Molec. gen. Genet. 162, 95-100 (1978) © by Springer-Verlag 1978

New Regulatory Mutations Affecting the Expression of the Threonine Operon in Escherichia coli K-12 Isabelle Saint-Girons* Unit6 de Biochimie Cellulaire, Departement de Biochimie et G6n+tique Microbienne, Institut Pasteur, 25 rue du Dr. Roux, F-75015, Paris (France)

Summary. The promoter of the threonine operon was joined to the structural genes of the lac operon in Escherichia coli K 12. The synthesis of fi-galactosidase was thus repressed by threonine plus isoleucine in the fusion strains. To isolate mutations which affect the expression of the threonine operon, alterations in the level of expression of the lacZ gene were selected. A new type of regulatory mutation was discovered.

previously found, are described. The isolation of strains in which lacZ is under the control of the thr promoter, according to Casadaban technique (Casadaban, 1976a) allows a new approach to the problems concerning the expression and the control of the tkr genes. Strains constitutive for the expression of lacZ and therefore for the threonine genes were then isolated.

Materials and Methods Introduction

Bacterial and Phage Strains

The threonine biosynthetic enzymes are multivalently repressed by threonine plus isoleucine (Freundlich, 1963). Their structural genes are organized as an operon thrABC (Th6ze, Saint-Girons, 1974). thrA is the structural gene for aspartokinase I-.homoserine dehydrogenase I (EC.2.7.2.4.) (EC. 1.1.1.3.). The two activities, carried by a single polypeptide chain (FalcozKelly etal., 1972) are inhibited by threonine, thrB and thrC respectively code for homoserine kinase (EC.2.7.1.39) and threonine synthase (EC.4.2.99.2). Mutations causing derepression of the thr operon have been mapped in two genes. One of the genes, thrO is located immediately proximal to the structural genes and has the properties of an operator-promoter region (Saint-Girons, Margarita, 1975; Gardner, Smith, 1975). The other gene thrS codes for the threonyl tRNA synthetase:threonyl tRNA or/and threonyl tRNA synthetase are implicated (Johnson et al., 1977) in the regulation of the threonine operon. In the present work, mutations affecting the expression of the threonine operon, distinct from those

Bacterial strains are described in Table 1. Phage strains are given in Table 2.

Media and Chemicals Minimal and complete media used have been described previously (Th6ze, Saint-Girons, 1974). Glucose and lactose were used at a concentration of 4 mg/ml. Amino acids were used at a concentration of 1 mM except when specified. Mac Conkey lactose plates were used for monitoring the expression of the lac genes (Miller, 1972). DL-~-amino-B-hydroxyvaleric acid (AVA) from Sigma was used at a concentration of 2.6 mg/ml. 5-Bromo-4-chloro-3 indolyl fi-D-galactoside (XG) from Sigma, was used at a concentration of 40 ~ag/ml.

Enzymes Assays fi-galactosidase (Gz) assay was performed according to Miller, 1972. Homoserine dehydrogenase and threonine deaminase assays were performed as previously described (Truffa-Bachi, Cohen, 1970; Szentirmai, Umbarger, 1968). Protein concentration was determined by the biuret method (Gornali et al., 1949).

Genetic Techniques * This paper is part of a thesis presented by Isabclle Saint-Girons in partial fulfillment of the requirements for the Dr. Sc. degree from the University of Paris

Mutagenesis. Mutagenesis was performed with ethyl-methane sup fonate followed by penicillin enrichment (MiIler, 1972).

0026-8925/78/0162/0095/$01.20

I. Saint-Girons: Expression of the Threonine Operon in Escherichia coli K-12

96 Table 1. Bacterial strains

The nomenclature used for the fusions strains is from Silhavy et al. (1976). The symbol 4~ designates fusions that place a gene (lacZ) under the control of another promoter. Operon fusions are designated cb (thrC-lacZ+). Fusions between structural genes which can result in the formation of hybrid protein are called protein fusions and designated q~ (thrC-lacZ) hyb. a

Strain

Genotype a

M C 4100 G T 415 GT 419 GT 432 G T 549 G T 550

F-, MC MC MC MC MC

G T 539

MC 4100 with q5 (thrA2_lacZ+)3_l

G T 527 G T 533

MC 4100 with ~b (thrAl-lacZ)hyb2_ ~ MC 4100 with ~ (thrA2-1acZ)hyb3_ ~

GT GT GT GT GT

GT GT GT GT GT

604 614 608 609 615

G T 64 GT 342

References and comments

araD139, A(lac)U169, strA, thi 4100 with thrClO59::Mu cts 4100 with thrA~lO46::Mu cts 4100 with thrA21057::Mu cts 4100 with cb (thrC, lacZ+)~_ ~ 4100 with ~b (thrC-lacZ+)~_2

533 533 533 533 533

Lac + constitutive of G T 533

"thrX" "thrX"

pro 1001, pyrA 53, serB22, lac-lO00 : : Mu- 1 metLM lO05, lysC lO04 trp his-proC, thrS1029, str mal, ara, gal, lac, metLMlO05, lysClO04

Bacterial D N A carried

Origin a n d Ref.

2pl (209)

laeA? YZO'-AW209trp 'AB': : ( + Mu') 2pl (209) with lacZUll8 thrOABC

Casadaban, 1976a

)AthrA1 kdthrA 2 2dthrB 2dthrC

Derivatives of kdthrC with mutations in different thr genes

Th6ze and Saint-Girons 1974 Johnson, Cohen and Saint-Girons, 1974

thrA

Phage

kdthr

GT 432 and G T 419 were lysogenized with 2pl (209)(118)

thrO1028 thrO1029 "thrX"

with with with with with

Table 2. L a m b d a transducing strains

kpl (209)(118)

Casadaban, 1976a This paper This paper This paper GT 415 was lysogenized with kpl (209)

serB .

.

.

.

.

ihrA.1 .

.

.

k \ \ \ \ "- . \ \ \ \ \ \ \ \ \

-thrA2

"~ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ~

osportokinose -homoserine

thrB ~

\

\

\

\

~

\

thrC \

\

\

\

\

\

\

\

\

\

\

\

pyrA \

~

.

.

.

.

.

.

.

homoserine

dehydrogenase I ki;ose synthelase

Casadaban, 1976a Schrenk and Weisberg, 1975 Th6ze and Saint-Girons, 1974

asp(~rt(Ite ~C> AspP~ASA! ~2~ h°m°serine i :=:C>HSP

0~m

* methionine

~=> threonine i

i isoleucine

i t

lysine Fig. 1. The threonine operon and the biosynthetic pathway of amino acids deriving from aspartate. 1 The reaction is catalysed also by aspartokinase II and aspartokinase III. 2 The reaction is catalysed also by homoserine dehydrogenase II

Transduction. Transduction with Plvir was performed according to Miller (1972) and transduction with Zdthr as previously described by Th6ze et al. (1974). Construction and Identification of Threonine Auxotrophs by Mu Insertions. Independent clones ol~ M C 4100, carrying a lac deletion were mutagenized by M u c t s (Boram, Abelson, 1971) at 30 ° C. M u cts contains a mutation in the repressor gene which results in a temperature sensitive repressor. A penicillin enrichment (Miller, 1972) was used and 24 independent threonine auxotrophs were obtained. The T h r - phenotype found is due to M u insertions in thrA as well as in thrB or thrC. The isofunctional enzymes, aspartokinase II-homoserine dehydrogenase II and aspartokinase III allow the biosynthesis of methionine and lysine in the T h r - m u t a n t s (Fig. 1). M u insertions in thrA abolish thrB and thrC expression and are therefore indistinguishable from insertions in thrB (which exert also a polar effect on expression of thrC). We therefore transduced

with a Plvir bacteriophage the thr mutations in a metLM, lysC strain, G T 64, i.e. devoid of the aspartokinase II-homoserine dehydrogenase II and aspartokinase III activities and also lysogenic for Mu. Set + transductants were selected on a medium containing uracil (0.5 mM), arginine, proline, threonine, methionine, lysine and diaminopimelic acid (0.1 mM). A m o n g those transductans, auxotrophs for threonine plus methionine plus lysine plus diaminopimelic acid (0.1 m M ) are isolated; the mutations were then identified as thrA, thrB or thrC by complementation analysis with the kdthr phages carrying different threonine mutations (Th6ze et al., 1974). The technique of M u mutagenesis does not preclude multiple insertions. A non essential gene can be affected impairing the selection of thr-lac fusion strains. We thus transduced the threonine auxotrophs to Thr + with a Plvir grown on M C 4100. Thr + transductants liberating M u c t s at high temperature reveals the presence of at least another bacteriophage M u outside the threonine locus: the corresponding threonine auxotrophs strains were not utilized.

I. Saint-Girons: Expression of the Threonine Operon in Escherichia coli K-12 /,(, PT

1

A1

PT

I

Table3. The thr-lac fusion strains were assayed for/Lgalactosidase following the introduction of the 2dthr bacteriophage

A2

A1

i

8

A2

I

C

B

I

Strain a

C ~ MU ~C ==~=i=

I

I

IPTI A l l

A2

I

B

I

Z

Cq M U

0 p

PA

/Lgalactosidase activity b MM MM MM + Thre+ Methioonine nine +Isoleu- +Lysine ° cine c

Xp

y

97

ee-

pression ratio d

B ~ MU q I

IB A* I0 Z Y I ~ I ~

l

Xp

N TMU

IC

...................... .........................

Fig. 2a-L Schema for isolating thrC-lacZ fusion strains, a The threonine operon ; arrows indicate directions of transcription. The threonine promoter is indicated PT. b A Mu (cts) phage is inserted in the thrC gene of a bacterial strain with a lac deletion, c The 2pl (lac, Mu') phage shown here is 2pl (209) which does not contain an intact attachment site. The lac promoter is missing. d The Mu lysogen (b) was lysogenized with this )0p (lac, Mu') phage (c). Recombination occurs between the homologous Mu DNA. Since the Mu (cts) phage is thermoinducible, selection for Lac ÷ survivors at 42 ° C will yield fusions of the lacZ gene to PT. Fusions resulting from deletions such as (e) possess an intact lacZ gene and are designated "operon fusions". Fusions between structural genes which can generate a hybrid protein result from deletions such as (f). These "protein fusion" come from lysogenization with 2pl (209)(118) carrying an early NcZ mutation U118. Primes as in Mu' or 'C indicate that the corresponding DNA is deleted on the side the prime is written

Selection of the thr-lac Fusion Strains, The general procedure described by Casadaban (1976a) was used to isolate strains carrying lacZ under the control of the threonine promoter. We describe briefly the characteristics of our system (Fig. 2). The mutants carrying Mu insertions in the thr operon were lysogenized with 2pl (209) or 2pl (209)(118). These lambda phages carry the lacZ gene as well as part of Mu; the ,t attachment site is missing as well as the lac promoter. Lysogens were selected using a 2 clear phage to kill non lysogenic cells (Shimada et al., 1973). Deletions of Mu which fused a new promoter to the lac genes are isolated as follows. The lambda lysogens were plated on a minimal medium containing lactose and threonine (0.5 raM). 0.5 mM satisfies the growth requirement of Thr- mutants. After incubation at 42°C for 24 h, the plates were shifted to 37° C. Lac + colonies appearing oi1 these plates were purified on a minimal medium containing glucose, XG and threonine (0.5 mM). The blue colonies (Lac +) were tested for having the lac genes regulated by the controls of the threonine genes, as described in Results. These fusion strains when obtained after lysogenization with )@1(209) were called "operon fusion"; when 2p 1(209) (118) carrying an early lacZ mutation UI18 was used, they were called "protein fusion". The generation of a hybrid protein requires a deletion which extend past the U118 mutation into lacZ. The deletion must however leave enough of the lacZ gene to allow for the production of active/%galactosidase.

GT 549 ~)(thrC-lacZ+ ) l - 1 GT 550 q~(thrC-lacZ+ )l _ ; GT 539 @(thrAz-lacZ+ )3 - 1 GT 527 ~b(thrA 1-lacZ)hyb z _ GT 533 4)(thrA2-lacZ)hyba _ 1

200

87

183

2.3

460 200

430

2.3

720 360

ND °

2

600 240

ND

2.5

160

ND

2.6

62

a ~(thrC-lacZ+): operon fusions; ~b(thrC-lacZ)hyb: protein fusions b /~-galactosidase activity is expressed as in Miller (1972) ° The cells are grown at least three generations in exponential growth; the amino acids are added to 5 raM. The carbon source used is glucose d Ratio between enzymatic levels in minimal medium (MM) compared to that in repressing conditions of the same strain N D : Not done

Results

Isolation and Characterization o f the thr-lac F u s i o n S t r a i n s T o f u s e t h e lac s t r u c t u r a l g e n e s t o t h e t h r e o n i n e p r o m o t e r in E s c h e r i c h i a coli, t h e lac g e n e s w e r e f i r s t t r a n s p o s e d n e a r to t h a t p r o m o t e r a n d t h e n deletions w e r e i s o l a t e d w h i c h j o i n e d t h e lac g e n e s t o t h a t p r o moter. The isolation of strains with bacteriophage M u i n s e r t i o n s in t h e t h r e o n i n e g e n e s w a s t h e f i r s t s t e p . T h e lac g e n e s w e r e t h e n t r a n s p o s e d t o t h e site of the insertion by lysogenizing strains containing Mu i n s e r t i o n s in t h r A , t h r B o r thrC, w i t h t w o d i f f e r e n t 2 p (lac, M u ' ) p h a g e s . W e o b t a i n e d t h e r m o r e s i s t a n t s t r a i n s in w h i c h M u w a s d e l e t e d f r o m s t r a i n s G T 4 1 5 ( t h r C : : M u cts), G T 4 1 9 ( t h r A 1 :: M u cts) a n d G T 4 3 2 ( t h r A z : : M u cts). S o m e of the t h e r m o r e s i s t a n t strains are s h o w n to carry l a c Z u n d e r the c o n t r o l o f the t h r e o n i n e prom o t e r in t h e f o l l o w i n g w a y . T h e y w e r e a s s a y e d f o r

I. Saint-Girons: Expression of the Threonine Operon in Escherichia coli K-12

98

Table 4./3-galactosidase activity from regulatory m u t a n t s and their parental strain

Table 6. Dominance studies in extracts from two regulatory mutants and their parental strain lysogenized with 2dthr

Strain"

/~-galactosidase activity b

Derepression ratio c

Strain a

Partial genotype b

GT GT GT GT GT GT

84 950 800 560 320 420

1 11 9.5 6.5 3.8 5

GT 533

thrAz-lacZ + ()~dthrA[A~ B+ C +) thrA2-lacZ + ()LdthrA~ A~ B+ C +) thrA~-lacZ + (2dthrA+ A+ B+ C +)

533 604 608 609 614 615

a Cells are grown in minimal medium containing glucose plus threonine (5 m M ) plus isoleucine (5 m M ) b /~-galactosidase activity is expressed as in Miller (1972) ° Ratio between enzymatic levels of the m u t a n t s compared to the parental strain, both in repressing conditions

(2dthr) GT 604 ()~dthr) GT 614

(2dthr)

/3-galactosidase c

Homoserine debydrogenase I t

60

80

570

60

350

70

a

See ~ Table 4 thrA 1 codes for aspartokinase I activity, thrA2 codes for homoserine dehydrogenase I ° See Table 5

b

Isolation of Threonine Regulatory Mutants Table 5. Homoserine dehydrogenase I and threonine deaminase activities in extracts from Thr + transductants of the regulatory mutants and their parental strain Thr + transductants of strain"

Homoserine dehydrogenase I activity b

Threonine deaminase activity b

GT GT GT GT GT GT

60 650 175 340 65 60

50 56 53 52 ND ND

533 608 609 615 604 614

a Cells are grown in the presence of threonine plus isoleucine (5 raM) b Activity is expressed in nanomoles of product/mn/nag/protein

/3-galactosidase (Gz) following the introduction of a lambda phage transducing the whole threonine opeton: 2dthr is inserted at the normal attachment site. The specific activity of Gz in the merodiploids grown in the presence of isoleucine (5 raM) plus threonine (5 raM) [repressing conditions[ and without addition are compared (Table 3). The repression by threonine plus isoleucine of the synthesis of the IacZ gene is of the same order (2 to 3) than that of the threonine genes observed in the parental strain (Table 3). Repression by threonine is specific since addition of methionine or lysine to the growth medium has no effect on the Gz specific activity. This implies that the Iac genes are regulated by the controls of the thr operon. The expression of lacZ varied widely from one thr-lae fusion strains to another as it was found in other fusion strains.

In strain GT 533, used to select constitutive mutants for Gz, the lacZ gene is inserted in thrA2, allowing the expression of the aspartokinase activity but not of the other threonine biosynthetic enzymes. It was ascertained that the level of Gz is indeed higher in the mutants selected as red colonies on Mac Conkey lactose (see Materials and Methods) than in G T 533 (Table 4). We distinguish regulatory mutations linked to the threonine operon from those located elsewhere, in the following way: Thr ÷ transductants were selected from the constitutive mutants by Pj grown on MC4100. To determine the presence or absence of the constitutive mutations in the transductants, the homoserine dehydrogenase activity ( H D H I ) was tested (Table 5). Three mutants (GT 608, 609, 615) showed no linkage between the threonine operon and the control mutation; two mutants (GT 604 and 614) had regulatory mutations linked to the thr locus. In addition, lambda phages were induced by UV light from those mutant strains as well as from G T 533. These lambda phages carrying the lacZ gene fused to the threonine operon were used to lysogenize MC4100 (Alac). The lysogens were streaked on Mac Conkey lactose: they were red in the case of G T 604 and G T 614 and white with a red center in the case of G T 533, 608, 609, 615. This was an additional proof that the regulatory mutations of G T 608, 609 and 615 are indeed outside the threonine operon, differing from those of 604 and 614. It should be added that lysogenization of MC4100 with the phages excised from the fusion strains occurs primarily by recombination with the homologous threonine region. Consequently, some lysogens, white with a red center, should be isolated by recombination with

I. Saint-Girons: Expression of the Threonine Operon in Escherichia coli K-12 Xp I

\

I

NI

/~

-I°'1

/ ~

A1 I A2'~'z

. . . . . I 0

i

AI i

A2

"

I J

!' YI .....

B

1

/

e

Fig. 3. Scheme of lysogenization of MC 4100 with 2thrO- thrA~ (thrA~-lacZ) hyb. 1 Crossing over yielding red lysogens on Mac Conkey lactose, carrying thrO- and lacZ. 2 Crossing over yielding white Iysogens on Mac Conkey lactose, carrying thrO + and laeZ. * Mutation in thrO

the phages isolated from GT 604 and 614. Those lysogenes carry the lacZ gene but not the linked threonine regulatory mutation (see Fig. 3).

Characterization of Regulatory Mutations Linked to the Threonine Operon A cis- trans test was performed to identify the mutations, in GT604 and 614, affecting the expression of the threonine operon. Merodiploid derivates of GT604 and GT614 were constructed by lysogenization with a lambda phage transducing the whole threonine operon. In such merodiploids grown in the presence of threonine plus isoleucine (repressing conditions), Gz and H D H I were assayed (Table 6). Gz is still at a high level whereas the level of H D H is not affected by the mutation. The mutation is active in the expression of genes located in the cis position and not in trans. They are moreover localized in the operator region (see following paper), thus presenting all the characteristics of operator constitutive mutations, cis-dominants.

Characterization of Regulatory Mu/ations Unlinked to the Threonine Operon The regulatory mutations in GT608, 609, 615 that are not lost in the corresponding Thr + transductants are therefore localized outside the threonine region. These transductants are resistant to AVA as are thrO mutants (Saint-Girons, Margarita, 1975) but in contrast to them, they are not threonine excretors. The synthesis of the threonine biosynthetic enzymes is not repressible by threonine plus isoleucine (Table 5). The threonine deaminase, the first enzyme of the isoleucine pathway, has the same specific activity in both the mutants and the parental strain (Table 5).

99

In a previous communication (Johnson et al., 1977), we have shown that the threonyl t R N A synthetase is involved in the regulation of the threonine biosynthetic enzymes. In order to determine whether the regulatory mutations from G T 608, 609, 615 isolated in the present work belong to similar mutational events, we performed the following experiments. The strain G T 342 is a threonine auxotroph due to a mutation in the thrS gene coding for threonyl tRNA synthetase. PI was grown on G T 608, 609 and 615, the recipient strain was G T 342. Thr ÷ transductants were selected and among them resistance to AVA was scored. Hundred transductants tested in each case were sensitive to AVA. This proves that the regulatory mutations from G T 608, 609 and 615 are not linked to thrS. Taken together, our experiments show that a new class of regulatory mutations "thrX" distinct from thrO and thrS has been discovered.

Discussion

The analysis of the regulation of gene expression was performed mainly in systems in which regulatory mutations are easily recognized. Such is the case of the catabolic pathways; for example, very sensitive solid media are used to monitor directly the expression of the lacZ gene. For anabolic pathways, a similar direct visualization of mutations causing derepression of genes is not available; one exception being the phenotypic character of the histidine constitutive mutants. The utilization of analogues is a powerful selection technique, however the biochemical characterization of the constitutive mutants is necessary. In the case of the threonine operon, the only methodology available is the resistance to AVA and the excretion of threonine. The transposition of segments of D N A to predetermined positions, resulting in the fusion of operons unfold a general approach to the study of gene expression. Using Mu insertions and 2 transducing phage, transposition and fusion of the lac genes to a new promoter were performed. In the case of arabinose and maltose, ara-lac fusion strains (Casadaban, 1976a) and mal-lac fusion strains (Silhavy et al., 1976) were isolated and new types of regulatory mutants were obtained (Casadaban, 1976b; M. Schwartz, unpublished results). In the fusion strains described in our paper, the lac genes are controlled by the regulation of the promoter of the thr genes. Selection of regulatory mutations consist then in the direct visualization and isolation of strains in which the level of the lacZ gene is altered; mutants were isolated on a lactose minimal medium in the presence of threo-

100

I. Saint-Girons: Expression of the Threonine Operon in Escherichia coli K-12

nine at high concentration. Two types of regulatory mutations were found: one localized in the threonine operator region [similar to thrO mutations already described (Saint-Girons, et al., 1975)], the other outside of this region. Three independent mutants of the last category were studied: they show similar properties, i.e. they are resistant to AVA and do not excrete threonine. However they can belong to different genes. We have shown that the " t h r X " mutation implicated is not cotransducible with the threonine locus or with the gene thrS coding for the threonyl t R N A synthetase. We demonstrate then, the existence of a new type of regulatory mutations " t h r X " distinct of thrO and thrS, causing derepression of the thr operon. Acknowledgments. J. Beckwith welcomed me in his laboratory and allowed me to isolate the thr-lac fusion strains. I thank Maxime Schwartz and Tom Silhavy for stimulating exchanges of ideas. I would like to thank P. Truffa-Bachi, D. Haas and A. Rambach for helpful suggestions about this manuscript. I wish to thank G.N. Cohen for his support.

References Boram, W., Abelson, J.: Bacteriophage Mu integration: On the mechanism of Mu-induced mutations. J. molec. Biol. 62, 171-178 (1971) Casadaban, M.: Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. molec. Biol. 104, 541-555 (1976a) Casadaban, M.: Regulation of the regulatory gene for the arabinose pathway, araC. J. molec. Biol. 104, 557-566 (1976b) Falcoz-Kelly, F., Janin, J., Saari, J., Veron, M., Truffa-Bachi, P., Cohen, G.N. : Revised structure of aspartokinase I-homoserine dehydrogenase I of Escherichia coli K 12. Evidence for four identical subunits. Europ. J. Biochem. 28, 507-519 (1972)

Freundlich, M. : Multivalent repression in the biosynthesis of threonine in Salmonella typhimurium and Escherichia coli. Biochem. biophys. Res. Commun. 10, 277-282 (1963) Gardner, J.E., Smith, O.H.: Operator-promoter functions in the threonine operon of Escherichia coli. J. Bact. 124, 161-166 (1975) Gornall, A.G., Bardawill, C.J., David, M.M.: Determination of serum protein by means of the biuret reaction. J. biol. Chem. 177, 751 756 (1949) Johnson, E.J., Cohen, G.N., Saint-Girons, I.: Threonyl transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia eoli. J. Bact. 129, 66-70 (1977) Miller, J.H. : Experiments in Molecular Genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory 1972 Saint-Girons, I., Margarita, D. : Operator constitutive mutants in the threonine operon of Escherichia coli K 12. J. Bact. 124, 1137-1141 (1975) Schrenk, W.J., Weisberg, R.: A simple method for making new transducing phage lines of coliphage 2. Molec. gen. Genet. 137, 101-107 (1975) Shimada, K., Weisberg, R., Gottesman, M.E.: Prophage lambda at unusual chromosomal locations. I I - M u t a t i o n s induced by bacteriophage lambda in Eseherichia coli K 12. J. molec. Biol. 80, 297-314 (1973) Silhavy, T.J., Casadaban, M.J., Shuman, J.A., Beckwith, J.R.: Conversion of fl-galactosiderase to a membrane bound state by gene fusion. Proc. nat. Acad. Sci. (Wash.) 73, 3423 3426 (1976) Szentirmai, A., Umbarger, H.E. : Isoleucine and valine metabolism of Escherichia coli. XIV-Effect of thiaisoleucine. J. Bact. 95, 1666 1671 (1968) Theze, J., Saint-Girons, I.: The threonine locus of Escherichia coli K 12. Genetic structure. Evidence for an operon. J. Bact. 118, 990-998 (1974) Truffa-Bachi, P., Cohen, G.N.: Aspartokinase I and homoserine dehydrogenase I (Escherichia coli K 12). Meth. in Enzymol. 17A, 694-696 (1970)

Communicated by G. O'Donovan Received December 27, 1977