Nitrite reduction with formate in Pseudomonas denitrificans ATCC 13867

Vol. 87, No. 1, 1979 March

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

15, 1979

Pages 140-145

NITRITE

REDUCTION

WITH FORMATE IN ATCC 13867

PSEUDOMONASDENITRIFICANS

Yushi

Nishimura,

Laboratory Industrial

Received

January

Teijiro

Kamihara

and Saburo

Fukui

of Industrial Biochemistry, Department Chemistry, Faculty of Engineering, Kyoto University, Kyoto, Japan

of

29,1979

Formate served as an electron donor in dissimilatory nitrite reduction in resting cells of a denitrifier, Pseudomonasdenitrificans. Pyruvate also donated electrons to nitrite. Pyruvate reduced nitrite at the same rate as formate after some lag period. Citrate, the carbon source for cultivation medium employed in this study, was less effective for nitrite reduction. Two distinct cytochromes were shown to be involved in the electron transfer from formate to nitrite. Denitrification electron a tool cal

acceptors

rate.

of oxygen

to waste

reductase

electron

tron

carriers

tion, rite

remains

reduction

that

though

However, there

formate

serves

cells

formate-dependent

Pseudomonasdenitrificans a synthetic

medium

containing

@ 1979 by Academic Press. Inc. of reproduction in ony form reserved.

only

as

as a microbiologinitrate

reducfrom nitgenerally

such as Escherichia coli

[l,

for

nitrite

reductase,

of nitrite.

of nitrite

the

role

reports

on the elec-

In this

communica-

electron

donor

Participation is

21

also

for

nit-

of cyto-

reported.

AND METHODS

ATCC 13867 was grown anaerobically at 30" C in (per 1): KNOB 5 g, NH&l 1 g, Nas-citrate 6 g,

0006-291X/79/050140-06$01.00/0 Copyright All rights

not

has been considered

of P. denitrificans. reduction

as

of denitrification

as an effective

MATERIALS

but

have been several

in the reduction

in resting

in the

formate

oxides

attention

Dissimilatory steps

in microorganisms

participating

we describe

chromes

unknown

earlier

[3].

nitrogenous

metabolism

treatment.

reductase,

and Pseudomonasdenitrifioans of formate

of energy

catalyze

donor

using

and has attracted

water

In the case of nitrate

as the major

of respiration

the regulation

applicable

and nitrite

a type

in place

of studying

system

tase

is

140

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

KH2PO4 0.5 g, K2HPOs 1 g, NaCl 0.5 g, MgSOs*7H20 0.2 g, FeSOb*7H20 10 mg, CaCl2 20 mg, CuS01+*5H20 1 mg, ZnS04*7H20 1 mg, MnClp.6H20 0.1 mg, CoC12 2 mg, NasMo04*2H20 2.5 mg and NazSeO4*nHaO 0.2 mg. Cell growth was monitored turbidimetrically at 580 nm. Cells harvested at the late logarithmic growth phase were washed three times with 33 mM sodium potassium phosphate buffer (PH 7.0), and suspended in the same buffer. Nitrite reductase activity was assayed by measuring decrease of nitrite at 30' C. The reaction mixture contained 1.5 mm01 of sodium potassium phosphate buffer (pH 7.0), 30 urn01 of sodium nitrite, 150 pm01 of sodium formate, sodium pyruvate or sodium citrate, and appropriate amounts of cells (wet weight 150 mg) in a total volume of 15 ml. The reaction was initiated by the addition of nitrite after incubation for 5 min. Aliquots of the mixture were withdrown with appropriate intervals and the concentration of nitrite remained was assayed spectrophotometrically by a diazo-coupling method according to Nicholas and Nason [4]. Ammonia formed was determined by the indophenol method [5]. The difference spectra of cells were obtained in Thumberg-type cuvettes (1 cm path length) under helium with a double-beam spectrophotometer equipped with double end-on type photomultipliers, Shimadzu MPS-5000 (Shimadzu Seisakusho Co., Kyoto, Japan). Cells incubated with formate and nitrite were referred to as "formate-reduced cells" and "nitrite-oxidized cells", respectively.

RESULTS Time-course resting

cells

occurred ty,

changes

linearly

(6.0

per mg-wet

nmolfmin

the

carbon

source

fold

(1.9

nmol/min

per mg-wet

No detectable

nitrite

shown).

disappeared)

duced

to compounds

ing

in the nitrite

taken

under

rite-oxidized tected reduced

several

after

reduction

than

(a twentieth

electron

except

that

reduction.

period

under

when lactate or lower

Most of nitrite

activi-

some

Citrate, rate

by three

the same condiwas used

(data

of the amounts

disappeared

of

would

be re-

ammonia.

with

conditions.

a shoulder with

on the electron

for-mate, Figure

spectrum.

cells

a lag

occurred

some information

reduction

at 553 nm, with against

cells)

(specific

at a lower

in

of nitrite

as an effective

to formate

nitrite

donors

Reduction

donor

of nitrite

reduced

produced.

other

difference

cells

medium,

electron

1.

served

comparable

initiation

of ammonia

were

to obtain

Pyruvate

cells)

the

in growth

Low amounts

nitrite

In order

for

various

was used as an electron

per mg-wet

was required

with

shown in Figure

cells).

period

not

are

when formate

lag

tions.

reduction

of P. denitrificans

6.8 nmol/min

donor

in nitrite

difference

carriers spectra

of cells

2-A shows formate-reduced

A peak of a cytochrome at 559 nm. no addition

141

participat-

Spectrum

had no detectable

minus

(o-band)

obtained

were

with peak

nit-

was deformate(Fig.

2-B),

BIOCHEMICAL

Vol. 87, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-

01

I

I

I

I I

20

10

OO

02

30

TIME(min)

B

\ I Wave 550

500

k I

I

650

600 lengthtnm)

Fig. 1 Reduction of nitrite by resting cells of P. denittif&ans sodium formate (O), sodium pyruvate (0) and sodium citrate (@. ctron donor was added at a final concentration of 10 mM.

with Each ele-

Fig. 2 A:Formate-reduced minus nitrite-oxidized difference spectrum of resting cells grown anaerobically with nitrate. Spectrum B was obtained with formate-reduced cells against cells with no addition. The reaction mixture in Thumberg-type cuvettes contained 150 umol of sodium potassium phosphate buffer (pH 7.01, cells (wet weight 45 mg) and 30 pmol of sodium formate or 30 pm01 of sodium nitrite in 3 ml. Base lines were obtained with the cell suspension under helium. The reductant, for-mate, and the oxidant, nitrite, were then added to each cuvette from side arms and the spectra were taken after incubation for 5 min.

indicating

that

nitrite

two cytochromes

even without

out nitrate

with

formate

Comparing

cytochromes that

mentioned

cytochrome

chrome

of these

peaks

spectra Spectrum

with

Figure above with

559 was nearly

553 was in reduced

state

state

Cells (data

of steady

not

+ nitrite)-had 2-A,

this

nearly completely in steady

formate.

142

a broad peak would equal

cells

with-

reducing

nit-

The shoulder

B in Figure peak

consist

heights.

oxidized state

aerobically

of

spectrum:(nitrite-oxidiz-

Spectrum

observed.

the absence

shown).

state

A-difference

in

grown

a peak at 553 nm.

2-A was not

(for-mate-reduced

in reduced

formate.

nitrite-oxidized-had

in Figure

minus

3).

were

added

difference

(Fig.

minus

559 nm shown

559 nm.

neither

we took

ed + formate)

reduced

exogenously

exhibited

Further, rite

these

These

reducing

3-formate-

from 553 nm to of two o-bands results

and some portion cells

at

nitrite

show of cytowith

of

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 560

I 500

I 600

I 660

Wave lergth(nm) Fig. 3 Difference spectra of steady state cells reducing nitrite with formate. Spectrum A:(nitrite-oxidized + formate) minus nitrite-oxidized. Spectrum B:Formate-reduced minus (formate-reduced + nitrite). The reaction mixture in cuvettes was same that in Figure 2. For spectrum A, a pair of cuvettes containing cells and nitrite were incubated anaerobically for 3 min at 30' C. Base line was obtained with the cuvettes. Formate was then added to one cuvette from the side arm and incubated for another 3 min for spectrum measurement. Formate and nitrite were added by reverse order for sepctrum B.

DISCUSSION The results effective

electron

produce first

gaseous

donor

for

clearly

demonstrated

dissimilatory

remained

reduction

strongly

suggests nitrite

than

citrate,

that

formate

were

to nitrite.

Recently,

chrome

had a role

glutamate

The cytochromes

reduction,

which

ATCC 13667.

It

various

fate

exhibited

source

has an important

leads would

to be the

in a

compounds

in cells higher

used in this role

as an

reduction carbon

their

for-mate

acts

reduction

in nitrite

but

the carbon

formate

have

has been

ability

of

study.

in physiology

This

of dissimi-

reduction.

Two cytochromes

b-559

nitrite

In P. denitrificans,

unknown.

nitrite

of formate

In many microorganisms,

to support

that

nitrite

in P. denitrificans

substance(s)

organism.

been employed

with

here

case to show the participation

denitrifying

latory

obtained

found

Walter

to participate

et al.

as electron

in the same strain participating

reported transport

that

in electron that

in nitrite

reduction

143

cytochrome

mediators

was employed

flow

from c-553

in nitrite

formate

and cytoreduction

in the present with

formate

study

would

[6].

be iden-

8lOCHEMlCAL

Vol. 87, No. 1, 1979

tical

with

cytochrome (Fig.

those

reported

b-559

in formate-reduced

2) was similar

ference

by them.

to that

mate was, results

however,

different

state

reported

cells

+ nitrite)-was rence

spectrum

hand,

state

from

with

with

not

to spectrum

nitrite-oxidized

dif-

of this of nitrite

ference

spectra

et aZ. [6].

(Fig.

state

and c-553

observed

ammonia

in E. coli whether of its

period

peak

contained

state.

cytochrome of which

in Figures

2 and 3.

was accompanied

by the re-

detected

in

but was not

mentioned

is now under

study.

bacterium

[7].

species

nitrite

which it

that

formate

by Walter

reduction

was tentatively

However,

the dif-

was shown that

have called

the bacte-

[7,8].

reported

in E. coli

[9].

and is different formate

cells

oxidized

some portion

in dissimilatory

strain)

e-t aZ.

reduction

in the

of this

in a certain

Abou-Jaud6

because

for-mate

as illustrated

system,

participating

to Alcaligenes

employed

with

state

3).

nature

nitrite

in E. cozi

cells

of the

diffe-

steady

completely

The same peak was also

(Iwasaki

be doubtful

in nearly

similar

(glutamate-reduced

the

for-mate.

intensively

is

that

with

studied

for

indicates

for-

nitrite-oxidized

some lag

carriers

Recently,

minus

requiring

Electron

donor

This

with

the spectrum

minus

peak

The precise

belongs

in which

glutamate-reduced

of glutamate-nitrite

P. denitrificans

[6],

nitrite

We obtained

glutamate-reduced

oxidized

state

reducing

glutamate.

of the cytochromes

completely

duction

source

c-553

difference

minus

cells

et al.

nitrite-reducing

in reduced

appearance

that

shown).

A peak at 503 nm was also

would

of cytochrome

nitrite-oxidized

of steady

glutamate-

had both

in nearly

remained

with

(data

glutamate

b-559

rite

minus

by Walter

identical

On the other

rium

The proportion

in glutamate-reduced

spectrum

to those

steady

been

RESEARCH COMMUNICATIONS

spectrum.

The difference

with

AND BIOPHYSICAL

plays

low efficiency

The reduction from

144

that

an important compared

culture.

acts

product

from nit-

in denitrifiers. role

with

as an electron

in nitrite

glucose,

the

It reduction carbon

The

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENT We are grateful to Dr. Makoto Ishimoto, Hokkaido University, Tateo Yamanaka, Osaka University, for their valuable discussion. was supported in part by the grant of No. 203035 from the Ministry cation, Japan.

and Dr. This work of Edu-

REFERENCES

111 Cole,

J. A., and Wimpenny, J. W. T. (1968) Biochim. Biophys. Acta 162, 39-48 C. H. (1978) In: Microbiology 1978 (D. Schlessinger eds) [21 MacGregor, pp. 330-333, American Society for Microbiology, Washington D. C. Radcliffe, B. C., and Nicholas, D. .I. D. (1970) Biochim. Biophys. Acta 131 205,

273-287

Nicholas, D. J. D., and Nason, A. (1957) In: Methods in Enzymology vol III (S. P. Colowick and N. 0. Kaplan eds) pp. 981-984, Academic Press, New York Solo'rzano, L. (1969) Lumnol. Oceanogr. 14, 799-801 [51 B., Sidransky, E., Kristjansson, J. K., and Hollocher, T. C. [61 Walter, (1978) Biochemistry 17, 3039-3045 Iwasaki, H. (1976) Seikagaku (Tokyo) 48,83-95 [71 M., Contopoulou, R., Kunisawa, R., and Palleroni, N. J. 181 Doudoroff, (1974) Int. .I. Syst. Bacterial. 24, 294-300 A., Chippaux, M., Pascal, M. C., and Casse, F. (1976) [91 Abou-Jaudg, Biochem. Biophys. Res. Comm. 78, 579-583 [41

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