Isolation of Vibrio, alginolyticus mutants defective in the respiration-coupled Na+ pump

Vol. 114, No, 1, 1983 July

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

18, 1983

ISOLATION

Pages

OF VIBRIO

ALGINOLYTICUS

MUTANTS DEFECTIVE Ns+ PUMP Hajime

Research

Institute

for

Received

May 23,

113-118

IN THE RESPIRATION-COUPLED

Tokuda

Chemobiodynamics, Chiba Chiba, Japan

University,

l-8-1

Inohana,

1983

When the respiration-coupled Na+ pump functions, V. alginolyticus is able to grow in the presence of an extremely high concentration of proton conductor, carbonylcyanide m-chlorophenylhydrazone. The mutants which became sensitive to the proton conductor were isolated and examined in regard to thy Na+ pump activity. Although the activity of a respiration-dependent H extrusion by the mutants is comparable to that by the wild type, the Naf pump activity of the mutants is significantly reduced. Furthermore, NADH oxidase of membranes isolated from the mutants is altered to be independent of Na+. It is concluded that the mutants have an altervion in the respiratory chain which simultaneously results in a lack of the Na pump.

Our extrudes 2).

recent not

studies only

Although

H ' but

the

pH values

place

at

electrochemical transport alkaline

of solutes(1,

the

marine

bacterium

Na+ as an immediate of

H+ by the

tested,

alkaline potential

that

also

extrusion

physiological mainly

revealed

the

pH.

of Na+ even 2) but

also

result

respiratory

of

Since

in the

presence

growth ' itself

Na+

at

extrusion

pump

all took

generates

of CCCP, not became

(1,

occurred

Na+

the

alginolyticus

respiration

chain

respiration-coupled

external

V. -

only

resistant

an active

to CCCP at

pH. The

interest

molecular since

respiration-coupled

'Tokuda,

H.,

mechanism it

may

give

H+ extrusion

and Unemoto,

T.,

of

the

respiration-coupled

another which

insight is

manuscript

Na' into

presently

pump is

the

mechanism

a subject

of

of

great of

the

controversy

in preparation.

m-chlorophenylhydrazone; TPP+, tetraAbbreviations: CCCP, carbonylcyanide TMPD, N,N,N',N'-tetramethyl p-phenylene diamine; MB, phenylphosphonium ion; acid; Tricine, tris(hydroxymethyl)methyl2-(N-morpholino)ethanesulfonic tris(hydroxymethyl)aminomethane; NTG, N-methyl-N'-nitro-Nglycine; Tris, nitrosoguanidine.

113

0006-291X/83 $1.50 Copyright 0 I983 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 114, No. 1, 1983

(3,

From this

4).

reason,

the

AND

BIOPHYSICAL

isolation

RESEARCH COMMJNlCATlONS

of mutants

defective

in the

Na+ pump

was attempted. NATERIALS

AND HKTHODS

1. alginolyticus 138-2 and its derivatives were grown aerobically at 37OC on either a rich (5) or a synthetic medium(6) buffered as specified. Mutants unable to grow at pH 8.5 on the rich medium containing 5 UM CCCP were induced by NTG (50 Mg/ml) at 37°C for 10 min. The mutants were isolated after two units/ml) enrichment (7) in the rich medium cycles of penicillin (8~10~ containing 5 kM CCCP and 50 mM Tricine-NaOH, pH 8.5. Selection was performed by replica plating. Glucose was omitted from the seletive plates to minimize a pH drop during growth. H+ flux induced by oxygen pulse and active extrusion of Na+ were assayed as reported(2) using K+-depleted and Na+-loaded cells prepared as described (8) from cells grown on the synthetic medium. Membrane fractions were isolated as described(5) from cells grown on the NADH oxidase activity was determined by following the synthetic medium. decrease in absorbance at 340 nm as described(5). 22?JaC1 (carrier-free) was obtained from NEN. CCCP and NADH (sodium salt) MES and Tricine were products of Nakarai Chemical were purchased from Sigma. co. RRSULTS The rationale coupled

highly

correlation

between

possesses in

expected

presence

unable

8.5.

1.

Naf

pump

and

As shown 6.5,

to

in

where

the

Na+

between

the wild

Nap

On the

other

activity,

the

were

significantly

results

are

permeable

not

type

of

similar

Na+ the

functions.

Close

Na+ pump was confirmed halophilic

-V.

respiration-

of V. alginolyticus

pump

bacterium,

alginolyticus

mutants

and is

lacking

has

than

the

the

little

by also

able

to

Na+ pump were

pH 8.5,

where

concentrations that

determined

concentrations among

of

were

mutants, the

114

strains

for

tested.

CCCP at

Nap 1 and

has

with wild

pH

significantly

Na+ pump

the

NTG

5 kM CCCP at

not

determined with

by

designated

of CCCP necessary

the

obtained

concentrations

activity, isolated

were

presence

inhibitory

and the at

medium

in

minimal

inhibitory

presented,

to H+ were

that

in the

growth

another

Therefore,

the

pump

lower

the and

by penicillin

hand,

minimal

the

on CCCP-containing

1,

different 2.

to

defective

growth.

grow

Table

when

costicola,

CCCP.

enrichment

that

growth

that

of

mutants

finding'

CCCP

to show CCCP-sensitive

mutagenesis

of

CCCP-resistant

an analogous the

on our to

finding

Mutants

pH

isolation

resistant

subsequent

grow

the

Na+ pump was based

becomes

the

for

a maximum the

type.

mutants Although

to make membranes The isolated

mutants

Vol. 114, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1. Minimal inhibitory concentration for CCCP. Aerobic growth of the listed strains in the presence of various concentrations of CCCP was examined in the rich medium buffered with 50 mM of either MES-NaOH, pH 6.5, or Tricine-NaOH, pH 8.5.

Table

CCCP (PM)

Strain pH 6.5

were

Wild

4

>80

Nap1

3

14

Nap2

2

11

able

to

grow

and required wild

examined

of

at to

was added

facilitate

transient

strains

tested

mutants

to

was pulsed

(Fig.

extrude in

the

pulse

C).

H+ was comparable presence

of

Hf

CCCP instead

at the

frequency

of

three

(2).

its to

results that

of of

that

the

type.

TPP+,

B

of 10 -8 .

strains

were

anaerobic

potential, in the

the

ability

of

When oxygen suspensions

C

0.5min

115

a

all

1 Respiration;dependent H+ extrusion (A to C) and Na+ pump-dependent H+ uptake (D to F). Na -loaded cells of the wild type (A and D), Nap 1 (B and E) and Nap 2 (C and F) were resuspended at final concentrations of 0.52, 0.66 and 0.59 mg protein/ml, respectively, in 2 ml of assay mixture containing 0.4 M NaCl, 20 mM glycerol, 0.2 mM Tricine-NaOH, pH 8.5, and either 2.5 mM TPP+ (A to C) or 20 BM CCCP (D to F). The cell suspensions were kept anaerobic at 25OC under the stream of nitrogen. Changes in medium pH were monitored by pH-electrode as reported (2). Oxygen pulse was performed at pH 8.5 by the addition of 100 ~1 of air-saturated 0.4 M NaCl. Fig.

was

TPP+ which

extent

indicate wild

of the

When oxygen

electrical

similar

of carbon

those

membrane-permeable

occurred

to

source to

as reported

against

These

sole

identical

chain

containing

H+ extrusion

A to

obtained

respiratory method

suspensions

of

were

were

Na+ by the

extrusion

as the

properties

revertants

the

1,

on lactose

i\-iikA

8.5

cell

not

These

by oxygen

anaerobic to

and

H+ and

pH 8.5

but

growth.

The spontaneous

Extrusion

rapid

on sucrose

Na+ for

type.

pulsed

pH 8.5

of

BIOCHEMICAL

Vol. 114, No. 1, 1983

;

-

AND

%.v-----*

BIOPHYSICAL

RESEARCH COMMUNICATIONS

--

OO

5

0

5 Time

0

5

(min)

Fig. 2 Sensitivity of Na+ extrusion to CCCP. Na+-loaded cells of the wild of type (A), Nap 1 (B) and Nap 2 (C) were resuspended at final concentrations 3.09, 2.31 and 2.40 mg protein/ml, respectively, in 50 mM Tricine-NaOH, pH 8.5, containing 0.4 M NaCl. The cell suspensions equilibrated with 22NaCl (1x104 cpm/kl) on ice were transferred to 25'C and incubated for 5 min in the presence of 20 mM glycerol. At 0 time, addition of none (O), 10 mM KC1 (0) or 10 mM KC1 plus 10 kM CCCP (A) was made. The level of 22Na+ retained by cells was determined at given times by filtering and washing an aliquot (50 ~1) of the cell suspension as reported (1, 2). Values are given in per cent of radioactivity at 0 time after correction for background radioactivity due to non-specific binding to filters.

the

wild

type

driven

by the

(Fig.

1,

Na+ pump-generated

loaded

V. -

previousy

alginolyticus

concentration

decreased

pump

at

gradient

upon

H+ uptake

potential

which

In contrast,

(2).

significantly

and,

the

extrusion

inhibited

by CCCP.

defective

performed

in only

2) and shown

the

portion

pH 0.5

contrast,

(1, quickly

a considerable

type.

in

characteristic

in

the

case

was

such a of

mutants

1, E and F). As reported

are

the

membrane

H+ uptake

Na+ pump-dependent (Fig.

D) demonstrated

the

addition

of K+ (closed

this

by

results

to

the

Na+ pump and that Na'/H'

antiport

alteration

in the

respiratory

membrane

fractions

(Table

2).

The

maximum

activity

specifically

required

Na+ for

its

In the by

and

that

in

C) the

was

the

Na+

A).

In

completely

isolated

of Na+ by the

wild

mutants mutants

is

system.

chain

116

Na+ against

circles).

(B

extrusion

Possible

Na+-

was performed

indicate the

and

CCCP (triangles

mutants

clearly

by a CCCP-sensitive

cytoplasmic

Na+ extrusion

insensitive Na+

of

K+-depleted

2,

most

therefore,

These

Fig.

extruded

of

of

in

of

the

NADH oxidase as reported

mutants of (5).

was examined the

wild K+,

type

or other

BIOCHEMICAL

Vol. 114, No. 1, 1983

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table

2. NADH oxidase activity of membranes isolated from the wild type and the mutants. NADH oxidase activity was measured at 30°C as described under "Materials and Methods" in 1 ml of assay mixture containing 20 mM Tris-HCl, pH 7.5, 0.2 mM NADH and 0.2 M of KC1 or NaCl. The assay was started by the addition of membranes to give a final concentration of 30 Ug protein/ml. oxidase

NADH

(umol/min/mg

protein)

Membranes KC1

NaCl

Wild

0.82

1.74

Nap1

1.61

1.64

Nap2

1.28

1.34

cations

(5),

was ineffective

NADH oxidase

activity

as a replacement

of

the

mutants

+ Na .

for

was completely

On the

other

independent

hand,

the

of Na+.

DISCUSSION As expected the

from

Na+ pump,

Na+

pump.

the

mutants

The

Na+

1 and 2).

H+.

of

the

membrane

independent

of

Na+

been

in

-V.

shown

resides

TMPD which pump (21, redox (9)

must

2). (5)

donates it

energy nor

altered

energization

of

data the

localized

at

to

cytochrome

found

that

re-reduction

the

Since the Na+

"TMPD-bypass"

involving

NADH:quinone

oxidoreductase

the

mutants pump the

Na+ pump.

this

to

Na+-dependent as the

Since

is

be

derived site.

also

possess

the

the

(91,

it

of

quite

the

dependent the

be reported

is

of Na+

TMPD by endogeneous

neither

Detailed

Na+-

addition

energization

from

be

Na+-dependence

Although

results),

Na+ pump will 117

segment

to has

that

this

of oxidized

(unpublished

to

indicate

c caused

TMPD oxidase

seems

was altered

found

segment.

mutants

The NADH

NADH oxidase

recently

and

to extrude

Na+ pump.

of

strongly

in the

the

ability

mutants

and

uptake

from

the the

oxidoreductase

electrons

in

retain

the

was

H+

absent

to

from

These

represents

occur.

did

be specific

NADH:quinone

was later did

completely

which

growth

to CCCP were defective

The Na+-requirement

costicola

Na+ pump is

CCCP-resistant

CCCP-dependent

mutants

isolated

Na+ pumpl.

the

sensitive

was almost

mutation

at

between

performs

the

of NADH oxidase

that

which

fractions

exclusively

likely

became

contrast,

(Table

respiration-coupled dependence

correlation

which pump

In

Therefore,

oxidase

close

Na+ extrusion

CCCP-insensitive (Figs.

the

the

on Na+

TMPD-dependent formation studies

elsewhere.

of on

the

BIOCHEMICAL

Vol. 114, No. 1, 1983

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOVLEDGhWVTS

This Science

work

was

and Culture,

supported

by

a grant

the

from

Ministry

of

Education,

Japan. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9.

Tokuda, 265-271. Tokuda, Mitchell, WikstrBm, Unemoto, 1389-1395. Tokuda, 4198-4203. Gorini, Tokuda, 788-794. Unemoto,

H., H.,

and

Unemoto,

T.

(1981)

Biochem.

Biophys.

Res.

Commun. 102,

and Unemoto, T. (1982) J. Biol. Chem. 257, 10007-10014. P. (1979) Eur. J. Biochem. 95, l-20. M., and Krab, K. (1980) Curr. Top. Bioenerg. 10. 51-101. T., Hayashi, M., and Hayashi, M. (1977) J. Biochem.

H.,

Nakamura,

T.,

and

Unemoto,

L., and Kaufman, H. (1960) Science H., Sugasawa, M., and Unemoto; T.,

and Hayashi,

M. (1979)

T.

131, 604-605. T. (1982) J.

J. Biochem.

118

(1981)

85,

Biochemistry Biol.

1461-1467.

Chem.

82, 20, 257,