tilisers for crop plants growing in stressful environments.
Our laboratory has been studying the re- sponses
of heterocystous
nitrogen-fixing An-
abaena strains to salinity stress and sucrose — hitherto
considered an
osmotic stress
in cyanobacteria. In Anabaena strains tolerance to
NaCl and sucrose are not obligatorily linked i.e. salt-sensitive strains exhibit significant tolerance
to sucrose and vice-versa [4]. Cyanobacterial halotolerance is primarily determined by the
ability of cyanobacteria to exclude Na
+
[7,8] while osmotolerance is facilitated by the specific
need-based expression of certain osmoresponsive genes and synthesis of osmotic stress proteins, or
OSPs [5,9,10] leading to the accumulation of compatible solutes like glucosylglycerol, sucrose,
trehalose and betaines [11 – 13]. Both halotoler- ance as well as osmotolerance of Anabaena
strains can be experimentally upgraded, respec- tively, by treatments which curtail Na
+
influx or induce expression of OSPs [5]. The most striking
difference in the response of Anabaena strains to ionicosmotic stresses relates to nitrogenase activ-
ity. We have shown earlier that exogenously added NaCl severely inhibits while sucrose re-
markably enhances acetylene reduction activity by 2.5- to 3-fold [4].
These findings have raised certain questions, such as: 1 do osmotic stresses affect cyanobac-
terial N
2
fixation the same way as sucrose? 2 If not, does sucrose act as osmotic stress or are
Anabaena cells permeable to sucrose? If sucrose is taken up. 3 Is it accumulated as compatible
solute? or 4 Is it utilised to support growth and nitrogenase activity in light and in dark? 5
Is the positive effect of sucrose on nitrogenase activity related to nitrogenase biosynthesis or its
activity? etc.
The present study has addressed the aforemen- tioned questions to reveal the molecular basis of
the differential effects of sucrose and NaCl on cyanobacterial nitrogenase activity. Our data
show that while NaCl represses the synthesis of dinitrogenase reductase Fe-protein, sucrose and
other permeable sugars enhance dinitrogenase re- ductase synthesis. Non-permeable osmolytes such
as polyethylene glycol PEG and mannitol nei- ther enhance dinitrogenase reductase synthesis
nor
affect nitrogenase
activity in
Anabaena strains.
2. Materials and methods
2
.
1
. Organisms and growth conditions The filamentous heterocystous nitrogen-fixing
cyanobacterium Anabaena sp. strain L-31 [14] iso- lated in this laboratory was used in axenic condi-
tion. The cultures were grown in combined nitrogen-free BG-11 liquid medium at pH 7.0 [15].
Salinity stress was applied as NaCl and osmotic stress either as mannitol or PEG. Sucrose, glucose
and fructose were added at the specified concen- trations. Rifampicin was added at 60 mM, 3-3,4-
dichlorophenyl-1,1-dimethylurea DCMU at 2
m M and NH
4
Cl at 3 mM concentration. All cul- tures were grown photoautotrophically in an or-
bital incubator
shaker at 25°C 9 2°C
under continuous illumination 2.5 mWcm
2
and with shaking 100 rpm. Growth was assessed by the
content of chlorophyll a measured in methanolic extracts as described previously [16]. Cultures of
Escherichia coli strain MC4100 were grown from single colonies in Luria Broth LB pH 7.5 in an
orbital incubator shaker at 37°C with shaking at 200 rpm.
2
.
2
. Measurement of nitrogenase acti6ity and medium osmolality
Nitrogenase activity was measured by the acetylene reduction technique at appropriate time
intervals. Culture aliquots 2-ml were transferred to 5-ml vacutainers and incubated with 0.1 atm
acetylene in air for 30 min under growth condi- tions. Gas chromatographic analysis of samples
was carried out as described previously [7]. Osmo- lality of the medium was determined by the freez-
ing point constant depression method using the Micro-osmette
Osmometer Precision
Systems Inc., Natick, USA as per the manufacturer’s
protocol.
2
.
3
. Uptake of radiolabeled sugars Radiolabeled [
14
C]glucose and [
14
C]sucrose spe- cific activity 305 mCimmol and [
14
C]fructose specific activity 170 mCimmol were obtained
from the Board of Radiation and Isotope Tech- nology BRIT, Mumbai, India. Three-day-old
Anabaena sp. strain L-31 cells were washed and resuspended in fresh BG-11 medium. Culture
aliquots 5-ml were supplemented with 0.5 mCiml of the required radiolabeled sugar and incubated
under the usual growth conditions wherein the uptake of sugars was linear for 60 min. Aliquots
1-ml were filtered Whatman GFC circles after 30 min, washed with 50 ml BG-11 medium and
dried. Filter paper circles were transferred to vials containing 10 ml of 2,5-bis-5-tert-butyl benzoxa-
zolyl-[2]
thiophene BBOT
in 0.4
wv toluene:methanol mixture 1:1 and radioactivity
was counted using an LKB Wallace 1217 Rack- beta Liquid Scintillation Counter.
Overnight LB-grown cultures of E. coli MC4100 were washed, transferred to the minimal medium
M63 with glucose and further grown at 37°C with shaking at 200 rpm. After 3 h, cells were washed
and resuspended in M63 medium without glucose. Cell suspensions 5-ml were incubated with 0.5
m Ciml of the required radiolabeled sugar at 37°C
and 200 rpm wherein the uptake of sugars was linear up to 30 min. Aliquots 1-ml were filtered
after 10 min, washed and collected onto Millipore filter paper circles 0.22 mm. The filter papers were
dried, transferred to scintillation vials containing BBOT and counted as described above.
2
.
4
. In 6i6o radiolabeling, electrophoresis and autoradiography of proteins
Logarithmic phase 3-day-old Anabaena sp. strain L-31 cultures were incubated with or with-
out 130 mM NaCl and 2.5 mCiml of either [
14
C]sucrose, [
14
C]glucose, or [
14
C]fructose for 2 h in an orbital incubator shaker under usual growth
conditions. Proteins
were extracted,
elec- trophoresed and autoradiographed as described
previously [17].
2
.
5
. Western blotting and immunodetection of dinitrogenase reductase
Cells were grown for 3 days under stress condi- tions. Proteins were extracted, electrophoresed on
5 – 14 gradient SDS-polyacrylamide gels and electroblotted onto Boehringer Mannheim posi-
tively-charged nylon
membrane Boehringer
Mannheim GmBH, Germany as described previ- ously [18]. Immunodetection was carried out with
an anti-dinitrogenase
Fe-protein antiserum
raised in rabbit against a combined preparation of Fe-proteins
from Azotobacter
chroococcum, Rhodospirillum rubrum, Bradyrhizobium japonicum
and Klebsiella pneumoniae. An anti-rabbit IgG conjugated to alkaline phosphatase Boehringer
was used as a second antibody and detected using 5-Bromo, 4-chloro, 3-indolyl phosphate X-phos
and Nitro blue tetrazolium chloride NBT as chromogenic substrates as per the manufacturers
,
protocol.
2
.
6
. Presentation of data The values of growth, nitrogenase acetylene
reduction activity and sugar uptake were calcu- lated as means of three replicates and in each
experiment the variation from the mean was less than 10. The data presented are representative of
three independent experiments.
3. Results
