Light-dependent ATP formation in a non-phototrophic mutant of Rhodospirillum rubrum deficient in oxygen photoreduction

Vol.

66,

No.

2, 1975

BIOCHEMICAL

LIGHT-DEPENDENT

ATP

RHODOSPIRILLUM

FORMATION

RUBRUM

S . del

AND

IN

Valle-Tascbn,

G .

de

IN

OXYGEN

COMMUNICATIONS

MUTANT

OF

PHOTOREDUCTION

and

Biologia

144,

RESEARCH

NON-PHOTOTROPHIC

Giminez-Gallego

Vel6zquez

July

A

DEFICIENT

Instituto

Received

BIOPHYSICAL

Juan

M.

Ramirez

Celular

Madrid-6,

Spain

15,1975

Summary : Intact cells and isolated membrane vesicles from a non-phototrophic mutant of Rhodospirillum rubrum carry out photophosphorylation at normal rates. In contrast, the same vesicles catalyze at decreased rates the photoreductions of oxygen and of Therefore it appears that the system which tetrazolium blue by exogenous donors. mediates these reactions, or part of it, is not required for photophosphorylation but is needed for the normal phototrophic growth of the organism. This conclusion is strengthened by the observation that the ability to catalyze the in vitro redox reactions is restored in vesicles prepared from a spontaneous, phototrophic revertant of the mutant.

The

light-dependent

donors

to

oxygen

tophores) of

electron (1),

isolated

the

cyclic

are of

of

the

least

a part

cyclic

chain

and

photosynthetic

* Abbreviations -dichlorophenol

Copyright AN rights

mutant of

the

of

reduced

reactions

defective

for

rubrum,

blue, (5)

tetrazolium metabolism

for

of

same

supports

reported

here,

blue of

in

isolated

R.

rubrum.

the the suggest

mediated

that

rubrum

and

also in

the is

far

the

in photo-

chloroplast

and

and

not

intact at

to

the

These

which for

that

belong

formation.

system required

obtained

suggesting

does ATP

a part

(2-4).

R.

rates,

(chromaby

preparations

normal

electron

vesicles

acceptor

light-dependent

chromatophores

514

oxygen

photoreductions

2,3,5,6-tetramethyl-p-phenylenediamine; used : DAD, indophenol ; TB, tetrazol ium blue . -

Q 19 75 by Academic Press, IRK. of reproduction irt nr!,j form reserved.

be

of

vesicle

also

artificial

photophosphorylation

at

mediates

to

electron

the

other

membrane

derivative

photophosphorylation which

by

photoreduction terminal

and

thought

mutant

another

which are

is

responsible

. I n contrast,

system

carriers

the

DCIP*

catalyzed

a non-photosynthetic

catalyze

which

observations,

the

system

reactions

cells

of

from

Rhodospirillum

tetrazolium

chromatophore

oxygen

from

from

laboratory

reduction

one

electron-transfer

Chromatophores our

transfer

reduces the

DCIP,

normal

2,6-

Vol.

66,

MATERIAL

No.

2, 1975

AND

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

METHODS

o facul tative phototrophic bacterium, and The wild type strain (Sl ) of R. rubrum, the culture medium were those used before (4). Non-phototrophic mutants were isolated after treatment of wild type cells with t+methyl -N’nitro-Nnitrosoguanidine (6) and penicillin. Cells surviving penicillin treatment we% plated% aerobic plates and the colonies resulting thereof were streaked both on aerobic and on anaerobic plates. Streaks failing to grow after 4 days on illuminated anaerobic plates were selected as possible non-phototrophic mutants and checked for phototrophic growth in liquid cultures (4). Aerobic liquid cultures were performed in erlenmeyer flasks filled up to 75% of their total capacity. The flosks were incubated at 30°C in the dark, and aeration was provided by girotory shaking. Cells were collected when the cultures reached 0.6 mg dry weight of cells per ml (about 3 nmoles of bacteriochlorophyll per mg dry weight) by centrifugation at room temperature (4 000 x g for 10 min). Chromatophores were prepared as described before (4) and their protsn and bocteriochlorophyll contents were estimated using the methods of Lowry and Clayton (8), --et al - (7) respectively. For estimation of ATP levels in intact cells, cells were suspended in fresh growth medium at 0.87 mg dry weight per ml. Two lo-ml aliquots of the suspension were placed in open test-tubes (15 mm internal diameter), covered with 4 ml of paraffin oil and incubated in total darkness for 30 min at 30° C. 1 ml of 10 N HCI was added to one of the suspensions at the end of this incubation. The other suspension was illuminated for 30 s between two 100-W incandescent lamps and then received also 1 ml of 10 N HCI . The suspension was stirred during illumination with a small magnetic bar, taking care not to disturb the layer of mineral oil . The aqueous phases of the acid mixtures were centrifuged in the cold (3 000 x g for 10 min) to remove insoluble material and the supernatant was adjusted to pH 7.2 i 0 *02 with NaOH . The ATP content of the neutralized supernatant was estimated with the aid of a crude extract of firefly lanterns (9). Photophosphorylative activity of chromotophores was assayed in a 3-ml reaction mixture containing : 47 mM Tricine-NaOH (pH 8-O), 0.5 mM ADP, 5 mM HK2P04, and 40-80 pg/ml of chromatophore protein. The mixtures, in

open

test-tubes

(11

mm

internal

diameter),were

placed

in

a water

bath

at

25OC,

incubated for 3 min in the dark and then illuminated for 1 min as described in a previous report (4) - At the end of illumination 0.3 ml of 10 N HCI were added to each assay tube and the mixtures were centrifuged in the cold (27 000 x g for 10 min) . The supernatants were neutralized to pH 7.4 ? 0.02 and assayed for-ATP, also by a luciferin-luciferase method (9). A duplicate of each assay mixture was kept in the dark and used to correct for background luminiscence. Light-dependent oxygen uptake by isolated chromatophores was estimated as previously described (4), using 50 pM DCIP. The reduction of tetrazolium blue was monitored with a Perkin Elmer spectrophotometer (model 356) in the split mode, following the light-dependent increase of absorption at 590 nm (5). The reaction mixture, in a cuvette of l-cm optical path, contained : 40 mM Tricine-NaOH (pH 7.5), 50 PM tetrazolium blue, 50 pM DCIP (or 50 pM DAD), 1 .7 mM sodium ascorbate, 27 mM glucose, 10 units of glucose oxidose (E C 1 .l .3.4) and chromatophores as indicated. The mixture was covered with 0.5 of paraffin oil and the cuvette was placed in the spectrophotometer chamber at room temperature 21-23°C. Actinic illumination (64 W . m-2) was provided by o tungsten-halide, 650-W source after filtration through a narrow-band interference filter (B-IR 888, Balzers, Liechtenstein) . The photomul tipI ier was shielded from the actinic light with the help of complementary color and interference filters.

515

Vol.

66,

No.

2, 1975

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS Strain

Fll

, a derivative

illuminated,

anaerobic

cultures Fll

when in

the

aeration

of

of

growth

parent

(Table

of

Nevertheless,

low

during

the

mutant

In

spite

of

in

strains

of

strain

inability

to

form

growth

in

In

contrast

,

mass

after

dark

aerobic

RF1 10,

is

not

their

R.

apparatus cells

of

the

cell

colonies

on

liquid

anaerobic

suspensions

of

incubation

conditions

strain

when

was

a spontaneous

to

the

observation

due

to

to

times

similar

to

phototrophic

In of

whole

revertant

ATP

cell

light,

its

of

of

the

lack

strain

if Fll

, grown and

and

parent of

the

of

and

isolated revertant

phototrophic

growth

photopigments.

intact

levels

oxygen.

obtained

suspensions in

the

by

bacteriochlorophyll

bands

of

the

the

that

be

Cultures

fact,

alteration

in

is repressed can

(10).

indicates

grow

rubrum

microorganism

corresponding

a gross

and

R.

low

spectra

This

inability

of

pigmented.

aborption

cells

of

phototrophic

strain

and

Fll

increased

non-phototrophic

rubrum.

time

ATP (nmoles/mg

dark aerobic

light anaerobic

230

240

(parent) (mutant)

RF1 10

under

is sufficiently

identical

shown).

Strain

Fll

I).

time

its

detectable

their

Doubling (min)

Sl

show

(Table

of

normally

the

Doubling

I.

for

not

increased

growth are

are

of

light

that

pigmented

chromatophores (not

and

aerobic

bands

strains

did

doubling

photosynthetic

oxygen,

carotenoid

Table

The

the

tension

under

the

medium

strain

selected

I).

synthesis

oxygen

rubrum

plates,

in

provided.

the

Fll

The

same

R.

agar

incubated

was

that

of

(revertant)

Increase of cell mass during with filter 66. Determination

192

2

10.000 235

210

growth of ATP

dark anaerobic

was levels

followed in is described

516

a

dry

weight)

I ight anaerobic

0.6

6.3

0.9

6.6

0.6

4.9

Klett-Summerson under Material

and

calorimeter Methods.

Vol.

66,

No.

their by

2, 1975

internal cells

was

levels

from

tested incubation

tions

the

low

by

the

to

be

cell

oxygen

upon

strain

in

under

values

(Table

functional

of

the

respiratory

in

the

mutant-

photophosphorylation

exogenous

redox

by

cofactors

in

endogenous

cyclic

chain

with

intact

cells,

chromatophores

ATP

formation

at

mutant

chromatophores

oxygen

by

revertant

reduced strains.

photoreduction

Table

of

II.

of

normal

DCIP

stressed

is

from

to the

the

Fll

shows

in

less

than

5 min

of

ATP

fell

to

could

not

the

of

bypassing

be

absence

wild

reduced

and

catalyze DCIP.

catalyzed of

that of

type

to

strains

ATP

the The

by

R . rubrum

Oxygen

Strain (nmoles

Sl

(parent)

per

min

per

mg

protein)

143

85

Fl 1 (mutant)

156

16

RF1 10

162

122

(revertant)

The assays were carried out as described under Material bacteriochlorophyl I content of chromatophores was (in strain Sl, 21 ; strain Fll, 15; strain RFllO, 23.

517

the

obtained

also

photoreduction

photoreduction

non-phototrophic

dota

shows

both

by

of

light-dependent

chromatophores blue,

of

the

results, the

caused

a part

from

those

condi-

appears

in

from

Fll

oxygen

and

up

catalyzed

mediate

tetrazolium

and

a prolonged those

expected

strain

of

accumulation to

levels

assayed

As

to

failure

reached

under

light

chromatophores

acceptor,

phototrophic

the

that

photophosphorylation

(11).

ability

used

possibility

which

a decreased

a different

the

II,

compared

in

was

from

Since

and

ATP

carriers

Table

ATP

was

Therefore

isolated

Photophosphorylotion

chromatophores

of

avoid

to

subjected

oil.

shown)

chromatophores

electron

as

paraffin

COMMUNICATIONS

similar

previously

(not

to

extent

light-dependent

suspension

chain.

RESEARCH

an

the

formation

order

rates. had

More

of

in

the

The been

a layer

electrode I),

to

I). had

present

oxygen

BIOPHYSICAL

illumination

which

min)

the

AND

(Table

suspensions

originally

operation

Cyclic

ATP

parent

(30

monitored

very

of

the

in

dark

as

BIOCHEMICAL

and Methods. The nmoles/mg protein) :

Vol.

66,

No.

2, 1975

reaction

was

sustained

(Fig.

1) . The

might

be

also

absorbs

This

interpretation

place

to at

even

however

transient

due

reduced

BIOCHEMICAL

the

by

increase

the

transient

BIOPHYSICAL

chromatophores of

wavelength

used

observed of

of

tetrazolium

blue,

as a donor

( Fig.

1) .

described

suggest

that

system

of

absence

phototrophic

at

the

oxidized

that but

the

not

COMMUNICATIONS

the

tetrazolium

observation

the

the

monitor

the

in

by

to

RESEARCH

from

absorption

accumulation

is supported

DCIP

AND

strains

onset

of

form

of

blue

reduction,

DCIP,which

transient

when

illumination

590

change

DAD

nm.

took

sustituted

for

DISCUSSION

The

results

the

same

just

electron-transfer

is directly that

the

related defects

selected

only

catalyze

the

present

moment

pleiotropic

to are

for

the

lack

due

to

phototrophic

mutation

and

tetrazolium

rubrum

and

that

phototrophic

independent

growth mutations

growth,

possibility affecting

that the

the

is

of

not

phenotypic

the to the than

possibility

ability

result one

system

strain

discard

by

this

. The

because

are more

reduced of

Fll

possible

of

are

alteration

strain

lesions

expression

Nild type

the

simultaneously it

normal

blue

is unlikely

recovered

Unfortunately

photoreductions. the

of

R.

oxygen

RFllO, to

at of gene.

the a Only

I RFIIO

DCIP

DCIP

/ I

I

0.01 A

I/ I

’

Figure 1 . Photoreduction of tetrazolium blue catalyzed by chromatophores from phototrophic and non-phototrophic strains of R. rubrum . The reaction was followed as described under Material and Methods. Arrows indicate the onset of actinic illumination. Protein concentration in the assay mixtures was : wild type strain, RF1 10, 44 pa/ml . Bacteriochlorophyll 48 Pg/ml ; strain Fll , 145 pg/ml ; strain content of chromatophores was as in Table II.

518

Vol.

66,

No.

the

isolation

2, 1975

and

presence

of

synthetic

growth

At

any

an

rate

-flow

cells

of

the

R. our

rubrum

process

results is

system

from

this

, a process

investigation

related

BIOPHYSICAL

may

is an

help

absolute

RESEARCH

COMMUNICATIONS

to

whether

discern

requirement

the

for

the

photo-

rubrum.

defective

system

preliminary

mutants

is

system this

other

clear

mutant.

photoreducing between

R,

seems

, since

AND

photoreducing

which

phorylation

of

active of

it

system

study

BIOCHEMICAL

This

the in

of

strain

Fll

conclusion intact

and

the

mechanism

on

has this

before

cell

are

conclusions

in

have NAD(

thought

great to

be

of

involved

the of

a

possible in

controversy interpretate

the in

function

photoreduction

difficult can

part

chromatophores into

P) of

the

directly

inquire

a matter

respect

that

is not

to

+ We of

been

report

normal

leads

the

which

this

is apparently

in

needed

data

electron-

photophos-

and

in

of

the

intact oxygen

relationship whole

cells

(11-13). and

a more

of

However, thorough

drawn.

ACKNOWLEDGMENTS This work was suggestions of E. V. Mar;n.

supported Dr. F.

F.

by the “III del Campo

Plan and

de the

Desarrollo” competent

. The authors appreciate technical assistance of

the Miss

REFERENCES 1 . Vernon, L. P., and Kamen, M. D . (1953) Arch. Biochem . Biophys. 44, 298-311 . and Gromet-Elhanan, Z. (1972) Proceedings of the llnd International 2. Feldman, N., Congress on Photosynthesis Research, vol. 2, pp. 121 l-1220, Dr. W. Lnk N . V. hbl ishers, The Hague . 3. Govindjee, R., Smith, W. R., Jr ., and Govindjee (1974) Photochem. Photobiol . 20, 191-199. 4. del Valle-Tascon, S., and Ramirez, J . M. (1975) Z . Naturforsch . 3Oc, 46-52. 5. Ash, 0. K., Zaugg, W. S., and Vernon, L. P. (1961) Acta Chem. Stand . 15, 1629-1638. 6. Adelberg, E. A., Mandel, W., and Chen, G. C. C. (1965) Biochem. Biophys. Res . Comm . 18, 788-795. 7. lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol . Chem . 193, 265-275. 8. Clayton, R. K. (1963) Bacterial Photosynthesis, pp. 495-503, The Antioch Press, Yellow Springs, Ohio. 9. del Compo, F. F . , Hernhndez-Asensio, M., and Ramfrez, J. M. (1975) Biochem. Biophys. Res. Comm. 63, 1099-l 105. 10. Cohen Bazire, G., Sistrom, W. R., and Stonier, R. Y. (1957) J. Cell Camp. Rysiol . 49, 25-68. 11 . Vernon, L. P. (1968) Bacterial . Rev. 32, 243-261 . 12. bckson , J . B., and Crofts, A. R . (1968) Biochem . Biophys . Res. Comm . 32, 908-915. 13. Govindjee, R., and Sybesma, C. (1970) Biochim. Biophys. Acto 223, 251-260.

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