Divergent transcription of the argECBH cluster of Escherichia coli K12. Mutations which alter the control of enzyme synthesis

J. Mol. Biol. (1976) 102, 205-220

Divergent Transcription of the argECBH Cluster of Escherichia coli K 12. Mutations which alter the Control of Enzyme Synthesis ANTHONY P. BRETSCHER~"AND SIMON BAUMBERG

Department of Genetics, University of Leeds, Leeds LS2 9JT, England (Received 13 November 1974, and in revised form 17 November 1975) A method is described for the selection of mutants with altered control of argE expression. Three mutations were characterized and mapped. The first, lying in argH, results in a partially functional enzyme and so limits the supply of arginine for repression. The second, the promoter mutation arg8, confers increased expression of argE and reduced expression of argCBH, although the wild type repression ratios axe retained. The third, the operator mutation arg11, leads to non-repressible expression of argE and reduced repressibility of argCBH expression. The effects of all three mutations are cluster specific. Arg8 and argll are dominant mutations and do not result from genetic duplication. These two mutations map close to, or at, the argE-argC boundary; arg8 lies between argE and arg11. The properties of these mutations support provions conclusions that the cluster is transcribed divergently from the argE-argC boundary. 1. I n t r o d u c t i o n The eight enzymes of Escherichia coli K12 involved in the biosynthesis of arginine from glutamate are specified b y nine structural genes (Fig. 1). The synthesis of these enzymes is subject to argR-dependent negative control in the presence of arginine (Maas et al., 1964; Maas & Clark, 1964). Only the four genes argECBH are clustered (Fig. 2) ; the rest are scattered around the chromosome. I t has been known for some time that argE expression is not controlled coordinately with argCBH, since the repression ratio (the ratio of the derepressed to repressed level of enzyme specific activity) for argE is only about a quarter of the ratio for argCBH (Baumberg et al., 1965 ; Glansdorff & Sand, 1965). Nonsense mutations in argB or C have a polar effect on the expression of argH, whereas argg nonsense and frameshift mutations do not, thus establishing that argE does not belong to the same operon as argCBH (Cunin et al., 1969; Baumberg & Asheroft, 1971; Jacoby, 1972 ; Elseviers et al., 1972). The deletion argSUPl02 (Fig. 2), which extends from argB into the argg-argC boundary, reduces the level of argE expression and alters control of argH expression (Cunin et al., 1969). The combination of argSUPl02 with deletion argDS11, which extends from the left into argE, has no further effect err the expression of argH: the necessary conclusion is t h a t argE and argCBH are transcribed divergently from their common boundary. I n addition, a low level t Present address : Department of Biochemistry, Stanford University Medical Center, Stanford, Calif. 94305, U.S.A. 205

206

A.P.

BRETSCHER

A N D S. B A U M B E R G

/

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FIe. 1. Genes a n d enzymes of arginine biosynthesis in E. coli K12. Glutamate ~ N-acetylglutamate _~B N-acetyl-~,-glutamyl phosphate c N-acetylglutamic ~,-semialdehyde ~ N-aeety], E o r m9t h m e ---) ornithine - ~ eitrulline ~ argininosuceinate ~ arginine.

MN42 EC(B] SUP/02 SUP400

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internal promoter for argH that is probably located at the beginning of this gene has been described (Elseviers et al., 1972). Recently DNA/messenger RNA hybridization experiments with separated strands of r DNA have shown that there are two components of messenger RbIA whose synthesis is repressible by arginine in strains carrying a functional argR + allele, and which specifically hybridize to different strands of the DNA (Panchal et al., 1974; Pouwels et al., 1974; Boyen et al., 1974). The pleiotropic effects of deletion argS UP102 and an analysis of revertants selected for restored argE activity, suggest that at least part of the control regions for the two operons overlap (Elseviers et al., 1972). These conclusions are substantiated by the isolation of mutations which reduce the repressibility of both argE and argCBH (Jacoby, 1972).

C O N T R O L M U T A T I O N S I~, E. coli a r g E C B H

207

To elucidate the s t r u c t u r e of t h e control regions a t t h e argE-C b o u n d a r y , f u r t h e r m u t a n t s altered i n p r o m o t e r a n d / o r operator f u n c t i o n s are necessary. I n this p a p e r we describe a m e t h o d for t h e isolation of m u t a t i o n s t h a t specifically increase t h e level of argE expression u n d e r c o n d i t i o n s of repression or reduce t h e level of argH expression. Analysis of these m u t a t i o n s s u p p o r t s the evidence for c o m m o n control of d i v e r g e n t t r a n s c r i p t i o n . A p r e l i m i n a r y a c c o u n t of these results has been p u b l i s h e d (Bretscher & B a u m b e r g , 1975).

2. Materials and Methods (a) Bacterial strains The gen0type a n d origin or construction of the E. coli K12 strains used are given in Table 1. 6P, 6G and 6H50 are derivatives of 619; 6PO4 is a spontaneous argR m u t a n t of 6P selected on medium containing aeetylhistidine plus ornithine (Baumberg, 1970).

(b) Media The minimal and n u t r i e n t media have been described (Baumberg, 1970). (c) Localized mutagenesis and the isolation of mutants Basically, the method of Hong & Ames (1971) was employed, b u t with phage Plkc in place of P22. Eight ml of phage (5 • l09 to 1011 plaque-forming units/ml) grown on strain 6P was centrifuged at 38,000 g for 90 m i n at 15~ in an MSE HS18 centrifuge (this leaves only about 0.1 ~/o of total phage in the supernatant). The s u p e r n a t a n t was gently removed and 3 ml mutagenesis mixture added. This mixture consisted of 0"9 ml phage buffer (Na2HP04, 7 g/l; KH2PO4, 3 g/l; NaC1, 5 g/l; MgSO~ and CaClr at 1 mM a n d 0.1 mM respectively); 1.8 ml 0.1 M-sodium phosphate buffer (pH 6), containing 1 mM-EDTA; and 0.3 ml of a fresh solution of 1 M-hydroxylammonium chloride containing 1 mM-EDTA a n d adjusted to p H 6 with 10 N-NaOH. The P l k c pellet was gently resuspended with a glass rod and incubated a t 37~ Mutagenesis was in general continued for 24 h, b u t was sometimes allowed to proceed up to 48 h. The phage was finally centrifuged as above, washed 3 times in phage buffer, resuspended in L-broth, and stored over a few drops of chloroform. During the hydroxylamine treatment, plaque-forming ability was lost variably b u t rapidly, typically by a factor of 10 every 4 h. The final mutagenized suspension was sometimes found to lose transducing ability after a few days' storage at 4~ and therefore was used immediately. The efficiency of mutagenesis was determined by transducing strain 22MN42 to ppc + (selecting on medium containing glucose) in the presence of arginine. Typically, 1% arginine auxotrophs were found among the transduetants, b u t this figure was variable; the highest rate found was 16%. Control mutations were selected by transducing 6G with the mutagenized lysate and plating on minimal glucose medium containing iV-acetylhistidine (10 t~g/ml), L-ornithine (25 ~g/ml) and a n y other necessary amino acid supplements. This method selects for the control m u t a n t phenotype, as explained below, and simultaneously demands t h a t the m u t a t i o n responsible be closely linked to ppc. (d) Conjugation and traneduction These techniques were performed as previously described (Elseviers et al., 1972). For localised mutagenesis and the transductional mapping crosses, Plkc was grown on 22MN42 for 6 cycles before growth on the donor. R e c o m b i n a n t clones were streaked onto a master plate and then replica plated for unselected markers. Recombinants carrying argB or argC markers were recognized by their ability to utilize ornithine; recombinants carrying argE or argH markers were unable to utilize acetylarginine or ornithine respectively. The presence of the argl, arg8 and arg11 mutations were tested on minimal glucose medium containing 10 ~g acetylhistidine/ml plus 50 tLg ornithine/ml or 50 ~g acetylhistidine/ml plus 50 t~g olmithine/ml, argE + argH + his recombinants carrying the argl or arg8 mutations

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grew on both these media, whereas those carrying the argll m u t a t i o n only grew on the l a t t e r medium, h/s recombinants wild t y p e for arginine control did n o t grow on either medium, argE + argH + recombinants could be simultaneously selected on m e d i u m conraining acetylmethionine and ornithine when the donor and recipient carried t h e same met m a r k e r : with this teclmique no selection was placed on argB or C markers in the cross. (e) Construction of merodiploids Strains diploid for the argECBH region were constructed according to the m e t h o d of Low (1968). The required alleles were transduced into the F - p r i m e of AB1206 F E 1 4 b y exchange with the argEld marker. The recipients were constructed from AT2681CB-1 b y first transducing in the reqttired allele a n d recovering metB arginine prototrophs, t h e n selecting spontaneous rifampicin-resistant m u t a n t s and finally m a t i n g with JG47 to give recA gly + reoombinants. The presence of recA was recognized where a p p r o p r i a t e b y sensitivity of clones to u.v. The F - p r i m e strains were crossed with these reeA metB rif derivatives and reeA Met + l%ifs transconjugants purified. These strains carry the entire argECBH cluster on the F ' , as metB and rif are on opposite sides of the cluster a n d are recessive to their wild-type alleles (Austin & Scaife, 1970). These merodiploids were maintained on supplemented minimal glucose m e d i u m lacking methionlne. (f) Preparation of cell extracts and enzyme assays To grow cells for enzyme assay, a clone was inoculated from a minimal plate into 5 ml of minimal glucose medium containing 1 rag arginine.HC1/m] plus a n y other necessary supplements. After overnight growth a t 37~ on an orbital incubator, 0.5 ml was inoculated into 100 ml of m e d i u m identical except in t h a t it only contained 100 ~g arginine.HC1/ml. I n c u b a t i o n was continued under the same conditions until the optical density reached a b o u t 0"5 a t 660 n m in a P y e Unlearn SP600 m a r k I I spectrophotometer. Cells were harvested a t 10,000 g for 10 rain a t 4~ The pellet was resuspended in 0.1 Mpotassium phosphate buffer (pH 7), containing reduced glutathione a t 1 mM, and the cells were then broken in an MSE 100 W ultrasonic disintegrator operating a t m a x i m u m amplitude for 20 s. E x t r a c t s were centrifuged a t 38,000 g for 15 rain a t 4~ to remove cell debris. Acetylornithinase (the enzyme specified b y argE) and/V-acetyl-7-glutamokinase (specified b y argB) were assayed immediately. The e x t r a c t was stored a t --20~ before performing the other assays. N-acetyl-7-glutamokinase, acetylornithinase a n d argininosuccinase (specified b y argH) were assayed as described b y Baumberg & Ashcroft (1971). /V-acetylglutamic ~,-semialdehyde dehydrogenase (specified b y argC) was assayed according to the m e t h o d of Glansdorff & Sand (1965) and ornithine transearbamylase assayed as described b y Davis (1962) b u t using the colorimetry of Prescott & Jones (1969). Protein was determined according to the m e t h o d of Lowry et al. (1951). E n z y m e activities are given in units/mg protein where 1 unit of enzyme catalyses the formation of 1 ~xnol product/h under the specified assay conditions.

3. Results (a) Isolation and characterization of mutants B a u m b e r g (1970) h a s d e s c r i b e d a m e t h o d for t h e i s o l a t i o n o f argR m u t a t i o n s in w h i c h selection was m a d e for relief f r o m r e p r e s s i o n o f a c e t y l o r n i t h i n a s e synthesis. A c e t y l o r n i t h i n a s e , t h e e n z y m e specified b y argE, is a b l e t o d e a c e t y l a t e N - a - a c e t y l ornithine, N - a - a c e t y l m e t h i o n i n e , N - ~ - a c e t y l l f i s t i d i n e a n d a n u m b e r o f o t h e r N-~a c e t y l a t e d a m i n o acids. H e f o u n d t h a t a c e t y l h i s t i d i n e c o u l d s a t i s f y t h e h i s t i d i n e r e q u i r e m e n t o f a n argR + s t r a i n i n t h e a b s e n c e o f o r n i t h i n e b u t n o t i n t h e p r e s e n c e o f sufficient o r n i t h i n e t o cause repression. M u t a n t s o f a h i s t i d i n e a u x o t r o p h a b l e t o grow on a c e t y l h i s t i d i n e p l u s o r n i t h i n e were f o u n d t o c a r r y a n argR m u t a t i o n a n d so were d e r e p r e s s e d for t h e e n t i r e p a t h w a y . S t r a i n s w i t h m u t a t i o n s in t h e argECBH region h a v i n g either a n i n c r e a s e d level o f a c e t y l o r n i t h i n a s e s y n t h e s i s u n d e r c o n d i t i o n s

C O N T R O L M U T A T I O N S I N E . coli arg~GBH

211

o f r e p r e s s i o n or a r e d u c e d level o f a r g i n i n o s u c c i n a s e s y n t h e s i s (to r e d u c e c o n v e r s i o n of o r n i t h i n e t o a r g i n i n e a n d so relieve r e p r e s s i o n b y lowerhlg t h e e n d o g e n o u s argln i n e pool) s h o u l d also b e selected. S i m u l t a n e o u s selection for localized m u t a g e n e s i s o f t h e argEGBH r e g i o n a n d a b i l i t y t o utilize sufficient a c e t y l h i s t i d i n e for g r o w t h in t h e presence o f o r n i t h i n e .yielded t h r e e t y p e s o f m u t a n t . S t r a i n 6-1 is a n e x a m p l e f r o m t h e m o s t c o m m o n class. T h e level o f a r g i n i n o succinase a c t i v i t y i n argR a n d argR + b a c k g r o u n d s w a s g r e a t l y r e d u c e d , a l t h o u g h a c e t y l o r n i t h i n a s e a n d o r n i t h i n e t r a n s c a r b a m y l a s e a c t i v i t i e s were n o r m a l (Table 2). S t r a i n 6-1 grows slowly on s u p p l e m e n t e d m i n i m a l glucose m e d i u m l a c k i n g a r g i n i n e as i t c o n t a i n s a n argH b r a d y t r o p h i c m u t a t i o n . M a p p i n g s t u d i e s (Table 3) show t h a t t h e m u t a t i o n m a p s close to, or in, t h e argH gene.

TABLE 2

Specific activities of ornithine transcarbamylase and the enzymes specified by a r g E C B H in mutants selected following localized mutagenesis and in the parent wild-type for the a r g E C B H cluster Strain

argE

6P (argR +) 6P04 (argR4) 6-1 (argR +) 6PO4-1 (argRd) 6-8 (argR +) 6PO4-8 (argR4) 6-11 (argR +) 6PO4-11 (argR4)

6.1 38.0 5.4 51.7 19.6 101 60.6 48.4

Enzyme specified by

argC

argB

<:0.2 1.1 ndJ" nd <:0-2 0.67 <:0.2 0.97

<:0.2 1-5 nd nd <:0.2 0.84 <:0.2 1-6

argH

Ornithine transcarbamylase

0,045 1,47 0.01 0.07 0-025 0.83 0.24 1.35

0-15 246 1.0 394 0-17 186 0.27 170

Cells were grown in supplemented minimal glucose medium containing arginine. Activities of ornithine transcarbamylase, especially at low levels, were found to be more variable than those of acetylornithinase or argininosuccinase. To transfer mutations into the argR background, the arginine cluster from 6-1, 6-8 and 6-11 was transduced into 6PO4MN42 to make strains 6PO4-1, 6P04-8 and 6PO4-11, respectively. nd, not determined. TABLE 3

Recombination of the a r g H bradytrophic mutation from 6-1 with del~ions Deletion strain

Wild type colonies

Total colonies

Recombination frequency (%)

P4XDS11 P4XEC(B) P4XCB-1

65 21 23

277 254 1223

23 8.3 1.9

Plkc was grown on 6-1 and used to transduce the deletion strains (see Fig. 2) to arglnine prototrophy. After 36 h incubation at 37~ wild-type transductants produced much larger colonies than those carrying the bradytrophic mutation. The recombination frequency is the percentage of large colonies.

212

A. P. BRETSCHER

AND

S. B A U M B E R G

Strain 6-8 is one of the two mutants which have enhanced synthesis of acetylornithinase and reduced synthesis of argininosuccinase in both argR + and argR backgrounds. The levels of N-acetylglutamokinase and N-acetylghitamie ~-semialdehyde dehydrogenase are also reduced in the argR background; however, the levels in the argR + strains were too low to measure. The effects are cluster specific, as control of ornithine transearbamylase is normal. As synthesis of all these enzymes has the same repression ratio as the wild type, the mutation in 6-8 (designated arg8) appears to be both an " u p " promoter for argE expression and a "down" promoter for argCBH expression. Finally, 6-11 is one of the two isolates in which the synthesis of acetylornithinase is no longer repressible and argininosuccinase less repressible than the wild type. Control of ornithine transcarbamylase is normal (Table 2). The enzyme levels in the argR derivative are the same as for the wild type argR control. This strain appears to contain an operator mutation (designated arg11) conferring constitutive expression of argE and partially constitutive expression of argH. (b) Genetic duplications in the argEC B H region I t has been amply demonstrated that the region of the chromosome in the vicinity of argEGBH is very prone to the formation of closely linked duplications (Hill etal., 1969a,b; Elseviers etal., 1969; Cunin etal., 1970; Hill & Combriato, 1973; Beeftinck etal., 1974). Closely linked duplications are generally unstable and can be expected to result in a reduced frequency of recessive mutations for the duplicated genes. These properties were used to demonstrate t h a t in neither 6-8 nor 6-11 was the phenotype due to a genetic duplication. Phage Plkc was g r o ~ on 6P, 6-8 and 6-11, mutagenized with hydroxylamine and used to transduce strain 221YIN42, which carries a deletion of the ppc-argECBH genes, to Tpc + in the presence of arginine. Table 4 records the numbers of arginine TABLE 4

Frequency of mutations in argEC B H and reversion to wild-type after localized mutagenesis Donor

6P 6-8 6-11

Number of isolates with mutations in argE argBor G argH

7

8

5

4 13

17 7

32 10

Number of isolates with altered ability to grow on acetylhistidine plus ornithlne Lostt Gained c.2005 1 0

Plkc was grown on the donors, mutagenized with hydroxylamine for 24 h at 37~ and then used to transduce 22MN42 to ppc + in the presence of arginine. Selection plates with about 200 colonies]plate were replica-plated to test for arginine auxotrophs and ability of the transduetants to grow on supplemented minimal glucose medium containing acetylhistidine (50 pg]ml) plus ornithine (50 pg]ml). About 5000 transductants from each cross were screened. Besides the argE and argH auxotrophs. :~As stated above, almost all of these will be bradytrophs carrying 'leaky' mutations in argH. It is interesting that only a fraction of transductants with a mutation in argH are recovered as auxotrophs.

CONTROL

MUTATIONS

IN E. coli argEGBH

213

auxotrophs (in the three classes differentiable b y plate tests) that appeared among the transductants, and the numbers of clones altered in their ability to grow on acetylhistidine plus ornithine, namely those able to grow on this medium in the cross with 6P as donor, and those unable to do so where 6-8 or 6-11 was donor. I t is seen that (a) the control phenotypes of 6-8 and 6-11 (as shown b y their ability to grow on acetylhistidine plus ornithine) are retained b y the transductants and (b) auxotrophs arise with similar frequencies in the three classes. The argB and C mutants isolated from the crosses with 6-8 or 6-11 as donor grew on aeetylhistidine plus ornithine, while those with 6P as donor did not. I t is therefore unlikely t h a t the m u t a n t control phenotypes of 6-8 and 6-11 reflect duplications of all or part of the argECBH cluster. The expression of arg8 and arg11 in merodiploids was examined. One copy of the gene cluster was carried on an F-prime, retained as it covered a methionine requirement on the chromosome, and the other copy was present on the chromosome. The activities of three arginine enzymes in these strains, after growth in miuimal glucose medium containing arginine, are shown in Table 5. Comparison of lines 6 and 7 and 6, 8, 9 and 10 show t h a t the effects of arg8 and arg11 are cluster-specific and dominant. The results are consistent with the description of arg8 as a promoter mutation and arg11 as an operator mutation, although they do not prove cis-dominance. TABLE 5

Enzyme activities in the merodiploid strains Genotype

1 2 3 4 5 6 7 8 9 10

E n z y m e specified by argE argH

Haploids arg + argll F14 arg + F14 arg8 F14 arg11

5-6 62 5.5 16 41

Merodiploid8 arg +] F14 arg + arg+l F14 arg8 arg+/F14 arg11 arg11/F14 arg + arg11] F14 arg11

18 36 89 88 171

Ornithine transcarbamylase

0-063 0.088 0.093 0.055 0.14

0-1 0"2 0"5 0"5 0"6

0-20 0"13 0"29 0-30 0.44

0.1 0.1 0.1 0.1 0.2

The merodiploid strains were constructed as described in Materials and Methods. Only the genotype of the markers in the argECBH cluster are given; the F14 haploids are derivatives of AB1206 FE14 and the merodiploids and other haploids are derivatives of AT2681CB-1. The cells were grown in supplemented minimal glucose medium containing arginine but lacking methionine. The results are the m e a n of two independent determinations.

(e)

Deletion mapping

Table 6 gives the results of mapping arg8 and arg11 against deletions argDS11, argEC-1, argSUP400 and argSUPl02 (Fig. 2). arg8 lies between the ends of deletions argDS11 and argSUPl02 (crosses 1 to 4) and arg11 between the ends of deletions argEC-1 and argSUP400 (crosses 5 to 9). These results indicate the order ppc-arg8,

arg11-argBH.

A. P . B R E T S C H E R

214

A N D S. B A U M B E R G

TABLE 6

Deletion mapping Cross number

1 2 3 4 5 6 7 8 9 10

Parents

619-13 22DS 11 22-8-C132 619-11 22-8-E 131 6-SUP400 22-8-E131 619-2 619-14 22D811 619-15 619-11 P4XH-15 P4XH-11 22-11-E133 6-SUP400 22-11 -E 133 619-2 619-15 22-11-E133

l~elevant markers in cross

arg8 argC53 argDS11 arg8 argC132 argEC-1 argH50 arg8 argE131 argS UP400 arg8 argE131 ppc argSUPl02 arg11 argO53 argDS11 arg11 argC1 argEC-1 argH50 arg11 argC1 argEC-1 argHSO arg11 argE133 argS UP400 arg11 argE133 ppc argSUPl02 arg11 argC1 arg11 argE133

Recombinants

arg8 15 % arg8 100%

arg8

arg8 + 85%

0% 55% arg8 + 5% 93% 1"7%

arg11 + 1%

65 %

35%

argll +

arg11 100%

150 604 409

argl l +

argl 1 100%

229

arg11 +

98.3% 99%

150

arg11 +

arg11 arg11

250

arg8 +

45 % 95%

10O

arg8 +

arg8 arg11 7% arg11

Total recombinants tested

0%

200 479

arg11 +

0%

ss8

For each cross the donor is listed first a n d is followed by the recipient. All crosses were performed by Plkc transduetion, with the exception of the mating presented in line 7. Recombinants were selected on minimal glucose medium laeldng arginino and were streaked onto master plates to test for urrsoloeted markers by replica plating.

The unexpected finding that argll recombines with argEC-1 (cross 6) suggests that arg11 maps in the argC gene. The result was repeated when the cross was performed by conjugation (cross 7). Six recombinants with wild-type control on the plate tests were further tested by enzyme assay. In supplemented minimal glucose medium containing arginine, they showed levels of acetylornithinase and argininosuccinase typical of those found in 6P, whereas six random recombinants from the same cross which gave an arg11 plate phenotype had enzyme levels typical of those found in 6-11. To eliminate the possibility that arg11 might for some reason be unstable in this cross, reverting to wild-type at the observed frequency, cross 10 was performed, with arg11 in both the donor and recipient. No recombinant with wild-type control (as judged b y plate tests) was recovered among 888 Arg + recombinants examined. This hypothesis therefore seems unlikely. The location of arg11 will be referred to again in the Discussion. (d) Four-point crosses A number of four-point crosses were performed; their results and interpretations as shown in Table 7. Crosses 1 and 2 illustrate the ordering of argE14, which maps close to the right-hand end of argE, and argC53, the leftmost marker in argC, and

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A. P. B R E T S C H E R AND S. BAUMBERG

also the characteristically high degree of negative interference. Cross 3 is consistent with, but does not by itself prove, the order ppc-E14-8-HSO; the strain carrying arg8 together with argHSO, required for the reciprocal cross, could not be obtained. Cross 4 suggests the order ppc-8-C53-HgO. Crosses 5 and 6 suggest the order ppc-Eld11-H50. Crosses 7 and 8 demonstrate the difficulty in ordering argll and argCg3. Cross 9 is in accord ~4th the order ppc-11-C1-H50. Cross 10 appears to decisively favour the sequence Eld-11-C53-H50; the cross, however, yields a remarkably lfigh recombination frequency between arg11 and argC53, and it is conceivable t h a t elevated negative interference is causing the true quadruple crossover recombinant class not to be the rarest. Finally, crosses 11 and 12 are consistent with the order

ppc-8-11. 4. Discussion Previous genetic studies have indicated t h a t the argE and argCBH operons are transcribed divergently from their common boundary (Jacoby, 1972; Elseviers et al., 1972). The isolation of further control mutations wlfich map at or near this boundary strongly supports the evidence for divergent transcription. The control regions for the argE and argCBH operons must overlap, at least partially, as arg8 increases ar!IE expression while reducing argCBH expression and arg11 makes argE expression fully constitutive and argCBH expression partially constitutive. The effect of arg11 on the expression of argE, C, B, and H is paralleled by its effect on mRNA levels as determined by DNA/mRNA hybridization (Cunin & Glansdorff, personal communication). We suggest that arg8 affects the promoter functions of both the argE and argCBH operons while argll affects the operator functions. Mapping both with deletions and b y four-point crosses indicates that arg8 lies to the left of arg11. A single four-point cross is consistent with arg8 mapping to the right of argE14, which maps close to the argE-C boundary. The position of argll is uncertain. A four-point cross suggests that it maps to the left of argC53, the closest available argC mutation to the argE-C boundary, yet deletion mapping puts it on the right of argC53, as it recombines with deletion argEC-1. As mapping with deletions in short regions is generally more reliable than by recombination frequencies between single-site mutations, it seems likely that arg11 maps to the right of argC53, namely in the argC structural gene. I t could be argued that deletion argEC-1 consists of two closely linked deletions, one covering arg8 and the whole of argE and the other covering argC53 but not arg11 ; however, heteroduplex mapping (Mazaitis, Palehaudhuri, Glansdorff & Maas, unpublished data) as well as genetic data (Cunin et al., 1969) make this unlikely. I t m a y be noted that the ar9C53 mutation, despite reports to the contrary (Baumberg & Ashcroft, 1969; Ashcroft & Baumberg, 1970 Abstr. lOth Int. Congr. Microbiol. Mexico City), has no affect on the other genes of the cluster (Bretscher, 1974; Bretseher & Baumberg, manuscript in preparation). Elseviers et al. (1972) have described the properties of the deletion argSUPl02. Control over argH is reduced, although the derepressed level of expression is unaffected; argE is expressed at a very low level with reduced repressibility. They suggested that argSUPI02 removes the normal promoter for argE and part of the operator for argECBH, and proposed the order argE-PcBn-OcsH/O~.-PE.argCBH for the topography of the control regions. A model in which a single operator controls

CONTROL M U T A T I O N S I N E. coli argEGBH

219

both directions of transcription cannot account for arg11 which differentially affects transcription in the two directions. The simplest interpretation of the behaviour of arg11, as well as argSUPl02, tl~e operator m u t a n t s of J a c o b y (1972), and m u t a n t s described in a preliminary account b y Crabeel et a/. (1974), is t h a t there are two overlapping operators, ~ possibility envisaged b y Elseviers e~ al. (1972). The arg8 m u t a t i o n increases expression of one direction of transcription (argE) and reduces t h a t of the other (argC.BH). This m a y be because arg8 lies in the overlap of the two promoters; or increased transcription in one direction m a y reduce it in the other direction for some different reason. Crabeel et al. (1974) also mention m u t a n t s with similar properties. Divergent transcription from common control sites is not unique to the argECBH cluster. Hofnung (1974) has demonstrated t h a t the ~nalB cluster of E. coli is transcribed divergently from common control sites. Guha et al. (1971) and Cleary et al. (1972) have shown t h a t the E. coli b i o A B F C D cluster is divergently transcribed from regions located between bioA a n d bioB. Moreover, promoter mutations have been isolated which increase transcription of bioA and reduce transcription of bioBFCD (Ketner & Campbell, 1975). These authors also describe operator mutations which render b o t h directions of transcription constitutive. We would like to thank Drs N. Glansdorff and R. Cunin for generous sharing of strains, stimulating discussions, and communication of unpublished results, and Dr W. K. l~Iaas for helpful comments on this manuscript. Preliminary estimations of argECBH mRNA levels in these control mutants were carried out collaboratively during a visit to Dr Glansdorff's laboratory made possible by a European Molecular Biology Organisation short-term fellowship to one of us (S.B.).

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