Possible Regulation of the Salmonella typhimurium Histidine ...

JOURNAL OF BACTERIOLOGY, Oct. 1980, p. 337-345 0021-9193/80/10-0337/09$02.00/0

Vol. 144, No. 1

Possible Regulation of the Salmonella typhimurium Histidine Operon by Adenosine Triphosphate Phosphoribosyltransferase: Large Metabolic Effects ROBERT K. GOITEIN AND STANLEY M. PARSONS* Department of Chemistry, University of California, Santa Barbara, California 93106

An effort to find growth conditions leading to conditional regulation of the histidine operon of Salmonella typhimurium by the allosteric first enzyme of the pathway, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), is reported. A strain deleting the enzyme, TR3343, behaved simply and predictably under all growth conditions, whereas histidine auxotrophs containing active enzyme behaved in complicated ways dependent upon the location of the histidine pathway lesion. hisE strains derepressed the operon only one-half as much as TR3343 when grown on limiting histidine and a poor carbon source, but they also grew more slowly, probably as a result of high N'-(5-phospho-,f-D-ribosyl)adenosine triphosphate levels in the cell. hisC strains exhibited oscillatory growth behavior and oscillatory histidine operon expression when grown on intermediate concentrations of the histidine precursor histidinol. This behavior probably was caused by synergistic in-phase variations in the histidine, purine nucleotide, and ppGpp pools of the cell. All of the growth and histidine operon expression effects associated with the presence of adenosine triphosphate phosphoribosyltransferase could be assigned to metabolic perturbation of the cell caused by unregulated enzymatic activity.

The biosynthesis of histidine in Salmonella typhimurium is catalyzed by 10 enzymes encoded by the histidine operon (10, 17). The pathway and operor are summarized in Fig. 1. ATP phosphoribosyinyansferase (EC 2.4.2.17), the first enzyme of the histidine pathway, has been implicated in the regulation of histidine operon expression, by both in vivo and in vitro experiments (5, 6, 8, 13, 22-24, 28). However, Scott et al. (32) showed that ATP phosphoribosyltransferase is not the primary regulatory agent of the histidine operon, in agreement with earlier inference (18). Rather, a regulatory model incorporating translation of a histidine-rich leader peptide interacting with transcription termination at an attenuator site recently has been formulated (2, 3, 9, 19, 21, 25, 34, 36, 37). Since a possible role for ATP phosphoribosyltransferase in gene regulation is restricted to an auxiliary function, it seemed probable that enzyme-specific regulation would be observed only under certain conditions or only transitorily after a shift in growth conditions. Thus, to clarify the possible role of ATP phosphoribosyltransferase in regulation of histidine operon expression, histidine auxotrophs containing either all of the structural gene for ATP phosphoribosyltransferase or a deletion of most of this gene were subjected to a wide variety of growth conditions in paired studies. The occurrence of interesting

large metabolic effects dependent on active ATP phosphoribosyltransferase is reported. MATERIALS AND METHODS Abbreviations used are as follows: VBC medium, Vogel-Bonner citrate medium; ppGpp, guanosine5'-diphosphate-3'-diphosphate; TA, thiazolalanine; PRibPP, 5-phospho-a-D-ribose-l-diphosphate; PRibATP, N'-(5'-phospho-,8-D-ribosyl)ATP; PRibAMP, N'-(5'-phospho-,8-D-ribosyl)AMP; AICAR, 5-aminoimidazole-4-carboxamide-1-ribonucleotide; Hol, L-histidinol. Bacterial strains used and their sources are listed in Table 1. Millipore filters were from Millipore Corp. Triethanolamine was from Mallinckrodt Chemical Works. Dowex-3 and ammonium molybdate were from J. T. Baker Chemical Co. L-Histidinol phosphate, Lhistidine, 2-thiazole-DL-alanine, and DL-norvaline were from Sigma Chemical Co. All other compounds were from usual commercial sources. The VBC medium referred to in the text is the E medium of Vogel and Bonner (35). The succinate growth medium is that of Meiss (27). Growth media generally were supplemented with small volumes of separately autoclaved concentrated stock solutions unless stated otherwise. Rich amino acid medium was prepared by making VBC medium 2 mM in the following 19 L-amino acids added as solids: Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asp, Asn, Glu, Gln, Lys, and Arg. The solution was adjusted to pH 7.0 with KOH, autoclaved, and supplemented as above. 337

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J. BACTERIOL.

GOITEIN AND PARSONS

HISTIDINE BIOSYNTHETIC PATHWAY PRIbATP PRIbAMP3BM-II pyrohopho-

ATP-

ATP

phorphoribosyltransferose

T+

r

pp

pp

Mg2+

I

J

cyclohydrolose

hydrolase

0

0

I~~~~~~~~~~~~~~~~~ HNb H N Niv HH OH

i3' c

PRibPP

OH

H2O0

1-

unknown

[]

5-(5'-phoephoribuloelaminoformimino)1-(5-pho plboeyl)-

5-(5'-phophorlbo-

PRibAMP

N' HNC.d

H2Z

OH OH

.ybminoformimino)ipWborlyl)-

Imidazoe4-carboxamide

imidole-

4-carboramde

(BBM -M)_ BBM-IE)_ ~~~~~~~~~~~(

_

_

Histidinol dehydrogenas

HP

IAP

phosphatase

transominase

NON-RP dehydrase H2N,C'C

I~x-

PRIbATP

__ , IG P

J.eo. H Hp N ~~~~~~~~HNy

N

H OH aOH OH

N$*N-RP Glutomine

NP^N-RP

N9'N-RP

N^N-RPPP Mg2+

I ! SOH

Isomerase

NH2

5-'mInolmldazole

4-carboxamide 4- rlbonucleotide (AICAR) +

Glutamate

-SH, Mn2+

%. N

HON

l$-CH

P D-er,tro

imidazole

2

_____

rt

N

0

N

HzN

OH

HN N

H2N CH2 HO 0

P

imidazole acetol

L-hlstidinol

phosphate

phosphate

phosphate

(IGP)

(IAP)

(HP)

glycerol

2NAD+

HN

N

L-histidinol

L-histidine

HISTIDINE OPERON

GIDIC IBIH_IFII E IT TIT TI-

ppGpp

promoter

leader

peptide

attenuator

FIG. 1. Histidine biosynthetic pathway and the histidine operon. The histidine biosynthetic pathway is drawn at the top. The name of the enzyme and its corresponding circled gene designation are given for each step of the pathway as well as the name and abbreviation (in parentheses) for each of the intermediates of the pathway. At the bottom is a genetic representation of the histidine operon. Preceding the hisG gene is the regulatory region of the operon. AICAR produced by the product of hisE gene is an intermediate in purine biosynthesis, and accordingly is shown as being recycled to give the ATP used in the first step. The following procedure was employed during the nutrient shift experiments. A 1-ml volume of a nutrient broth culture arising from a single colony was added to 20 ml of VBC medium containing 0.1 mM histidine and 0.25% glucose. This culture was grown for 12 h at 370C with shaking. A 1-ml volume of this culture was added to 100 ml of VBC medium containing 0.1 mM histidine and 0.25% glucose and incubated at 37°C with shaking. After 4 h of growth, the absorbance of the culture at 650 nm was determined on a Gilford

2000 spectrophotometer. A portion of this culture was added to 100 ml of an adaptive growth solution which was identical to that employed in the actual experimental growth. The volume added was determined by the density of the 4-h culture and the anticipated rate of growth so as to result in a cell density of about 1.0 absorbance units at 650 nm after 12 or 20 h of adaptive growth. A portion of the adaptive growth culture then was added to 100 ml of the experimental growth solution so that the initial cell density corresponded to

REGULATION OF S. TYPHIMURIUM HISTIDINE OPERON

VOL. 144, 1980

TABLE 1. Salmonella typhimurium LT2 strains used Genotype Origin/source (reference) B. N. Ames (32) Ahis_8476a dhuAlb (TR3343) B. N. Ames (14) hisC115c B. N. Ames (14) hisC120c J. R. Roth (14) hisElld J. R. Roth (14) hisE164e J. R. Roth (14) Ahis-640f B. N. Ames (14) hisG46e hi8G52e B. N. Ames (14) B. N. Ames (14) hisG70 B. N. Ames (14) hisA30e a Nonpolar internal deletion of most of hisG. b Allows growth on D-histidine (1). c Low reversion frequency missense mutation. d Nonpolar leaky missense mutation. eNonpolar complete missense mutation. f Deletes hisI and hisE. an absorbance of 0.1 at 650 nm. The experimental growth was conducted at 370C with shaking for 6 to 24 h. The experimental growth was monitored by removing 3-ml portions periodically, adding sufficient histidine to make it 1 mM, and placing the sample on ice. Absorbance at 650 nm was determined immediately. The samples were stored on ice up to 20 h before the determination of histidinol phosphate phosphatase activity. A nutrient shift growth and a control growth not involving a change of conditions were simultaneously conducted for all strains. TR3343 and a strain expressing hisG were subjected to identical growth conditions simultaneously, for a total of four cultures in one experiment. A nutrient was removed from the culture (downshift) by centrifugation at 2,500 x g for 10 min with resuspension of the bacteria or by depletion caused by cell growth in the case of histidine. To determine the level of expression of the histidine operon, the activity of histidinol phosphate phosphatase (EC 3.1.3.15) in washed toluenized cells was determined similarly to the method of Ely (11). The assay measures phosphate liberated from histidinol phosphate using a Fiske-Subbarow type technique. Histidinol phosphate in water was pretreated with Dowex-3 at pH 4.0 to remove contaminating phosphate. Absorbance of the ammonium phosphomolybdate complex was determined at 820 nm on a Gilford 2000 spectrophotometer with a red filter. Units of specific activity are given in A820 phosphate per 15 mi

per A6ew cells.

RESULTS

Uptake of Hol is rate limiting for growth of S. typhimurium dependent on exogenous Hol. Thus, growth of histidine auxotrophs on exogenous Hol results in slow growth and depression of the histidine operon. Since expression of all of the enzymes of histidine biosynthesis is coordinate (6, 13), determination of the hisB enzyme activity, histidinol phosphate phosphatase, provides

a

convenient

measure

of relative operon

339

expression. Figure 1 summarizes the pathway and operon structure. When the hisG deletion strain TR3343 was grown under histidine upshift and downshift conditions in VBC medium plus 0.25% glucose, histidine operon expression began repressing or derepressing, respectively, immediately after the shift (not shown). Qualitatively similar results were found when the histidine auxotrophs hisEll, hisE164, and Ahis640 were subjected to the same experimental conditions. Thus, no evidence for a kinetic role of ATP phosphoribosyltransferase in "efficient restoration of repression" (2) was found. A survey was conducted in which strains TR3343 and hisE164 were subjected to histidine downshifts under various growth conditions (Table 2). Growth rates and histidine operon expression were similar before and after the shift for both strains growing under very poor or otherwise rich conditions (experiments 1 and 2). However, for three growth conditions, strains TR3343 and hisE164 responded to histidine downshift differently. Growth on glucose or citrate as the carbon source or under purine-limiting conditions gave up to threefold slower growth rates for the hisE164 strain after the downshift (experiments 3, 4, and 6). The growth rate of hisE164 was increasingly slower at longer times after the downshift (experiment 3). In the case of growth on citrate, histidine operon expression was significantly lower in hisE164 compared to that of TR3343 (experiment 4). The effect of the Hol concentration during growth on citrate was investigated more thoroughly. The behavior of strain hisE164 differed from that of TR3343 at low Hol concentrations, whereas its behavior at high concentrations became identical to that of TR3343 (Fig. 2). When the two strains were grown in the presence of TA, the hisE164 strain behaved more like the TR3343 strain (Table 2, experiment 5). TA hinds to ATP phosphoribosyltransferase at the histidine site and inhibits its catalytic activity (10). TA had no effect on the growth rate or operon expression of TR3343 under repressing or derepressing conditions, suggesting that its only target in the ceil indeed is ATP phosphoribosyltransferase. Thus, the quite slow growth rate of hisE164 on Hol is consistent with an effect arising from the ATP phosphoribosyltransferase enzymatic activity. Similar results were obtained from hisEll. Since the hisE strains exhibited complicated postshift growth behavior on Hol, several strains carrying mutations in other genes of the pathway were surveyed in an effort to find a better control which contained ATP phosphoribosyltransferase and which would exhibit "normal"

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J. BACTERIOL.

growth and histidine operon control that could zymatically inactive hisG strains grew as well as be compared with those of TR3343. Table 3 TR3343 (experiments 6 and 7). Finally, hisC115 summarizes some results for growth patterns exhibited very unusual and dramatic oscillatory after adaptation to growth on Hol for 20 h. Of growth involving rapid growth alternating with note first is that hisEll grew as well as TR3343 slow growth (experiment 8). on Hol after adaptation in contrast to its poor hisC120 behaved similarly (Fig. 3A). At the growth rate several hours after a histidine down- beginning of the experiment, which did not inshift (experiments 1 and 2). Thus, the hisE volve a change in growth conditions, the hWCl2O strains apparently could adjust their metabolism strain was growing quite rapidly with an approxafter a lag period to overcome the "excess" imate doubling time of only 60 min. After 1 h growth limitation occurring during an interme- the growth rate spontaneously slowed to a doudiate period after histidine downshift. The rela- bling time of about 170 min. After about 3 h tively long time required for this adjustment, more the growth rate spontaneously increased which was some time longer than the 9 h moni- to a doubling time of about 60 min again. This tored in Table 2, suggests that a gene level was qualitatively reproducible behavior, and in response might be required. Second, hisA30 some cases the oscillatory growth rate behavior was even more extreme than shown in Fig. 3A, grew slowly even after adaptation (Table 3, experiment 3). The growth of hisA30 was stimu- with one experiment exhibiting doubling times lated appreciably by TA (experiment 4) and of 85 and 900 mip (essentially no growth for 5 slightly by adenine (experiment 5). Third, en- h)! This behavior should be compared to the TABLE 2. Effects of carbon, general amino acid, or purine nutritional states on histidine downshift time Doubling Doublngtime (min) Expt

Nutrient conditiona

Strain

TR3343

hisE164 J TR3343 2

hisE164 TR3343

hisE164 J

TR3343

hisE164 J TR3343 hisE164 J

Succinate +0.1 mM His

-*

succinate

+0.1 mM Hol VBC +0.25% Glc + 2mM 20AA VBC + 0.25% Glc + 2 mM 19AA +0.1 mM

Hol VBC + 0.25% Glc + 0.1 mM His VBC + 0.25% Glc + 0.1 mM Hol

VBC+0.1mMHis-.*VBC+0.1mM Hol VBC + 0.1 mM His VBC + 0.1 mM Hol + 0.1 mM TA

Histidine opexpressio b

eron

sOn Pre- Postshift shift

Postshift dtri

nation (h)c

Preshift

Postshift

450

450

1.5

9.0

7

450

450

1.5

9.0

7

30

750

0.1

10.0

7

30

960

0.1

11.5

7

50

135.

2.5

15.0

7

50

235 460 460

2.5 15.0 13.0

7 9

100

180

2.0

16.5

7

90

450

2.0

10.0

7

100

180

2.0 16.0

90

250

2.0

16.5

7

7

5 130 2.5 16.0 VBC + 0.25% Glc + 0.1 mM His + 20 80 mMATd -VBC + 0.25% Glc + 0.1 5 mM Hol + 20mM AT 2.5 14.0 90 410 hisE164J a Cultures were grown in the preshift medium for 12 to 20 h to no more than 1.8 absorbance. Portions of these cultures were then transferred to the postahift medium. Growth was monitored for at least 7 h after the shift. All cultures achieved linear growth by at least 3 h subsequent to the shift except hisE164 in experiment 3. Abbreviations not previously identified: His, histidine; Glc, D-glucose; 20AA, the 20 common amino acids; 19AA, the 19 common amino acids except histidine; AT, aminotriazole. b Histidinol phosphate phosphatase specific activity. 'The doubling time and histidine operon expression were determined at the indicated time after the shift in medium. d AT inhibits endogenous purine biosynthesis (16). TR3343

6

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.Growth

REGULATION OF S. TYPHIMURIUM HISTIDINE OPERON

0

a;EE ._

.0

0 a

341

0 0)

-.

0 0

-C

0c a.

HISTIDINOL, mM

FIG. 2. Growth behavior and histidine operon expressionof TR3343 and hisEl64 on a poor carbon source as a function of Hol concentration. Doubling times (-0-@-) and histidinol phosphate phosphatase levels (-0-U-) for strains TR3343 (-0-) and hisE164 (-U-) are plotted for growth on VBC medium supplemented with different concentrations of histidinol. Cultures were grown for 12 h on VBC medium containing 0.1 mM histidine, centrifuged, and resuspended in VBC medium, and a portion was added to VBC medium containing Hol. Data was determined 5 h after the shift to Hol. TABLE 3. Growth behaviors of adapted histidine auxotrophsa Expt

Strain

1 2 3 4 5

TR3343 hisEll hisA30 hisA30 hisA30

Addition(s) .

pattern

Linear Linear Linear 0.1 mM TA Linear 1 mM adenine Linear + 0.1 mM thiamineb

Doubling time (min) 125 100 420 190 340

hisG46 Linear 125 hisG52 Linear 115 hisC115 Oscillatory 60,170 a Strains were grown on VBC medium containing 0.25% glucose and 0.1 mM histidinol for 20 h and then transferred to fresh medium. Growth was monitored for at least 7 h. b Thiamine is required by adenine-supplemented cultures (7). 6 7 8

growth of TR3343 which was linear under the same conditions (Fig. 3A). The hisC120 strain alternately grew faster and slower than the hisG deletion strain. Furthermore, the rapid growth phase of hisC120 had a doubling time nearly as short as that of either strain growing on histidine, 50 min, even though growth was on Hol. The apparently "abnormal" oscillatory growth of hisC120 did not result in a long-term growth rate disadvantage relative to TR3343 since hisC120 actually overtook TR3343 in culture density in this experiment.

Figure 3B shows that histidine operon expression also was oscillatory in hisC120. Operon expression was significantly repressed during the two periods of fast growth, whereas it became more derepressed during the period of slow growth. The above results are consistent with the presence of excess intracellular histidine during the bursts of near maximal growth rate, and with limitation of the growth rate during the slow phase by depletion of this internal pool of histidine, at the same time some other factor slowed the growth rate below that of TR3343. The oscillatory growth behavior of hisC120 was dependent upon the Hol concentration. Linear growth was observed at Hol concentrations both lower and higher than 0.05 to 0.10 mM Hol (Table 4). At high Hol concentrations the growth rate increased to the same as that in the presence of exogenous histidine (experiments 1, 5, and 6) similarly to what occurred with hisE164 at high Hol concentrations. At low Hol concentration the growth rate was similar to that of other well behaved strains (Table 4, experiment 2; Table 3, experiments 1, 2, 6, and 7). Thus, internediate availability of exogenous Hol was a critical determinant ofthe oscillatory behavior, and, at extremes of exogenous Hol concentration, hisC120 behaved normally. When hisC120 was grown under oscillatory conditions in the presence of TA, linear growth with a normal doubling time resulted (Table 4, experiment 7). Thus, oscillation probably was

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GOITEIN AND PARSONS

J. BACTERIOL. B

E

C

0) U) 0

0 to) (0

-0

4C

0. CL U)

0

00.01 L

V

I

I

I

a

I

I

I

a ARA7 u a I

Qv

In0v

Time, Hours

0

1

2

3

Time,

4

5

6

7

Hours

FIG. 3. Growth behavior and histidine operon expression of TR3343 and hisC120 on internediate Hol. Both strains were grown for 20 h on VBC medium containing 0.25% glucose and 0.1 mM Hol before transfer to fresh medium. (A) Cell densities are plotted. Doubling times of hisCI20 (.4-) for the initial rapid growth, intermediate slower growth, and final rapid growth were 60, 170, and 60 min, respectively. The duration of the slower phase was 170 min. The doubling time for TR3343 (-0-) was 135 min. (B) Histidinol phosphate phosphatase specific activity for hisCI20 (-@-). The specific activity for TR3343 was constant at 17.

sulted (experiment 8). Since adenine supplementation lowers PRibPP levels (31), this result indicates that the oscillatory growth pattern did Dounot arise from PRibPP depletion. Thus, the bling Growth Expt Growth conditionsb pattern time oscillatory growth behavior of hisC mutant (min) strains can be made linear either by preventing 1 0.1 mM His Linear 50 drain of the purine nucleotide pool through in2 0.015 mM Hol Linear 120 hibition of ATP phosphoribosyltransferase or by 3 0.05mM Hol Nonlinear, simflar 55,155 compensating for the drain. to Fig. 3A 4 0.1 mM Hol Nonlinear, Fig. 3A 60,170 Inhibition of ATP phosphoribosyltransferase 5 0.5 mM Hol Linear 55 also was achieved using another approach. 6 1 mM Hol Linear 50 was grown under oscillatory conditions, hisC120 7 0.1 mM Hol + 0.1 Linear 100 and norvaline was added in the early part of the mM TA 8 0.1 mM Hol + 0.1 Linear 100 slow growth phase (Fig. 4). Norvaline causes mM adenine + starvation for isoleucine and valine by repressing 0.01 mM thiaacetohydroxy acid synthetase (17). A concentrarninec tion of norvaline was used which caused only All strains were grown on the indicated medium for 12 to slight inhibition of growth of TR3343 (Fig. 4). 20 h and then transferred to fresh medium. Growth was then However, with hisC120, the addition of norvamonitored for at least 7 h. bAll were grown on VBC medium containing 0.25% glucose line actually stimulated growth by inducing onplus the supplements listed. His, Histidine. set of the rapid phase of growth about 3 h sooner 'Thiamine is required by adenine-supplemented cultures than it otherwise occurred. This behavior is (7). readily understood as resulting from synergistic inhibition of ATP phosphoribosyltansferase in vivo by ppGpp and histidine (30). Addition of. coupled to ATP phosphoribosyltransferase cat- norvaline would cause an increase in intracellualytic activity. One possible mechanism for this lar ppGpp levels due to isoleucine and valine effect would involve oscillatory drain of the in- starvation. Buildup of intracellular levels of histracellular purine nucleotide pool caused by the tidine during the slow phase of growth, which ATP phosphoribosyltransferase reaction. In- must occur before the rapid phase of growth can deed, when hisC120 was grown under oscillatory occur, would lead to inhibition of ATP phosphoconditions in the presence of adenine, linear ribosyltransferase sooner in the presence of ingrowth with a normal doubling time again re- creased ppGpp levels. TABLE 4. Effect of histidine nutritional state, TA, and adenine on growth behavior of hisC120a

VOL. 144, 1980

REGULATION OF S. TYPHIMURIUM HISTIDINE OPERON

0

U,C.) E c

0

CD

-f

Time, Hours FIG. 4. Effect of norvaline on oscillatory growth behavior of hisC120. hisC120 was grown for 20 h on VBC medium containing 0.25% glucose and 0.1 mM Hol. Two portions were transferred to fresh media at time 0. The cell density represented by absorbance at 650 nm is shown versus time. One culture was made 0.4 mM in norvaline (-0-) at the arrow. TR3343 was treated similarly.

DISCUSSION Since a role by ATP phosphoribosyltransferase in histidine operon regulation has been restricted to an auxiliary one, we sought in this study to find growth conditions under which conditional regulation might be expressed. The general hypothesis was that a moderately low intracellular histidine level insufficient to support maximal growth rate could be sufficient under slow growth conditions limited by some other factor. ATP phosphoribosyltransferase, in fact, possesses such an integrative capability for control of histidine biosynthesis at the metabolite level. High AMP levels, a signal of low energy charge, and high ppGpp levels, a signal of amino acid (26) and other limitations (10, 12), act synergistically with histidine to inhibit ATP phosphoribosyltransferase enzymatic activity (29, 30). Extension of this behavior to the gene level suggests that ATP phosphoribosyltransferase bound to histidine and AMP or to histidine and ppGpp might act negatively to decrease the histidine threshold for "derepression" of the operon so that derepression would not occur as readily if AMP or ppGpp levels were high. This role for the enzyme might be evident only under condi-

343

tions of moderate histidine limitation, since extreme histidine limitation would eliminate the enzyme-6istidine-AMP or enzyme-histidineppGpp complexes and excess histidine might result in full effectiveness of the primary attenuator mechanism which could not be further augmented by the enzyme. Histidine auxotrophs containing intact hisG and blocked in different parts of the histidine biosynthetic pathway were examined in an effort to find a suitable control strain presumably expressing normal operon regulation. The behavior of the hisG deletion strain then was to be compared to the control strain. Ironically, all "control" strains containing active ATP phosphoribosyltransferase exhibited growth behavior complications whereas TR3343 behaved straightforwardly. The hisE strains expressed the histidine operon at lower levels than TR3343 when grown on low and intermediate histidine concentrations, but this can be explained by their slower growth rates. The progressively slower growth after histidine downshift appeared to be due to ATP phosphoribosyltransferase catalytic activity, and was probably a result of progressive buildup of abnormally high intracellular PRibATP concentrations. Slow growth of hisE strains was not likely to be due to a drain on the ATP pool, per se, because the equilibrium constant for formation of PRibATP is l0' (4). A drain on the PRibPP pool was not likely of importance either since even when an ATP drain did occur PRibPP apparently was not limiting (see below). A reasonable explanation for slow hisE growth on Hol is that high PRibATP con* centrations inhibit some other ATP utilizing enzyme. Apparently, the inhibition could be overcome, perhaps by derepression, since adapted cultures grew at a normal rate. The slowed growth rate of hisE strains after a histidine downshift could account for lower levels of operon expression since intracellular levels of histidine derived from transported Hol would be higher as a result of slow incorporation into protein. Some of the most interesting behavior was exhibited by hisC strains which had been adapted to growth on Hol. Extremely oscillatory growth rate behaviors occurred when growth was supported by intermediate concentrations of Hol. hisC specifies a step in the pathway after formation of AICAR, which is an intermediate in the de novo biosynthesis of purines. Effectively, AICAR cycles through the histidine pathway to carry a CN fragment into histidine biosynthesis (Fig. 1). Derepressed hisC mutants growing on Hol are free to divert large quantities of ATP to AICAR. Suppression of the oscillatory

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GOITEIN AND PARSONS

growth pattem of hisC strains by histidine, TA, or adenine and shortening of the oscillatory period by presumed higher ppGpp levels all indicate that the phenomenon is caused by ATP phosphoribosyltransferase-catalyzed drain of the purine nucleotide pool. A model which explains this is as follows. During the slow growth phase a derepressed level of uninhibited ATP phosphoribosyltransferase causes a large drain on the ATP pool. This could inhibit the growth rate but not the uptake rate for Hol. The intracellular histidine concentration begins to rise, since it cannot be utilized rapidly for protein biosynthesis. When the histidine concentration becomes sufficiently high enough, ATP phosphoribosyltransferase becomes inhibited, the drain on the ATP pool ceases, and rapid growth characteristic of histidine excess conditions is allowed. Some histidine might also have been secreted during slow growth so that the combined intra- and extracellular histidine pools were substantial enough to allow one to two doublings. Rapid growth could continue until the histidine pools became depleted, ATP phosphoribosyltransferase became fully active, the drain on the ATP pool began, and the cycle would begin again. This interpretation is supported by recent results from Johnston and Roth (20), indicating that a fully active histidine pathway causes adenine auxotrophy. This model also explains why oscillatory growth occurred only at intermediate Hol concentrations. At high Hol concentrations the high intracellular level of histidine derived from transported Hol apparently inhibited ATP phosphoribosyltransferase enough to stop the ATP drain. At low Hol concentrations the intracellular level of histidine probably could not increase enough to allow periods of rapid growth. Also, the intracellular ppGpp concentration should be oscillatory in this model as the histidine levels rise and fall. Since ppGpp inhibits adenylosuccinate synthetase (33), it is probable during periods of high rates of conversion of ATP to AICAR that recycling of AICAR to adenine nucleotides was inhibited. When the histidine level built up, the ppGpp level would fall, and large amounts of AICAR (or IMP) could be converted to ATP. Thus, in addition to histidine and ATP, ppGpp and purine nucleotide precursor levels probably are strongly oscillatory in hisC strains. These oscillations apparently are "in phase" in hisC and, combined with yet other consequences of oscillatory ATP and ppGpp levels, result in spontaneous strongly oscillatory growth patterns for hisC strains. In summary, growth circumstances resulting in conditional regulation of the histidine operon

J. BACTERIOL.

by ATP phosphoribosyltransferase, either transiently or in steady state, have not been found. Rather strong metabolic consequences of inadequately regulated histidine biosynthesis have been observed in other organisms as well (15). ACKNOWLEDGMENTIS This work was supported by Public Health Service grant GM23031 from the National Institute of General Medical Sciences. We thank Bruce Ames and John Roth for supplying bacterial strains and phage used in this work. LITERATURE CITED 1. Ames, G. F., K. D. Noel, H. Taber, E. N. Spudich, N. Nikaido, J. Afang, and F. Ardeshir. 1977. Fine structure map of the histidine transport genes in SalmoneUa typhimurium. J. Bacteriol. 129:1289-1297. 2. Artz, S. W., and J. R. Broach. 1975. Histidine regulation in Salnonella typhimurium: an activator-attenuator model of gene regulation. Proc. Natl. Acad. Sci. U.S.A. 72:3453-3457. 3. Barnes, W. M. 1978. DNA sequence from the histidine operon control region: seven histidine codons in a row. Proc. Natl. Acad. Sci. U.S.A. 75:4281-4285. 4. Bell, R. M., and D. E. Koshland, Jr. 1971. Allosteric properties of the first enzyme of the histidine operon. Bioorg. Chem. 1:409-423. 5. Blasi, F., R. W. Bouton, J. S. Kovach, and R. F. Goldberger. 1971. Interaction between the first enzyme for histidine biosynthesis and histidyl transfer ribonucleic acid. J. Bacteriol. 106:508-513. 6. Brenner, M., and B. N. Ames. 1971. The histidine operon and its regulation, p. 349-387. In H. J. Vogel (ed.), Metabolic pathways, vol. 5. Academic Press, Inc., New York. 7. Dalal, F. R., R. E. Gots, and J. S. Gots. 1966. Mechanism of adenine inhibition in adenine-sensitive mutants of Sabnonella typhimurium. J. Bacteriol. 91:507-513. 8. DiNocera, P. P., A. Alessandra, and F. Blasi. 1975. In vitro transcription of the Escherichia coli histidine operon primed by dinucleotides. J. Biol. Chem. 250: 8376-8381. 9. DiNocera, P. P., F. Blasi, R. DiLauro, R. Frunzio, and C. B. Bruni. 1978. Nucleotide sequence of the attenuator region of the histidine operon of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 75:4276-4280. 10. EdHin, G., and P. Doini. 1971. Synthesis of guanosine 5'diphosphate, 2'-(or 3'-) diphosphate and related nucleotides in a variety of physiological conditions. J. Biol. Chem. 246:4371-4373. 11. Ely, B. 1974. Physiological studies of SalnoneUa histine operator-promotor mutants. Genetics 78:593-606. 12. Friesen, J. D., N. P. Fiil, and K. von Meyenburg. 1975. Synthesis and turnover of basal guanosine tetraphois phate in Escherichia coli. J. Biol. Chem 250:304-309. 13. Goldberger, R. F., and J. S. Kovach. 1972. Regulation of histidine biosynthesis in SalmoneUa typhimurium, p. 285-308. In B. L Horecker and E. R Stadtman (ed.), Current topics in cellular regulation, vol. 5. Academic Press, Inc., New York. 14. Hartman, P. E., Z. Hartman, and R. C. StahL 1971. Classification and mapping of spontaneous and induced mutations in the histidine operon of SalmoneUa, p. 134. In E. W. Caspari (ed.), Advances in genetice, vol. 16. Academic Press, Inc., New York. 15. Henderson, J. F. 1972. Regulation of purine biosynthesis, p. 214-217. In American Chemical Society monograph 170. American Chemical Society, Washington, D.C.

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16. Hilton, J. L., P. C. Kearney, and B. N. Ames. 1965. ole Mode of action of the herbicide, 3-amino-1,2,4-triazole (amitrole): inhibition of an enzyme of histidine biosynthesis. Arch. Biochem. Biophys. 12:544-547. 17. Holzclaw, W. D., and L. F. Chapman. 1975. Properties of some norvaline-resistant mutants ofBacillus subtilis. J. Gen. Microbiol. 88:289-294. 18. Ino, L, P. E. Hartman, Z. Hartman, and J. Yourno. 1975. Deletions fusing the hisG and hisD genes in Salmonella typhimurium. J. Bacteriol. 123:1254-1264. 19. Johnston, H. M., W. M. Barnes, F. G. Chumley, L. Bossi, and J. R. Roth. 1980. Model for regulation of the histidine operon of Salmonella. Proc. Natl. Acad. Sci. U.S.A. 77:508-512. 20. Johnston, H. M., and J. R. Roth. 1979. Histidine mutants requiring adenine: selection of mutants with reduced hisG expression in Salmonella typhimurium. Genetics 92:1-15. 21. Kasai, T. 1974. Regulation of the expression of the histidine operon in Salmonella typhimurium. Nature (London) 249:523-527. 22. Kovach, J. S., M. A. Berberich, P. Venetianer, and R. S. Goldberger. 1969. Repression of the histidine operon: effect of the first enzyme on the kinetics of repression. J. Bacteriol. 97:1283-1290. 23. Kovach, J. S., J. M. Phang, F. Blasi, R. W. Bouton, A. Ballesteros-Alma, and R. F. Goldberger. 1970. Interaction between histidyl transfer ribonucleic acid and the first enzyme for histidine biosynthesis of Salmonella typhimurium. J. Bacteriol. 104:787-792. 24. Kovach, J. S., J. M. Phang, M. Ference, and R. F. Goldberger. 1969. Studies of the repression of the histidine operon. II. The role of the first enzyme in control of the histidine system. Proc. Natl. Acad. Sci. U.S.A. 63:481-488. 25. Lawther, R. P., and G. W. Hatfield. 1978. Effects of altered rho gene product on the expression of the Escherichia coli histidine operon. J. Bacteriol. 136:12011204. 26. Lazzarini, R. A., M. Cashel, and J. Gallant. 1971. On the regulation of guanosine tetraphosphate levels in stringent and relaxed strains of Escherichia coli. J.

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Biol. Chem. 246:4381-4385. 27. Meiss, H. K., W. J. Brill, and B. Magasanik. 1969. Genetic control of histidine degradation in Salmonella typhimurium, strain LT-2. J. Biol. Chem. 244:53825391. 28. Meyers, M., F. Blasi, C. B. Bruni, R. G. Deeley, J. S. Kovach, M. Leventhal, K. P. Mullinix, T. Vogel, and R. F. Goldberger. 1975. Specific binding of the first enzyme for histidine biosynthesis to the DNA of the histidine operon. Nucleic Acid Res. 2:2021-2036. 29. Morton, D. P., and S. M. Parsons. 1977. Inhibition of ATP phosphoribosyltransferase by AMP and ADP in the absence and presence of histidine. Arch. Biochem. Biophys. 181:643-648. 30. Morton, D. P., and S. M. Parsons. 1977. Synergistic inhibition of ATP phosphoribosyltransferase by guanosine tetraphosphate and histidine. Biochem. Biophys. Res. Commun. 74:172-177. 31. Sadler, W. C., and R. L. Switzer. 1977. Regulation of Salmonella phosphoribosylpyrophosphate synthetase activity in vivo. J. Biol. Chem. 262:8504-8511. 32. Scott, J. F., J. R. Roth, and S. W. Artz. 1975. Regulation of histidine operon does not require hisG enzyme. Proc. Natl. Acad. Sci. U.S.A. 72:5021-5025. 33. Stayton, M. M., and H. J. Fromm. 1979. Guanosine 5'diphosphate-3'-diphosphate inhibition of adenylosuccinate synthetase. J. Biol. Chem. 254:2579-2581. 34. Stephens, J. C., S. W. Artz, and B. N. Ames. 1975. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino-acid deficiency. Proc. Natl. Acad. Sci. U.S.A. 72:4389-4393. 35. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 36. Winkler, M. E. 1978. Expression of the histidine operon in rho mutants of Escherichia coli. J. Bacteriol. 136: 721-725. 37. Winkler, M. E., D. J. Roth, and P. E. Hartman. 1978. Promotor- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon. J. Bacteriol. 133:830-843.