Differential regulation of nitrogenase activity by ionic and osmotic
stresses and permeable sugars in the Cyanobacterium
Anabaena
sp.
strain L-31
Tonina A. Fernandes, Shree Kumar Apte *
Cell Biology Di6ision,Bhabha Atomic Research Centre,Trombay,Bombay400 085,India
Received 28 April 1999; received in revised form 14 September 1999; accepted 14 September 1999
Abstract
Nitrogenase (acetylene reduction) activity inAnabaenasp. strain L-31 is significantly enhanced by the addition of sucrose, but is inhibited upon addition of sodium chloride. The positive effect of sucrose is not a general osmotic stress effect since non-permeable osmolytes (mannitol or polyethylene glycol (PEG)) do not influence nitrogenase activity. Unlike enteric bacteria,
Anabaena cells take up and metabolise sucrose and incorporate products of its catabolism into proteins. Cultures inhibited in photosynthesis retain the ability to take up sucrose but do not show acetylene reduction activity, even when supplemented with sucrose. Addition of an inhibitor of transcription (rifampicin) or of a repressor of nitrogenase biosynthesis (ammonium chloride) abolishes the positive effect of sucrose on acetylene reduction activity. Cultures grown with permeable sugars (glucose, fructose and sucrose) show significantly higher levels of dinitrogenase reductase (Fe-protein of nitrogenase complex) while those grown with NaCl lack the protein. Fe-protein content is not affected by non-permeable solutes. Thus, exogenous sucrose elevates dinitrogenase reductase synthesis but does not appear to support the requirement of reductant for nitrogenase activity. The data substantiate our previous finding that the ionic and osmotic stresses differentially regulate cyanobacterial nitrogenase activity and explain the relatively superior osmotolerance of diazotrophic cyanobacterial strains, as compared with their sensitivity to salinity stress. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Anabaena; Ionic/osmotic stress; Differential effects; Nitrogen fixation
www.elsevier.com/locate/plantsci
1. Introduction
Among the environmental stresses, salinity and drought (osmotic stress) rank as the most detri-mental for crop productivity [1]. Apart from the crop plants, these stresses also adversely affect the growth and metabolism of agriculturally impor-tant bacteria (especially nitrogen fixers, phosphate solubilisers, etc.) thereby further limiting crop pro-ductivity. Both salinity and drought impose a wa-ter stress on all living cells and cause turgor loss.
Salinity stress, in addition, has an ionic compo-nent that is especially deleterious to plant cells [2]. Generally, heterotrophic microbes (like enteric bacteria and yeast) show an identical response to ionic and osmotic stresses [3]. In contrast, pho-toautotrophic microbes like cyanobacteria exhibit differential sensitivity to ionic and osmotic stresses [4]. In view of the close similarity of their physiol-ogy and metabolism to plants and their possible phylogenetic relationship with chloroplasts, cyanobacteria are considered good model systems for analysis of plant responses to environmental stresses [5,6]. In addition, information on the sen-sitivity of cyanobacterial nitrogen fixation to envi-ronmental stresses has an important bearing on the potential of these microbes as nitrogen biofer-* Corresponding author. Tel.: +91-22-550-5000/2348; fax: +
91-11-5505-151.
E-mail address:[email protected] (S. Kumar Apte)
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 osmotolerhalotoler-ance 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 ionic/osmotic 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 N2 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
mM and NH4Cl at 3 mM concentration. All
cul-tures were grown photoautotrophically in an or-bital incubator shaker at 25°C92°C under continuous illumination (2.5 mW/cm2) 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 usfreez-ing the Micro-osmette Osmometer (Precision Systems Inc., Natick, USA) as per the manufacturer’s protocol.
2.3. Uptake of radiolabeled sugars
Radiolabeled [14C]glucose and [14C]sucrose
(spe-cific activity 305 mCi/mmol) and [14C]fructose
(specific activity 170 mCi/mmol) were obtained from the Board of Radiation and Isotope Tech-nology (BRIT), Mumbai, India. Three-day-old
aliquots (5-ml) were supplemented with 0.5mCi/ml
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 GF/C 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% w/v toluene:methanol mixture (1:1) and radioactivity was counted using an LKB Wallace 1217 Rack-beta Liquid Scintillation Counter.
Overnight LB-grown cultures ofE.coliMC4100 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
mCi/ml 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 (0.22mm). 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 mCi/ml of either
[14C]sucrose, [14C]glucose, or [14C]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 mosm/kg, nitrogenase activity was reduced to only 2% during salt stress. In compari-son, at 50% growth inhibitory osmolalities (80 – 174 mosm/kg) mannitol and PEG did not adversely affect nitrogenase activity. At higher osmolality (305 mosm/kg) mannitol did inhibit acetylene reduction but less severely than NaCl. Effects of sugars on nitrogenase activity were quite the opposite. At 130 mosm/kg, 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 mosm/kg), significantly enhanced acetylene reduc-tion activity by 2 – 3-fold (Table 1). Combined addition of NaCl+sucrose (295mosm/kg) seri-ously impaired nitrogenase activity and growth in
Anabaena sp. strain L-31.
3.2. Uptake of sugars
Uptake of [14C]-radiolabeled sugars in Anabaena
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. [14C]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 [14CO
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 ofAnabaenasp. strain L-31a
Osmolality (mosm/kg)
Growth condition Growth Nitrogenase activity
Control 100 100
120
82 82
NaCl (50 mM)
2
214 50
NaCl (130 mM)
0 20
NaCl (200 mM) 300
116
80 52
PEG (10% w/v)
160
PEG (20% w/v) 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) 303 65 290
NaCl (100 mM)+
Sucrose (150 mM) 295 26 12
aGrowth 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
chlorophylla/ml and 90.94 mmol of C2H4/mg chlorophylla/h.
Table 2
Comparative uptake of sugars inE.colistrain MC4100 andAnabaenasp. strain L-31a
Sugar uptake (nmol/mg protein) Treatment
Organisms
Glucose Fructose Sucrose
119
E.coli 35 4
AnabaenaL-31 Light 21 22 24 (100)
Dark – – 21 (88)
15 (64) –
– DCMU (2mM)
Glucose (150 mM) – – 7 (31)
18 (75)
– –
Fructose (150 mM)
Sucrose (150 mM) – – 432×103 (20)
aE.coli cultures were grown as described in Section 2. TheAnabaenacultures treated either with 150 mM glucose, 150 mM fructose or 150 mM sucrose for 1 h, or subjected to dark incubation or DCMU (2mM) 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 [14C]sucrose taken up/
mg protein, expressed as a
Table 3
Accumulation of [14C]-radiolabeled sugars in normal and salt-stressed cultures ofAnabaenasp. strain L-31a
cpm/ml culture aliquot (% initial radioactivity)
Treatment Additions
Day 1 Day 2
Supernatant Cells Supernatant Cells
14.0 32.0
Control [14C]glucose 19.0 30.0
NaCl (130 mM) [14C]glucose 14.8 28.0 15.7 25.0
Control [14C]fructose 92.3 5.6 76.0 9.9
84.0 7.0
[14C]fructose 63.0
NaCl (130 mM) 9.7
13.0 38.0 14.4 34.0
Control [14C]sucrose
15.6 32.0 18.4 35.0
NaCl (130 mM) [14C]sucrose
aThree-day-oldAnabaenacultures were washed, resuspended in combined nitrogen-free BG-11 medium supplemented with or without 130 mM NaCl and 0.5mCi/ml 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 cpm/ml) added to the medium.
lular glucose/sucrose 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 (B15% in 24 h andB40% 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 and/or 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
Table 4
Effect of photosynthetic inhibitors on sucrose-mediated en-hancement of nitrogenase activity in Anabaena sp. strain L-31a
Nitrogenase activity (% Assay
Growth
condi-control) tion
tion
Day 3 Day 5
100
Control (light) Light 100
65 76
Dark
18
Light 18
Control (dark)
00 00
Dark
Light 00 00
Control (DCMU)
00 00
Dark
200
Sucrose (light) Light 284
79 95
Dark
25
Light 25
Sucrose (dark)
Dark 00 00
Light 34 15
Sucrose (DCMU)
00 00
Dark
aCultures were grown either in light, or in dark, or with 2
mM 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
C2H4/mg chlorophylla/h, 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 nitrogedinitroge-nase) (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-grownAnabaenasp. 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 2mM 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 60.9mmol C2H4/mg chlorophyll
a/h 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 NH4Cl, on acetylene reduction
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 NH4Cl (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 88mmol C2H4/mg chlorophylla/h.
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 N2 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
-abaenasp. 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
occurs initially and declines during prolonged ex-posure [10].
In some cyanobacteria sucrose has been shown to accumulate as a compatible solute during salin-ity stress [23]. In salt-stressed as well as unstressed cultures of Anabaena sp. strain L-31, instead, the absorbed sucrose appears to be catabolised fur-ther. Some of its downstream products are incor-porated into proteins also (Fig. 1). Intracellular sucrose can also serve as a source of reductant for nitrogenase (acetylene reduction) activity in two ways. First it can be catabolised via the oxidative pentose phosphate pathway and produce NADPH in a light-independent manner [24,25]. This does not seem to happen since the dark-grown cultures, which do take up sucrose, show no nitrogenase activity (Table 4). Second, sucrose could be catabolised to organic acids which can feed elec-trons into the PS-I and produce NADPH in a light-dependent manner as has been shown for some Anabaena strains [26]. The inability of su-crose to support nitrogenase (acetylene reduction) activity in DCMU-grown cultures discounts this possibility (Table 4). These results, along with the fact that in Anabaena strains energy for N2
fixa-tion is largely derived from photophosphorylafixa-tion [27], clearly suggest that effects of sucrose do not relate to nitrogenase (acetylene reduction) activity directly.
A well-established effect of exogenous sucrose in
Anabaena strains is the induction of osmorespon-sive genes, leading to selectively enhanced tran-sient synthesis of several proteins [10,22]. Several lines of evidence indicate that sucrose enhances nitrogenase synthesis in Anabaena. First, the gen-eral transcriptional inhibitor rifampicin or the ni-trogenase synthesis repressor NH4+ inhibits
nitrogenase (acetylene reduction) activity both in control as well as in sucrose-grown cultures at comparable rates (Fig. 3). Second, in rifampicin-treated cells, addition of sucrose does not enhance nitrogenase activity up to 30 h (data not shown). Finally, immunodetection analysis clearly shows that Fe-protein content is remarkably enhanced in the presence of sucrose and other permeable sug-ars (Fig. 4). It remains to be ascertained whether it is due to induced transcription/translation of nifH mRNA or altered turnover of mRNA/Fe-protein. We have not tested the effects of sucrose on nitrogenase (MoFe protein) biosynthesis due to unavailability of a suitable antibody. However, it
is well-known that the dinitrogenase reductase is the rate-limiting factor in nitrogenase catalysis and an increase in its level alone is sufficient to explain enhancement of nitrogen fixation.
As mentioned earlier, sucrose is neither accumu-lated as a compatible solute (Table 3) nor offers any osmoprotection to Anabaena sp. strain L-31 exposed to salinity stress [Table 1]. The effects of sucrose on nitrogenase activity in Anabaena, thus, appear to be quite distinct from those of osmopro-tectants like glycine betaine in K. pneumoniae[19]. Glycine betaine has been shown to alleviate re-pression of nitrogenase synthesis by NaCl but does not per se induce nitrogenase biosynthesis. In con-trast, sucrose induces Fe-protein biosynthesis in
Anabaena (Fig. 4) but does not block repression of nitrogenase synthesis caused by NaCl (Table 1, last treatment). Another well-known compatible solute of bacteria, proline, has rather adverse ef-fects on acetylene reduction activity in Anabaena, both in the absence and in the presence of NaCl [8].
In conclusion, this study supports the con-tention that sucrose enhances nitrogenase content (especially that of dinitrogenase reductase) but does not appear to contribute to its activity. The results elucidate the biochemical basis of differen-tial effects of NaCl (inhibition), sucrose/glucose/
fructose (enhancement) and non-permeable osmolytes (no effect) on cyanobacterial nitroge-nase activity and reveal it to be in complete agree-ment with their effects on dinitrogenase reductase synthesis. The study also suggests that as biofer-tilisers of nitrogen, cyanobacteria hold greater promise in drought situations than in saline environments.
Acknowledgements
The authors wish to thank Professor Paul Lud-den (Department of Biochemistry, University of Wisconsin, Madison, USA) for kindly providing the anti-dinitrogenase reductase antibody.
References
[2] R. Serrano, R. Gaxiola, Microbial models and salt stress tolerance in plants, Crit. Rev. Plant Sci. 13 (1994) 121 – 138.
[3] L.N. Csonka, A.D. Hanson, Prokaryotic osmoregula-tion: genetics and physiology, Annu. Rev. Microbiol. 45 (1991) 569 – 606.
[4] T.A. Fernandes, V. Iyer, S.K. Apte, Differential re-sponses of nitrogen-fixing cyanobacteria to salinity and osmotic stresses, Appl. Env. Microbiol. 59 (1993) 899 – 904.
[5] S.K. Apte, T.A. Fernandes, V. Iyer, A. Alahari, Molec-ular basis of tolerance to salinity and drought stresses in photosynthetic nitrogen-fixing cyanobacteria, in: K.K. Tewari, G.S. Singhal (Eds.), Plant Molecular Bi-ology and BiotechnBi-ology, Narosa, New Delhi, 1997, pp. 258 – 268.
[6] F. Joset, R. Jeanjean, M. Hagemann, Dynamics of the response of cyanobacteria to salt stress: deciphering the molecular events, Physiol. Plant. 96 (1996) 738 – 744. [7] S.K Apte, B.R. Reddy, J. Thomas, Relationship
be-tween sodium influx and salt tolerance of nitrogen-fixing cyanobacteria, Appl. Environ. Microbiol. 53 (1987) 1934 – 1939.
[8] B.R. Reddy, S.K. Apte, J. Thomas, Enhancement of cyanobacterial salt tolerance by combined nitrogen, Plant Physiol. 89 (1989) 204 – 210.
[9] T.J. Close, P.J. Lammers, An osmotic stress protein of cyanobacteria is immunnologically related to plant de-hydrins, Plant Physiol. 101 (1993) 773 – 779.
[10] V. Iyer, T. Fernandes, S.K. Apte, A role for osmotic stress-induced proteins in the osmotolerance of a nitro-gen-fixing cyanobacterium, Anabaenasp. strain L-31, J. Bacteriol. 176 (1994) 5868 – 5870.
[11] E. Blumwald, R.J. Mehlhorn, L. Packer, Studies of osmoregulation in salt adaptation of cyanobacteria with ESR spin probe technique, Proc. Natl. Acad. Sci. USA 80 (1984) 2599 – 2602.
[12] M.A. Mackay, R.S. Norton, L.J. Borowitzka, Organic osmoregulatory solutes in cyanobacteria, J. Gen. Mi-crobiol. 130 (1984) 2177 – 2191.
[13] R.H. Reed, J.A. Chudek, R. Foster, W.D.P. Stewart, Osmotic adjustments in cyanobacteria from hypersaline environment, Arch. Microbiol. 138 (1984) 333 – 337. [14] J. Thomas, Absence of the pigments of photosystem II
of photosynthesis in heterocysts of a blue-green alga, Nature (London) 228 (1970) 181 – 183.
[15] R.W. Castenholz, Culturing of cyanobacteria, Methods Enzymol. 167 (1988) 68 – 93.
[16] G. Mackinney, Absorption of light by chlorophyll solu-tions, J. Biol. Chem. 140 (1941) 315 – 322.
[17] S.K. Apte, A. Bhagwat, Salinity-stress-induced proteins in two nitrogen-fixing Anabaena strains differentially tolerant to salt, J. Bacteriol. 171 (1989) 909 – 915. [18] A. Alahari, S.K. Apte, Pleiotropic effects of potassium
deficiency in a heterocystous, nitrogen-fixing cyanobac-terium, Anabaena torulosa, Microbiology 144 (1998) 1557 – 1563.
[19] L. Bouillard, D. Le Rudulier, Nitrogen fixation under osmotic stress: enhancement of nitrogenase biosynthesis in Klebsiella pneumoniae by glycine betaine, Physiol. Veg. 21 (1983) 447 – 457.
[20] G.D. Smith, R.M. Lynch, G. Jacobson, C.J. Barnes, Cyanobacterial nitrogen fixation in arid soils of Central Australia, FEMS Microbiol. Ecol. 74 (1990) 79 – 90. [21] A. Peat, N. Powell, M. Potts, Ultrastructural analysis
of the rehydration of desiccated Nostoc communeHUN (cyanobacteria) with particular reference to the im-munolabeling of NifH, Protoplasma 146 (1988) 72 – 80. [22] A.A. Bhagwat, S.K. Apte, Comparative analysis of
proteins induced by heat shock, salinity and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena
sp. strain L-31, J. Bacteriol. 171 (1989) 5187 – 5189. [23] R.H. Reed, D.L. Richardson, S.R.C. Warr, W.D.P.
Stewart, Carbohydrate accumulation and osmotic stress in cyanobacteria, J. Gen. Microbiol. 130 (1984) 1 – 4. [24] S.K. Apte, P. Rowell, W.D.P. Stewart, Electron
dona-tion to ferredoxin in heterocysts of the N2-fixing alga
Anabaena cylindrica, Proc. R. Soc. Lond. B200 (1978) 1 – 25.
[25] W. Lockau, R.B. Peterson, C.P. Wolk, R.H. Burris, Modes of reduction of nitrogenase in heterocysts iso-lated from Anabaena species, Biochim. Biophys. Acta 502 (1978) 298 – 308.
[26] T. Murai, T. Katoh, Photosystem I-dependent oxida-tion of organic acids in the blue-green alga Anabaena
6ariabilis, Plant Cell Physiol. 16 (1975) 759 – 797.
[27] W.D.P. Stewart, Some aspects of structure and func-tion in N2-fixing cyanobacteria, Annu. Rev. Microbiol. 34 (1980) 497 – 536.
