesof TRA-resistant mutants served as donors inall transductions. Three kinds of crosses wereperformed with TRA resistance as the selectedmarker. Results of these crosses are presentedin Table 7. TRA-resistant colonies that ap-peared on the selective plates were picked astransductants and purified by single-colony iso-lation on the same medium as the selective plate.The transductants obtained in the experi-

ments of Table 7 were examined for their prop-erties. Most transductants ofthe crosses HdTr23x HdMHr581 and HdTrl42 x HdMHr581 (rep-resented by strains HT-2253 and HT-2604, re-spectively) produced more than 12 mg of L-his-tidine per ml (Table 8). All had markedly in-creased enzyme levels of histidine biosynthesisand a phosphoribosyltransferase that was insen-sitive to L-histidine. On the other hand, all trans-ductants ofthe cross HdTr51 x HdMHr581 (rep-resented by strain HT-2429) produced about 8mg of L-histidine per ml. Although they had

TABLE 7. Transductional crosses between TRA-resistant mutants and a 2MH-resistant mutant with

phage PS20ONo. of

No. of phage- TRA-re-Donor Recipient MOjb infected cells sistant col-

spread/plate oniesfound/plate

HdTr23 HdMHr581 150 5.7 x 107 32HdTr51 HdMHr581 150 7.5 x 107 35HdTrl42 HdMHr581 180 8.3 x 107 43

a TRA resistance was used as the selected marker.bMOI, Multiplicity ofinfection, as estimated by the number

of plaque-forming units of the phage lysate per recipient cell.

increased enzyme levels, their phosphoribosyl-transferase was as sensitive to 10mM L-histidineas that of the donor strain HdTr51. Thus, theyhad only the genetic alteration of the donorstrain.

In transductants of the crosses HdTr23 xHdMHr581 and HdTrl42 x HdMHr581, the lev-els of phosphoribosyltransferase were extremelylow compared with those in transductants ofthe cross HdTr51 x HdMHr581 (Table 8). Thisdifference may be due to the instability of theenzyme in the recipient HdMHr581 as a resultof desensitization, since the difference was notas extreme when cell-free extracts were usedwithout dialysis, and no such difference wasobserved in histidinol phosphate phosphataseand histidinol dehydrogenase.L-Histidine production. Investigations were

made to improve the cultural conditions for L-histidine production by strain HT-2604. As aresult, a medium containing 7% glucose, 10%dextrin (Matsutani Chemical Co., Ltd., no. 3),2% urea, 0.1% K2HPO4, 0.05% MgSO4- 7H20,0.7% corn steep liquor, and 3% CaCO3 was foundto be most favorable for L-histidine production.Typical changes by strain HT-2604 are shownin Fig. 4. L-Histidine production paralleledgrowth and reached a maximum production of16.7 mg of L-histidine per ml at 96 h. As by-

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TABLE 8. Accumulation of L-histidine and lack offeedback controls in transductantsL-Histidine accumu- Sp act Inhibition of transferase (%) by

lated (mg/ml) L-histidineTransductant (cross)

48 h 72 h Transferase Phospha- Dehydro- 0.1 mM 1 mM 10 mMtase genase

HT-2253 (HdTr23 xHdMHr581) 7.9 12.1 1.7 54.5 12.1 0 0 0

HT-2429 (HdTr51 xHdMHr581) 6.0 8.3 30.0 57.9 12.4 0 0 19

HT-2604 (HdTrl42 xHdMHr581) 6.1 12.9 3.1 59.2 14.7 0 0 0

=200 20 7

E E5gCP 501l

E

0 24 4872 620 OM100- .10 E

0~~~~~~~1

:5~~~~~~~~~

+_0

0 24 48 72 96

Culture time (hr)FIG. 4. Changes during L-histidine production by

strain HT-2604. Symbols: (0) L-histidine; (0) growth(dry cell weight); (V) pH; (A) total sugar; (A) glucose.

products, trace amounts of L-valine, L-leucine,and L-glutamic acid were detected.

DISCUSSIONAs stated above, we succeeded in the construc-

tion of histidine producers by using transduc-tional crosses between analog-resistant mutantsof S. marcescens Sr4l.

In 2MH-resistant mutants of S. marcescens

Sr4l, the first enzyme of the histidine biosyn-thesis was insensitive to feedback inhibition, butthe levels of histidine biosynthetic enzymes werealmost equal to that of the wild-type strain. Thistype of regulatory mutant was reported in 2-thiazole alanine-resistant mutants of S. typhi-murium. In these S. typhimurium mutants, a

defect in the first enzyme rendered it insensitiveto inhibition by L-histidine, and the mutationwas mapped in the hisG gene, the structuralgene for the first enzyme (30). From this fact,the mutation responsible for 2MH resistance inS. marcescens Sr4l is assumed to lie in thestructural gene of the first enzyme.On the other hand, genetic analysis of TRA-

resistant, derepressed mutants of S. typhimu-rium has revealed that the mutations are locatedin six different regions. One of the genes, hisO,

is closely linked to the nine histidine structuralgenes and has the properties of an operator-promoter region. None of the other five genesare linked to the histidine operon (6, 7, 12, 29).TRA-resistant mutants of S. marcescens Sr41were also found to be derepressed mutants.Among them, mutants possessing a partiallyfeedback-insensitive first enzyme were found. Areasonable explanation for this type of mutantmight be that it carries at least two separateregulatory mutations; presumably, one is locatedin the structural gene of the first enzyme andthe other is located in a certain regulatory gene.

In our attempt to construct histidine pro-ducers, three kinds of crosses were performedbetween three TRA-resistant mutants (as do-nors) and a 2MH-resistant mutant (as recipient).TRA-resistant transductants of the crossesHdTr23 x HdMHr581 and HdTrl42 xHdMHr581 had genetic alterations in both thecorresponding donor and recipient strains (Ta-ble 8). This suggests that in strains HdTr23 andHdTrl42, the mutation(s) causing derepressionis unlinked to the structural gene of the firstenzyme. On the other hand, as far as we coulddetermine, all the transductants of the crossHdTr51 x HdMHr581 had only a genetic alter-ation of the donor strain. This indicates that instrain HdTr51 the mutation causing derepres-sion may be very closely linked to the structuralgene of the first enzyme. Therefore, this muta-tion in strain HdTr51 may be located in thehistidine operon, presumably in the operator-promoter region, assuming the arrangement ofhistidine genes in S. marcescens to be very sim-ilar to that in S. typhimurium. Thus, it seemslikely that in S. marcescens Sr4l, more thantwo genes are responsible for the repressionprocess of histidine biosynthesis as in S. typhi-murium.From the above considerations, we can con-

clude that the following reasons made it possibleto construct the histidine producers: (i) the iso-lation of two kinds of regulatory mutants usingtwo histidine analogs with different antagonistic

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472 KISUMI ET AL.

actions, and (ii) the absence of linkage betweenthese two regulatory mutations with PS20-me-diated transduction.Based on the results obtained in the present

study, it is anticipated that histidine producerscan be modified to urocanic acid producers byrestoring histidase activity and removing uro-canase activity. Such urocanic acid producerscan be readily constructed by introducing histi-dine productivity of the histidine producers intoa urocanase-less mutant by transduction. More-over, the transductional procedure will facilitatethe construction of various mutants of S. mar-cescens Sr4l, which would produce a variety ofamino acids and other valuable metabolites (19;M. Kisumi, T. Takagi, and I. Chibata, Abstr.Symp. Amino Acid Nucleic Acid, 25th, Tokyo,Japan, p. 8-9, 1976).

ACKNOWLEDGMENTSWe are grateful to T. Takayanagi, former Senior Manager

of Research and Development Division, Tanabe Seiyaku Co.We also thank F. Murakami for technical assistance.

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