Protein degradation during diauxic growth of Escherichia, coli

Vol. 20, No. 6, 1965

BiOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROTHN DmRADATION DURIN@ DIAUXIC OabWTH OP ESCHERICHIA COLI

N. S. Wlllette national

Inetltute

for Medical Rerearoh,

all

Hill,

London, N.W.7.

Received August 3, 1966 The phenomenon of diauxlo (1945).

He gr8r B. &

and found that

in a

growth

wae fket

contalnlq

medium

demonetrated both glucose

there were tm pheree of growth

eeparated

during We

period en intermediary

and lactoee,

by a lag period.

A mechauiemproposed to explain this le that the cello flret glucose:

by &mod

utilized

the

metabollte me prodwed aoh

repreered the eyntheeie of the ensyma neceeeary for laotore utillratlon, even in the pre8ence of laetoee.

When the gluoore use exheueted, the aelle

had neither the entgmee neceseary for lactoee utilieatlon, for

their

nor amino aclde

Then emlno aoide were derived from Intracellular

eptherie.

breekdoun of protein

(We

lncmaeing in rate when growth rtopped) and ueed

for the aynthe8i~ of proteine -oh

Included the now denpmeed

neoerv

Exponential growth war then reeumed

for la&ore

utlllration.

erwue

(Maudeletem, 1963). A rimllu

growth 4

~110 were tmtaoferred fsotore thedse; nthlonine mixture

whbh

they

har

alm

to a m

wili\n

had the ge~typlo,

e.g. arglnlne

(tkwinl,

been okerred

In other -tam

fmm one eon-

but not pb~Qpie,

acide

and

growth

Ruth ability

to w

9undermn and Rurser, 19611, wmteiae and

(Bourgeois, Wan and UUnaehle~lle, of amlno

where

fadore

1960).

692

prement in a

1960); or the eo*er broth

medium(u,

BIOCHEMICAL

vol. zo,No.6,1965

pirect

conflrmstion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the connection between di8uxic

growth

rind protein deg%sdatlon haa been obtained during growth of g. & on 8 mixtum of glucoosecud either lactate

E. Coli XL 328~ lsc+leu- w8e1grorar. in a synend Methoda. mm

Yeteriale thetic

medium(Mandelstem, 1960))

p&l),

or glycerol.

8nd containing

of Lv8llne

supplemented

nith

DLleudne

the carbon eouroee end different

described below.

Emline,

&iWL

At

extracted protein

end then followlng

the begimlq

Its liberation

ecid.

radioactivity

into the culture eemples were

acid (TCA) at 90’ C for 30 mln.

were flltered

on Oxoid membranefilters

eucceeeively ulth 5%TCA cont8iniq 1% acetic

cello nith radio-

of the experiment, culture

with 5% trichloroacetic preclpitatee

conoentrntiona

All lncubatione were at 35' C.

Protein degradation me measured by labelllng active

(300

Emline

( 150 v&l),

end nrarhed 5% TCA, and

They were then glued tc alundnium plenchettee end their

determlned, giving 8 measure of the original

of the cell protein.

radioactlvzlty

Culture remplee were t&en during the experiment,

oooled to 0’ C tc prevent further

metabolitm, end centrifuged.

rupenzatent w8e rexwed,

and ite emino aoidr adeorbed onto

diluted,

coluume of Zeo-K8rb 225 in the Ii+ form. with water tc remcve inorganic 0x080s of 2 2 EH,OH.

eelte,

The eclutionfi

8ud the amino 8cide eluted with en were evaporated to drgnsee at 100’

There were dried end their

radioactivity

menwureof the nmcunt of radioactive Comparison of thb eetimte

The

The columua were rrarrhed through

and the residue6 dieeclved in w8ter end trenefemd ettee.

The

Lv8llne

with the radioactivity

to 8lumlnlum planchdetermined, giving a

in the culture

mapernntnnt.

of the cell protein gave (UI

of protein degrpdation.

Prellmlmry experlmnte elmwed thet radioactive Lmline w8e Incorporated only into Lmline in the cell protein, end that cell lyeie followed by extracellular

protein

degmd8tion

002iditl0ar umd in theae l xperlmente.

693

C,

did

not occur under thg

The liber8tion

of intmmllul~

Vol. 20, No.6, 1965

BIOCHEMICAL AND BWHYSlCAl

&galaotoeidaee

Into

the culture

RESEARCH COMMUNICATIONS

medltm use umed ae en index of cell

lyrlr. Correotiona preparation

were mede for loor

of radioaotivlty

of the eamplee, end for their of their

detemination

radioaotirity

dudng

eelf-abeorptlon

duz%ng

(in an Automatlo Sample Changer

Counter with a thin end-ulndon Geiger tube dote&or).

Beeult8.

Cells were grownfroma

mallinoculum

mediumcontaining glucore (1 m&l) 0.1 p&l).

and L-f4CJ

(1%)

reeuepended in amediumcontaining (200 &l).

We latter

radloaatit&

(35 &ml;

valine

during exponentld

They were hamooted

inamediumcontalnlngL-valine

in the synthetic

growth, mahed tuioe

p&l)butno

carbon~ource,

gluaoee( 150 &l)arrcZ

Errline

wae included to prevent reinoorporation

L-saline liberated

culture

and -lea

a period of 9C min. for the detezmlnation of

deneity and protein

degradation.

almoot exhaueted, and the following to oulture(a)

of

by protein degradation.

Thl8 culture wae divided into eix part6 ((a) to (f)), t&en from eacshover

and

glucose wa8

Atthispolntthe

addltione uere tie:

eodiumla&ate(SCC

&I.)

n

(b) @y-=1

w

(0) no addition

W

(d) glwo-

w

(0) gluooee (SW p&l)

and eodium la&ate

n

(f)

and gl@erol

(@C M@)

(eo0 da)

glwo~e (see II&&

(WC &ml)

(sao 44.

The additions were delayed to eneure mlnlmel 8yntheslm of the en-0 fint

neoeeeaq for the utilleation

of laotate

or glycerol

drprine

the

growth period. Sampler were taken for

the determlnatlon of suture

and protein degradation for afurthor The result8

are

ohown in pig.

duuiw

150 min. 1.

694

Those

of experimnte

(d),

(e),

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 6, 1965

$20 i c 35 I -10 5 =5 0a 0

12

3

4 TIME

Fig. 1. Protein degradation during growth on: A, end lactate (SO0 &ml); B, glucose (150 &ml) and p&l); C, glworo (150 p&l); D, &woee (950 with la&ate (SO0 pg/lil), or glyoerol (SO0 &ml). Symbole: 0, protein degradation; o , culture deneity.

and (f)

were similar,

neither

lactate

aud are shown In Fig.

nor glycerol

1, D.

per ee &fected

Thle indicated

the rate

that

of protein

degradation.

of protein

Comparison (c) aud (d)

&owe

expdnentially

degradation

that the rate

was slow (about

about 6% per hour)

and cell

of protein

0.6s

degradation

per hour),

when the glueore

there wae a diauxie

of expmential

growth.

at an lnureared

rate,

wherear

degraded at the #low rate Three reeulte intracellular ayntheeia

support

protein end that

of the wchaniam

In oelle

thi8

protein

of diauxlo

leg period,

both before

charaoterietlc

in experlmentm

rapidly

(to

euppllee

degradation growth.

695

uaa degraded

and aitmmrds

that

(a) and

the two perioda

protein

of grouiug

the mgge6tion

degradation

growing

WELBexhawted.

leg of about 30 ndn. between During

in experiments

but lnareased

When a eecood carbon Bource mu3 pxwent, (b),

growth

it

wea

oelle.

during

a diauxic

lag

the emlno a&de for protein

is therefore

an eeeential

part

Vol. 20, No. 6,1965

Aeknowledmaent. adrioe

throughout

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The euthor rriehee to thank Dr J. kndeletem

for hie

this w*.

Referenaee. Ii. (1960) Bioehlm. Bourgeole, S., Whme, J. Y., and Lelouchler-Degrmlie, Biophye. Aota 38, 136. Gorini, L., Ounderaen, W., and Burger, Y. (1961) Cold Spr. Harb. Sump. quent. Blol. 26, 173. Maalgfe, 0. (1960) Symp. Soo. Oen. Hlcroblol. IO, 272. Meadeletem, J. (1960) Blochem. J. 6& 103. Mandeletem, J. (1963) Ann. I?. Y. Aead. 801. 102. 626. Mod, J. (1945) Ann. In&. Peeteur 111, 37.