Indian Journal of Experimental Biology Vol 45, June, 2007, pp 563-567
Iron mediated regulation of growth and siderophore production in a diazotrophic cyanobacterium Anabaena cylindrica Ruchika Raghuvanshi & Surendra Singh* School of Studies in Microbiology, Jiwaji University, Gwalior 474 011, India and
Prakash Singh Bisen Institute of Life and Allied Sciences, Seedling Academy of Design, Technology and Management , Jagatpura , Jaipur 302 004, India Received 27 October 2006; revised 27 February 2007 Iron mediated regulation of growth and siderophore production has been studied in a diazotrophic cyanobacterium Anabaena cylindrica. Iron-starved cells of A. cylindrica exhibited reduced growth (30%) when the cells were growing under N2-fixing conditions. In contrast, NO3-, NO2-, NH4+ and urea grown cells exhibited almost 50% reduction in their growth in the absence of iron as compared to their respective counterparts cultured in the presence of iron. However, at 60 μM of iron, A. cylindrica cells exhibited almost equal growth regardless of the nitrogen source available. Siderophore production in A. cylindrica was started after day 2nd of the cell growth and attained its optimal level on day 5th when the cells were at their mid-log phase. No siderophore production was, however, recorded on day 2nd at all the concentrations of iron tested. The production of siderophore in A. cylindrica further increased with increase in iron concentration and attained its optimum level on day 5th at 60 μM iron. A. cylindrica cells took at least 3 days for initiation of siderophore production and produced about 60% siderophore on day 5th even under iron-starved condition. A. cylindrica produced dihydroxamate type of siderophore. Keywords: Anabaena cylindrica, Cyanobacterium, Iron, Siderophore
Diazotrophic cyanobacteria are oxygenic, photosynthetic prokaryotes which have long been recognized as having potential in biotechnology. A better understanding of the cyanobacterial nitrogen metabolism is, however, prerequisite for enhancing the potential of these organisms as a biofertilizer. Iron is considered to be an essential trace element for the growth and metabolism in cyanobacteria. However, what should be the concentration of iron to support the optimum growth and metabolic activities in cyanobacteria is a subject of much debate. Microorganisms synthesize high affinity ferric-ion transport compounds collectively known as siderophores1. They are produced by a wide range of prokaryotic and eucaryotic microorganisms under iron limiting conditions and serve as virtually iron specific extracellular ligand to aid in the solubilization and assimilation of iron. Whereas, these iron acquisition systems are well understood in E. coli2 and other ______________ *Correspondent author Phone : (0751) 4016737 (O), (0751) 2345338 (R) E-mail :
[email protected] [email protected]
bacteria3, comparatively little is known about cyanobacteria4,5 in this regard. Nitrogen metabolism and siderophore production appeared to be mutually exclusive processes in cyanobacteria6. Growth of Anabaena variabilis in the absence of a combined nitrogen source actually resulted in higher levels of siderophore production than growth in a medium containing nitrate7. Furthermore, cells grown without combined nitrogen appeared to have much greater demand for iron, since siderophore production occurred at higher initial concentration of iron7. Thus, relationship between iron stress and nitrogen metabolism may be quite complex and will be an interesting subject for further studies. The apparently conflicting results for siderophore production and nitrogen metabolism in different cyanobacterial species point out two difficulties in the literature on iron deprivation in cyanobacteria. Firstly, limited number of studies in any one area are often done on different species, this makes the comparison difficult and generally slows the progress of research in this particular area. Secondly, in many studies on iron stress, the cells
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cultivated in the absence of iron have been used; under these conditions cells may simply die. Keeping the above facts in mind, the present study was undertaken to gain further insight into the iron mediated regulation of growth and siderophore production in a diazotrophic cyanobacterium Anabaena cylindrica. Materials and Methods Organism and culture conditions⎯ Anabaena cylindrica (obtained from National Centre for Conservation and Utilization of Blue Green Algae, IARI, New Delhi) was axenically grown in BG-110 medium8 devoid of combined nitrogen source. The medium was buffered to pH 7.5 with 10 mM of 4-(2-hydroxyethyl)-1-peperazine ethane sulphonic acid (HEPES)/NaOH. The cultures were maintained at 25°±1° C and illuminated with day-light fluorescent tubes having the photon fluence rate of 50 μmol m-1 s-1 at the surface of the vessel with 18/6 hr light/dark cycle. BG-110 medium devoid of iron was passed through a chelex 100 (Biorad) column and filtered through acid-washed polycarbonate filter (pore size 0.4 μm) to make it completely free from iron. For experiments, FeCl3 was added to the basal medium to obtain Fe concentration of 20, 40, 60, 80 and 100 μM in the culture. NO3-, NO2-, NH4+ and urea in the form of KNO3, NaNO2, NH4Cl and urea (1 mM each; final concentration) were added to the growth medium as required. Measurement of growth⎯ Growth of A. cylindrica was measured by measuring the increase in its protein and chlorophyll a (chl a) content, and was expressed in terms of specific growth rate (k) which was computed by following the method of Guillard9. Whereas, chl a was estimated following Mackinney10, protein was assayed using the method of Lowry et al.11 with lysozyme (Sigma) as the standard. Detection of hydroxamate nature of siderophores⎯ Exponentially growing (6 day old) A. cylindrica cells were harvested by centrifugation and the cell-free supernatants were used for assay of hydroxamate nature of siderophores by following the spectrophotometric method of Neilands12. Spectrophotometric assay ⎯To 1 ml of culture supernatants, 3 ml of freshly prepared 2% of aqueous FeCl3 solution was added and the absorbance between 200-600 nm was recorded. A peak at 420-450 nm indicated the presence of ferrate hydroxamate siderophores.
Detection of catecholate nature of siderophores⎯Cell-free supernatants were used for the assay of catecholate nature of siderophores by following two tests. Spectrophotometric assay ⎯To 1 ml of cell-free supernatant, 3 ml of freshly prepared 2% of aqueous FeCl3 was added. The formation of wine color complex that absorbed maximally at 495 nm, indicated the catecholate nature of siderophores. Arnow test 13⎯ To 1 ml cell-free supernatant, 1ml each of 0.5 N HCl and nitrite-molybdate reagent (10 g each of NaNO2 and (NH4)2 MoO4) were added. If yellow colour appeared at this point, 1 ml of 1 N NaOH was added (a red colour appeared) and the volume was made 5 ml with distilled water. The absorbance was recorded at 500 nm using 2,3 dihydroxybenzoic acid as standard. Detection of mono, di- and tri-hydroxamate nature of siderophores⎯ pH-dependent absorption maxima of ferrate siderophores of A. cylindrica have been used to distinguish ferric complexes of mono-, di- and trihydroxamates. Ferric complexes were detected spectrophotometrically for a shift in λ max (nm) at different pH14. Trihydroxamates showed little or no shift, when pH of the growth medium varies from 4 to 7. Dihydroxamates dissociated at pH 4-5 and showed wide shift. However, monohydroxamates showed a shift when pH drops to 4 (500-520 nm). Results and Discussion Relative contribution of different nitrogen nutrition on the growth of A. cylindrica in response to iron⎯ Since, iron starvation resulted in a significant decrease (70%) in the growth of A. cylindrica when the cells were growing in nitrogen-free medium (N2-fixing conditions) (Table 1), it was therefore, desirable to see the relative contribution of different Table 1 ⎯ Relative contribution of different nitrogen sources on the growth rate (K) of Anabaena cylindrica in the presence and absence of iron Nitrogen sources N2 NO3NO2NH4+ Urea
K (Division day-1) - Iron* 0.63 0.99 0.96 1.10 0.98
+ Iron** 1.89 1.96 1.92 2.12 2.05
* The medium was made iron-free by passing it though chelex100 column **Iron (60 μM) in the form of FeCl3 was added to the sterilized growth medium
RAGUVANSHI et al : SIDEROPHORE PRODUCTION IN ANABAENA CYLINDRICA
nitrogen sources (N2, NO3-, NO2-, NH4+ and urea) on the growth of A. cylindrica in response to 60 μM of iron, the concentration which supported the maximum growth in A. cylindrica15. Iron-starved cells of A. cylindrica exhibited reduced growth (30%) when the cells were growing under N2-fixing conditions (Table 1) However, there was a three fold increase in the growth of A. cylindrica under N2 fixing conditions in the presence of iron. In contrast, NO3-, NO2-, NH4+ and urea grown cells exhibited almost 50% reduction in their growth in the absence of iron as compared to their respective counterparts cultured in the presence of iron. Thus, the cells grown without combined nitrogen (N2 medium) appeared to have a much greater demand for iron, since siderophore production occurred at higher initial concentration of iron in the medium. These results also support the contention of the earlier report7. At optimum iron concentration (60 μM), A. cylindrica cells, however, exhibited almost equal growth regardless of the nitrogen source available. Relationship between growth and siderophore production⎯ Since, iron (60 μM) supported the optimal siderophore production as well as the cellular activities in A. cylindrica15, the relationship between growth and siderophore production in response to iron (60 μM) has, therefore, been studied in A. cylindrica (Fig.1). Growth (in terms of μg protein ml-1) and siderophore production (μg ml-1 culture filtrate) of A. cylindrica were measured simultaneously. Siderophore production in A. cylindrica was started after day 2nd of the cell growth and attained its optimal level on day 5th when the cells were at their mid-log phase. This suggested that A. cylindrica cells
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supported maximum siderophore production in response to optimal concentration of iron (60 μM) when they were actively growing. Further increase in growth, however, could not support the increase in siderophore production and siderophore production declined to its minimal level on day 8th. These results indicated that siderophore took at least 3 days period for its initiation in A. cylindrica in response to 60 μM of iron. This result is also in agreement with those reported in Anabaena sp.16,17 and A.quadruplicatum18. Siderophore production in response to iron⎯ Since the threshold level of iron required for optimum growth vary greatly for different cyanobacteria19-22, what should be the threshold level of iron for optimum siderophore production is still a subject of much debate. Further to ascertain and also to find out more conclusive evidence regarding the role of various iron concentrations on siderophore production in A. cylindrica, siderophore production in response to various concentrations of iron (20-100 μM) was measured in A. cylindrica (Table 2). A. cylindrica cells could not produce siderophores on day 2nd at all the concentrations of iron tested. However, a significant level of siderophore production in response to iron was recorded on day 5th in A. cylindrica. The cells of A. cylindrica produced about 60% siderophore on day 5th even in the absence of iron (during iron starvation). The production of siderophore in A. cylindrica further increased with increase in iron concentration and also attained its optimum level on day 5th at 60 μM iron. Iron at 20 μM concentration (the concentration of iron which is used for routine culturing of cyanobacteria) supported only 75% siderophore production in A. cylindrica Table 2 ⎯ Siderophore production in Anabaena cylindrica in response to various concentrations of iron Iron(μM)
0 (without iron) 20 40 60 80 100
Fig. 1⎯ Relationship between growth ( ) and siderophore ( )production in Anabaena cylindrica in the presence of iron (60 μM). Hundred percent siderophore production corresponds to 1.4 μg ml-1 culture filtrate.
Siderophore production (μg ml-1 of culture filtrate) 2nd day
5th day
ND ND ND ND ND ND
0.84 1.05 1.19 1.40 0.84 0.49
Anabaena cylindrica cells starved of iron for 7 days were resuspended in BG-110 medium (devoid of iron and citrate) containing 20-100 µM iron. Since, siderophore production in A. cylindrica was found to be maximum on day5th, siderophore production was therefore, assayed on 2nd and day 5th of growth. ND = Not detectable
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(Table 2). This further supported that 60 µM was the best suited concentration of iron for optimum growth and siderophore production in A. cylindrica and it must, therefore, be used for the routine culturing of A. cylindrica cells. Moreover, iron, (100 µM) resulted in a reduced level (35%) of siderophore production in A. cylindrica, suggesting that iron at higher concentrations possibly acted as cytotoxin, leading to inhibition of siderophore production in A. cylindrica. Initiation of siderophore production under iron starvation⎯ Since A. cylindrica exhibited significant growth on 4-6th day even in the absence of iron (during iron starvation) (data not shown), and the role of siderophore was proposed to support the growth of A. cylindrica in this condition, it was, therefore, desirable to see the role of incubation on the siderophore production in A. cylindrica under iron deprived condition (Fig. 2). A. cylindrica cells took at least 3 days for the initiation of siderophore production and maximum siderophore production (100%) was recorded on day 5th which continued upto day 6th under iron starved condition. Further increase in incubation period, however, resulted in a decrease in siderophore production in A. cylindrica. This suggested that 3 days iron starvation was required for siderophore initiation in A. cylindrica, however, it functioned with its full capacity at day 5th (Fig. 2). Identification of the nature of siderophores⎯ To ascertain and to find out the conclusive evidence regarding the nature of the siderophores produced during iron starvation, the culture supernatants of
Table 3⎯ Nature of the siderophores produced by Anabaena cylindrica Nature of siderophores
Neilands
Arnow
Shift in λmax (pH 4-7)
Hydroxamate (i) Dihydroxamate (ii) Trihydroxamate+ Catecholate
+ + -
-
+ -
The culture supernatants of A. cylindrica grown under iron starved conditions were used for the assay of siderophores. + = Positive test - = Negative test
A. cylindrica grown under iron-starved condition were analyzed for the identification of the nature of siderophores (Table 3). The culture supernatants of A. cylindrica cells showed positive signal against Neilands test12, a test for hydroxamate type of siderophores and thus, provides an evidence for the production of hydroxamate type of siderophores by A. cylindrica. In contrast, negative signal was recorded against Arnow test13, a specific test for catechols. Further to confirm whether the hydroxamate type of siderophores produced by A. cylindrica was dihydroxamate or trihydroxamate in nature, the shift in λmax on various pH (4-7) from 420-430 nm was recorded. A shift in λmax on various pH (4-7) was recorded in cell-free supernatant of A. cylindrica, further confirming that the siderophores produced by A. cylindrica was dihydroxamate type. Similar results were also reported in other cyanobacteria 18,21,22. Acknowledgement The authors thank University Grants Commission, New Delhi for providing financial assistance to the author (RR). References
Fig. 2⎯ Effect of incubation on siderophore production in Anabaena cylindrica under iron starvation. Hundred percent siderophore production corresponds to 0.84 μg ml-1 culture filtrate
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