Heterogeneous amino acid transport rate changes in an E. coli unsaturated fatty acid auxotroph

Vol. 81,No.2, 1978 March 30, 1978

BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS Pages

HETEROGENEOUS AMINO ACID TRANSPORT RATE CHANGES IN AN r.

588-595

COLI

UNSATURATED FATTY ACID AUXOTROPH Joseph

T. Holden, James Bolen, Joyce and John de Groot Division of Neurosciences of Hope National Medical Duarte, CA 91010

City

Received

January

A. Easton

Center

31,1978

SUMMARY - Amino acid transport rates in an E. coli unsaturated fatty acid auxotroph were non-uniformly affected by en%chment of membrane lipids in various unsaturated fatty acids. Proline and threonine transport rates were depressed much more than lysine and asparagine rates by trans unsaturated acids. Myristoleate and linolenate enrichment also produced non-uniform but lesser rate reductions. Although changes in the relative numoer of effective transport catalysts could account for these findings, comparisons of proline and lysine transport rates over a broad temperature range indicated that non-uniform alterations in transport catalyst reaction rates account at least partly for the activity changes associated with membrane lipid alterations. Several

laboratories

by membrane ported

lipid

fatty

primarily

in fatty

of various

lipid

investigations and little rates

order/disorder utilized

attention

of a variety

different

fatty

Using tarum

plots

to reduce

cellular

different

amino

transitions

is

is sup-

of membrane correlative

temperatures occurred.

on comparative

in

rate at which

Most of these

changes

as a function

lipids changes

displayed

or at most a few transport

systems

affected

conclusion

system

relative

has been focused of transport

substrates, in absolute

of growth

with

acids.

several

cultured

a single,

produced

a transport

and the

This

enrichment

points

phase

transport

(l-4).

that

at which

in Arrhenius

solute

composition

melting

temperatures

transitions membrane

acid

by the observation

acids

the relative

have shown that

E. coli

under

conditions lipid

acid

lipid-overproducing

levels

transport

of pantothenic (6), systems

acid

did

588

not

(5)

and L. plan-

restriction

we have previously

0006-291X/78/0812-0588$01,00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

mutants

respond

reported uniformly

sufficient that when the

EIOCHEMICAL

Vol. 81, No. 2, 1978

membrane

lipid

demonstrate several activity is

composition that

of several

the

indication

binding

proteins

in fatty

acids

was modified.

nutritional

unsaturated

acids

amino

acid

transport

may be less that

The studies

enrichment

fatty

that

AND’ 8lOPHYStCAL

reduce

of E. coli

produced transport systems

severely membrane

differential

RESEARCH COMMUNKATIONS

described membranes

here in one of

changes

in the

systems.

Of especial

dependent

on shock-releasable

affected

by membrane

interest

enrichment

fluidity.

MATERIALS AND METHODS Organisms: Experiments were carried out with E. coli unsaturated fatty acid auxotroph K1062 (provided by Dr. P. Overath). This strain also carries an altered fad E gene which limits its ability to degrade fatty acids, although there was a measurable conversion of 18:l to 16:l fatty acid in these experiments. Stock cultures were maintained on glucoseyeast extract-agar supplemented with Brij 35 (0.5%) and oleic acid (0.05%). Growth conditions: Cells were grown in Vogel Bonner medium E (VB) (7) using glycerol as carbon source. Ethanol solutions of unsaturated fatty acids were added to 0.15 mM immediately prior to inoculation. Fatty acid solubility was facilitated and toxicity minimized using bovine serum albumin (1.5 mg/ml; essentially fatty acid-free, fraction V, Sigma), Cultures were grown at 37'C with shaking. An initial overnight tube culture, using inoculum cells from an agar slant, was used to inoculate a second overnight tube culture. The inoculum was adjusted so that growth reached a cell density of 0.30 mg/ml after approximately lo-12 divisions at a convenient time on the following day. The final large-scale log phase culture was harvested at 0.30 mg/ml after lo-12 divisions (20-22 hrs). Strict control of growth conditions was required to attain reproducible (+ 5-10%) transport rates from day to day. Prior to harvest, the cultures were cooled and then centrifuged at 2,200 x 6. The cells were washed twice with cold VB lacking glycerol, fatty acid and serum albumin. Washed cells were resuspended in this medium to a density of 12 mg/ml and stored in ice until used ( cl.5 hrs). The lipid fatty acid content of chromatography. Cells grown with the as indicated in the respective acid: 60%; myristoleic acid, 29%; linolenic fatty acid.

the mutant was determined by gas following fatty acids were enriched palmitelaidic acid, 51%; elaidic acid, acid, 25% as combined 16:3 and 18:3

Transport rate measurements: Cells were preincubated for 15 minutes at 27°C in 0.9 ml of VB supplemented with glycerol (0.4%), chloramphenicol (0.1 mg/ml), and aminooxy acetic acid (2 mM). The suspension was bubbled with oxygen for 10 seconds at 30 seconds prior to initiation of the reaction. The final cell concentration was 1.2 mg/ml. The reaction was started by adding 0.1 ml of radioactive amino acid using a pipetter attached to an event marker which recorded amino acid addition, subsequent removal of 0.1 ml reaction aliquots, and their expulsion onto millipore filters covered with 1.0 ml of VB at room temperature (Q 21"~). Filters were rinsed once with 3 ml of VB. For each reaction, five consecutive

589

BtOCHEMICAL

Vol. 81, No. 2, 1978

AND 8IOPHYStCAL

RESEARCH COMMUNICATIONS

TABLE I Effect of Growth with Trans Unsaturated Fatty Acids on Amino Acid Transport by r. & Fatty Acid Auxotroph K1062 Uptake Fatty Acid Supplement Cis-Vaccenic acid (1B:l)

Rate

(9)

Proline

Threonine

Asparaqine

Lysine

100

100

100

100

Palmitelaidic acid (16:lt)

6.2

6.8

45

41

Elaidic acid

1.5

2.6

14

14

(18:lt)

Transport rates observed in cis-vaccenate-enriched cells were arbitrarily set at 100% for each amino acid. The absolute transport rates observed in were as follows (all values are pmoles Tin proline, 3.35 ? 0.19; threonind, 11.18 _ 1.04; asparagine, 0.26 + 0.043; lysine, 2.97 2 0.15.

samples were taken within the initial 12-16 seconds of incubation. Filters were immediately transferred to counting vials containing 2 ml of absolute ethanol. Radioactivity was measured by scintillation counting, and isotope content was converted to amounts of amino acid, using appropriate standards. Transport rates were determined graphically from plots of cellular amino acid content vs. incubation time. Chemicals: 14C]-labeled amino acids were obtained from New England Nuclear. [ 16 C]-Asparagine was purified as described previously (8). acids were obtained from Applied Sciences Laboratory and Sigma. All chemicals were the highest grade commercially available.

Fatty other

RESULTS Cells fatty than

grown

acids those

The rate

agine

relatively

transported observed

reductions

asparagine less

with

and lysine.

sensitive

than

several in the mutant were

greater Aspartic

proline

high

melting

point

amino

acids

at rates

grown for acid

with

proline

and lysine.

590

unsaturated

substantially

cis-vaccenic

acid

and threonine

and leucine

and threonine,

trans

(data

than

lower (Table

I).

for

not

shown)

but more sensitive

than

were aspar-

VoL81,No.

8lOCHEMlCAl AND BIOPHYSICALRESEARCH COMMUNICATIONS

2,1978

TABLE II Effect of Growth with Low Melting Point Fatty Acids on Amino Acid Transport by g. coli Fatty Acid Auxotroph K1062 Uptake Fatty Acid Supplement

Alanine

Cis-Vaccenic

acid

Myristoleic Linolenic

(18:l)

Rate

(%)

Arginine

Asparagine

100

100

100

7':

2

125 104

acid (14:l) acid (18:3)

Transport rates are expressed as a percentage of the rate observed with cis-vaccenate grown cells whose values were arbitrarily set at 100%. The transport rates observed with cis-yaccenate cells were as follows (all values are pmoles min-1 gm' ): alanine, 6.98 ? 0.46; arginine, 3.31 ? 0.44; asparagine, 0.26 + 0.043.

Lipid

enrichment

generally

had a less

Some amino like

in

acids

arginine,

more than

lower

melting

pronounced

effect

on transport

asparagine

were

not affected

like were

affected

any other

amino

myristoleate-enriched acid

melts

behavior acyl

in terms

does not

effects

of transport

tion,

lysine

slightly.

below

whether

these

of exogenously

and proline

on the

synthesis

effects.

transport

behavior

Since

in

this

of this

rates

compared

transport

rate

fatty

fatty

dichotomous

changes

acids

lipid

fatty

a broad cells

systems

591

showed

catalysts,

to answer

in cis-vaccenateover

reflected

on the reaction

of transport

In an effort

of cis-vaccenate-enriched

and proline

were

supplied

Using

lysine

was reduced

of membrane

heterogeneous

cells

the

others,

uptake

O"C, an explanation

telaidate-enriched

son,

II).

(Table

vs. asparagine

ratios

acids

significantly;

heterogeneous.

of order/disorder

of both

the response

fatty

rates

Alanine

The alanine

was clearly

catalysts,

or some combination

unsaturated

seem tenable.

was not clear

differential rates

cells

solely

It

acid.

at temperatures

chains

only

point

this

ques-

and palmi-

temperature

range.

as a basis

of compari-

different

responses

BIOCHEMICAL

Vol. 81, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

0

9r--G--

d

40

OC 4.0 -

0

/

/

a

I

I

A

3.0 -

/

0

A

a

2.0 -

/I

0

&

A/

/

0

a'

/

i'-'-

\ A

r'

A

0 1.0 -

0

9/

0’ A-

./\ A-0

*:84

20

30

TEMPERATURE

to temperature proline

transport

1.

l‘t-.

AlA

10

Fig.

.

40

("C)

Effect of temperature on proline (0,)) and lysine (a,~) transport rates in E. coli K1062 grown with cis-vaccenic acid (open symbols) or palmitelaidic acid (filled symbols). Inset: variation with temperature in the ratio; transport rate in cis-vaccenate (CV) cells/transport rate in palmitelaidate (16:lt) cells for proline (0) and lysine (A).

in palmitelaidate-enriched rates

exceeded

cells lysine

rates

592

(Fig.

1).

in cis-vaccenate

Whereas cells

at

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

all

temperatures

up to 4O"C,

enriched

cells.

declined

in activity

is

in the

acid.

for

seconds

rates

of carriers

is

modification

composition

is altered

transport

for

lipids

lysine,

unlikely

of the

variation

was modified.

to be metabolically rates,

reaction

these

rates

as an explanation

fatty of

and palmitelaidate-enriched

temperature

transport

which

this

the differential

as the

system

cells,

containing

in cis-vaccenate-

to measure

non-uniform

these

in palmitelaidate-

but not the

illustrates

and proline

required

was reversed

proline,

transition

directly

transport

complement

the

of a phase

lysine

pattern

above 32°C in palmitelaidate-enriched

1 inset

the relative cells

Furthermore,

range

Fig.

this

Since

modified data

in the 15

strongly

implicate

when the membrane

of the

the

non-identical

lipid

behavior

of

catalysts. DISCUSSION

We have previously acid

transport

lipid

systems

or bacterial

reports

acid

lipid

in several

rates

The magnitude transport

were

the most

appears,

reduced

that

case, the

lysine

either

point

trans

considerably

the

different the

fluidity

a possible

proline

were

catalyst

lysine than

is the

molecular undergoes

for

the

lipids,

or that

explanation

593

they

system transport of this

a more extensive

fatty

proline least

is

behavior. enriched

different

and proline

proline

in cells

unsaturated

study

of

of this

reduced

of

unsaturated

modifications

lysine

transporting

This

in an E. coli

examined,

and asparagine

that

distinctly but that

varied

acids

amino

(5,6).

investigation

Among the systems

sensitive,

membrane

melting

effect

systems.

with

environments

latter

of this

therefore,

associate

high

several

when the amount

to directed

facilitate amino

for

is modified

phenomenon

amenability

all

rates modified

composition

will

for

uptake

of this

whose

relatively

acid

acid

composition

Transport

that

heterogeneously

examples

auxotroph

membrane

are

fatty

additional

fatty

reported

acids. amino

and threonine

sensitive.

It

transport

systems

occur

in similar

less

affected

system. behavior conformational

by

In the might

be change

BIOCHEMICAL

Vol. 81, No. 2, 1978

than

is

required

of the

to a reduction ently

does

in membrane not depend

and possibly (9).

our

survey

dependent

systems

ment than

is

suggested

have noted

out

effects

which

demonstrate

membrane alter

fatty

the number

composition

of "carriers"

in addition

to,

temperature

change

affecting

to changes

to non-uniform since

in membrane

alterations

presumably

changes is

ments.

The finding,

of course,

effects

on "carrier"

synthesis

acid

lysine

other

groups

acid

composi-

not

alterations

rather

than,

catalysts.

1 show that

must

or

The at least

transport be attributed

of these

was fixed

in

may differentially

and proline

rates

rule

such as these

growth

component

of "carriers"

during

fatty

acids

of these

in Fig.

of the

does

is

lipids.

with

during

rates

described

fatty

enrich-

at temperatures

several

the fatty

in the reaction

the complement

rate

associated

that

the reaction

responses

acid

to reduced

experiments

synthesized

experiments

some of the dichotomous systems

rate

fatty

(4,10,11).

in interpreting

transport acid

transport

one

binder-

unfavorably

in membrane

reactions

One of the difficulties

that

activity

laboratory,

of changes

and transport

whereas

of palmitelaidate-enriched in this

appar-

such a component

in addition

system

transition

include appears

factors

system

component,

to trans

in the proline

carried

on enzymatic

it

sensitive

transport

fall

transport

systems

That

system.

phase

non-uniform

less

is more vulnerable

binding

incomplete,

are

may affect

the studies

therefore,

The proline

transport

is still

by the marked

Besides

tion

lysine

the proline

above the expected

and,

fluidity.

generally

fluidity

catalyst

on a shock-releasable

two of the

While

membrane

lysine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

catalysts, in such experi-

out additional

heterogeneous

growth.

ACKNOWLEDGMENTS These studies were supported by grants No. GM 20395 and CA 11186 from the U.S. Public Health Service and by the Aerospace Industry Research Fund. The authors are indebted to Mr. William Laffin and Dr. Nedra M. Utech for their assistance during these studies and to Dr. Peter Overath for the r. coli fatty acid auxotroph.

8lOCHEMlCAL AND EIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

REFERENCES H. U.,

and Overath,

J. Mol.

Schairer,

2.

Wilson, Comnun.

3.

Esfahani, M., Limbrick, (1971) Proc. Nat. Acad.

4.

Shechter, Biochem.

5.

Holden, J. T., Utech, N. M., Hegeman, G. D., Biochem. Biophys. Res. Comnun. 50, 266-272.

6.

Holden, J. T., Easton, Acta 382, 657-660.

7.

Vogel,

8.

Holden, 640-655.

9.

Oxender,

G., Rose, S., 38, 617-623.

and Fox,

J. T.,

Biochem.

L.,

and Gulik-Krzywicki,

J. A.,

and Bunch,

T.

44,

(1974)

and Kenyon,

J. M. (1975)

and Bunch,

J. M. (1973)

Biochim.

Biophys.

and Vagelos,

P. R. (1972)

J.

Biophys.

97-106.

Acta

307,

777-814.

10.

Mavis,

11.

Morrisett, J. D., Pownall, H. J., Plumlee, R. T., Smith, L. C., Zehner, Z. E., Esfahani, M., and Wakil, S. J. (1975) J. Biol. Chem. 250, 6969-6976.

595

J.

Eur.

Biochim.

Chem. 218,

41,

S. J.

C. N. (1973)

J. Biol.

Rev. Biochem.

Res.

and Wakil,

D. M. (1956)

Ann.

209-214.

Biophys.

and Bonner,

D. L. (1972) R. D.,

C. F. (1970)

Eiol.

A. R., Knutton, S., Oka, T., Sci. U. S. 68, 3180-3184.

E., Letellier, 49, 61-76.

H. J.,

P.

(1969)

1.

Biol.

Chem. 247,

652-659.