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Free-Living Rhizobium Strain Able To Grow on N2 as the Sole Nitrogen Source

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Abstract

A Rhizobium strain isolated from stem nodules of the legume Sesbania rostrata was shown to grow on atmospheric nitrogen (N(2)) as the sole nitrogen source. Non-N(2)-fixing mutants isolated directly on agar plates formed nodules that did not fix N(2) when inoculated into the host plant.
APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Feb.
1983,
p.
711-713
0099-2240/83/020711-03$02.00/0
Vol.
45,
No.
2
Free-Living
Rhizobium
Strain
Able
To
Grow
on
N2
as
the
Sole
Nitrogen
Source
B.
L.
DREYFUS,'*
C.
ELMERICH,2
AND
Y.
R.
DOMMERGUES1
Office
de
la
Recherche
Scientifique
et
Technique
Outre-Mer,
B.P.
1386
Dakar,
Senegal,'
and
Unite
de
Physiologie
Cellulaire,
Institut
Pasteur,
75724
Paris,
Cedex
15,
France2
Received
7
September
1982/Accepted
23
November
1982
A
Rhizobium
strain
isolated
from
stem
nodules
of
the
legume
Sesbania
rostrata
was
shown
to
grow
on
atmospheric
nitrogen
(N2)
as
the
sole
nitrogen
source.
Non-N2-fixing
mutants
isolated
directly
on
agar
plates
formed
nodules
that
did
not
fix
N2 when
inoculated
into
the
host
plant.
Rhizobium
spp.
normally
fix
atmospheric
ni-
trogen
(N2)
only
within
nodules
formed
as
a
result
of
their
symbiotic
association
with
specif-
ic
host
plants
of
the
family
Leguminosae.
Some
strains
belonging
to
the
slow-growing
Rhizobium
group
(Rhizobium
spp.
of
the
cowpea
group
and
R.
japonicum)
are
also
able
to
express
nitrogen-
ase
under
free-living
microaerobic
conditions
(4-6,
8,
9),
but
they
require
a
source
of
com-
bined
nitrogen
to
support
their
growth.
There-
fore,
the
use
of
conventional
bacteriological
techniques
to
isolate
symbiotic
nitrogen
fixation
Rhizobium
mutants
has
not
yet
been
possible
without
screening
each
of
the
potential
mutants
on
individual
plants.
In
previous
papers
(2,
3),
Dreyfus
and
Dom-
mergues
indicated
that
a
fast-growing
Rhizobi-
um
strain
(ORS
571)
isolated
from
stem
nodules
of
the
tropical
legume
Sesbania
rostrata
forms
nodules
on
both
the
stem
and
the
roots
of
the
host
plant.
We
report
here
that
this
strain
not
only
expresses
nitrogenase
activity
in
culture
but
also
is
able
to
grow
on
N2
as
the
sole
nitrogen
source.
Preliminary
experiments
showed
that
the
growth
of
strain
ORS
571
in
a
defined
medium
was
dependent
on
the
addition
of
three
vitamins:
biotin,
pantothenic
acid,
and
nicotinic
acid.
We
studied
the
growth
of
the
strain
in
three
media:
a
nitrogen-free
medium
(LO
medium),
LO
medi-
um),
and
a
complete
medium
(YL
medium,
i.e.,
LN
medium
supplemented
with
1
g
of
yeast
extract
per
liter).
The
composition
of
LO
medi-
um
was
as
follows
(per
1000
ml):
sodium
lactate,
10
g;
K2HPO4,
1.67
g;
KH2PO4,
0.87
g;
NaCl,
0.05
g;
MgSO4
*
7H20,
0.1
g;
CaCl2,
40
mg;
FeCl3,
4
mg;
Mo04Na-
2H20,
5
mg;
biotin,
10
mg;
nicotinic
acid,
20
mg;
pantothenic
acid,
10
mg;
and
trace
elements.
The
pH
was
maintained
constant
at
6.8.
The
strain
was
grown
at
37°C
in
a
1.5-liter
Biolafitte
fermentor
that
contained
1
liter
of
medium
and
was
inoculated
with
bacteria
previ-
ously
grown
under
aeration
in
YL
medium
and
washed
twice
in
LO
medium.
Internal
agitation
of
the
broth
was
at
400
rpm,
and
the
gas
mixture
was
constantly
bubbled
through
at
a
rate
of
1
liter/min.
Growth
of
strain
ORS
571
on
N2
or
NH4
under
different
gas
phase
conditions
is
reported
in
Fig.
1.
When
grown
in
LN
medium
under
air
or
3%
02-97%
N2,
the
strain
exhibited
a
generation
time
of
3
h
at
37°C.
When
grown
in
nitrogen-free
LO
medium
under
3%
0297%
N2,
strain
ORS
571
showed
exponential
growth;
the
optical
den-
sity
at
570
nm
increased
from
0.15
to
2.30,
and
the
generation
time
was
6
h
at
37°C.
It
exhibited
nitrogenase
activity
only
2
h
after
inoculation.
Specific
nitrogenase
activity
(not
shown
in
Fig.
1)
was
maximum
(29
nmol
of
C2H4
mg
of
pro-
tein-1
min-1)
for
an
optical
density
of
0.6.
No
ammonium
accumulation
could
be
detected
in
.-
.00
*--
*
*-*
t";0°2:"0225%:
97%
O
/
/
0ot;2^$:7
°
/
i-
b_
^
A_
~~~~~~~~~LO;
02:
12
20:
90%
%)
2
1.
tO
t
OR22
51
on
N
oh
NH0
FIG.
1.
Growth
of
strain
ORS
571
on
N2
or
NH4.
711
FIG.
2.
(A)
Nitrogen
and
non-nitrogen-fixing
colonies
on
LO
medium
incubated
for
5
days
under
3%
02-97%
N2.
Colonies
of
the
parent
strain
(ORS
571),
which
actively
fixed
N2,
appear
as
large
dark
spots;
colonies
of
non-
nitrogen-fixing
mutant
strain
5740,
whose
growth
was
very
poor,
appear
as
small
pale
spots.
Previously
tested
strains
had
been
mixed
before
plating.
(B)
Ability
of
non-nitrogen-fixing
mutant
5740,
isolated
from
a
plate
culture,
to
nodulate
and
fix
nitrogen,
as
determined
by
the
root
inoculation
test
(2):
left,
uninoculated;
center,
inoculated
with
parent
strain
ORS
571;
right,
inoculated
with
mutant
5740.
Root
nodules
formed
by
ORS
571
fixed
nitrogen,
but
root
nodules
formed
by
5740
did
not.
Roots
were
inoculated
when
plants
were
3
days
old,
and
the
photograph
was
taken
when
the
plants
were
3
weeks
old.
(C,
D,
and
E)
Ability
of
mutant
5740
to
nodulate
and
fix
nitrogen,
as
determined
by
the
stem
inoculation
test
(2).
(C)
Absence
of
nodules
on
uninoculated
stems,
(D)
nitrogen-fixing
nodules
on
stems
inoculated
with
parent
strain
ORS
571,
(E)
non-nitrogen-fixing
nodules
on
stems
inoculated
with
mutant
5740.
Stems
were
inoculated
when
plants
were
3
weeks
old,
and
the
photographs
were
taken
3
weeks
later.
712
I
NOTES
713
the
medium.
The
total
nitrogen
content
of
the
culture
broth
(which
included
nitrogen
from
the
vitamin
supplement)
increased
from
4
to
50
mg/
liter.
When
the
percentage
of
oxygen
in
the
gas
mixture
was
increased
(20o
02,
80%
N2)
or
when
N2
was
replaced
by
argon
(3%
02,
97%
Ar),
the
maximum
observed
optical
density
was
ca.
0.3.
These
results
clearly
show
that
strain
ORS
571
was
able
to
grow
in
nitrogen-free
LO
medium
with
N2
as
the
sole
nitrogen
source,
provided
that
the
02
tension
was
appropriate.
Under
the
conditions
which
permitted
the
growth
of
ORS
571
on
N2,
strain
CB
756,
a
cowpea
strain
known
to
express
nitrogenase
activity
in
culture
(1),
neither
grew
nor
exhibited
any
acetylene
reducing
activity.
Purity
of
the
cultures
was
assured
by
repeated
use
of
single
colony
isolates,
and
the
identities
of
the
isolates
were
checked
by
plant
infection
tests.
Strains
reisolated
from
nodules
obtained
under
aseptic
conditions
exhibited
the
same
characteristics
as
those
shown
by
strain
ORS
571.
Since
strain
ORS
571
in
pure
culture
can
grow
on
N2,
it
was
possible
to
isolate
symbiotic
nitrogen
fixation
mutants
by
standard
bacterio-
logical
techniques.
Ethyl
methane
sulfonate
mu-
tagenesis
(200
,ug
of
ethyl
methane
sulfonate
ml-')
was
performed
as
described
by
Miller
(7).
Mutagenized
bacteria
(5%
of
the
cells
survived
and a
5-
to
10-fold-increase
occurred
in
the
number
of
streptomycin-resistant
mutants)
were
grown
overnight
in
LN
medium,
washed
once
with
LO
medium,
and
plated
on
solid
LO
medi-
um.
After
5
days
of
incubation
under
a
3%
02-
97%
N2
gas
phase,
40,000
colonies
from
two
mutagenized
cultures
were
examined.
Fifty
small
colonies
were
plated
on
LO,
LN
and
YL
media
with
toothpicks.
Clones
which
exhibited
the
same
growth
on
YL
and
LN
media
but
which
did
not
grow
on
LO
medium
were
checked
for
nitrogenase
activity.
Of
the
50
colonies
tested,
five
mutants
showed
no
or
very
little
nitrogenase
activity
in
culture.
In
liquid
LN
medium,
the
growth
rate
of
these
nitrogen
fixation
mutants
was
similar
to
that
of
the
parent
strain
(ORS
571),
but
in
liquid
LO
medium
under
3%
02-97%
N2,
there
was
no
significant
growth,
compared
with
strain
ORS
571.
Under
the
same
gas
mixture
but
on
solid
LO
medium,
the
growth
of
the
mutants
was
very
poor,
compared
with
that
of
the
parent
strain
(Fig.
2A).
The
colony
size
of
the
mutants
grown
on
solid
LO
medium
under
3%
02-97%
N2
was
similar
to
that
of
the
parent
strain
grown
on
the
same
medium
but
under
non-nitrogen
fixation
conditions
(3%
02,
97%
Ar).
Each
of
the
five
mutants
was
then
inoculated
onto
the
roots
or
stems
of
S.
rostrata
to
check
nodulating
and
nitrogen
fixing
ability.
Nodules
appeared
at
the
same
time
(4
to
5
days
after
inoculation),
regardless
of
whether
the
plants
had
been
inoculated
with
the
parent
strain
or
the
mutants.
Root
and
stem
nodules
formed
by
the
parent
strain
fixed
nitrogen
actively
(Fix').
In
contrast,
nodules
formed
on
both
stem
and
roots
by
the
mutants
were
ineffective
(Fix-):
no
acet-
ylene
reduction
could
be
detected,
and
the
plants
inoculated
with
the
mutants
remained
as
yellow
as
the
uninoculated
control
and
quite
different
from
the
green
healthy
plants
inoculat-
ed
with
the
parent
strain.
Figure
2
(B,
C,
D,
and
E)
shows
the
results
of
the
infection
tests
per-
formed
with
strain
5740,
one
of
the
five
mutants.
Thus,
the
analysis
of
the
nif
(nitrogen
fixation)
genes
of
certain
Rhizobium
strains
can
be
car-
ried
out
as
if
these
strains
were
classical
free-
living
nitrogen-fixing
bacteria,
such
as
Klebsiel-
la
spp.
The
possibility
of
using
plate
screening
for
obtaining
symbiotic
nitrogen
fixation
Rhizo-
bium
mutants
could
accelerate
investigations
of
the
genetics
of
this
agronomically
important
bacterial
genus.
We
thank
J.
Beringer,
A.
H.
Gibson,
and
J.
P.
Aubert
for
helpful
discussions,
D.
Melck
and
L.
Fall
for
technical
assist-
ance,
and
M.
Boureau
for
the
photographs.
This
research
was
supported
by
DGRST
grant
Ecar
81.G.1451.
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VOL.
45,
1983
... However, rhizobia are generally thought to be capable of N 2 fixation only in nodules [16,17]. The exceptions are found in some members of Bradyrhizobium and Azorhizobium which were shown to be able to fix N 2 in both the symbiotic and free-living states [18,19]. Such cases in Bradyrhizobium were mostly explored in photosynthetic members [18,19], but have recently been found in other strains. ...
... The exceptions are found in some members of Bradyrhizobium and Azorhizobium which were shown to be able to fix N 2 in both the symbiotic and free-living states [18,19]. Such cases in Bradyrhizobium were mostly explored in photosynthetic members [18,19], but have recently been found in other strains. For instance, Bradyrhizobium sp. ...
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An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
... It also could infect cereal crop wheat (Triticum aestivum L.) and form para-nodules to provide 16-23% nitrogen for host wheat (Kennedy et al., 1997). What makes it more unique is that A. caulinodans could fix N 2 in the free-living state (outside of the nodule) (Dreyfus et al., 1983). The wide adaptability and applicability in agriculture of A. caulinodans are reasons for its great attractions. ...
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Bacterial signal transduction pathways are important for a variety of adaptive responses to environment, such as two-component systems (TCSs). In this paper, we reported the characterization of a transcriptional regulator in Azorhizobium caulinodans ORS571, ActR, with an N-terminal receiver domain and one C-terminal OmpR/PhoB-type DNA binding domain. Sequence analysis showed that ActR shared a high similarity with FtcR regulator of Brucella melitensis 16M known to be involved in flagellar regulation. The structural gene of this regulator was largely distributed in Alphaproteobacteria, in particular in Rhizobiales and Rhodobacterales, and was located within clusters of genes related to motility functions. Furthermore, we studied the biological function of ActR in A. caulinodans grown at the free-living state or in association with Sesbania rostrata by constructing actR gene deletion mutant. In the free-living state, the bacterial flagellum and motility ability were entirely deleted, the expression of flagellar genes was downregulated; and the exopolysaccharide production, biofilm formation, and cell flocculation decreased significantly compared with those of the wild-type strain. In the symbiotic state, Δ actR mutant strain showed weakly competitive colonization and nodulation on the host plant. These results illustrated that FtcR-like regulator in A. caulinodans is involved in flagellar biosynthesis and provide bacteria with an effective competitive nodulation for symbiosis. These findings improved our knowledge of FtcR-like transcriptional regulator in A. caulinodans .
... While the unique feature of a symbiotic association of A. caulinodans with its associative host plant, tropical legume Sesbania rostrata is the capability of stem nodulation besides root nodulation (de Bruin, 1989). Another characteristic is its ability to fix nitrogen nonsymbiotically also and are closely related to the diazotroph, Xanthobacter (Dreyfus et al., 1983). Two novel Mesorhizobium sp. ...
... The alphaproteobacterium Azorhizobium caulinodans ORS571, as a gram-negative nitrogen-fixing bacterium, has the dual ability to fix nitrogen both under free-living conditions and in a symbiotic interaction with the tropical legume Sesbania rostrata, which forms both stem nodules and root nodules [1]. The nitrogen-fixing symbiosis of soil bacteria with legume plants is a multistep process that involves an exchange of signals between compatible partners. ...
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Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the Δ acfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the Δ acfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the Δ acfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium -host plant interaction.
... The symbiosis between A. caulinodans and its typical host, the tropical legume Sesbania rostrata, is unique in the sense that nodules are elicited not only on the roots but also on stems (for a review, see reference 87). Furthermore, A. caulinodans, unlike R. meliloti and B. japonicum, is able to grow in pure culture with molecular dinitrogen as the sole nitrogen source (109). Thus, A. caulinodans shares features of both free-living and symbiotic diazotrophs. ...
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This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans. Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54). The characteristic features of these proteins and their functions within species-specific regulatory pathways are described. Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA. FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL. In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription. This type of control includes FixLJ in R. meliloti and FixLJ-FixK in A. caulinodans or is brought about by autoregulation in B. japonicum. NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation. The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators. Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions. In B. japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.
... Azorhizobium caulinodans ORS571 is a rhizobium belonging to alpha-Proteobacteria uses chemotaxis for plant colonization. It fixes nitrogen with the host Sesbania rostrata by forming stem or root nodules (Dreyfus et al., 1983). A. caulinodans ORS571 has only one chemotaxis pathway including one gene cluster (cheA, cheW, cheY2, cheB, and cheR) and two orphan genes (cheY1 and cheZ) (Jiang et al., 2016). ...
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