J. MoZ. Biol. (1971) 61, 681-604

Complementation Studies in the Maltose-A Region of the Escherichia coli K12 Genetic Map

MAURICE HOFKUXG: MAXIME SCHWARTZ AND DOLPH HATFIELD

iXpartntent de Biologic mole’culaire Iwtitut Pasteur, Paris, France

and National Cawzr Institute, National Ir&it,utes of Health

Bethesda, Md., U.S.A.

(&eceived 14 Decenhher 1970, and irb revised form 26 May lY71)

Three genes, m&T, nzalY and wu~l&, have been defined previously in the nuL4 region. malP and malQ belong to the same operon, and are the structural genes for maltodextrin phosphorylase and amylomaltase, respectively. m&T has been attributed the role of a positive regulatory gene for the maltose system.

Complementation analysis in the m&A region performed in this study u-ith stable merodiploids demonstrates that :

(1) the m&T gene is outside the malP-mat& operon ; (2) the promoter and initiator of the m&P-malQ operon are located between

nm?T and m&P ; (3) mET and m&Q each probably consist of a single cistron. Results of assays for the maltose enzymes in merodiploids provide evidence

that: (1) the wwlT gene product is present in near limiting concentration in wild type

for full expression of the m&P-m&Q operon. (2) the rnalT gene product is not used stoichiometricaily.

1. Introduction In Escherichia coli, maltose induces the synthesis of the three catalytic proteins known to have a role in its metabolism, and the synthesis of the bacterial receptor sites for adsorption of phage h (Monod & Torriani, 1950; Wiesmeyer & Cohn, 1960; Schwart,z & Hofnung, 1967; Schwartz, 1967c). Mutations specifically affecting the ability of E. coli to utilize maltose as a sole carbon source, occur in two regions, r&A and muZB, respectively, located at 65 minutes and 79 minutes on the genetic map (Schwartz, 1966 ; Taylor, 1970) (Fig. 1). A summary of what is known about the maltose syst.em is given in Figure 2.

The m&B region is believed to contain two genes belonging to the same operon (Schwartz, 1967b,c). One is the structural gene for maltose permease (or part of the permeation system), and the other is a gene involved in the synthesis of the receptors for phage h.

The m&A region is composed of at least three genes (Hatfield, Hofnung $ Schwartz, 1969a; Schwartz, 1967a). Two of them, maZP and ma@, belong to the same operon. The polarity of this operon is maZP--wnaZQ (Hatfield, Hofnung $ Schwartz, 19693). w&P is the structural gene for maltodextrin phosphorylase, and m&Q is the structural

681

682 RI. HOPNUNG, 31. SCHWARTZ AND 0. HATFlELD

arg G \ Hfr KLl6

FIG. 1. Location of the ntuZA and rnaZB regions on the genetrc map of EJ. coli K12. The drawing is a simplified version of the genetic map of E;‘. co& K12 (after Taylor, 1970)

graduated in IO-min units and giving the locations of the mutations referred to in this paper. Origins of transfer of Hfr strains are indicated by arrows.

ma/ B region mat A region

melA ma/E /amB uvr A ma/T ma/P ma/O bio H sir A

Permfase Am ylotjaltase

I Maltodextrin phosphorylase

;

$;;;;’ - Internal i multose p

i i

Maitodextrin -&+ glucose - I - phosphate

Glub, I

7- glucose - 6-rphate

AT P mtermedtary metabolism

Fm. 2. The maltose system in E. coli EC12 The struct,ure of the muZA region is discussed in this paper. The structure of the n&B region

was discussed previously (Schwartz, 19676). Gene maZB is probably the gene for maltose permeate. Gene ZamB, previously called XrecB (Schwartz, 1967c), is involved in the synthesis of bacterial receptors for phage A.

gene for amylomaltase. The third gene, mulT, is defined by pleiotropic mutations which almost completely abolish the ability of the cell to synthesize permease, amylomaltase and phosphorylase, and to adsorb phage A.

Previous work suggested that malT is outside the nbcd~-nd& operon (Hatfield et al., 1969u,b) and that its product, which is a protein, has a positive role in the regulation of the expression of the malA and malB operons. In the presence of mal- tose, the malT gene product would activate transcription (or translation) ofthe maltose operons.

In this paper, the role and mode of action of the mulT gene product are further examined by complementation studies between different mut,ants in the m&A region.

2. Materials and Methods (a) Strains, media,, nrnd bacteriological techniques

Bacterial strains are listed in Table 1. Genetic markers are named after Taylor (1970) and their location on the E. coli map is shown in Fig. 1.

tit43 MALTOSE-A GESE COMPLEMENTATION IN E. COLl

TABLE 1

Bacterial strains

Name mnZd region Main characteristics Origin

l’A505MS33

PA505MS33MAd-- >i MT-

. . MQ-

. . M&-v

./ YM

PA505 Iv18 15 l’A505WAd 13” A .d

HfrG 6 HfrGBME’ll

HfrG6MTIMPII

liL16 -1. KLl61 + KLltir! 1.

KLl66 -L

Hfr701

JCI553(KLF41)

Deletion Point mutant in mdT Point mutant in mtrlh)

I-

Deletion Point mutant, in w~alT Point mutant in mcdQ

_.

-1.

Deletion

:- Point mutant in maW

Double point mutant in mulP and mull’

argG metA his thy 8tr” SU- F-

argG metA his thy 8tT’SlL- p-

urgG- meld - atr’ au - rec.4 p-

his 8trY Hfr ,1

,,

at@ Hfr thy St?.= Hfr thy 8te rijP Hfr

at@ ri;f’ red Hfr

rec.4 atrs Hfr

F’ argG + 8tP maEA + leu metB his recA

0rgG str’ maL4

Transduction of PA505MS33 by 1’1 grown on a mulA strain (see Materials and Methods)

Rccombinants be- tween the above strains and KL166 (se0 Mat,crials and Methods)

See Hatfield et ~1. (1969a)

Dr Matney Ethyl methane sulpho- natc mutagenesis on HfrG6 (see Hatfield et ccl., 1969a) Selection of a /\- resistant m&A dcrira- tivc of HfrGGMP 11

thy- derivative of KL16 Rifampicin-resistant derivative of KL163 Recombinant between Hfr701 (donor) and KL162 (recipient)

Materials and Methods)

Dr Brooks Low (see

All mcdA mutations in this Table arc shown in Fig. 5, except MT1 which has not been mapped precisely within mcrlT.

When no origin is indicated, the strain was taken from the laboratory collection.

recA derivatives of 7/u&l mutants were constructed as follows. Pl stocks were grown on mulil mutants and used to transduce PA4505MS33 for ad+. a.sd +mulA derivatives of PA505MS33 were crossed with Hfr KL 166, and his+ tiny + str + recombinants were selected : about 90% of these recombinants were recA aa shown by their sensitivity to U.V. light.

Media, transduction and sexual crosses have been described previously (Hatfield et al., 1969a; Schwartz, 1967a).

Phages

&SOCII is a clear plaque mutant of $80 ; XimmAattSOhSO is a recombinant between X + and &O+ which has the immunity region of h and the attachment site, integraso, and

684 M. HOFNUNG, M. SCHWARTZ AND 1). HATFltiLll

host range of $80 ; hV is a virulent mutant of h + used to test different strains for rcsistanca to pha,ge I\. These phages come from the collection of F. Jacob.

Transducing phages #SOdmuEA are discussed below.

(b) Not&orbs for rnerodiploids

The alleles on the opisome are given on the upper line and F’ indicates F’ factor, + indicates phage 480, no indication means F’ or 4. The alleles on tho chromosome are given on the lower line.

For example :

F’ A+B-C+ # A+B-C+ A+B-C+ A+B+(=-,,+ ’ A+B+C-,,+ ’ A+B+C-D+ ’

(c) Construction and WE of F’ merodiploids The F’ factor KLF41 carries a portion of the chromosome extending approximately

from minute 60 to minute 67 and including in particular the alleles orgG+, .strS, aroB+, mulA + , asd + (Fig. 3) (unpublished results; Brooks Low, personal communication).

.---. c ” 0 ” ! “I orgG chromosome

----- 67 66 65 64 63 62 61 60

FIG. 3. Epiaome KLF41 and shorter episomes derived from it. The map graduated in minutes is shown on the lower line. Episome KLF41 is sho\+n on 1110

upper line. Shorter episomes derived from it, are presented under KLF41 (see text; and Jean-l’itul Thirion, unpublished results).

(i) Isolation of episonws currying melA mutations

Merodiploids constructed by introduction of wild-type episome into a malA mutant sire always complemented to Mal+. Starting from such a merodiploid of structure F’ strS mulA+ asd +

str= w&A a.sd ’ mnuld homogenotes can be obtained by plating samples of a culture

grown in complete medium on TTZ (Hatfield et al., 1969a) plates containing streptomycin. Since these plates do not contain meso-a-E-diaminopinelic acid, only str’ asd + colonies will grow. About 10 y. of the str’ asd + colonies are Mal- and subsequent genetic analysis shows

that they are of the type F’ strr malA aad +

str’ wu.zlA asd ’ The KLF41 episome is unstable in Ret + strains. Its muZA derivatives are more unstable

for an unknown reason. Therefore, complementation tests with such episomes were only performed in recA strains.

(ii) Test for complementation

Two mutations in the amylomaltase gene, MQG and MQ7, were transferred to KLF41 by the procedure described above. The two corresponding episomes were introduced into w&A argG metA recA strains. Plating was done on minimal eosin-methylene blue-maltose agar (Hirota, 1960) (EM-M&l), and on minimal glucose plates, both supplemented with methionine. Glucose plates demonstrated the efficiency of transfer and nmltose plates demonstrated whether complement&ion between m&A mutations did (growth in 48 to 60 hr) or did not occur (no growth in 120 hr).

Enzyme assays in F’ merodiploids were not reliable even though recA strains were used. In liquid culture, m&A episomes derived from KLF41 undergo extensive deletions. The bacterial population is then enriched with merodiploids herbouring shorter episomes.

MALTOSE-A GENE COMPLEMENTATION IN E. COLl 685

These episomes carry only the alleles which are specifically selected for in the culture (here rtr@ + ) and lose the rnaZA region?.

For this reason, enzyme assays were done only in the merodiploids constructed with transducing pheges. C!omplementat,ion on plates. however, gave the same results with rithcr F’ or 480 merodiploids.

(d) CovLstruct,ion rend we of 480 vn~rodiploids

The original transducing phage (called 480dmnE, or 4 T + P+ Q ) carries only part of the mrrlzl region, namely the malT and m&P genes and a portion of the ,nnlQ gene (an- l~nl~lished result,s; and Schwartz Rr Beckwith, 1970). A phage carrying all known genes of the mrrlil region (cnllpd 480&mrrl, or 4 T + P+ Q’) (Fig. 4) wa,s derived from it, by 7).

g/p8 ma/T ma/P ma/ 0 &o/i + 00dma12 ---.- +-*- I I -____

Fro. 4. Structure of tho maZA regions of phages 48OtlmaZ, and 480dm&. Thr portions shown in interrupted lines may or may not be carried by the phage.

Schwartz & J. Beckwith in the following way. A strain carrying a deletion of the att480 and tonB loci was lysogenized with 480dma2, and 480h (use of a 48Odmal, stock made with 480h as a helper was necessary because the ton.B mutation results in resistance t.o 48Oh+). Integration of both phages occurred in the mdA region of the chromosome. Lysates were made by U.V. induction and phage 48Odm& was selected for its ability to transduce to Mal+ a strain carrying the MAdl08 deletion. Both phages (designated as 48OdmaZA) give stable lysogens without helper. They integrate into the bacterial chro- mosome at the att480 locus as shown by extensive curing of lysogens t)hrough heteroimmune superinfection (Signer, 1968) with XimmXattHOh80.

(i) Isolation of 4rYOdmalA phnges cnrr?/inq a malT mutntion

Cultures of 4 n&T + lnalT heterogenotes, which are phenotypically Mal+ and X6, are grown

in complete medium and streaked onto EM-Ma1 plates covered with about 10ghV particles and 10R 48OCII particles.

Colonies on developed plates are m&T homogenates. They have 480 immunity. Trns- duction with the phages contained in these strains shows that these phages carry the expected m.nlT mutation.

(ii) Test for com@emenLtntion

The m&A mutants were grown in complete medium and streaked on EM-Ma1 plates containing the necessary supplements. Drops of lysates containing about 200 transducing particles were spotted on the streak. Appearance of colonies in the spot within three days indicated complementation.

Phage 480dm& which complemented all the muL4 mutants tested was always used as a( control.

(iii) Enzyme c~98wys

Cells were harvested in exponential phase of growth at a concentration of 2 to 5 x lo8 cells/ml., resuspended in cold phosphate buffer (pH 7, 0.1 M) and the O.D.CCC of the suspension ww immediately recorded. Permeate, amylomaltase and phosphorylase assays were performed as previously described (Schwartz, 1967a). Protein concentration was

t Taking advantage of this phenomenon, we later obtained smaller F’ from KLF41 (Fig. 3). Some of the smaller F’ C8r137 the maZA region, are more stable than the original episome and could hopefully be used for quantitative complement&ion t,ests.

686 M. HOFNUNG, M. SCHWARTZ AND D. HATFIELD

measured by the method of Folin and Lowry (Lowry, Rosebrough, Farr & Randall, 1951). The error for a single assay is about 25%. This error is less than loo/, when assays are

repeated in the same extract. Possible segregation of the 480dmaZA phage was determined in each case by streaking

a sample of the culture on EMB-Ma1 (Hatfield el! al., 1969a) plates, or by testing for $80 immunity in at least 100 clones. No segregants were found.

3. Results First, experiments will be presented which give some information on the structure

of t,he m&A region. Second, t,he dose effects of the malT gene product will be considered.

(a) Complementation and malA structure

All of the point mutants and deletions shown on Figure 5 were submitted t,o the ccmplementation test on plates. In some cases, as indicated below, enzyme assays were performed on the merodiploids.

(i) Point mutants

Number of cistrons in malT and malQ. Complementation tests between point mutants in mulT and m&Q were performed in order to determine whether malT and malQ each constituted a single cistron (Benzer, 1957). This study was not done for the malP gene, since malP mutants are able to utilize maltose as a sole carbon source and their analysis by complementation would require a special technique. malT: none of six point mutants (MTlOl, MT103, MT106, MT153, MT160, MT162) mapping in six different regions within malT complemented each other (all 36 possible diploids were constructed). Thus m&T is likely to be a single cistron.

malQ : none of the mutants M&6, M&7, M&10, M&l 1 and M&20 was complemented by phage $80 T+ P+ Q-, or by an episome bearing the M&6 or the M&7 mutation. m&Q is therefore also likely to be a single cistron.

Complementation of malT point mutants bg a T + P+ Q- epitome. Alit of the malT point mutants shown in Figure 5 are complemented by a T+ P+ Q- episome. malT and malQ are therefore two different cistrons.

Furbhermore, the induced level of amylomaltase in merodiploids is the same for malT nonsense mutants as it is for malT mutants non-suppressible by two amber suppressors (su2 and su3) or by an ochre suppressor (su4) (Fig. 2). There is thus no polar effect of m&T nonsense mutations on the expression of the malP-malQ operon, which strongly suggests the existence of two different messenger RNA molecules, one for m&T, and one for the malP-malQ operon.

(ii) Deletion mutants

(a) All deletions shown in Figure 5 were tested on plates for complementation. The following conclusions are derived from this study (Tables 3 and 4). Haploid strains

t Two mutants, MT163 and MT173, mapped in w&T by deletion mapping (Hatfield et rrl., 1969a) were not complemented by episome T + P + Q -. They were later shown to be double mutants of malT and malQ by separating the malT mutation from the malQ mutation by Pl transduction. The m&Q mutations are called MQIO and MQll and the original double mutants are now called MT153MQlO and MT173MQll. The resulting single maZT mutants, MT153 and MT173, arc’ complemented by episome T + P+ Q- .

Occurence of such double mutants is probably due to the fact that maZQ mutants are slight,ly inhibited when grown in complete medium supplemented with maltose. Selection t)hen favors strains harboring a secondary mutation in m&T or in malB (unpublished results).

MALTOSE-A GESE COMPLEME~TATIOiK IN E. COLI 687

FIG. 5. Genetic map of the maltose-A region.

T, The malT, malP and malQ genes are represented on the line near the top and designated as P and Q, respectively. I and P designate the initiator and the promoter of the mnEP-malQ operon.

It in not known whether the point mutations MT105 and MT162 are located to the left or to right of MA4131 (as indicated by the arrows).

The hatched bars represent deletions and each deletion is representetl by it.s number.

t Indicates that t,he corresponding mutation is an amber mutation. 1 Jndicatrs t,hat the corresponding mutation is an oohro mutation.

TABLE 2

Induced amylomaltase levels in HfrG6 and Mal- derivatives com,plemented by +80dmal,

C’hromosome Episome Amylomaltase c$T+P+Q- (unitsjmg)

Wild type MT201 MT202 MT203 MT205 MT151 MT103 MT171 MT101 MT101

-

+ + + I-

+ -t + -

+

265 227 185 207 240 170 I 6.5 22.5 <I 210

Mut,ations 201, 202, 203 and 206 are suppressible by ~2, ~19 and szrl. Mutations 17 103 and 151 are not suppressible by the above suppressors.

101, 1,

TABL

E 3

Sum

mur

y of

th

e pr

oper

ties

of

dele

tion

mut

ants

in

mal

A an

d of

the

m

erod

iplo

ids

con&

uded

by

in

trodu

ctio

n of

an

ep

isom

e T

+ P +

& -

in

to

thos

e m

utan

ts

Grou

p fro

m

map

ping

data

Nam

e of

de

letion

Ph

enot

ype

of

haplo

id Ph

enot

ype

in

pres

ence

of

a

T +

P +

Q -

episo

me

(com

plete

na

me

is

Grow

th Am

ylom

altas

e Re

spon

se

Grow

th Am

ylom

altas

e Re

spon

se

MAP

follo

wed

by

on

(%

of

to

on

(%

of

to

the

num

bers

be

low)

malt

ose3

wi

ld

type)

ph

age

/\ m

altos

et

wild

typ

e)

phag

e h

102,

10

3,

138,

15

6,

105,

15

5,

118,

12

0,

- En

d in

side

mal

T 14

2,

145,

15

4,

104,

13

6,

111,

10

1,

115,

13

1

End

insid

e m

&P

112

122

114

> 14

9 1

- 10

7:

109’

End

beyo

nd

malP

13

3,

110,

13

2,

108

I

157

and

161

End

insid

e m

alT,

in

side

m&P

, or

3

-

betw

een

mal

T an

d m

dP

L 5

Non-

induc

ible

l-2%

R

f In

ducib

le to

N

85%

S

Non-

induc

ible

from

no

n-de

tec-

ta

ble

to

15%

Non-

dete

ctable

Non-

induc

ible

l-2%

Non-

induc

ible?

N

40%

Non-

induc

ible?

N

40%

R I

Non-

induc

ible

S

Non-

dete

ctable

Indu

cible

to

N 85

%

Leve

l of

40

%

supe

rindu

cible

to N

10

0%

Leve

l of

40

%

not

supe

rindu

cible

t Ph

osph

oryla

se

and

amylo

malt

ase

are

both

pa

rtiall

y co

nstitu

tive

in

this

strain

. 1

+ Co

lonies

pr

esen

t in

48

hr

or

le

ss;

I co

loni

es

pres

ent

in

48

to

60

hr;

- co

loni

es

not

pres

ent

in

120

hr.

R is

re

sista

nt

and

S is

sen

sitive

in

re

spon

se

to

phag

e /\.

Emym

e lev

els

iu

hete

roge

aote

s ob

tain

ed

itL R

CA

deriu

atiae

s of

PA

505M

S3;5

Chro

mos

ome

Amylo

malt

ase

Phos

phor

ylaso

Pe

rmea

se

Episo

me

(unit

s/mg)

(u

nits/m

g)

(pm

ole/h

r x

mg)

+T

+P+Q

- Un

induc

ed

Indu

ced

Unind

uced

In

duce

d Un

induc

ed

Indu

ced

Wild

typ

e -

13

240

11

195

55

460

MAd

I +

25

230

16

220

36

220

MAd

156

+ 25

24

5 20

18

5 35

37

0 MA

A 10

5 +

25

230

27

245

21

230

MAd

ll8

+ 25

21

5 24

22

0 28

29

0 MA

A +

25

220

36

262

50

430

MAA

l +

13

230

6 18

0 65

42

0 MA

A 12

0 11

0 MA

A +

138

255

85

265

i2

590

MAA

- 67

51

MA

A +

100

88

34

216

120

300

MAA

l -

92

61

MAA

1 2.

64

68

76

20

0 27

0 71

0 MA

A +

52

61

11

290

110

350

MAA

+ 42

58

6

180

160

550

MAA

llC

+ 48

51

11

23

0 83

33

0 M

AAl

+ 45

55

3

238

60

500

MT2

02

+ 22

21

0 13

31

0 33

70

0 M

T204

+

19

210

30

280

80

440

MT2

05

+ 16

19

0 11

20

0 76

65

0 M

T206

+

19

215

12

225

34

420

In

colum

n 1,

the

na

me

of

the

malt

ose

mut

ation

of

th

e ba

cteria

l ch

rom

osom

e is

giv

en;

wild

typ

o is

PA5

05M

S33D

Mv.

In

colum

n 2,

pr

esen

ce

( +)

or

abse

nce

( -)

of

the

phag

e +S

Odm

aZ,

is g

iven.

To

ru

le ou

t th

e po

ssib

ility

that

th

e re

sults

ob

taine

d we

re

depe

nden

t on

th

e str

ain

used

, co

mple

men

tatio

n of

th

ese

r&A

mut

ants

was

perfo

rmed

in

a

diffe

rent

ge

netic

ba

ckgr

ound

wi

th

iden

tical

re

sults

.

ci!Jo ill. HOFSUIUG, 111. SCH\Villb’l’% ASL, 11. HA’I’h’lEl,l,

carrying deletions terminating inside mall’ have very low and non-inducible amylo- maltase and phosphorylase activities (Hatfield et al., 1969a). In merodiploids con- structed with a T +Q- episome, inducibility of amylomaltase is restored. Therefore, the promoter of the malP-malQ operon is not located to the left end of malT (see Fig. 5).

Since polarity of the operon is malP--+malQ (Hatfield et al., 19698) the promoter of this operon must be located between malT and mulP (i.e. malT is outside the m&P-m&Q operon) .

(b) Haploid strains carrying deletions terminating inside m&P or beyond have variable, but always non-inducible, levels of amylomaltase (Hatfield et al., 1969a). In the presence of the T+Q- episome, inducibility of amylomaltase is never restored. This agrees with the above conclusion that the promoter is located between mal!!!’ and

malP.

(c) Previous studies did not determine whether deletions MAA and MAA terminated within, or beyond, malT. In the presence of a T+Q- episome, they behave like deletions terminating within m&T. Therefore, they presumably also end in t)his gene.

(d) Previous studies have shown that strains carrying deletions MAA and MAA were partially constitutive for the m&P-m&Q operon. Genetic mapping did not separate them from each other or from MAA 157 and MAA discussed above (Hatfield et al., 1969a). They show a striking difference in their complementation pattern. Inducibility of amylomaltase to wild-type level is restored by introduction of a malT + allele in trans position to MAA3; this is not true for MAA5. This suggests that MAA is longer than MAA and that it cuts out an essential part of the genetic structure necessary for the initiation of the expression of the m&P-m&Q operon, while MSA3 does not (Table 4 and Fig. 5).

(iii) Complementation groups and the malB operon

It can be seen in Table 3 that introduction of T+ P+ Q- episome into all maid mutants (including strains carrying a deletion of the whole malA region) restores sensit,ivity to phage h and inducibility of the maltose permease. This is compatible with a model (Schwartz, 1967c) where the m&T product activates expression of the n&B operon.

(b) Bnulysis of enzyme levels ,in merodiploids

In merodiploids of the type +T+ P+ Q-

T- P+ Q’ ) the average induced enzyme levels arc

about 85% of wild-type haploid for amylomaltase, and 135% for phosphorylase (Tables 2 and 4, and lines 3 and 4 in Table 5), instead of 100% and 200% as could bc expected from the number of gene copies per cell. In order to explain this discrepancy, an analysis of enzyme levels was performed in various other types of merodiploids. A summary of the results is presented in Table 5; in the Discussion, enzyme levels are expressed as a percentage of the induced wild-type level. The following conclusions are drawn from Table 5.

(a) The presence of an extra target site for malT product action in merodiploids does not lead to a titration of the malT product. This is shown in lines 10 and 6 of

Table 5 where the level of phosphorylase is the same in a T+ P+ Q-

T- P- Q’ merodiploid as

&IALToSE-A GESE: CGMPLEMESTATION IN E. COLI

TABLE 5

Induced amylomultase and phosphorylase levels in sowbe diploids

ti91

Strain Chromosome Episomc Bmylomaltase Phosphorylase

2 3 4 5 6 7 s 9

IO

HfrG6 HfrG6MT173MQI l(&lnrctZ,) HfrG6MTlOl(~dmuZ,) HfrG6MT202(c#,dmuZ,) HfrGGMPll(+T-P+L)‘)t HfrGGMTlMPl l(&l,cctrZ,) HfrGG(&imtrZ,) HfrGG(+dmrtZ,) l’A.505MS15 I’A505MAd 1:12(+ti,/roZ2)

100 100 55 133 Y8 136 83 133

102 57 90 54

101 153 148 147 160 100

54 53

Enzyme levels are expressed as a percentage of the wildtype induced level. Sssays were repeated from two to five times in different extracts and the reported value is the average of the values found. ,4 comparison of the averages using the standard t-test of Fisher & Yates (Schwartz, 1963) showed that a difference of 10 between two averages was significant at s level of confidence of above 99%. Only differences of 10 or more are considered in t.he discussion.

In both wild-type strains (HfrG6 and PA505MS15) enzyme specific activkies in units per mg protein were 310 for amylomaltase and 200 for phosphorylase.

jThc mtrZT amber mutation carried by this phage (line 5) is MTLOO.

in a T+ P+ Q’

total delet,ion of n&A merodiploid. Also (lines 1 and 5) the level of amylomaltase

is identical in a T + P + Q + haploid as in a T- P+ Q-

T+ P- Q’ merodiploid.

(b) The level of amylomaltase (whose gene is on the chromosome) is significantjly

lower in a T+ ‘+ ‘- T- I’- Q’

merodiploid (where the malT gene is on the phage), than in a

T- P+ Q-

T+ P- Q’ merodiploid (where the m&T gene is on the chromosome) (lines 6 and 5).

(c) When the malP-malQ operon is carried by #Ndmal, or +30dmul,, the production of maltose enzymes is lower than when the opercn is at its normal location on t,he chromosome. A comparison of the enzyme levels in the merodiploids of lines 6 and 10 shows that the level of n&P expression is t*he same for both phages, and that co-ordination of amylomaltase and phosphorylase is conserved when the genes

are carried by the phage. As shown on line 5, in a T- P+ Q-

T+ P- Q’ merodiploid, the level

of WULE~ operon expression on the phage (measured by phosphorylase) is 5’iSb of that on the chromosome (measured by amylomaltase). A similar conclusion is obt’ained by

comparing the phosphorylase levels in a T- P+ Q-

T+ P- Q’ merodiploid (line 5) and the wild-

type haploid, or the amylomaltase levels in T+ P+ Q’

T- P+ Q- and T+ P+ Q-

T- P+ Q’ merodiploids

(lines 2 and 3). The only apparent exception occurs in strains having a delet*ion ending in nzolP and promoting a low constit,utive amylomaltase activity. When 4 T + P + Q-

69% M. HOFNUNG, M. SCHWARTZ AND L). HATYIELI)

is coupled with this type of deletion mutant, the amount of phosphorylasc produced by the phage m&P gene reaches 120% (Table 4). In such a strain, amylomaltase activity is low (15% of the induced wild-type level or less) and the situation is therefore similar to the one which is present in m&Q mutants, or in a weakly suppressed m&Q amber mutant where the phosphorylase level is much higher than in the wild type (Hatfield et al., 19693; Schwartz? 1967a)T.

It is not clear why the level of expression of the malP-malQ operon of the phage should be 54% of the level of the bacterial operon. However, one explanation is t,hat if replication of the chromosome initiates near 50 minutes and progresses clockwise, as suggested by several authors (Wolf, Pato, Ward & Glaser, 1968; Cerda Olmedo & Hanawalt, 1968), then there would be on the average about twice as many copies of the m&A genes when they are at their normal location (65 mm) as when they are integrated at the att480 sit,e (25 min).

The results of the above studies suggest an explanation for t’he unexpectedly low

levels of phosphorylase and amylomaltase in the T+ PC Q-

T- P+ Q’ merodiploids. malP and

malQ are expressed at a lower level when they are carried by the phage. It seems reasonable then that malT may also be expressed at a reduced level. Previous studies suggested that the malT gene product is present in limiting concentration in the haploid wild-type strain. Therefore, the reduced amount of malT product made from the phage would be insufficient to fully activate the chromosomal malA operon. Then, due to the deficiency in malT product, the malP and malQ genes carried by

the chromosome in a zr s’ z- + + merodiploid will be expressed only at 85 t’o 90% of

the haploid level. Furthermore, the phage-carried malP gene will be expressed only at about 55% of the haploid level. This accounts for the observed average 85% amylo- maltase and 135% phosphorylase in such merodiploids.

An additional observation may be made from the data in Table 5 (lines 7 and 8).

The level of amylomaltase in a s: EI z: merodiploid is the same as in the wild-type

haploid. Also, in the %: g: ii and f: z: i: merodiploids, the level of phosphor-

ylase is 150% as would be expected from a contribution of about 100% from the chromosome and about 50% from the phage. Therefore, the level of expression of a malA operon is not increased above that of the haploid wild type by adding an extra copy of the m&T gene.

4. Discussion m&T has been attributed the role of a positive regulatory gene (Schwartz, 1967b).

The fact, shown herein, that n&T + is dominant to m&T-, supports this conclusion. Also m&T is shown most likely to consist of a single cistron.

Complementation studies with deletions demonstrate the existence, between m&T and m&P, of a genetic structure necessary for the expression of the malP and malQ

t In all these strains, maltose metabolism is non-existent or very low, which may result in a higher intracellular maltose concentration or in a lower concentration of cetabolites effecting oatabolite repression. For any of these two reasons, the rate of expression of the maltose operon would become higher (see analogous situation in Lee & Englesberg (1963)).

MALTOSE-A GENE COMPLEMENTATIOS IN E. COLI 693

genes located in the cis position. This result confirms earlier data (Hatfield et al., 1969a) suggesting that m&P and malQ belong to the same operon and that malT is outside this operon.

Expression of a positively regulated operon is controlled in the c&s position by at least two genetic elements: a promoter, P (Ippen, Miller, Scaife & Beckwith, 1968), defined as a site necessary for initiation of transcription by RNA polymerase, and an initiator, I, defined as the target for positive regulation {(Englesberg, Sheppard, Squires & Meronk, 1969a). In malA these two structures are most certainly between malT and malP : deletion of the left part of malT (see Fig. 5) does not prevent induction of the malP-mal& operon located cis to the deletion in merodiploids carrying a malT + allele; also deletion of the right side of mulQ, as in phage $80dnaal,, does not prevent induction of m&P.

The st,ructure of the m&A region therefore appears to be very similar to that of the ara region (Englesberg et al., 1969a), with the target for positive regulation between a positive regulatory gene and the structural genes. So far, contrary to what has been observed in the ara system (Englesberg, Squires BE Meronk, 19696), no evidence has been found for a repressive effect of the regulatory gene product in the absence of inducer.

The analysis of enzyme levels in various merodiploids suggests that when malT is carried by +80dmulA, t,he level of m&T product’ in the cell is only about 55% of that in the wild-type haploid, and that this results in a lower level of induction of a malP- malQ operon located on the chromosome. This is in agreement with previous data showing that suppression of m&T nonsense mutations resulted in a restoration of the malP-m&Q operon expression proportional to the efficiency of the suppressor (Hatfield et al., 19696). It was then concluded that the malT gene product is limiting in the cell for malA operon expression. However, it is shown here that increasing the number of malT copies per cell does not increase the level of expression of the operon. It seems, therefore, that the malT gene product is present in only near limiting amounts in wild type for full expression of the operon.

When the m&T gene is provided by the phage, i.e. when it is in limiting amounts, it will promote expression of a given malA operon (carried by the phage on the chromo- some) at the same level whether or not another m&A operon is also present. Thus the malT gene product does not act stoichiometrically.

Studies of cell-free systems have led to proposals of plausible mechanisms for positive regulation of gene expression at the level of transcription. They all display the common feature that a factor (u: Burgess, Travers, Dunn & Bautz, 1969; Bautz, Bautz & Dunn, 1969; &: Travers, Kamen & Schleif, 1970; Cap: Eron, Arditti, Zubay, Connaway & Beckwith, 1971; de Crombrugghe et al., 1971; p: Roberts, 1969) modifies the behaviour of RNA polymerase toward a DNA sequence: either a start signal or a stop signal. In the latter case, positive regulation would be achieved through inhibition of p action by the positive regulatory gene.

There is no argument at the moment to decide what could be the exact mode of action for malT. However, if the malT product operates through binding to RNA polymerase, this could justify the observation reported herein, that the malT product is present in near limiting amount. Indeed, it would then seem reasonable that any positive regulatory gene product be synthesized in limiting amount, so as not t,o mobilize uselessly RNA polymerase molecules.

694 M. HOFNUNG, M. SCHWARTZ AND D. HATFIELD

We thank D. Schwartz and J. Beckwith for the gift of the transducing phages &3OdmaZA and Dr Brooks Low for the gift of episome KLF41. Permanent or temporary members of the DBpartment de Biologie Mol&ulaire are acknowledged for discussion, criticisms and help. We are indebted to F. Jacob and J. Monod for their constant interest in this work as well m their material support.

This work was supported by grants from the Centre National de la Recherche Scientifique, t,he Commissariat B 1’Energie Atomique, the DBldgation G&&ale it la Recherche Scienti- fique et Technique and the National Institutes of Health.

REFERENCES

Bautz, E. K. F., Bautz, F. A. & Dunn, S. S. (1969). Nature, 223, 1022. Benzer, S. (r95’7). In The Chewvicul Basis of Heredity, ed. by W. D. McElroy & B. Glass,

p. 70. New York: Academic Press. Burgess, R. R., Travers, A. A., Dunn, S. S. & Bautz, E. K. F. (1969). Nature, 221, 43. Cerda Olmedo, E. & Hrtnawslt, P. C. (1968). Cold Spr. Hurb. Symp. Qua&. Biol. 33,

599. Crombrugghe, de B., Chen, B., Gottesman, M., Pastan, I., Varmus, H. F., Emmer, M.

& Perlman, R. L. (1971). Nature, 230, 37. Englesberg, E., Sheppard, D., Squires, C. & Meronk, F., Jr. (1969a). J. Mol. BioE. 43, 281. Englesberg, E., Squires, C. & Meronk, F., Jr. (1969b). Proc. Nat. Acad. Sci., Wash. 62,

100. Eron, L., Arditti, R., Zubrty, G., Connaway, S. & Beckwith, J. R. (1971). Proc. Nat. Acad.

Sci., Wash. 68, 215. Hatfield, D., Hofnung, M. & Schwartz, M. (1969a). J. Bact. 98, 559. Hatfield, D., Hofnung, M. & Schwartz, M. (1969b). J. Bmt. 100, 1311. Hirota, Y. (1960). Proc. Nat. Acad. Sci., Wash. 46, 57. Ippen, K., Miller, J. H., Scaife, J. & Beckwith, J. (1968). Nature, 217, 825. Lee, N. & Englesberg, E. (1963). Proc. Nat. Acad. Sci., Wash. 50, 696. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 193,

265. Monod, J. & Torriani, A. M. (1950). Ann. Inst. Pmteur, 78, 65. Roberts, J. W. (1969). Nature, 224, 1168. Schwartz. D. (1963). Me’thodes statistiques ci l’wage des middicins et des biologistes, p. 157.

Paris: Flammarion. Schwartz, M. (1966). J. Bmt. 92, 1083. Schwartz, M. (1967a). Ann. Inst. Pasteur, 112, 673. Sohwsrtz, M. (19673). Ph.D. Thesis, Universitb de Paris. Schwartz, M. (1967c). Ann. Inst. Pusteur, 113, 685. Schwartz, D. & Beckwith, J. R. (1970). In The Lactose Operon, p. 417. New York: Cold

Spring Harbor Laboratory. Schwartz, M. & Hofnung, M. (1967). Europ. J. Biochem. 2, 132. Signer, E. R. (1968). Ann. Rev. Microbial. 22, 451. Taylor, A. L. (1970). Bact. Rev. 34, 155. Travers, A. A., Kamen, R. I. & Schleif, R. F. (1970). Nature, 288, 748. Wiesmeyer, H. & Cohn, M. (1960). Biochim. bkphys. Acta, 39, 417. Wolf, B., Pato, M. L., Ward, C. B. & Glaser, D. A. (1968). Cold Spr. Hnrb. Sym,p. Quant.

BioZ. 33, 575.