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
3
.
1
. Effect of exogenously added solutes The effect of different solutes on growth and
nitrogenase activity of Anabaena sp. strain L-31 is shown in Table 1. NaCl strongly inhibited growth
and nitrogenase acetylene reduction activity at higher osmolalities. At 50 growth inhibitory os-
molality of 214 mosmkg, nitrogenase activity was reduced to only 2 during salt stress. In compari-
son, at 50 growth inhibitory osmolalities 80 – 174
mosmkg mannitol
and PEG
did not
adversely affect nitrogenase activity. At higher osmolality 305 mosmkg mannitol did inhibit
acetylene reduction but less severely than NaCl. Effects of sugars on nitrogenase activity were quite
the opposite. At 130 mosmkg, glucose, fructose and sucrose all enhanced nitrogenase activity 1.7 –
2.25-fold and did not inhibit growth. Sucrose, even at 35 growth inhibitory concentration 303
mosmkg, significantly enhanced acetylene reduc- tion activity by 2 – 3-fold Table 1. Combined
addition of NaCl + sucrose 295mosmkg seri- ously impaired nitrogenase activity and growth in
Anabaena sp. strain L-31.
3
.
2
. Uptake of sugars Uptake of [
14
C]-radiolabeled sugars in Anabaena was compared with that in E. coli, which does not
permeate sucrose. In E. coli, glucose uptake oc-
curred at much higher rates than in Anabaena, while the fructose uptake rates were comparable in
the two bacteria Table 2. Unexpectedly, An- abaena displayed 6-fold higher rates of sucrose
uptake than E. coli. Sucrose uptake in Anabaena cells was only partially inhibited in dark-incubated
or DCMU-treated cultures and strongly inhibited in the presence of glucose, but not that of fructose.
[
14
C]sucrose uptake was significantly inhibited in the presence of an excess of non-radioactive su-
crose in the medium.
3
.
3
. Fate of absorbed sugars Attempts were made to investigate if the sucrose
taken up by the Anabaena cells was accumulated during normal or salinity-stressed conditions, or
used up to support metabolic activities. Table 3 shows that 90 of exogenously supplied glu-
cose and sucrose were taken up within 24 h. Much of it was lost as [
14
CO
2
] in cellular respiration and could be recovered by absorbing the gas phase in
aqueous KOH solution data not shown. Intracel-
Table 1 Effect of different osmotic stresses on the growth and nitrogenase activity of Anabaena sp. strain L-31
a
Osmolality mosmkg Growth condition
Growth Nitrogenase activity
Control 100
100 120
82 82
NaCl 50 mM 2
214 50
NaCl 130 mM 20
NaCl 200 mM 300
116 80
52 PEG 10 wv
160 PEG 20 wv
51 109
102 55
Mannitol 200 mM 174
305 Mannitol 350 mM
35 60
110 225
Glucose 150 mM 130
130 Fructose 150 mM
100 200
Sucrose 150 mM 130
115 169
Sucrose 350 mM 290
65 303
NaCl 100 mM+ Sucrose 150 mM
295 26
12
a
Growth and nitrogenase activity were measured 3 days after exposure to various stresses. All values are expressed as percentages of unstressed controls. The respective control values for growth and nitrogenase activity on day 3 were 12.0 mg
chlorophyll aml and 90.94 mmol of C
2
H
4
mg chlorophyll ah. Table 2
Comparative uptake of sugars in E. coli strain MC4100 and Anabaena sp. strain L-31
a
Sugar uptake nmolmg protein Treatment
Organisms Glucose
Fructose Sucrose
119 E. coli
35 4
Anabaena L-31 22
21 Light
24 100 Dark
– –
21 88 15 64
– –
DCMU 2 mM Glucose 150 mM
– –
7 31 18 75
– –
Fructose 150 mM Sucrose 150 mM
– –
432×10
3
20
a
E. coli cultures were grown as described in Section 2. The Anabaena cultures treated either with 150 mM glucose, 150 mM fructose or 150 mM sucrose for 1 h, or subjected to dark incubation or DCMU 2 mM treatment for 24 h were compared with
light-grown control cultures. Both bacterial cultures were washed and re-suspended in respective fresh medium before incubation with radiolabeled sugars. Values in parentheses represent cpm of radiolabeled [
14
C]sucrose taken upmg protein, expressed as a percentage of light-grown controls.
Table 3 Accumulation of [
14
C]-radiolabeled sugars in normal and salt-stressed cultures of Anabaena sp. strain L-31
a
cpmml culture aliquot initial radioactivity Treatment
Additions Day 1
Day 2 Supernatant
Cells Supernatant
Cells 14.0
32.0 Control
19.0 [
14
C]glucose 30.0
NaCl 130 mM [
14
C]glucose 14.8
28.0 15.7
25.0 Control
[
14
C]fructose 92.3
5.6 76.0
9.9 84.0
7.0 [
14
C]fructose 63.0
NaCl 130 mM 9.7
13.0 38.0
14.4 34.0
Control [
14
C]sucrose 15.6
32.0 18.4
35.0 NaCl 130 mM
[
14
C]sucrose
a
Three-day-old Anabaena cultures were washed, resuspended in combined nitrogen-free BG-11 medium supplemented with or without 130 mM NaCl and 0.5 mCiml of one of the radiolabeled sugars and grown as described in Section 2. After the specified
time, 1-ml aliquots were rapidly filtered and washed. Radioactivity present in the cell pellet and in the filtrate was counted. Values are expressed as percentages of initial radioactivity 1 302 600 cpmml added to the medium.
lular glucosesucrose levels ranged between 30 and 38 in controls and between 25 and 35 in salt-
stressed cultures. In contrast, fructose utilisation by Anabaena cells was very low B 15 in 24 h
and B 40 in 48 h and intracellular fructose levels in both control and salt-stressed cells were only
5 – 10 of the sugar added. Fig. 1 shows that cells incubated with any of the radiolabeled sugars
incorporated the radiolabel into newly synthesised proteins in vivo, indicating that exogenously sup-
plied glucose, fructose or sucrose were not only taken up, but were also metabolised by Anabaena
cells. Protein profiles visualised with all three sug- ars were quite identical, both under normal and
salt-stress conditions.
3
.
4
. Dependence of nitrogenase acti6ity on light metabolism andor sugars
Nitrogenase activity acetylene reduction in Anabaena sp. strain L-31 was totally light-depen-
dent and was strongly inhibited during growth in the dark or with DCMU Table 4. Provision of
sucrose in the dark- or DCMU-grown cultures did not support nitrogenase acetylene reduction ac-
tivity. Light-grown, sucrose-supplemented cultures showed a significant loss of activity when shifted
to the dark for 30 min; the activity in such cultures was comparable with that of control without
sucrose cultures shifted to dark for 30 min Table 4. Thus, even in the sucrose-supplemented cul-
tures, reductant and ATP for nitrogenase activity seemed to be primarily derived from photosyn-
thetic reactions. Fig. 2 shows that when sucrose-grown 3-day-
old cultures were washed off the external sucrose, resuspended in fresh medium and grown with
photosynthetic inhibitors, they rapidly lost nitro- genase acetylene reduction activity in 3 h like
the controls. However, if such cultures were grown in light, they retained higher nitrogenase
activity for at least 6 h, subsequent to which
Fig. 1. Incorporation of catabolic products of exogenously added [
14
C]-labeled sugars into cellular proteins of Anabaena sp. strain L-31, under normal and salinity-stressed conditions.
Unstressed lanes 1, 3 and 5 and 130 mM NaCl-stressed lanes 2, 4 and 6 cells were radiolabeled with either
[
14
C]sucrose lanes 1 and 2 or [
14
C]glucose lanes 3 and 4 or [
14
C]fructose lanes 5 and 6 for 2 h. Proteins were extracted, equal TCA-precipitable radioactivity was resolved by 5 – 14
gradient SDS-PAGE and visualised by autoradiography. Ar- rows on the right side denote salinity-stress-induced proteins
and their molecular mass.
Table 4 Effect of photosynthetic inhibitors on sucrose-mediated en-
hancement of nitrogenase activity in Anabaena sp. strain L-31
a
Nitrogenase activity Assay condi-
Growth condi- control
tion tion
Day 3 Day 5
100 Control light
100 Light
65 76
Dark 18
Light 18
Control dark 00
00 Dark
Light 00
00 Control
DCMU 00
00 Dark
200 Sucrose light
284 Light
79 95
Dark 25
Light 25
Sucrose dark Dark
00 00
Light 34
15 Sucrose
DCMU 00
00 Dark
a
Cultures were grown either in light, or in dark, or with 2 m
M DCMU in light, and nitrogenase assays acetylene reduc- tion activity were carried out on day 3 and 5 both under light
and dark conditions. All the values are expressed as percent- ages of unstressed light control. Nitrogenase activities of
control cultures on days 3 and 5 were 43 and 121 mmol C
2
H
4
mg chlorophyll ah, respectively.
3
.
6
. Effect of exogenously added solutes on cellular Fe-protein content
Western blots of the proteins extracted from stressed cells and unstressed cells were subjected to
immunodetection for dinitrogenase reductase lev- els. Sucrose-, glucose- and fructose-grown cultures
all exhibited a much higher content of dinitroge- nase reductase Fe-protein of nitrogenase Fig. 4.
In contrast, in NaCl-stressed cultures, Fe-protein could not be immunodetected lanes 5 and 6.
Cells stressed with NaCl + sucrose showed no Fe- protein while those stressed with non-permeable
osmolytes PEG, mannitol showed no significant change in the levels of Fe-protein compared to
unstressed controls data not shown.
Fig. 2. Effect of osmotic downshock on nitrogenase acetylene reduction activity of sucrose-grown Anabaena sp. strain L-31.
Cultures grown with 350 mM sucrose for 3 days were washed off the external sucrose, resuspended in fresh BG-11 medium
without combined nitrogen and incubated either in light or in dark or with 2 mM DCMU in light
. Unstressed
control cultures incubated in light or in dark or with DCMU
are also included for comparison. All the values
are expressed as percentages of unstressed control grown in light . Nitrogenase activities of the control culture at 3, 24
and 48 h were 30.0, 40.0 and 60.9 mmol C
2
H
4
mg chlorophyll ah respectively.
activity declined to levels comparable with those found in controls. Thus, the sucrose-induced en-
hancement of nitrogenase activity was strictly light-dependent and was sustained only in the
continuous presence of external sucrose Fig. 2.
3
.
5
. Effect of nitrogenase biosynthesis inhibitors Nitrogenase acetylene reduction activity was
sensitive to protein synthesis inhibitors in An- abaena sp. strain L-31. Effects of the transcrip-
tional inhibitor rifampicin and of the nitrogenase synthesis repressor NH
4
Cl, on acetylene reduction activity are shown in Fig. 3. Addition of these
inhibitors to 3-day-old control or sucrose-grown cultures inhibited acetylene reduction activity at
comparable rates, though the sucrose-supple- mented cultures, at all points, showed higher
acetylene reduction activity. Addition of sucrose to rifampicin-pretreated 1 h cells, did not result
in higher nitrogenase activity data not shown.
Fig. 3. Effect of rifampicin and ammonium chloride on the nitrogenase acetylene reduction activity in Anabaena sp.
strain L-31. Cells were grown in media without open symbol or with 150 mM sucrose closed symbol for 3 days. Ri-
fampicin 60 mM , or NH
4
Cl 3 mM , was added at 0 h. Nitrogenase activities in the control and su-
crose-grown cultures, at the start of the experiment, were 35 and 88 mmol C
2
H
4
mg chlorophyll ah.
earlier that in NaCl-grown Anabaena cultures cel- lular energy is diverted away from nitrogenase to
Na
+
efflux resulting in the loss of N
2
fixation. In conformity with this, growth conditions which
curtailed Na
+
influx and conserved energy expen- diture on subsequent Na
+
efflux were shown to protect nitrogenase activity in Anabaena [7]. The
present study shows that NaCl also represses the synthesis of dinitrogenase reductase Fe-protein
Fig. 4. Repression of MoFe and Fe-proteins by NaCl has earlier been reported in K. pneumoniae
[19]. Thus, repression of nitrogenase synthesis to- gether with reduced availability of ATP appears to
be responsible for the loss of nitrogenase activity in Anabaena during salt stress.
Unlike salinity stress, osmotic stresses do not adversely affect cyanobacterial nitrogenase activity
[20]. At eco-physiologically relevant osmolalities, drought imposed by mannitol or PEG shows no
adverse effect on nitrogenase acetylene reduction activity Table 1. This insensitivity appears to be
a consequence of lack of repression of nitrogenase synthesis data not included and is in conformity
with the earlier reports that osmotically-stressed cyanobacteria retain Fe-protein and quickly revive
N
2
fixation upon rehydration [21]. In contrast to the non-permeable osmolytes, exogenously added
sugars significantly enhance Fe-protein synthesis Fig. 4 and acetylene reduction activity Table 1.
Thus, the positive effect of sucrose on nitrogenase activity reported earlier in Anabaena [4] is not a
general effect of osmotic stress but is specifically related to sugars. This raises the question, ‘are
Anabaena cells permeable to sucrose?’.
The present study clearly shows that Anabaena cells are permeable to all the sugars tested i.e.
glucose, fructose and sucrose Table 2. Ability to take up sugars is constitutively expressed in An-
abaena and does not show an obligatory require- ment for the presence of sugars or light for its
induction. Based on competition studies, glucose and sucrose, but not fructose, appear to share the
same permease Table 2. Thus, unlike E. coli, sucrose does not act as an osmotic stress for
Anabaena, though sudden exposure to a high con- centration of sucrose may cause short-term water
stress and does induce expression of osmorespon- sive genes as has been shown previously [10,22].
This supports our earlier finding that OSP expres- sion following sucrose up-shock occurs only tran-
siently in Anabaena, i.e. maximal OSP expression
Fig. 4. Immunodetection of dinitrogenase reductase in An- abaena sp. strain L-31. Cultures were grown under different
osmotic stresses for 3 days. Proteins were extracted, elec- trophoresed and electroblotted on to Boehringer Mannheim
positively charged nylon membrane and probed with an anti Fe-protein rabbit IgG. An anti-rabbit IgG conjugated to
alkaline phosphatase was used as second antibody and de- tected by using X-phos and NBT as chromogenic substrates
as described in Section 2. Various lanes contained 150 mg protein from the following treatments: control lane 1; 350
mM sucrose lane 2; 150 mM glucose lane 3; 150 mM fructose lane 4; 100 mM NaCl lane 5 and 130 mM NaCl
lane 6. Arrow on the right side denotes detection of the 33 kDa NifH protein.
4. Discussion
