Evidence for the order promoter-operator-first structural gene in the tryptophan operon of Salmonella

J. Mol. Biol. (1970) 51, 709-715

Evidence for the Order Promoter-Operator-First Structural Gene in the Tryptophan Operon of Salmonella Genetic evidence suggests that the order, with respect to the first structural gene, of the genetic elements controlling the expression of the tryptophan (trp) operon of Salmoneucr &yphimurium is PI-tvO-trpA (promoter-operator-first structural gene). Two independent lines of evidence are consistent with this order: (1) the deletion 8~~x38, which removes Pl, fails to recombine with some trpOc mutations but recombines with one of them suggesting that it covers only part of trp0 ; (2) we have been unable to flnd .YU~X to trp0 deletions after several attempts, but have easily obtained multisite mutations with the characteristics of deletions extending from trp0 to bypA. The promoter has been defined as a discrete region of DNA at which transcription of an operon is initiated (Jacob, Ullman Sz Monad, 1964). Beokwith and his coworkers have recently shown that the promoter for the lac operon of Eaherichiu coli is separable from the lac operator and that these elements are arranged in the order promoter-operator-first structural gene (Ippen, Miller, Scaife & Beckwith, 1968 ; Miller, Ippen, Scaife & Beckwith, 1968). The tryptophan (trp) operon of both E. coli and Salmonella typhimurium consists of a cluster of five structural genes repressed as a unit by the end-product of the pathway, tryptophan (Ito & Crawford, 1965; Bauerle

& Margolin, 1966a). The order of these genes is the same in both organisms but the nomenclature is different. In S. typhimurium this order can be written PO-ABEDC (PO representing the promoter-operator region). In addition to the main promoter (Pl) located in the proximity of the operator, this operon also has a low eficienoy promoter element (Pi?) located between the second and third structural genes, B and E (Bauerle & Margolin, 1966a,1967; Balbinder, Blume, Weber & Tamaki, 1968; Morse & Yanofsky, 1968). In this report we will present evidence suggesting that PI and the ttp operator (trp0) are arranged in the same order with respect to the first structural gene (trpA) as that found for the promoter and operator in the lac system. Operator constitutive (gC) mutations were obtained in mutants for tvA by selecting for strains resistant to the tryptophan analogs S-methyltryptophan and 6-fluorotryptophan in the presence of anthranilic acid. Mutants altered in the trpA gene lack the first enzyme of the tryptophan biosynthetic pathway, ASaset, and require a,nthranilic acid for growth. The 0” mutations showed about 90% co-transduction with the trpA markers and were further characterized by the standard criteria of cisdominance, constitutive synthesis of the trp enzymes and map position. Details of the isolation and characterization of these mutations are presented in a separate report (Balbinder et al., 1970). The map position of 0” mutations was determined to a large extent in crosses to the deletion 8~~x38. This deletion was isolated by Margolin & Bauerle (1966) who found that it removed PI and probably the trp operator but did not extend into tqA. The strain carrying this deletion lacks the AS&se-PRTase t Abbreviations used : ASaae, anthranilate synthetase ; PRTasc phosphoribosyl transferase @TSase and aTBase, /I and OLcomponents of tryptophan synthetase; MeTrp, 5-methyltryptophan; FTrp, B-fluorotryptophan. 709

;

710

R.

CALLAHAN

III,

A. J.

BLUME

ND

E.

BALBINDER

enzyme complex, whose components are determined respectively by the first and second structural genes of the operon (trpA and trpB), but makes the last three trp enzymes at a low constitutive level (about 0.5 of repressed wild type, see Table 2) due to initiation at PZ (Bauerle & Margolin, 19663,1967). This level does not change when the strain is grown under conditions of limiting tryptophan which normally lead to derepression of the trp operon. Table 1 shows the results of several crosses between trpA trp0 mutants and supX38. The rationale behind these croasea is simple: if a trp0 mutation falls within the segment covered by the deletion no 0 + (MeTrp sensitive) recombinants will be obtained ; if it falls outside, both MeTrp-resistant and -sensitive recombinanta will result, In these crosses both donor and recipient were auxotrophic and selection was exclusively for prototrophic recombinanta. Control platinga of both donor and recipient were carried out. The results clearly show that one of the MeTrp-resistant mutations, TABLE

1

of trp0 mutations against the deletion supX38

Mapping

Recombinants A+S A+R trpA49 trpA512 trpO518 trpA49 trpA512 trpO519 trpA8 trp0522 trpA49 trpO525 aupX38

x x X

x x

supX38 supX38 supX38 wpX38 trpA49 trp0525

The recipients were infected with and plated on minimal agttr (Blume recombinants (trpA+) were selected recombinants were then tested for colonies in 1 ml. of sterile saline and of the analogue/ml. Abbreviations: recombinants.

0 0 0 18 26

384 432 430 262 174

Total

tested 384 432 430 280 200

transducing lysates of phage P22 grown on each donor strain & Balbinder, 1906). Under these conditions only prototrophic and resistance or sensitivity to MeTrp was unselected. The resistance or sensitivity to the analog by suspending the streaking each suspension on minimal agar containing 100 pg A+S, MeTrp-sensitive recombinants; A+R, MeTrp-resistant

maps outside the segment covered by the deletion while the other three map inside this segment. To determine whether the location of trp525 was operator proximal or operator distal with respect to trpA49, (the structural mutation in trpA which maps closest to trpO), we performed the cross diagrammed in Figure 1. The results given in the Figure favor the order trp525,trpA49-trpB. Since trp525 exhibits the properties expected of an operator mutation (&-dominance, elevated enzyme levels under conditions of repression) and it maps in the aame region as similar MeTrp-resistant markers, it can reasonably be considered as an 0” mutation. We have tentatively designated it aa trp0525. If this characterization of trp0525 is correct, our results clearly mean that supX38 deletes PI and only part of trp0, a situation compatible only with the order PI-tyvO-trpA. If this order of the regulatory elements with respect to the first structural gene is indeed the right one, we can predict that deletions from trpA to trp0 will be found but deletions from supX to trp0 will not (see Fig. 2). Both types can be selected directly. Deletions of the supX region suppress the mutation Zeu-,500(in t,he leucintt operon) and can extend into the trp operon (Margolin & Bauerle, 1966). Thus, if trp525,

LETTERS (a 1 Order

0525, 0525

D

, ‘1 1

Ra

1’

supx3tl Exchange 1

2 3 t-2-3

+

TO

THE

A49

EDITOR

711

A-W, 0525

( b) Order

Ads , ‘1 2 \

A49

: 3

,

:

I ’

#

+

Sup x 38

O+A+ (A+9 A+ (A+R, 052s A49 (A-R) O+A49 (A-S)

0525

mipsrJty

7

0525 :

2

O+A+ CAtSI O+Ae (A-S) h-25 Aa (A-R 0525 A+ (A+R,

: 3 +

+

1

Results

A+$

A+R

A-S

A-R

Total

-i

7

-ii

336

350

FIG. 1. Deterniination of the order of trp0525 with respect to the structural mutation trpA49 by a three-point test. The oroa wag performed aa described in the legend to Table 1, with the difference that the infected bacteria were plated on minimal sgar supplemented with 10 pg of anthranilio acid/ml. Recombinants were then tested for sensitivity or resistance to MeTrp aa described in the legend to Table 1 by using properly supplemented minimal media. The mutant 8~~x38 oannot utilize anthranilic acid as B growth faetor since it lacks PRTase a~ a result of missing the promoter PI (Margolin & !BBeuerle, 1966). Abbreviations: A+S, MeTrp-sensitive prototrophs; A+R, MeTrp-resistant prototrophs; A-S, MeTrp-sensitive anthrenilio aoid auxotrophs; A-R, MeTrp-resistant snthranilic acid ctuxotrophs.

supX to trp0 deletions are possible, they could be isolated as revertants of leu-500 which are simultaneously resistant to MeTrp. Deletions from trpA to trp0 can be recovered from strains carrying extreme polar mutations of trpA or mutations in the “unusual” region (see legend, Fig. 2). These grow extremely slowly on anthranilic acid supplement since they possess very low levels of PRTase activity (Bauerle & Margolin, 1966b; Balbinder et al., 1968). Secondary mutants of such strains selected for improved utilization of anthranilic acid carry deletions of various portions of the trpA gene (Balbinder et al., 1968). By selecting simultaneously for normal growth on anthranilic acid supplement and resistance to MeTrp or FTrp we should be able to obtain deletions from trpA to trp0. Although such deletions would remove the translation initiating signal present at the beginning of trpA, they could be obtained if translation initiators allowing for the synthesis of a sufilcient level of PRTase and the remaining trp enzymes are available within trpA or at the beghming of trpB. Some of these could be equivalent to the translation restart signals found within the lac z gene of E. coli (Newton, 1969; Michels & Zipser, 1969). In attempts to isolate supX to trp0 deletions, a mutant carrying ku-500 was mutagenized with nitrous acid and portions plated on minimal agar plates containing 100 pg of MeTrp/ml. The same procedure was followed with a double mutant, leu-500 trpA512, carrying an internal deletion in trpA (Fig. 2), but in this case 10 pg of authranilic acid/ml. was added to the plates since trpA512 leads to a requirement for this growth factor. After several experiments involving a total of about lOlo

712 cys B

R.

CALLAHAN

III,

AND

supx

E.

BALBINUER

fJ-P ~518

II

A. .I. BLUME

I

519

522

II sUPX

” , I I I

38

OA 528 OA 526 OA 673

A 525 ,

49 278 I 1 I I I I

111 47 I I 1 I I 1 j572 I I I I I

8 I

“unusual” muts. 46 < t c 2 t I I i I I I

B

E PZ 1t---

I I I I

Fro. 2. Partial map of the operator proximal region of the trp operon. The extent of the deletions mentioned in the text is indicated, as well as the map position of the various trp0 mutations. The “unusual” mutations (Bauerle & Margolin, 19666) are deficient in both A&se and PRTese activities and map between &pA and tr~~bl, the first and second structural genes of the operon (Bauerle & Margolin, 1966b; Balbinder et al., 1968). The Oc mutations give no MeTrp-sensitive recombinants when crossed to each other, but such trp0.518 and trp0519 recombinants have been found in crosses of trp0518 to trp0522 and trp0525. The ardor shown for the trp0 mutations is based on the frequency of sensitive reoombinants obtained in these crosses and agrees with that found by deletion mapping. Full data are presented in a separate publication (Balbinder et al., 1970).

mutagenized bacteria, no supX MeTrp-resistant mutants were obtained. In contrast, when strains carrying the extreme polar mutations trpA49 or the “unusual” mutations trpA28 or trpA46 were treated with nitrous acid and plated onto minimal agar supplemented with 10 pg of anthranilic acid/ml. plus 100 pg of MeTrp or FTrp/ml., secondary mutants with characteristics of trpA to trp0 deletions (requirement for anthranilic acid, MeTrp resistance, inability to revert to prototrophy) were recovered at a frequency of about one per IO8 mutagenized bacteria. Three of these, containing the multisite mutations trpOA526, trpOA528 and trpOA673 have been analyzed. The multisite mutations were mapped by P22-mediated transduction against 8~~x38, various point mutations in trpA and the unusual region, and the trp0 mutations already discussed. Figure 2 summarizes the results of these crosses. As indicated, these three mutations failed to yield prototrophic recombinants in crosses to 8~~x38 or the various point mutations shown, and gave no MeTrp-sensitive recombinants in crosses to trp0 trpA+ donors in a total of 4000 prototrophic recombinants teated. If these strains have lost the trp operator but retain PI intact, we should expect that in each strain the trp enzymes will be synthesized at the same level under represGon or derepreasion conditions, although levels might differ among strains depending upon the efficiency of their translation initiating signals. In contrast, strains carrying 0” mutations not associated with a deletion might retain some capacity for repressor recognition and could show an increase in enzyme level when grown under conditions of derepression. Table 2 shows the levels of PRTase and both the /3 and a components of TSaae, products of the second, fourth and fifth genes, respectively. As predicted, the. three trpOA multisite mutants have stable enzyme levels while the 0” mutants show a distinct capacity to derepress. Two of the three trpOA mutants, trpOA526 and trpOA528, show conatitutive levels for the TSase j3 and a components which are comparable to those found in fully derepressed cells (see trpA8, no. 9 in Table 2). The third trpOA mutant, trpOA673 only shows a six- to sevenfold constitutive synthesis of these enzyme components. Those observations may indicate that both high and low eEoioncy translation initiating sites exist within trpA or near the

LETTERS

TO

THE

EDITOR

713

TABLE 2 Levels of phosphoribosyl transjerase, typtophan synthetaae in trp0 and trpOA mutants NO.

Genotype

1 2 3 4

@AS trpA8 trpA8 trpA8

b 6 7

Deletion Deletion Deletion

8 9 10 Standard

Relative speoifio aotivities Repression oonditions Derepression PRTase p TSase ccTSase /I T&se

St&n

trpO518 tqXM19 trpO622 trp0525 trpOA526 trpOA528 trpOA673

Wild type (LT2) trpA8 eupX38 speoifio aotivity

conditions a TSase

lb 20 10 n.d.t

7 10 7 3

8 9 7 2

33 33 30 n.d.

30 33 30 n.d.

2

69 76 6

75 68 7

66

73 8

70 70 6

6 70

b 90

b

2 1 1 0

1 1 0.5

1 1 0-b

0.02

0.36

o-33

Speoifio activities are expressed as relative to those of repressed wild type (LT2) taken as unity. The standard specific aotivities for the three enzymes are shown at the bottom of the Table. Repression oonditions consisted of growth in minimal medium supplemented with excess (50 PgLgl ml.) L-tryptophan (Balbinder et al., 1968). Standard derepression oonditions oonsisted of overnight growth in the same medium supplemented with a limiting amount (6 H/ml.) of tryptophan. In oases where we wanted to determine only whether certain strains were oepable of derepressing, rather than their maximum derepressed enzyme levels, the bacteria were 5rst grown under standard repression oonditions (above), harvested by oentrifugetion, wasbed and resuspended in minimal medium without tryptophan and inoubated on a rotary shaker at 37°C for 4 hr. Strains 1 through 7 in this Table were treated in this fashion. PRTase was assayed in repressed oultures only. The prooedurs employed was that of Corder-o, Levy & Balbinder (1968). The /l and Q subunits of T&se were assayed by the procedure of Smith & Yanofsky (1962). Since wild type cannot derepress oompletely due to repression by endogenously synthesized tryptophan (no. 8 in the Table) the non-polar mutation trpA8 was introduoed into all strains oarrying trp0 mutations to block synthesis of this amino acid. In the presence of trpA8 full derepression can be obtained (no. 9 in the Table). t n.d., assay not done.

beginning of trpB. Another interpretation of these result8 is also possible, as we shall point out later. The behavior of PRTase shows a different pattern in the Oc and trpOA mutants: in the former it is normal but in the latter PRTase activity is well below the level of the TSase components. PRTase normally forms a complex with ASase but it is enzymically active when free (Ito & Yanofsky, 1966; Bauerle & Margolin, 19663). However, it seems to be less stable in the free state than ~EJpart of the complex, and we believe that the low PRTase activities we observe are a result of this decreased stability in the absence of ASase. This awaits further investigation. The enzyme levels shown by the trpOA mutants are much too high to be explained as the result of expression initiated at P2 (compare to ~4~x38, no. 10 in Table 2). In addition, 8~~x38 lacks PRTase entirely while the trpOA mutants have distinctly measurable levels of this enzyme. The results we have presented are consistent with the order Pl-trpO-trpA for the elements controlling the expression of t,he trp operon of 8. typhimurium, but do not prove it conclusively. Our first line of evidence is based on the characterization

714

R.

CALLAHAN

III,

A. J.

BLUME

AND

E.

BALBINDER

of trp0525 as an 0” mutation, and the fact that three-point tests position it to the left of trpA49 (as Fig. 2 is drawn). We cannot exclude the possibility that trp525 is not a mutation of the operator but it represents a mutationally created “promoter” in the early portion of trpA. Mutations of this type have been reported and have many of the properties of 0” mutations (Bauerle, 1968; Morse & Yanofsky, 1969; Callahan & Balbinder, 1969,197O). Furthermore, three-point tests are not always reliable in determining the position of two mutations with respect to each other (Balbinder, 1962). As Table 2 shows, trp0525 leads to a lower constitutive level of expression than the other Oc mutations studied. It has also some exceptional characteristics which are described elsewhere (Balbinder et al., 1970). For these reasons our characterization of trp525 as an 0” mutation is only tentative. The second line of evidence is based on our ability to obtain presumed trp0 to trpA deletions while being unable to recover deletions from supX to trp0. This is also open to some objections. It is possible that our procedure for selecting supX to trp0 deletions was not technically correct, or that we did not screen a sufiicient number of bacteria for occurrence of such deletions. Also, while our multisite trp0A mutants have the characteristics expected of deletions moving from trpA to trp0 and leaving PI intact, we cannot exclude, either the possibility that these deletions join the trp genes to a functional promoter to the left of the trp operon (as Fig. 2 is drawn), or that they are not deletions at all but insertions of some extraneous genetic material containing a functional promoter (Jordan, Saedler BEStarlinger, 1968 ; Shapiro, 1969). These last two alternatives would require additional assumptions such as (1) this “foreign” promoter is as highly efficient as PI (i.e. trpOA526 and trpOA528) and (2) polypeptide synthesis may be started by a foreign translation initiator located near the foreign promoter. On this interpretation the differences in level of expression between trpOA673 and the other two tqvOA mutants could be explained by assuming that the genetic alterations (deletions or insertions) in trpOA526 and trpOA528 do not change the phase of reading of the genetic message, but trpOA673 ends “out of phase” thereby generating a nonsense triplet which has a polar effect (Whitfield, Martin Q Ames, 1966). We do not have sufficient information at the present time to eliminate these alternative interpretations of our results. Nevertheless, the fact that two independent lines of evidence are consistent with the order PI-trpO-trpA is not without significance. Conclusive proof for this order should come from (1) the confirmation of the status of trpOA525 as an 0” mutations, and (2) the demonstration that mutations of the promoter (PI) fail to recombine with supX38 but recombine with the trpOA multisite mutations. Work on this problem is continuing. The nature of the translation starters in the trpOA mutants is also under investigation. This work was supported by grants

GB4903

and

GB7296

of the

National

Science

Foundation.

ROBERTCALLAHAN III ARTHUR J. BLTJME~ ELIAS BALBINDER

Biological Research Laboratories Department of Bacteriology and Botany Syracuse University Syracuse, N.Y.13210, U.S.A.

Received 12 January

1970, and in revised

t Present address: Department Md 20014, U.S.A.

form

of Biochemical

20 April

Genetics,

1970 National

Heart

Instit,ute,

Bethesda,

LETTERS

TO THE

EDITOR

716

REFERENCES Balbinder, E. (1962). Ge%&ic-g, 47, 646. Balbinder, E., Blume, A. J., Weber, A. & Tam&i, H. (1968). J. Bad. 95, 2217. Balbinder, E., Callahan, R., McCann, P. P., Cordaro, J. C., Weber, A. R., Smith, A. M. & Angelosanto, F. (1970). Ueenetica, in the press. Bauerle, R. H. (1968). Proc. 12Q Id. Gong. Gene&&, Tokyo, Japan, vol. II, p. 44. The Science Council of Japan. Bauerle, R. H. & Margolin, P. (1966o). Proc. Nat. Acud. Sci., Wc&. 56, 111. Bauerle, R. H. & Margolin, P. (1966b). Cold Spr. Harb. Sump. Quad. BioZ. 31, 203. Bauerle, R. H. & Margolin, P. (1967). J. Mol. Biol. 26, 423. Blume, A. J. & Balbinder, E. (1966). Genet&, 53, 677. Callahan, R. & Balbinder, E. (1969). Bad. Pmt. p. 66. Callahan, R. & Balblnder, E. (1970). Science, in the press. Cordaro, J. C., Levy, H. R. & Balbinder, E. (1968). Biochtm. Biqvhys. Rea. &mm. 33, 183. Ippen, K., Miller, J. H., Scaife, J. G. & Beckwith, J. R. (1968). Nature, 217, 826. Ito, J. & Crawford, I. P. (1966). Qen&ice, 52, 1303. Ito, J. & Yanofsky, C. (1966). J. BioZ. Chern. 241, 4112. Jacob, F., Ullman, A. & Monod, J. (1964). C.R. Acud. Sci. Par& 258, 3126. Jordan, E., Saedler, H. & Starlinger, P. (1968). Mol. &n. Uenetica, 102, 363. Margolin, P. & Bauerle, R. H. (1966). Cold Sp. Hurb. Symp. Quad. Biol. 31, 311. Michels, C. A. & Zipser, D. (1969). J. Mol. Biol. 41, 341. Miller, J. H., Ippen, K., Scaife, J. G. & Beckwith, J. R. (1968). J. Mol. BioZ. 88, 413. Morse, D. E. & Yanofsky, C. (1968). J. Mol. BioZ. 88, 447. Morse, D. E. & Yanofsky, C. (1969). J. Mol. BioZ. 41, 317. Newton, A. (1969). J. Mol. BioZ. 41, 329. Shapiro, J. A. (1969). J. Mol. BioZ. 40, 93. Smith, 0. H. & Yanofsky, C. (1962). In Methods in Enzymology, ed. by S. P. Colowick & N. 0. Kaplan, vol. 6, p. 794. New York: Academic Preaa Whitfield, H. J., Martin, R. R. & Ames, B. N. (1966). J. Mol. BioZ. 21, 336.

46