Cloning and characterization of the mutated threonine operon (thrA15A25BC) of Serratia marcescens

Gene.

151

151-158

57 (1987)

Elsevter GEN 02108

Cloning and characterization of the mutated threonine operon (thrA,SA,5BC) of Sewatia lttarcescens (Recombinant

Takahisa

DNA;

TnlOOO mutagenesis;

Sugita, Sahuro Komatsubara

gene dosage;

and Masahiko

maxicell;

threonine

production)

Kisumi

Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Osaka 532 (Japan) Received Revised

25 March

1987

27 May 1987

Accepted

30 May 1987

SUMMARY

The entire threonine operon (thrA,5_4,5BC) of Serratia marcescens TLr156, which lacks threonine-mediated feedback inhibition of both aspartokinase I (AK I) and homoserine dehydrogenase I (HD I), was cloned on a multicopy plasmid pLG339. Hybrid plasmid pSK301 carried a 6.5kb chromosomal DNA. Several derivatives of pSK301 with TnlOOO insertions were obtained. By examining the phenotypes and the physical maps of these plasmids, we could define the loci of the thrA,SA,S, thrB, and thrC genes. The thrA,SA,S and thrC gene products were identified by the maxicell method as proteins with M,s of 85000 and 43000, respectively. The thrA,SA,S genes encode a single polypeptide similar to the thrA,A, genes of Escherichia coli. Plasmid pSK301 was introduced into S. marcescens T-l 112, in which both AK I and HD I are produced constitutively. The resulting transformant carried five to six copies of pSK301 per chromosome and produced the AK I and HD I enzymes at three to four times higher level than control strain T-11 12[pLG339]. Strain T-l 112[pSK301] produced four times higher levels of threonine than strain T-1112[pLG339], yielding about 35 mg of threonine per ml of a medium containing sucrose and urea.

INTRODUCTION

S. marcescens resembles E. coli K-12 in the regulatory mechanism and genetic background for Correspondence Applied Kashima,

to: Dr. S. Komatsubara,

Biochemistry,

Tanabe

Yodogawa-ku,

Abbreviations:

Osaka

AK, aspartokinase;

serine dehydrogenase;

streptomycin; transposon; state;

Laboratory

Co.,

532 (Japan)

Ltd.,

a, sensitivity;

bp, base pair(s);

UV, ultraviolet

electrophoresis;

SDS, sodium

Tc, tetracycline; light;

of

3-16-89,

Tel. (06)300-2571.

kb, 1000 bp; Km, kanamycin;

cation; PAGE, polyacrylamide-gel r, restriction;

Research

Seiyaku

m, modifiR, resistance;

dodecyl

thr, threonine

[ 1,designates

HD, homo-

sulfate; (operon);

Sm, Tn,

plasmid-carrier

::, novel joint.

0378-l 119/87/$03.50

0 1987 Elsevier

Science Publishers

B.V. (Biomedical

threonine biosynthesis. The thr operon is composed of four genes: thrA 1, thrA,, thrB, and thrC, which code for AK I (EC 2.7.2.4), HD I (EC 1.1.1.3), homoserine kinase (EC 2.7.1.39), and threonine synthase (EC 4.2.99.2), respectively (Komatsubara et al., 1979). AK I and HD I are feedback-inhibited by threonine and repressed by threonine plus isoleucine. We have obtained the thrA,S and thrA,S mutants which lacked the feedback inhibition of AK I and HD I by isolating threonine-resistant mutants from a strain sensitive to this amino acid (S.K. and M.K., manuscript in preparation). The hnrA 1 mutant which led to derepressed expression of AK I, HD I, and ilv enzymes was selected among Division)

152

/I-hydroxynorvaline-resistant

mutants

(Komatsu-

bara et al., 1978a; 1979). These regulatory were transductionally tions

releasing

combined

feedback

threonine-producing

to

(a) Strains, plasmids and media

construct

(Komatsubara

Derivatives of E. coli K-12 and S. marcescens Sr41 (Matsumoto et al., 1975) are listed in Table I.

et al.,

1983). To construct

threonine-hyperproducing

strains by

amplifying mechanism

the thr operon and to understand the for feedback controls more clearly, we

intended

to

clone

the

entire

thr

Plasmid

operon

tration at

of

both

AK I and HD I. This paper describes cloning of the entire thr operon of S. marcescens TLr1.56, identitication of the thr operon gene products by the maxicell method, and a gene dosage effect on threonine pro-

obtained

from

Dr.

N.G.

to 0.5%. Required

1 mM.

acids were used

~-amino

DL-/&Hydroxynorvaline

was

used

at

0.1 mM. Antibiotics were added at the specified concentrations: Km, 100 pg/ml; Sm, 50 pg/ml; Tc, 10 pg/ml. The medium for threonine production contained 15% sucrose, 1.5% urea, 0.05 y0 (NH&SO,, 0.1% K,HPO,, 0.1% MgSO, .7H,O, 0.02% corn steep liquor, and 1% CaCO,.

duction.

TABLE

was

Davis and Mingioli (1950) was modified by omitting the Na * citrate and increasing the glucose concen-

threonine-mediated

inhibition

pLG339

Stoker (Stoker et al., 1982). The minimal medium of

(thrA ,5A,5BC) of S. marcescens TLr 156 which lacks feedback

AND METHODS

mutations

with other muta-

controls

strains

MATERIALS

I

Bacterial

strains

used

Strain

Genotype

Source

a

or reference

E. coli Bachmann

thr-1 IeuBl thi-1 supE44 lacy1 tonA

GT12

thrB metLM

IysC pro ser

GT13

thrC metLM

1ysC pro ser

GT15

thrA, metLM

IysC pro ser

Theze et al. (1974)

GT16

thrA , metLM

IysC pro ser

Thbze et al. (1974)

GT121

As GT12 except for SmR

SmR derivative

of GT12

GT131

As GT13 except for SmR

SmR derivative

of GT13

GT151

As GT15 except for SmR

SmR derivative

of GT15

GT161

As GT16 except for SmR

SmR derivative

of GT16

GT1211

As GT121 except for srlC300::Tn10

recA56

Hfr cross: JClO240

x GT121

GT1311

As GT131 except for srlC300::TnZO

recA56

Hfr cross: JC10240

x GT131

GT1515

As GT151

except for srlC300::TnZO

recA56

Hfr cross: JC10240

x GT151

GT1614

As GT161

except for srlC300::TnZO

recA56

Hfr cross: JC10240

x GT161

JC10240

HfrC(P045),

srlC300::TnZO

hsdR hsdM

(1972)

C600r - m

Theze et al. (1974) Thtze

et al. (1974)

Laszlo

recA56 thr-300 ilv-318 rpsE300

and Alvin (1980)

Mark (1978)

MG1063

F’,

MM294

thi hsdR

red1

Backman

N1790

recA99 uwA54

Horii et al. (1981)

et al. (1976)

s. mareeseens D-60

ilvA

Komatsubara

T-1112

thrA,S thrA,5 hnrA 1 ilvA

S.K. and M.K.

TLr156

thrA,S thrA,S ilvA

TT392

NW-

(manuscript

thrA,S, lack of feedback

Takagi

r - ApS Kms

a In E. coli K-12, the thrA gene specifies a single polypeptide (Thtze

inhibition

and Saint-Girons,

in preparation)

SK. and M.K. (manuscript

to AK I and HD I, respectively

et al. (1978b)

chain but is composed 1974). Genotype

of HD I; hnrA 1, derepression

of two cistrons,

designations:

(1985)

thrA, and thrA,, which correspond

thrA,S, lack of feedback

of AK I, HD I, and ilv enzymes.

in preparation)

and Kisumi

inhibition

of AK I;

153

(b) General methods

RESULTS

Standard procedures for DNA manipulations were as described by Maniatis et al. (1982). The maxicell method (Sancar et al., 1979) was employed for labelling proteins encoded by plasmids. SDS-PAGE of proteins was carried out according to Laemmli (1970). Plasmid copy number was determined by fluorescence densitometry as described by Projan et al. (1983). Transformation of E. coli or S. marcescens was carried out following the method of Takagi and Kisumi (1985). Plasmids were introduced into S. marcescens by transformation first into strain TT392, which is deficient in DNA restriction enzymes, but proficient in modification. They were re-isolated and transformed into strains D-60 and T-1112.

(a) Cloning

(c) Assay of enzyme activity and threonine productivity The activities of AK and HD were measured as described by Kisumi et al. (1977). Threonine productivity was determined according to Komatsubara et al. (1979).

TABLE

AND DISCUSSION

of

the

mutated

threonine

operon

(thrA,SA,SBC) The chromosomal DNA of S. marcescens TLr 156, which lacks feedback inhibition of both AK I and HD I, was prepared and hybrid plasmids carrying the chromosomal fragments on the Sal1 site of the cloning vector pLG339 were constructed. E. coli C600r - m - (thrB) was transformed with the mixture of these hybrid plasmids, and then spread on minimal agar plates supplemented with leucine, thiamine, and threonine analog p-hydroxynorvaline. Since thrA,SA,S mutations render strain TLr156 resistant to fi-hydroxynorvaline, /I-hydroxynorvaline-resistant and ThrB + clones were selected as the candidates carrying cloned thrA,SA,SBC genes. Subsequently, these clones were examined for threonine excretion by spotting cells on a minimal agar plate seeded with a threonine auxotroph. Thus, we obtained one transformant showing threonine excretion. This strain was designated C600rm[pSK301] and tested for AK and HD (Table II). These enzymes of C600r-m- [pSK301] were insensitive to feedback inhibition by threonine as were those of strain TLr156. On the contrary, the two enzymes were feedback-inhibited by threonine in strains D-60 (wild type) and C600r-m-[pLG339]. These results convinced us that this transformant carried the chromosomal fragment carrying thrA,5A25 genes.

II

Lack of threonine-mediated

inhibition

of aspartokinase

and homoserine

dehydrogenase

activities

in an Escherichiu coli strain carrying

pSK301 Inhibition

Strain

(%)

Aspartokinase

a

Homoserine

dehydrogenase’

E. coli C600r _ m _ [pLG339]

91

68

C600r -m

11

18

85

19

[pSK301]

S. marcescens D-60

-23

TLr156 a Assayed

as described

lysine-sensitive b L-Threonine

in MATERIALS

AK activity. was added

L-Threonine at 10 mM.

9 AND METHODS, was added

section c. L-Lysine was added at 50 mM to the reaction

at 50 mM.

mixture to inhibit

154

The plasmid from this strain, designated pSK301, was introduced into E. coli MM294(r- m +). It was re-isolated and transferred into E. coli GT16(t/zrA,), GTlS(thrA,), GT12(thrB), and GT13(thrC). The transformants were purified and tested for the presence of the thrA,S, thrA,S, thrB, and thrC genes by complementation tests. Since pSK301 complemented all four mutations, we concluded that the cloned segment carries the entire thr operon (thrA,SA,SBC) of S. marcescens TLr156.

5A25BC

fhrB, and ~/WCgenes

(b) Location of the thrA,5A,5, on pSK301

The restriction map of pSK301, which consists of a 6.5-kb chromosomal DNA fragment and 6.2-kb pLG339 DNA, was constructed (Fig. 1). To locate the thrA,SA,SBC genes on pSK301, its derivatives with TnlOOO insertions were obtained and the sites of insertions were mapped by restriction enzyme cleavage patterns (Fig. 2). The phenotypes of these plasmids were also determined by examining their ability to complement the thrA , , thrA,, thrB, and thrC

h

s2

thrB

\ I

of the thrA,5A,SBC

on plates and pSK301

by Sal1 and Hind111 digestion

are oriented Insertions ThrA,5

Insertions

on pSK301

42 and 43 are pLG339

20-27,

28-37,

and

+ A,5 + B + C + phenotypes,

under the map of TnZOOO insertions. bottom.

orientations

are indicated

SalI,-SalI,

fragment

The positions

insert

from

represents

the

of the genes and their

on the outer circle. pSK304 carries the

on pLG339.

Abbreviations

S, SalI; EI, EcoRI;

tion enzymes:

EV, EcoRV;

for the restricH, HindHI;

M,

MluI; X, XhoI.

pLG339 h

with TnlOOO insertions

38-41

respectively.

were isolated.

of the thr genes on pSK301

Phenotypes

with the yend of the transposon l-19,

pLG339.

sector

gene loci on pSK301. Transposon TnZOOO(y6) was inserted into pSK301 as described by was mated with GT1211, GT1311, GT1515, and GTl614. KmR and SmR exconjugants

derivatives

patterns.

tests. The sites of TnZOOO insertions the plasmid.

vector

and the solid-line

>

thrC

Linder et al. (1983). Strain MGl063[pSK301] were selected

plasmid

TLr156

The double-line

DNA

s4

s3 . I

thrAISA25 Fig. 2. Determination

S. marcescens

and pSK304.

chromosomal

1

I I

1 I

Sl

map of pSK301 the 6.5kb

represents

TLr156 chromosome I\

Strain

I

Fig. 1. Restriction sector

or pLG339

The sites of TnZOOO insertions

derivatives

are shown by the vertical

were also determined

lines. Each number

: :T&000 and others are pSK301:: TnZOOO. Insertions to the left. Insertions showed

Appropriate

The direction

shown below the horizontal

ThrA,S-A,S-B+C+, locations

of transcription

and translation

clone number

above the horizontal

line are in the opposite

ThrA,S+A,S+B-C+,

of thrA,SA,5BC

indicates

shown

were determined

by complementation

orientations.

ThrA,S+A,5+B+C-,

genes and Sal1 sites (S,-S,)

of the thr genes is indicated

of line and

are represented

by the arrows

at the

155

mutations. Derivatives with insertion numbers 1 to 19, 20 to 27, 28 to 37, and 38 to 41 showed ThrA,-A,-B’C’, ThrA,+Az+B-C’, ThrA,’ A,+B+C-, and ThrAi+A,‘B+C+ phenotypes, respectively. Based on these results, the correlated physical and genetic map of the cloned thrA ,SA,SBC region was constructed as shown in Fig. 2. The approximate sizes of the zhrA,5A,5, thrB, and thrC genes seemed to be 2.4 kb, 1.0 kb, and 1.3 kb, respectively. Among the 27 independent derivatives, there were none that showed any polar effect although TnlOQO was inserted into the thrA,SA,S or thrB gene. The deletion plasmid pSK304, which carries SatI,-S&I, fragment and lacks most of the thrA 15A,5 genes, also complemented the thrB and thrC mutations. These results indicate that the thrA ,5A,5, thrB, and thrC genes of S. marcescens are transcribed, at least partially, from separate promoters. However, a possibility cannot be ruled out that the promoters on TnlOOO or pLG339 are related to the expression of the downstream thr gene(s).

thrC gene product. Only a small amount of the thrA,SA,5 gene product was produced in the cells bearing the ThrA,+AZ+B+CCplasmids. This might result from the difference of the effects of UV radiation, because, in another expe~ment, the from the thrA,5A,5 gene products ThrA, + A, + B + C - plasmids were expressed at the same level as those produced from other ThrA, + AZ+ plasmids (not shown). Cells bearing the ThrA, + A, + B - C + plasmids produced all of the three kinds of unique proteins as cells bearing pSK301 did (Fig. 3, lanes j and k); an additional 29-kDa protein was produced from the pSK301 derivative with insertion number 24. The maximal size of the coding region of the thrB’ gene on the pSK301 derivative with insertion number 24 is about 800 bp, which is very similar to the deduced size of the coding region of 29-kDa protein. Therefore the 29-kDa protein is likely to be the truncated poly peptide of the thrB gene product. When plasmid pSK30 1 was introduced into S. marcescens T-l 112, in which both AK I and HD I are produced constitutively, the unique proteins with Mrs of 85 000,43 000,

(c) Ide~ti~cation of the br operon gene products To identify the thr operon gene products, proteins encoded by pSK301 and its derivatives were labelled with [ 35S]methionine in maxicells, and the cell extracts were analyzed by SDS-PAGE, followed by &orography. In cells bearing pSK301, unique proteins of 85,43 and 3 1 kDa were synthesized whereas similar proteins were not observed in cells bearing pLG339 or pLG339: : TnfOUO (Fig. 3, lanes a-c). The above proteins are similar in size to the thr operon gene products of E. co&(Parsot et al., 1983). Cells bearing the ThrA, - A, - B + C + plasmids produced unique smaller proteins, which are probably the truncated polypeptides of the 85-kDa protein (Fig. 3, lanes g-i). By correlating the sizes of truncated pol~eptides with the sites of Tn~~U~ insertions, the tr~sc~ption~ direction of the thrA,SA,S genes was determined to be from left to right in Fig. 2. The thrA,SA,S genes could not be further divided into cistrons by TnlOOO insertions. These results suggest that the thrA,SA,S genes encode a single 85-kDa protein. When plasmids in which the TnlOOOinsertion inactivated the thrCgene were used as templates, the 43-kDa protein was not observed (Fig. 3, lanes l-n), suggesting that this protein is the

abcdetg‘hi

jklmno

Mrxlo-”

WrMv ‘=‘414yr;u*

-g4thrA,5Ar5 -67

-3

thrC

Fig. 3. [35S]Methionine-labelled proteins produced in maxicells by plasmids derived from pSK30 1or pLG339. Proteins produced in maxi&Is were analyzed by SDS-PAGE, followed by fluorography. E. cob N1790 was used as a host for each plasmid to be tested. Lanes: (a) pLG339; (b) 42; (c) pSK301; (d) 38; (e) 39; (f) 1; (g)5; (h) 11; (i)4; (j)20; (k)24; (1)29; (m)30; (n)31; (a) pSK304. These clone numbers of pSK301::TnlOOO or pLG339 : :TnlOOOare shown in Fig. 2. The arrowheads indicate the truncated polypeptides of the thr gene products (lanes g, h, i, and k). Symbols thrA,SA,5 and thrC represent chrA,SA,S and thrC gene products with M,s of 85 000 and 43 000, respectively. The markers are phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa).

156

a

b

c

sis of the nucleotide sequences will provide information for understanding this problem.

d

MKKY3

(d) Expression of the t/w operon genes from plasmid pSK301 in Serratia marcescens

5‘A25

Fig. 4. Overproduction of the thr operon gene products in S. mnrce~cen~ T-l 112. Cell extracts used in Table III were analyzed by SDS-PAGE, followed by Coomassie brilliant blue staining. Lanes: (a) T-l 112; (b) T-l 112[pLG339]; (c)T-l 112[pSK301]; (d)M, markers (see Fig. 3). Symbols thrA,SA,S and thrC represent thrA,SA,S and thrC gene products with M,s of 85 000 and 43 000, respectively.

and 3 1000 were produced in large amounts as seen on an SDS-polyacrylamide gel stained with Coomassie brilliant blue (Fig. 4, lane c). These proteins were not amplified when pSK301 was introduced into D-60, which is of wild type for the thr operon gene expression (not shown). These results suggest that these unique proteins are from the thr operon genes and the 31-kDa protein is likely to be from the thrB gene. But cells bearing the ThrA, + A, + B - C + plasmid produced the 3 l-kDa protein. There is a possibility that another gene which encodes the 31-kDa protein exists on pSK301 and this protein and the thrB gene product comigrate on an SDS-polyacrylamide gel. The analy-

Plasmid pSK301 was introduced into S. marcescens D-60 and T- 1112, which are of wild type and constitutive for the thr operon gene expression, respectively. The AK and HD activities were assessed to see whether the thr operon genes on pSK301 were expressed in these transformants (Table III). When the cells were grown with excess isoleucine, where the wild type thr operon genes are repressible, the HD level of D-60[pSK301] was about five times as much as that of D&O[pLG339]. The HD level of T-1112~pSK301] was about four times as much as that of T-l 112[pLG339] and about 20 times as much as that of D&O[pLG339]. In strain D-60[pSK301], the HD activity was not inhibited by threonine. Similar results were obtained for the AK. Cell extracts of these transformants were analyzed by SDS-PAGE, followed by Coomassie brilliant blue staining (Fig. 4). Strain T-1112[pSK301] produced large amounts of proteins with M,s of 85000, 43 000, and 31000 as mentioned above. The copy number of pSK30 1 was assessed to find the gene dosage of the cloned thr operon. Plasmid pSK301 existed at five to six copies per 5’. marcescens chromosome (Table III). Since strain T-l 112 lacks feedback repression of AK I and HD I, the thr operon genes on pSK301 were expressed at higher levels in this strain, reflecting the gene dosage. (e) Threonine pSK301

production

by

strains

carrying

Threonine production by strains carrying pSK301 was examined (Table IV). Strain T-1112[pSK301] produced 35 mg of threonine per ml, whereas strain T-l 112[pLG339] produced 8 mg of threonine per ml. The threonine productivity was markedly improved by introducing pSK301 into a threonineproducing strain. The fmal cell amount of strain T-l 112 was slightly decreased by introducing pSK301 (Table IV). But even when Km was not added to the medium, pSK301 was almost stably maintained in the cells during the entire period of

157

TABLE

III

Aspartokinase

and homoserine

Strain

dehydrogenase

activities

Specific

a

activity

Aspartokinase

in Serru~iu marcescens strains

carrying

the plasmids

Copy number e

b

Homoserine dehydrogenase

D-60[pLG339]

17 (

7S)C

10 (

D-60[pSK301]

37 (

12)

48 (- 15)

T-l 112[pLG339]

38 (-22)

T-11 12[pSK301]

120 (- 16)

a Assayed

as described

b Assayed

in the presence

in MATERIALS

49 ( 202 ( AND METHODS,

NT NT

4) 1)

5.4 f 0.4

section c and expressed

(%) by 50 mM L-threonine

(minus values might depend

d Inhibition

(%) by 10 mM L-threonine

(minus value might depend

expressed

NT

in nmol product/mg

proteimmin.

of 50 mM L-lysine.

’ Inhibition

e The copy number

77)d

per chromosome

was calculated

on stabilization

of the enzyme

on stabilization

of the enzyme

by t_-threonine). by L-threonine).

by taking the M, of the S. marcescens chromosome

to be 2.5 x 109. The value is

as mean + SD (n = 4). NT, not tested.

incubation (not shown). Strain T-1112[pSK301] is useful for the industrial production of threonine. We have succeeded in constructing S. marcescens strains producing this amino acid in much larger amounts by using the hybrid plasmid described here.

REFERENCES

Bachmann,

B.J.: Pedigrees

coli K-12. Bacterial. Backman,

K., Ptashne,

plasmids Acad. Davis, ACKNOWLEDGEMENTS

carrying

of same mutant

strains ofEscherichiu

Rev. 36 (1972) 525-557. M. and Gilbert,

W.: Construction

the cI gene of bacteriophage

of

1. Proc. Natl.

Sci. USA 73 (1976) 4174-4178.

B.D. and

requiring

Mingioli,

methionine

E.S.:

of Escherichiu co/i

Mutants

or vitamin

B,,. J. Bacterial.

60 (1950)

17-28. Horii, T., Ogawa,

We are grateful to Dr. G.N. Cohen for providing GT12, GT13, GT15, and GT16, to Dr. N.G. Stoker for pLG339 and to Dr. A. Nakata for JC10240 and MG1063. We also thank Dr. I. Chibata and Dr. T. Tosa for encouragement.

TABLE Threonine

T. and Ogawa,

H.: Nucleotide

sequence

of the

IexA gene of E. coli. Cell 23 (1981) 689-697. Kisumi,

M., Komatsubara,

isoleucine

S. and Chibata,

hydroxamate-mediated

provement

of isoleucine-producing

cescens. Appl. Environ.

Microbial.

I.: Enhancement

growth strains

inhibition

of

and im-

of Serratiu mur-

34 (1977) 647-653.

IV production

Strain

D-60[pLG339] D-60[pSK301] T-l 112[pLG339] T-1112[pSK301]

by strains

carrying

Growth

the plasmids

a (mg/ml)

L-Threonine produced b (mg/ml)

120h

144 h

120h

144 h

38 37 36 30

NT” NT 35 31

0 15 8 29

NT” NT 8 35

a A loopful of cells grown overnight on a nutrient agar slant supplemented with Km was inoculated into a 15 ml liquid medium in a 500-ml Sakaguchi shaking flask and was incubated at 30°C with reciprocal shaking (140 rev./min, 7-cm stroke). The growth is expressed as dry cell weight. b L-Threonine was determined by bioassay with Leuconostoc mesenteroides P-60, as described by Komatsubara et al. (1979). ’ NT, not tested.

158

Komatsubara,

S., Kisumi,

Threonine

production

M., Murata,

K. and

by regulatory

mutants

marcescens. Appl. Environ. Komatsubara,

S., Murata,

Threonine

Microbial.

Chibata,

35 (1978a)

K., Kisumi,

I.:

of Serruriu

M. and

834-840.

Chibata,

M. and Chibata,

I.: Transductional

of a threonine-producing

murcescens. Appl. Environ. Komatsubara,

S., Kisumi,

construction

Microbial.

38 (1979) 1045-1051.

M. and Chibata,

murcescens: lack of feedback

controls

dehydrogenases.

U.K.:

assembly

Cleavage

of three aspartokinases

of the head of bacteriophage

proteins

Micro-

during

T4. Nature

Laszlo, N.C. and Alvin, J.C.: Construction recA

J. Bacterial.

Linder, P., Churchward, cation mutants

the

227 (1970)

mutations

ofan Hfr strain useful Escherichiu coli

between

G. and Caro, L.: Plasmid

generated

by insertion

T., Fritsch,

A Laboratory Spring Harbor, Mark,

SC.:

E.F. and Sambrook,

Manual.

pSClO1 repli-

of transposon

TnZOOO.

J.: Molecular

Cloning.

Laboratory,

Cold

The y 6 sequence

A., Hack,

of F is an insertion

sequence.

termination

R.P.: Determination

by fluorescence

A.M. and Rupp, of plasmid-coded

densitometry.

of

Plasmid

W.D.:

Simple method

for

proteins.

J. Bacterial.

137

(1979) 692-693. N.G.,

Fairweather,

low-copy-number coli. Gene

murcescens mutant

K-12:

Mapping

M.: Isolation

structure

B.C.:

Versatile

of a versatile cloning

Serrutiu of the

161 (1985) l-6.

I.: Threonine and

locus ofEscherichiu coli

evidence

for an operon.

J.

118 (1974) 990-998. D., Cohen, G.N., Borne, F. and Patte, J.C.:

of the structural

and of two homoserine

genes of the three aspartokinases dehydrogenases

117 (1974) 133-143.

J. Communicated

Spratt,

for cloning in Escherichiu

as a host and molecular

gene. J. Bacterial.

genetic

Bacterial.

and

vectors

18 (1982) 335-341.

T. and Kisumi,

aspartase

N.F.

plasmid

K-12. J. Bacterial.

NY, 1982.

Mol. Biol. 126 (1978) 347-365.

T.:

operon in Escherichiu coli K-12. Nucl.

S. and Novick,

copy number

Theze, J., Margarita,

Cold Spring Harbor

Tazaki,

I. and Cohen, G.N.: Nucle-

of thrC and of the transcription

Theze, J. and Saint-Girons,

143 (1980) 529-530.

J. Mol. Biol. 170 (1983) 287-303. Maniatis,

Sancar,

Takagi,

for transferring

P., Saint-Girons,

S.J., Carleton,

identification

680-685.

strains.

Projan,

Stoker, of structural

K. and

9 (1983) 182-190.

strain of Serrutiu

biol. 45 (1983) 1445-1452. Laemmli,

C., Cossart,

plasmid

I.: Transductional

Appl. Environ.

Suzuki,

Acids Res. 11 (1983) 7331-7345.

of Serrutiu

strain

of a threonine-hyperproducing

and two homoserine

Parsot,

region of the threonine

S., Kisumi,

construction

H.,

cluster ofSerruriu murcescens. Jpn. J. Microbial.

otide sequences

135 (1978b) 318-323. Komatsubara,

H., Hosogaya,

Argininegene

19 (1975) 35-44. I.:

by Serruriu murcescens. J. Bacterial.

degradation

Matsumoto,

by H. Yoshikawa

of Escherichiu coli