Vol.

78,

No.

2, 1977

INTERACTION K12

BIOCHEMICAL

BETWEEN AND

THE NITRATE

THE NITROGEN

Mary Department Australian

L.

of Genetics, National

Received

August

AND

BIOPHYSICAL

RESPIRATORY

FIXATION

GENES

Skotnicki

and

Research University,

RESEARCH

SYSTEM

OF ESCHERICHIA

OF KLEBSIELLA

Barry

G.

of

Biological A.C.T.

School Canberra,

COMMUNICATIONS

COLI

PNEUMONIAE

Rolfe Sciences, Australia

2601,

The

lo,1977

coli K12 chl mutants defective SUMMARY: Hybrids were constructed between g. __ in nitrate respiration and an F' plasmid carrying nitrogen fixation genes from K. pneumoniae. Examination of these hybrids showed that expression of nif+ -genes does not require a functional nitrate respiratory system, but -7 tha nitrate reductase and nitrogenase do share some MO-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the MO-): cofactor encoded by chl genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity. INTRODUCTION: Evidence genase

suggesting

enzyme

presented this

6,

complexes

(1,

2,

proposed The 7).

have

been

can

reduce

3),

Mutants

defective

isolated to

because toxic

from

the the

-K.

Bacterial

other

studies

complex

has

in

their

inducible

of

resistance (8,

possible

relationship

and

of

various the

pneumoniae

MATERIALS

a common

chlorite

nitrogenase

between (chl-)

membrane-bound

share

although

respiratory

constructed ation

may

nitrate

and

the

nitrate

reductase

molybo-protein have

and

subunit

provided

has

little

nitrobeen

support

for

commonality(4).

Therefore, system

that

FN68 (nif -P

plasmid

well-studied

in

formate-nitrate to

chlorate,

g.

coli

K12

reductase which

nitrate

between

pneumoniae

coli

K12 which

was

the

nitrate

reductase

respiratory

investigated

mutants

defective

carries

the

by in

nitrogen

using

nitrate

hybrids respir-

fixation

genes

carrying

the

+ 1.

AND METHODS strains:

E.

coli

K12

strain

SB1801

his_-

ma1 -

(5,

complex

9).

-K. E. --

been

BIOCHEMICAL

Vol. 78, No. 2, 1977

CbR) re 4;-uctase chlmutants

FN68 plasmid (F'nif:, experiments with nitrate with pleiotropic nif__-

(10) was used as the donor in conjugation and also (chl-) mutants of E. & K12, of &. pneumoniae strain M5al.

efK

Media: Nitrogen-free medium Growth in liquid NFM was measured When necessary, NFM was supplemented amino acids at 25 vg/ml 100 w/ml, E. coli Conjugation: in conjugation experiments were selected and purified tested for nitrogen fixation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(NFM) was that of Cannon et al. (11). by following turbidity in a nephelometer. with vitamin-free Casamino acids at and vitamins at 10m3 UM.

K12 strain with mal+ as described by growth

SB1801 (FN68) was used as the donor recipient strains. Mal' CbR hybrids previously (12), andwere then on NFM and by acetylene reduction.

Rates of acetylene Acetylene reduction assay: were measured by the method of Tubb & Postgate (13). to the medium repressed nitrogenase of 50 mt3 (NH~)~ so4 by acetylene reduction. RESULTS

AND

None

DISCUSSION

of

expression gene

the

of

for

+

cofactor

(5)

mutants

of

was

indicating To

the

carrying

were

tested

levels

of

the

coded

fixation,

cofactor

by

-chlB

indicating

nitrogenase nitrite

for

and is

acetylene

not

in

grown

that nitrate

are

for

necessary

reduction

in

hybrids,

nitrate

for the

inhibition,

nif -

NFM

chlA __

(5)

for

nitrate

prevent

for

addition

with

KNO3 chlB-

hybrids nitrate and

hybrid.

Similarly

still

inhibited

nitrogen

-chlD-

the

fixation, or

KNO2,

of

in

the

showed

gave

similar

(Table

1).

association

are

still

and

hybrids

inhibition

nitrate

of

functions.

conversion

chlC-

a MO-X

in

functions

since

not

nitrogen

chlcand

molybdenum-processing

necessary

+

without

However,

structural

coding

these

by

the

activity

in

or

phenotypic

However

and

the

with

reductase.

did

gene,

activity; NFM

(5),

inhibits

Only

coded (5)

chlD

were

nitrogenase

*C,

nitrogenase

nitrate

reduction.

reduction

MO-X

full

which

plasmid

acetylene

acetylene

Thus, factor

FN68

of

reduced

activity.

between by

loss

chlA __

nitrogenase for

or

enzyme

a functional for

of

the

reductase

interaction

inhibition

prevented

Even

nitrate

mechanism

the for

nitrate

I).

essential

an

examine

hybrids

the

unnecessary

ions

4

mutants

(Table

Likewise, is

2-

x00

reductase

genes

a subunit fixation.

no

nitrate

nif

nitrogen

10-4M

reduction to ethylene In all cases, addition activity as measured

of shared nitrate caused -chlD-,

nitrogen by to loss _chlE-,

and

-chlC

727

fixation.

Since

the

of

Vol. 78, No. 2, 1977

TABLE.

1.

Phenotypic rn

E.

BIOCHEMICAL

coli hybrid

expression

nitrate

of

reductase

K12

chl

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nif+ ~

mutants

genes

KP of

E.

on

coli

plasmid

K12

Acetylene

mutation

Reduction

NFM

C181(FN68)

chlAA

FN68

NFM+

NFM+

KNO3

fQJO2

39.0

38.0

0.9

54.4

58.6

0.6

KB70(FN68)

chlA

C183(FN68)

chlB

43.0

40.8

0.08

DDlls(FN68)

chlB-

31.2

32.2

0.08

Puig

chlC-

35.4

0.08

co.01

Clll(FN68)

chlD*

46.0

co.01

co.01

C113(FN68)

chlD-

46.3

10.01

co.01

c123(FN68)

chlD-

39.1

co.01

co.01

C197(FN68)

chlE-

49.0

co.01

co.01

C202(FN68)

chlE-

45.0

co.01

co.01

DD38(FN68)

chlG-

56.8

0.3

0.1

40.2

40.2

0.2

426cFN68)

SA291(FN68)

chlA __

Acetylene KNOX were

reduction added at

R

various

chl __

(6),

inhibition

of

this

system.

through

mutations

Nitrogenase However, low by

level nitrite

in

also

cause

nitrogenase

chlA-,

acetylene (Table

chlD* -

measured in concentrations

activity the

of

was final

A

loss

run01 of

of

activity

in chlB reduction

C2Hq/min/mg 50 mM.

the by

formic

nitrate

all

hybrids

and

chlG__

hybrids,

which

may

tested

1).

728

protein.

there indicate

hydrogenlyase is

was

KNO2

unlikely

inhibited was incomplete

and

system to

by

be

KNO2.

a reproducible repression

acting

Vol. 78, No. 2, 1977

TABLE

2.

Growth

BIOCHEMICAL

of

&

coli

Kl?.

(FInif+ or

Hybrid

chl __

AND BIOPHYSICAL

KP)

RESEARCH COMMUNICATIONS

in

hybrids

NFM

Growth

NFM

chlAn

NFM+

NFM+

mo:,

m07

100

47

64

100

93

49

KB70(FN68)

chlA

C183(FN168)

chlB-

100

220

106

DD115(FN68)

chlB-

100

110

15

Puig

chic-

100

172

57

100

155

45

426(FN68)

A

Clll(FN68)

chlD

C113(FN68)

chlD-

100

192

75

C123(FN68)

chlD-

100

148

46

c197(~pJ68)

chlE-

100

44

61

C202(FN68)

chlE-

100

46

73

DD38(FN68)

chlG-

100

109

31

Growth in Pankhurst incubation, and is and KN02 were added

All were

KN03

KNOL

mutation

C181(FN68)

with

four

observed

(a) (b)

possible when

classes hybrids

inhibition

of

inhibition (chlA)

(cl

tubes was measured in a nephelometer after expressed as a percentage of the NFM control. to give final concentrations of 50 mu.

were both

growth grown

in

growth

of

growth

of

nif ----KP

but

and not

response

and

NFM

nitrate

nif *P of

with '

nif -

acetylene

phenotypic + KP

reduction

(Tables

1 and

expression

phenotypic

D,

+

phenotypic

expression

G);

729

but

of

2):

(chlE);

expression

not

hr KiY03

;

inhibition (chlC,

of

24

growth

Vol. 78, No. 2, 1977

Cd)

no

BIOCHEMICAL

inhibition

of

either

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

growth

or

t

nif KP

phenotypic

expression

CchlB). Genetic exists,

and

and

tory

that

nitrate

it

the

piration

ever, have

When phenotype of

of

of

the

same

was

completely

growth

in

hybrids

was

were

and

reduced

(Table

also

of

E. coli -__

K12

defective

energization

of

the

membrane,

such

phorylation,

mutants

affecting

the

cases

systems

are The

FN68

E.

coli

K12

in

the

nitrate Two

nif -

did and

mutants

defective

in

nitrate

and

were

then no

expression mutation.

of

the

assimila-

their

NFM

and

chlE

(14). nitrate

res-

nitrate

appear

pathway.

mutants

How-

appear

to

+ nitrite,

except

for

generally the

was

t

Nif

mutants,

examined

particular defective

the

or

in

nitrate

nitrogenase inhibition

not

of nitrate functions

strain

M5a1,

made

anaerobic

oxidative

phos-

complex,

reduction,

and but

activity.

Thys

nitrogenase

activity.

reductase

carry

hybrids

for

reductase

fumarate

any

in

systems

fumarate-nitrate

does

chlB-

the

these

activity to

in

to

complement

chl-

defects

system. of

-K.

in

nitrogenase

pneumoniae

nitrogen was

plasmid-borne expected,

component were

(15),

reduction

As

and

mutants

restore

thus

for

acetylene

as

nitrate

not

reduction tested

in

reductase

NFM

in

either

for

respiratory

tive

nif -

plasmid

t

in

activity

inhibited

required

hybrids,

areprobably

hybrid

in

completely

not

the

2).

nitrogenase

important

nitrate

in

inhibited,

mutants

all

chlA-

grown

of

mutants

growth

reductase

system

defective

nitrate

the

nitrate

respiration

are good

one

complexes.

inhibition

quinone

only components:

nitrate

assimilatory

Nitrate with

that

distinct

the

their

mutations both

two

mutants

because

pleiotropic

indicate

and

a functional

perturbated

extent

a complex pathway

but

retained the

studies

chlorate-resistant

system,

have

is

reductase

All

to

biochemical

as

(Table

detected,

which

t

genes

acetylene

730

and

and which

recipients

fixation

nif ---KP no

used

II,

nif88 __

is

are

for In

3).

the the

indicates abolished

reduction

by was

nifl05,

which

also FN68

defecplasmid

nif88(FN68) that

phenotypic

the

chromosomal

obtained

by

this

Vol. 78, No. 2,1977

TABLE in

3.

mutants

BIOCHEMICAL

Phenotypic of

-K.

expression pneumoniae and

nif88(FN68)

defective nitrate

+

nif _

genes

KP in

both

plasmid

nitrogen

fixation

Growth

'0.01

100

NFM

+ NO;

CO.01

77

NFM

+ + NH,+

x0.01

208

8.0

100

NFM

+ NOCi

30.0

98

NFM

t + NH4

-0.01

141

51.3

100

NFM NFM

+ NO;

'0.01

84

NFM

+ + NH4

co.01

201

added to NFM in Pankhurst tubes to NO-3 and NH: were trations of 50 mM. Growth was assessed after 24 hr measuring turbidity in a nephelometer, and is expressed of the NFM control.

hybrid

in

contrast, also

showed 3).

growth

with

(Table

3).

been

reported

(17).

The -K.

presence

hybrid

(Table

of

the

of

either

nitrate

nif105(FN68)

This

could since

A similar for

KP

growth

stimulation

soybean +

not

be was

was

a low

stimulation

similar

of

give final concenincubation, by as a percentage

(Table

at

explained

simply in

nitrogenase

bacteroids

plasmid

ammonia

acetylene

four-fold

result

nitrate,

or

reduced

a nitrate-dependent,

F'nif -

EN68

Acetylene reduction (nmol C;Hq/min/mg protein)

NFM

nif+ __

on

reduction

NFM

nif105(FN68)

M5al

of

Medium

Hybrid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(16) expressed

and

NFM

3). level

normally

for

in

NFM,

results,

using

nif -

t KP

hybrids

defective

731

but

acetylene

reduction

on

the

of

with

basis

or

without by

Spirillum

in

other

in

nitrate

better nitrate

nitrate

has

lipoferum nif~-

chlS

pneumoniae. These

marked

of

activity also

In

respiration,

mutants

Vol. 78, No. 2, 1977

show

that

BIOCHEMICAL

regulation

of

inhibition

of

nitrogen

functional

or

organizational

reductase brane

systems

also

(20),

where

ase. presence

by

the

hybrid

nitrate

such

the

The

results

obtained

reductase

and

commonality

could

where

Since

some

these

appears

that

nitrate

to or

nitroqenase in

et

Rhizobium

in

al.

concluded

reduction

were

also the

Klebsiella, by

altered

also

in

more

complicated,

result

addition carriers

activated.

hybrids

in

suggest

that This

Rhizobium

meliloti

nitrogenase

activity.

capacity,

pathway

is

be

electron

nodulation

respiration

proposed

but

fully

lacked

their

the

functions.

observed

mutants

nitrate

of

chlD-

nitrogen-

in

to

system

be

of

able

less

slo-processing

pleiotropy

vinelandii

this

is

and

A. -

of

formed

now

interpretation

Klebsiella

explanation

competition

mem-

may

it

also

affect

the

interactions.

Pagan 32H1,

the

in

the

a functional

even

activity

chlBshare

reductase

defects

Rhizobium-plant

chlA-, __

nitrogenase

nitrate

is

can

nitrate

formation

situation

complex

with

the

the

proposed

nitrogenase

explain

mutants

the

and in

of

affect

transport

affects

mutant

directly

complex

proposed

This

acetylene

simplest

the

without

reduced

the

Nitrate

carriers

(19). R

not

nitrogenase

enzyme

modified

but

The

fumarate

fumarate.

a __ chl

However,

electron

the

with

must

hybrid.

presumably

electron

occurs

functions

stimulated

normal

by

phenotype

complex.

affecting

the

to

carriers.

that

nitrate

(11,

these

by

transport

hybrids

is

between

activity

chlB

nitrogenase

by

manifested

obtained the

and

niflOLi(FN68)

activated of

-chlA-

actually

pneumoniae that

results

electron

nitrate

be

activity

competition

a Nif-

inhibited

nitrate,

competition since

the

the

of

that

electron

nitrate

Since

may

such

system,

explain

nitrogenase

relationship

efficient

reduction

would

and

fixation

(18)

prevents

fumarate

growth

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

using

(4), that

nitrate

nitrite,

competed

activity.

they with

These to

the

lack

reductase

probably

although

nitrite

due

nitrate

two of

mutants

inhibited did

not

nitroqenase possibilities well-defined

732

of

Rhizobium ___-

sp.

activity

by

nitrogenase exclude for

the

possibility

electrons, could

mutants

not available

that

thereby be

its

lowering

distinguished in

E. -__ coli

Vol. 78, No. 2, 1977

K12,

which

have

BIOCHEMICAL

allowed

a clearer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

analysis

of

the

problem.

ACKNOWLEDGEMENTS Drs. strains. Fellowship

B. Bachmann, M.L.S. is the Plan Award.

D. Dykhuizen recipient

and W. Venables a Commonwealth

of

are thanked Scholarship

for and

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