J.MoI.Evol.

8,317-328

Journalof MolecularEvolution

(1976)

© by Springer-VerlagI976

Selective Disadvantage of Non-Functional Protein Synthesis in Escherichia coil KEN J. ANDREWS* and GEORGE D. HEGEMAN** Bacteriology and Immunology Department, University of California, Berkeley, Ca. 94720 and Department of Microbiology, Indiana University, Bloomington, Indiana 47401, USA Received August 4, 1976 Summary. Comparison of growth rates of isogenic strains that synthesize varying levels of 8-galactosidase during continuous culture on non-inducing

medium indicates that synthesis of low levels of non-functional protein has a small but possibly significant effect upon growth rate. Key words: Selection/Continuous Culture/Non-Functional Escherichia coli/Metabolic Evolution

Protein Synthesis/

INTRODUCTION In

1964,

Horowitz

unified

and

extended

Lewis'

(1951)

theory

of

t h e e v o l u t i o n of m e t a b o l i c pathways. Lewis had suggested that new gene function could arise by a process involving the duplic a t i o n of g e n e t i c m a t e r i a l c r e a t i n g r e d u n d a n t g e n e s t h a t c o u l d t h e n u n d e r g o m u t a t i o n a n d s e l e c t i o n to n e w f u n c t i o n . H o r o w i t z c o m b i n e d h i s e a r l i e r t h e o r y (1945) c o n c e r n i n g the retrograde e v o l u t i o n of b i o s y n t h e t i c pathways with the gene duplication i d e a s of L e w i s , w h e n he i m a g i n e d t h a t as an e s s e n t i a l n u t r i e n t in t h e m e d i u m w a s e x h a u s t e d , a v a r i a n t a r i s e s in t h e p o p u l a t i o n which possesses an e n z y m e t h a t c o u l d c r e a t e the n u t r i e n t f r o m a pre-existing immediate chemical precursor in t h e m e d i u m . P a t h w a y s a r i s e in a r e t r o g r a d e m a n n e r as a d d i t i o n a l n u t r i e n t s become exhausted and variants, a b l e to s y n t h e s i z e the n u t r i e n t from precursors in t h e m e d i u m , a r e s e l e c t e d . C a t a b o l i c p a t h w a y s c o u l d a r i s e b y a s i m i l a r p r o c e s s of g e n e

Present address: Harvard Medical School, Microbiology and Molecular Genetics, 25 Shattuck St., Boston, MA 02115. ** To whom offprint requests should be sent. 317

duplication and mutation to new function thus allowing cells to catabolize immediate precursors of utilizable compounds. The duplication and subsequent modification of genetic material, often entailing the expression of the duplicate gene(s) prior to their full assumption of a new role, is also a feature of virtually all models for the evolution of new protein function or specialization (e.g., Clarke, 1974; Rigby et al., 1974; Waley, 1969; Ycas, 1974; and Zuckerkandl, 1975). It can be argued that these theories about the evolution of enzyme systems are not particularly attractive because the variants, with their wasteful excess synthesis of the duplicated gene products, would be culled from the population by selection. Establishment of such variants would depend either upon periodic selection and/or immediate isolation of the variant population. Some circumstantial evidence (Horiuchi et al., 1962) suggests that indeed high levels of excess protein synthesis (25% of the total cellular protein being non-functional) are detrimental to a cell. However, recent reviews of the evolution of enzyme systems in bacteria (Hegeman & Rosenberg, 1971; Clarke, 1974) suggest excess synthesis of proteins to play a major role in allowing cells to gain new metabolic capabilities. Additionally, comparisons of growth rates of cells in batch culture (A.G. Mart, personal communication) have suggested that cells performing wasteful synthesis at lower levels (-5% total cellular protein being non-functional) may not be stringently selected. Thus allowing such variants to successfully compete within the population. We used the lac operon in E s c h e r i c h i a coli as a model to test the validity of the objection raised to the Horowitz-Lewis theory of pathway evolution and more recent models that entail synthesis of non-functional protein. We also hoped to gather information concerning growth rate regulation in bacterial cells (Maal~e, 1969) growing with long generation times.

MATERIALS

AND METHODS

Strain C o n s t r u c t i o n and R e s u l t a n t Genotype. D r . Jack Sadler kindly furnished us with a series of lac constitutive strains that were isogenic in all known respects, except the lac operator region, to W 42, a lac 0 + strain. The O c mutations in D 531, D 533, D 534, and D 536 were produced spontaneously in the operator region of a lac operon on an F' lac episome. The O c mutation in D 532 was produced by 2-amino purine mutagenesis. In order to minimize any additional genetic differences among the strains, this "D" series was constructed by the transfer into the F- female of the original F' lac + episome. Recipient isogenicity for all of the crosses was insured by using samples, for the individual episome transfer experiments, of a culture of recipient cells cloned from a single colony. The

318

Table 1 Strain

Genotype

D 531

F' lac-pro B

+

+ i

c 0

z

+

y

+

/

A

lac-pro B rec A

-

s str , spcR

3-4

D 532

"

Oc 116 Oc IO3

D 533

"

D 534

"

0c IO

D 536

"

W 42

"

Oc 12 + O

ii

Ii

,t

Table 2 o o Generation time and Z/B min at 20 C and 37 C for all strains on R-glycerol medium a

Generation time

Z/B min 20°C

37°C

20°C

(h)

(±O.12)

37°C (±O.I)

531

0.420

0.415

6.7

2.O

532

O.O99

0.iO1

6.6

2.O

533

0.092

0.099

6.6

2.1

534 536

O.120 0.038

O.123 0.040

6.7 6.6

2.1 2.1

W 42

0.001

O.O01

6.6

2.0

a

Z/B min is a measure of the constitutive rate of expression of B-galactosidase in a culture. It is the ratio of absorbance at 420 nm/ml of culture

(o-nitrophenol released in the standard 8-galactosidase assay) to

the turbidity of the culture measured at 350 nm. The Z/B min has dimensions of specific activity

(enzyme per mg cell material), and is a measure

of cellular enzyme content as well as the rate of the lac gene products synthesis under steady-state conditions of growth in non-inducing medium.

donor column study;

sex

factor

was

the

same

in

each

of Table I lists the genotype those genetic symbols left of

case

(Table

I).

The

of the strains used the slash represent

center in t h e the

genotype of t h e F' lac O c, w h i l e t h o s e t o t h e r i g h t o f t h e s l a s h represent the chromosome. The recipient s t r a i n w a s recA- t o prevent recombination. T h e proB + m a r k e r o n t h e e p i s o m e i n s u r e d the

continual

presence

of

the

episome

in

cells

grown

in minimal

media. Each of the mutants had a characteristic induced operon expression. The cells were grown glycerol medium and assayed for 8-galactosidase. of B - g a l a c t o s i d a s e in the culture per milliliter

level of unin minimal The activity divided by the

turbidity of the culture l i s t e d i n T a b l e 2.

minimal

(A350

nm ) gave

the

Z/B

values

319

Strain Storage and Maintenance. StOck s u s p e n s i o n s as i n o c u l a for e x p e r i m e n t s w e r e p r e p a r e d by f r e e z i n g 0.5 ml samples of high d e n s i t y R - g l y c e r o l c u l t u r e s of the strains in v i a l s c o n t a i n i n g 3 to 5 drops of d i m e t h y l s u l f o x i d e (DMSO). Such (5 to 6) small screw c a p p e d v i a l s of each s t r a i n w e r e p r e p a r e d and s t o r e d at -70°C. I n d i v i d u a l 5 - b r o m o - 4 - c h l o r o - 3 - i n d o l y l - B - D - g a l a c t o s i d e (BCIG) i n d i c a t o r m e d i u m c u l t u r e s of each strain w e r e m a i n t a i n e d at 20°C for day to day use.

The m i n i m a l m e d i a u t i l i z e d in this w o r k w e r e m a d e from the m i n e r a l base (R-buffer) of S m i t h & Sadler (1971). In the t u r b i d o s t a t (Nephelostat) e x p e r i m e n t s d e s c r i b e d b e l o w and for cells g r o w i n g u n d e r n o n - i n d u c i n g conditions, 0.2% (v/v) f i l t e r - s t e r i l i z e d g l y c e r o l was a d d e d as sole c a r b o n and e n e r g y source. All liquid c u l t u r e s w e r e s h a k e n at 150 cycles per m i n u t e in 125 ml flasks fitted w i t h a 15 m m × 125 m m test tube side arm, on a r o t a r y shaker or a r e c i p r o c a t i n g w a t e r b a t h shaker. App r o x i m a t e l y 10% of the v o l u m e of the flask c o n t a i n e d m e d i u m . T u r b i d i t y was used as a m e a s u r e of growth. P e r i o d i c a l l y d u r i n g an e x p e r i m e n t the c u l t u r e fluid was t i p p e d into the side arm and its t u r b i d i t y was m e a s u r e d in a K l e t t - S u m m e r s o n c o l o r i m e t e r fitted w i t h a n u m b e r 66 filter. Media and Conditions of Cultivation.

Solid m i n i m a l m e d i a w e r e p r e p a r e d by a d d i n g 1.0% (w/v) I o n a g a r (Wilson D i a g n o s t i c Inc.) to the l i q u i d media. The agar and m i n e r a l s o l u t i o n s w e r e each p r e p a r e d at d o u b l e strength, a u t o c l a v e d s e p a r a t e l y , and m i x e d just prior to d i s p e n s i n g . B C I G i n d i c a t o r m e d i u m was u s e d to d i f f e r e n t i a t e c o l o n i e s of c o n s t i t u t i v e m u t a n t s from t h o s e of W 42. This m e d i u m is b a s e d on R - b u f f e r and has the a d d i t i o n a l i n g r e d i e n t s per liter: 25 mg BCIG, 2.0 g d i s o d i u m succinate, 2.5 g c a s a m i n o acids and 10 g Ionagar. P l a t e s w e r e i n c u b a t e d at 37°C and e x a m i n e d after 24 h. W 42 p r o d u c e d a w h i t e c o l o n y on this m e d i u m , w h e r e a s the lac O C m u t a n t s p r o d u c e d b l u e colonies. The assay p r o c e d u r e of S m i t h & Sadler (1971) was used, e x c e p t that cells (0.5 to 1.O ml of an e x p o n e n t i a l l y g r o w i n g culture) w e r e lysed w i t h 20 ~i of a m i x ture: 1.O ml toluene, 1.O ml 10% (w/v) s o d i u m lauryl s u l f a t e (SLS), 1.O ml 0.02 M M n S O 4 - H 2 0 and 5.0 ml 8 - m e r c a p t o e t h a n o l (Putnam & Koch, 1975). E n z y m e u n i t s per ml of a s s a y c u l t u r e (Z) w e r e c o m p u t e d as net a b s o r b a n c e at 420 n m / ( i n c u b a t i o n time) (ml c u l t u r e assayed), w h i l e Z/B is s i m p l y Z / a b s o r b a n c e at 350 nm per ml of the c u l t u r e assayed. The s p e c i f i c a c t i v i t y of pure B - g a l a c t o s i d a s e was d e t e r m i n e d u t i l i z i n g these assay c o n d i t i o n s and the v a l u e o b t a i n e d a g r e e d w i t h p r e v i o u s l y p u b l i s h e d v a l u e s (Craven et al., 1965). Assay Conditions for B-Galactosidase.

Protein Determinations. The m e t h o d of L o w r y et al. (1951) was u s e d to d e t e r m i n e p r o t e i n c o n c e n t r a t i o n s w i t h b o v i n e s e r u m a l b u m i n as a standard. The m e t h o d of Hu et al. (1959) was u s e d to de-

320

termine the concentration of B-galactosidase when the specific activity of the pure enzyme was measured. Disc-gel electrophoresis (Ornstein, 1964) was used to ascertain the purity of the B-galactosidase. Chemicals. All inorganic chemicals were of highest purity comm e r c i a l l y available. From the Fisher Scientific Company, Fairlawn, New Jersey were obtained: dimethyl sulfoxide, disodium succinate, glycerol, SLS, TRIS, and toluene. Sigma Chemical Company, St. Louis, Missouri was the source of BCIG and 8-mercaptoethanol; from Difco Laboratories, Detroit, Michigan, yeast extract, casamino acids (vitamin free) and agar. The Ionagar No. 2S came from Wilson Diagnostic Inc., Glenwood, Illinois; the bovine serum albumin from Pentex, Inc., Kankakee, Illinois and chloramphenicol was supplied by Calbiochem, San Diego, California. h e Nephelostat and Modifications. We felt there were several advantages to comparing the growth rates of strains in continuous culture apparatus with its constantly controlled conditions of m e d i u m composition, cell density and oxygen level. The Lab-Line Mark I Nephelostat was used for all continuous culture experiments. Preliminary experiments resulted in several modifications of the basic apparatus: (I) the replacement of the 30 ml overflow collection tube with a 500 ml round bottomed flask, (2) the addition, to compensate for fluctuations in both the quality and quantity of air bubbled through the culture vessel, of an activated carbon filter and a flow controller, respectively, (3) the taping of a black cloth over the overflow assembly to prevent light in the room from interfering with the turbidity m o n i t o r i n g system inside the growth cabinet, (4) the addition of an auxillary recorder to monitor the culture density and the rate of addition of m e d i u m to the growth vessel, and finally (5) the growth vessel walls were treated with trimethyl silyl chloride to retard the growth of bacteria on the vessel wall. This treatment delayed wall growth, but did not prevent it. All experimental runs eventually were terminated by vessel wall growth.

Samples of both a minimal glycerol culture of a mutant and a minimal glycerol culture of W 42 were used to inoculate the growth vessel for each mixed culture experiment. During an experimental run, samples were periodically taken from the culture and plated on BCIG indicator medium. When growth occurred, the ratio of mutant to W 42 was determined. The flow rate of the system was also m o n i t o r e d and the cell generation time was calculated using the following assumptions and equations: (a) in t u r b i d o s t a t s the rate of which the culture vessel volume is replaced by new medium at constant flow rate (e) is equal to the specific growth rate (~). ~ = ~. Mixed Culture Growth Experiments and Data Treatment.

321

1.7 1.6 1.5

mu

unf

R (pa~e.t)VS,Tlme

\

Fig.l Ratio (R) of inducible parent to constitutive mutant 531 cells in the effluent of the Nephelostat as a function of time during growth on non-inducing glycerol minimal medium during a typical experiment. The calculation of the percent difference in growth rate is shown

Ln R1/Ro X

1.4

z'~ =~

a:: 1.3

.Ln .98/I.7 x 21 gen I00= -.551 .,',, tO0=

~ ~{'k~

1.2

~l~,k

-.026x I00= - ?..6%

I.I 1.0

0.9 0.80

I

20

I

40

I

60

I

80

I

i

1%1

I00 120 [40 160 Time ( hrs~

(b) ~ = W / V O, w h e r e W = f l o w culture volume. (c) T = c e l l g e n e r a t i o n time

rate (min)

(ml/min), = In

a n d V O is t h e

2/a

0.693 -

T

L0

The percent difference in g e n e r a t i o n time between the mutant a n d W 42 w a s c a l c u l a t e d u s i n g t h e e q u a t i o n of B a i c h & J o h n s o n (1969): in R t / R O # of g e n e r a t i o n s (R o + Rt)

x 100 = % d i f f e r e n c e

R

in g e n e r a t i o n

times

= ratio mutant/W 42 at s o m e f i n a l time R = ratio mutant/W 42 at s o m e i n i t i a l o time. T o a c c u r a t e l y d e t e r m i n e R o a n d R t for an e x p e r i m e n t , a graph of R w i t h r e s p e c t to t i m e w a s m a d e , a n d a l i n e r e p r e s e n t i n g the best fit through the data points was drawn and R o and R t were determined f r o m t h a t l i n e ( F i g u r e 1). A t y p i c a l e x p e r i m e n t r e q u i r e d 192 h. R (± 3%) w a s d e t e r m i n e d a b o u t 30 t i m e s . T h e cell generation t i m e (± 10%) w a s c a l c u l a t e d approximately 10 times. A l i n e a r r e g r e s s i o n a n a l y s i s of t h e d a t a i n d i c a t e d t h e l i n e s h o w n in F i g u r e I h a d a K e n d a l l c o r r e l a t i o n coefficient of + 0 . 9 8 ( N o e t h e r , 1971). S i m i l a r a c c u r a c y w a s o b t a i n e d for a l l experiments. t

RESULTS

Preliminary Experiments to Establish the Conditions of Growth in the Nephelostat°

322

Preliminary

experiments

with

the Nephelostat

led

to

Fig.2 The effect of culture density upon growth rate of the parental strain in the continuous culture apparatus

G e n e r a t i o n T i m e V S . C u l t u r e {Density

i

190(

/

/

I I

100(

I I I I I'-"

I

6c~

/

$

// // 40C

-I

22"

celJs/ml) (m/n) Density Gen.Tirne

I 5X106 2X10 7 7)