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Research Paper Influence of iron and chelator on siderophore production in Frankia strains nodulating Hippophae salicifolia D. Don
Anju Singh1, Arun Kumar Mishra1, Satya Shila Singh1, Hridip Kumar Sarma2 and Ekta Shukla1
1 2

Department of Botany, Banaras Hindu University, Varanasi 221005, India Department of Biotechnology, Dibrugarh University, Dibrugarh, Assam, India

Effect of iron and chelator on the growth and siderophore production in the ten newly Frankia strains isolated from the root nodules of Hippophae salicifolia D. Don and the two reference strains were studied. Growth of the strains was greatly affected when grown in the iron and EDTA deprived conditions. All the strains were capable of producing both the hydroxamate and catecholate type siderophore that was detected using the Csaky and Arnow assays. Production of siderophore was enhanced in the EDTA replenish condition in contrast to the iron supplemented medium suggesting that EDTA reduces the availability of other free metals and hence creates the stress condition for which the secretion of siderophore is enhanced. A decrease in siderophore production was observed with an increase in iron concentration. Strains HsIi2 and HsIi10 were found to be producing more siderophore than the other strains.
Keywords: Frankia strains / Hippophae salicifolia D. Don / Chelator / Hydroxamate / Catecholate type siderophore Received: August 14, 2007; accepted November 05, 2007 DOI 10.1002/jobm.200700262

Introduction

Iron is an essential trace element for most of the living organisms. It is the major component of various biological activities such as DNA synthesis, electron transport and nitrogen fixation and possess a mechanism to acquire it from environment [1, 2]. The enzyme nitrogenase required for the nitrogen fixation is composed of multiple protein compounds and 36 iron atoms is required between the iron sulphur protein dinitrogenase reductase and the Fe and Mo cofactor (Fe MoCo) containing dinitrogenase [3, 4]. The iron containing protein haemoglobin and leghaemoglobin which are involved in the regulation of O2 pressure have been found in nodules of some actinorhizal plants e.g. Casuarina and Myrica in amounts comparable to those present in the Rhizobium-legume symbiosis [5, 6]. Although iron is present in abundance but it is unavailable due to its presence as the insoluble iron oxyhydroxide polymers

Correspondence: Dr. Arun Kumar Mishra, Department of Botany, Banaras Hindu University, Varanasi 221005, India E-mail: akmishraau@rediffmail.com; akmishraau@hotmail.com Phone: 91-542-3296111/91-542-2307147 Fax: 91-542-2368174 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

under aerobic conditions at biological pH. Ferric ions (Fe3+) solubility under these conditions is 1017 M, whereas cytoplasmic iron concentration is approximately 107 M in metabolically active microbes [7]. Due to differences in concentration, uptake by the cells is not an option for these microbes. Such a high demand of iron from the unavailable source is made available by secretion of high affinity low molecular mass iron chelating agents termed siderophore [6, 8, 9, 11]. It binds iron with an extremely high affinity. It is specifically recognized by a corresponding outer membrane receptor protein, which in turn actively transports the complex into the periplasm of the cell [2, 12, 13]. Siderophore production is strain specific [14]. There are three types of siderophore on the basis of chemical functional groups i.e. Carboxylate, Hydroxamate and Phenolate (catechol) type [14]. Siderophore production has been reported in many members of rhizobiaceae [10], several N2 fixing microorganisms such as cyanobacteria [15], Azotobacter [16], Azospirillum [17] and Frankia [18, 19]. Frankia, is microaerophilic, gram positive, filamentous, symbiotic actinomycete capable of fixing nitrogen in the root nodules of non-leguminous actinorhizal plants. Although a number of Frankia strains have been isolated and experimented on the
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siderophore production [4, 6] but till date no work has been reported on the identification of siderophore in the strains isolated from Hippophae salicifolia D. Don growing in the high altitude (~1400 m asl) areas of the eastern Himalayas (Sikkim and the neighbouring areas) where natural resources is limited. Therefore, the present work is to investigate the influence of different concentrations of iron and chelator on growth and siderophore production in different Frankia strains and also to identify the type of siderophore produced by these strains under the same conditions.

in the experiment. Both FeCl3 and EDTA was filter sterilized (0.45 m) before adding to the medium. The experiment was set up for 12 d as the 8th 10th day was observed to be the exponential phase. Maximum growth was observed in BAP medium containing iron (20 M) and EDTA (40 M). Growth measurement in terms of protein Cultures were harvested by centrifuging at 13,000 rpm for 15 minutes. The pellet obtained were homogenized with sterile distilled water. A known amount of the above cultures (0.5 ml) were taken for the estimation of protein as described by Lowry et al. [24] with Bovine Serum Albumin as a standard. Protein was measured at different time intervals (0, 2, 4, 6, 8, 10 and 12 d). Siderophore production The presence or absence of siderophore was measured in Frankia strains using Universal Chrome Azurol S assay (CAS) as described by Schwyn and Neilands [25]. Strains were grown in iron and EDTA deficient conditions for 12 days at standard growth condition. Cultures were filtered through Whatman no. 1 filter paper to remove hyphae and centrifuged at 13,000 rpm for 15 min. Supernatant was concentrated five times by lyophilization to 1 ml. After lyophilization, cultures were centrifuged and the supernatant (0.5 ml) was taken for the siderophore production. An equal amount of blue CAS solution was added to the cultures and was left for 6 h. A reference solution was prepared with uninoculated medium. Absorbance was measured at 630 nm. Detection of hydroxamate type siderophore The Csaky Assay (Csaky 1948) was used to detect the hydroxamate type siderophore. Culture supernatants were concentrated to five fold by lyophilization to 1 ml and obtained aliquots were boiled with 1 ml concentrated H2SO4 (6 N) for 5 10 min to release the bound hydroxamate. After cooling the aliquots, 3 ml sodium acetate (35%), 1 ml sulphanilic acid and 0.5 ml iodine solution was added. The aliquots were shaken vigourously after adding each and every reagent and were left for 5 min. Further, sodium arsenite (1 ml) and -naphthylamine (1 ml) was added to the above aliquots with a vigourous shaking. Final volume was maintained to 10 ml with double distilled water and was incubated for 20 30 min for the complete colouration and was referred to a standard curve prepared with unhydrolysed hydroxylamine HCl. Reference solution was prepared with uninoculated medium and absorbance was measured at 526 nm.
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Materials and methods

Maintenance of cultures Ten different Frankia strains (HsIi2, HsIi4, HsIi5, HsIi8, HsIi9, HsIi10, HsIi11, HsIi12, HsIi13, HsIi14) were isolated from root nodules of Hippophae salicifolia D. Don (Seabuckthorn) growing at high altitude (at Lachen and Zema) areas in North Sikkim, India [20] and two reference strains (Ag4b and CpI2) that were isolated from Alnus glutinosa [21] and Comptonia peregrina [22], respectively, were obtained from Dr. Johannes Pasi Haansuu, Department of Biosciences, University of Helsinki, Finland. All the strains were maintained in the sterilized liquid BAP medium [23] pH 6.9, containing filter (0.45 m) sterilized sodium pyruvate (10 mM), autoclaved ammonium chloride (5 mM) as a sole carbon and nitrogen source respectively. Antibiotics cyclohexemide (50 g ml1) and nalidixic acid (10 g ml1) were added to the autoclaved medium after filter (0.45 m) sterilization to prevent any bacterial and fungal contamination. Phosphate buffer (10 mM) from a stock (1 M K2HPO4 + KH2PO4) and Fe-EDTA (20 M) were autoclaved separately and added to the cooled medium. Strains were grown in properly washed and autoclaved 250 ml Erlenmeyer flask containing 150 ml BAP medium with a 1000 l suspension of Frankia cells growing at a concentration of 35 g protein ml1. All the strains were incubated in dark at 29 0.5 C in BOD throughout the experimental setup. Strains were subcultured once a week to obtain uniform inocula. Growth conditions Various Frankia strains were grown in properly washed and autoclaved Erlenmeyer flasks containing BAP medium with or without supplemented iron and EDTA for 12 days in dark at 29 0.5 C and cultures were maintained in BOD incubator fitted with rotary shaker. The concentrations of iron (FeCl3) used were 0, 3, 5, 10 and 20 M whereas 0, 5, 10, 20 and 40 M EDTA were used
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Detection of catechol type siderophore Arnow assay was performed for detection of catecholate type siderophore [26]. Frankia strains were concentrated to five fold by lyophilization to 1 ml. To the obtained aliquots, concentrated HCl (0.5 N) (1 ml), nitrite molybdate reagent (10 g sodium nitrite +10 g sodium molybdate in 100 ml double distilled water) (1 ml) and 1 N NaOH (1 ml) were added with vigourous shaking. Volume was maintained to 5 ml by adding 1 ml double distilled water and incubated for 10 15 minutes. Absorbance was measured at 510 nm with 2,3-dihydroxy benzoic acid (2, 3 DHBA) as a standard.

Results
Effect of iron on the growth Growth (in terms of protein) was compared in two reference strains CpI2, Ag4b and ten different strains of Frankia isolated from the root nodules of Hippophae salicifolia under iron replete (+Fe) and deplete conditions (Fe) (Fig. 1). The strains showed a better growth in iron replete condition in comparison to the strains growing in iron deplete conditions. Maximum biomass production (in terms of g protein ml1) was observed in HsIi14 (63.5) followed by HsIi9 (61.7), HsIi11 (58.8), CpI2 (57.9) HsIi4 (57.4), Ag4b (57.2), HsIi8 (54.4), HsIi13 (53.7), HsIi12 ( 51.9) and HsIi5 (51.2) while a minimum

was recorded in HsIi10 (39.7) followed by HsIi2 (39.0) in the iron replete conditions. A decrease in biomass production was observed in the iron deplete condition. Maximum biomass in terms of g protein ml1 was recorded in CpI2 (53.7), Ag4b (53.0), HsIi14 (51.4) followed by HsIi9 (49.7), HsIi11 (44.6), HsIi8 (43.5), HsIi4 (48.2), HsIi12 (42.8), HsIi13 (42.8) and HsIi5 (42.1) while a minimum production reached in HsIi10 (28.5) followed by HsIi2 (27.8). Comparing the reference strains with ten other strains in the iron replete (+Fe) and deplete (Fe) conditions, one thing is very clear that presence of iron (20 M) in the BAP medium increased the biomass production slightly in reference strains whereas a greater enhancement in biomass production was observed in ten other strains in the same conditions. Detection of siderophore Strains inoculated in iron and EDTA deplete BAP medium for 10 days showed a change in colour from blue CAS to orange after 6 hours of incubation. All the strains showed a positive reaction to the CAS assay (Table 1). All the strains showed an affinity with Fe3+, which was confirmed by the change of colour from blue, to orange. Strains HsIi8 and CpI2 showed the highest affinity for Fe3+ followed by HsIi2, HsIi5, HsIi9, HsIi10, HsIi11 and HsIi14 whereas weak reaction was observed in HsIi4, HsIi12, HsIi13 and Ag4b. Effect of iron on siderophore production Two different assays for the type of siderophore production were performed [26, 27]. Csaky assay was performed for the detection of hydroxamate type siderophore. Maximum siderophore production in terms of g hydroxamate mg1 protein was observed in
Table 1. Siderophore production by Frankia strains as estimated by CAS assay, after 10 days of incubation in iron deplete BAP medium. Frankia strains HsIi2 HsIi4 HsIi5 HsIi8 HsIi9 HsIi10 HsIi11 HsIi12 HsIi13 HsIi14 Ag4b CpI2 Chrome Azurol S(CAS) Assay ++ + ++ +++ ++ ++ ++ + + ++ + +++

Figure 1. Growth of twelve strains of Frankia cultivated in BAP medium with or without iron after 10 days of incubation. Bars indicate the standard error of six replicates (n = 6).
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+++ strong reaction; ++ moderate reaction; + weak reaction.

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Figure 2. Hydroxamate type siderophore production in twelve strains of Frankia cultivated in BAP medium with different concentration of iron after 10 days of incubation. Bars indicate the standard error of six replicates (n = 6).

Figure 3. Catecholate type siderophore production in twelve strains of Frankia cultivated in BAP medium with different concentrations of iron after 10 days of incubation. Bars indicate the standard error of the six replicates (n = 6).

the control condition (Fe) in strain HsIi10 (424.7) followed by HsIi2 (416.5), HsIi8 (216.8), HsIi13 (209.5), HsIi5 (203.1), HsIi12 (202.1), HsIi11 (201.7), whereas a minimum siderophore production was recorded in HsIi4 (180) followed by HsIi9 (172.8) and HsIi14 (162) at the same condition (Fig. 2). As the concentration of iron was increased in the medium, the siderophore production was decreased proportionately and maximum decrease was observed at 20 M Fe3+ (which was best suited concentration for the growth). Maximum production of siderophore in terms of g hydroxamate mg1 protein was recorded in HsIi2 (268.1) followed by HsIi10 (262.5), HsIi5 (140.4), HsIi12 (140.3), HsIi13 (131.5) and HsIi9 (125.7) whereas minimum siderophore production was observed in HsIi4 (120.4) followed by HsIi11 (116.4) and HsIi14 (112.1) in the presence of 20 M iron. Almost an equal amount of siderophore production (g hydroxamate mg1 protein) was observed by Ag4b (203.4) and CpI2 (202.7) in the control condition (Fe) but a decrease in production was observed in Ag4b (161.9) and CpI2 (161.9) in the iron replete conditions (20 M). Comparing the ten strains with the reference strains, maximum siderophore production was recorded in the strains isolated from Hippophae salicifolia. Catecholate type siderophore was iden 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tified by Arnow assay [26]. Production of siderophore was more pronounced in the control (Fe) as compared to the iron replete condition (+Fe). Maximum siderophore production in terms of g catecholate mg1 protein was observed in HsIi2 (214.2), followed by HsIi10 (167.1), HsIi11 (155.4), HsIi4 (153.5), HsIi12 (153.0), HsIi5 (152), CpI2 (152.1), Ag4b (151.3), HsIi14 (109.3), HsIi9 (105.6), HsIi13 (90.9) and HsIi8 (86) in the control condition (Fig. 3). As the concentration of Fe3+ was increased gradually up to 20 M, a proportionate decrease in the production was observed. Maximum siderophore production in terms of g catecholate mg1 protein was recorded in HsIi5 (130.4) followed by HsIi12 (101.0), HsIi2 (75.3), HsIi11 (74.5), HsIi4 (71.2), HsIi10 (64.2), HsIi9 (61.3), CpI2 (58.9), Ag4b (57.0), HsIi14 (53.7), HsIi8 (53.0) and HsIi13 (50.9) in the iron replete (20 M) condition. Comparing the reference strains with ten newly isolated Frankia strains HsIi2, HsIi10 and HsIi11 were more siderophore producing strains followed by the reference strains Ag4b and CpI2. Effect of chelator on the growth Growth in terms of protein was examined in all the strains growing in the BAP medium containing various concentrations of EDTA after ten days of incubation.
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Figure 4. Growth of twelve strains of Frankia cultivated in BAP medium (Fe) with or without EDTA after 10 days of incubation. Bars indicate the standard error of six replicates (n = 6).

Figure 5. Hydroxamate type siderophore production in twelve strains of Frankia cultivated in BAP medium (Fe) with different concentration of EDTA after 10 days of incubation. Bars indicate the standard error of six replicates (n = 6).

A very different result was obtained when the same strains were growing in the iron replete conditions (20 M). Biomass production was more in the control ( EDTA) in comparison to EDTA replete condition (40 M). Maximum biomass production in terms of g protein ml1 was observed in HsIi14 (51.4) followed by HsIi9 (49.7), HsIi11 (44.6), HsIi8 (43.5), HsIi4 (43.2), HsIi12 (42.8), HsIi13 (42.8), HsIi5 (42.1), HsIi10 (28.5) and HsIi2 (25.8) in the control condition (EDTA). In contrast, a decreasing trend was observed in biomass production when cultures were grown in EDTA replete condition i.e. HsIi14 (41.3) followed by HsIi13 (40.3), HsIi9 (40.2), HsIi12 (35), HsIi5 (34.7), HsIi8 (34.7), HsIi11 (33.6), HsIi4 (32.8), HsIi10 (20.3) and HsIi2 (19.9) (Fig. 4). While both the reference strains showed no significant difference in biomass production either grown in EDTA replete or deplete condition. Results suggest that the biomass production remained higher in the reference strains compared to the Frankia strains isolated from Hippophae salicifolia despite of the conditions provided. Effect of chelator on the siderophore production Siderophore production was determined as per the protocol provided by Csaky [27] and Arnow [26] under the EDTA deplete and replete conditions. Fig. 5 shows the maximum hydroxamate type siderophore production (in terms of g hydroxamate mg1 protein) in HsIi10 (424.7) followed by HsIi2 (416.5), HsIi8 (216.8),
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HsIi13 (209.5), HsIi5 (203.1), HsIi12 (202.1), HsIi11 (201.7), HsIi9 (172.8), HsIi4 (171.2) and HsIi14 (162.0) in the control. As the concentration of EDTA was gradually increased up to 40 M, the production of hydroxamate type siderophore showed a proportionate increase. Siderophore production rose in HsIi2 (673.7) followed by HsIi10 (659.6), HsIi11 (471.0), HsIi8 (365.8), HsIi13 (333.9), HsIi5 (292.6), HsIi12 (292.3), HsIi9 (281.7), HsIi4 (280.3) and HsIi14 (263.3) in EDTA replete conditions. While reference strains showed a similar trend but with a lesser increase in the siderophore production under control (EDTA) and +EDTA (40 M) conditions. Both Ag4b and CpI2 showed a production of about 203 g hydroxamate mg1 protein in the control condition and 256 g hydroxamate mg1 protein in the Fe + EDTA condition. Catecholate type siderophore production in terms of g catecholate mg1 protein was recorded maximum in HsIi2 (189) followed by HsIi10 (167.14), HsIi11 (155.4), HsIi4 (153.5), HsIi2 (153), HsIi5 (152), HsIi14 (109.3), HsIi9 (105.6), HsIi13 (90.9) and HsIi8 (86.0) in the control (EDTA) (Fig. 6). Like hydroxamate type, with the increase in EDTA concentration to 40 M, a gradual increase in catecholate type siderophore was also observed. Maximum siderophore production in terms of g catecholate mg1 protein was observed in HsIi2 (1637.7) followed by HsIi10 (1205.6), HsIi11 (335), HsIi4 (320), HsIi9 (252.4), HsIi5 (237.8), HsIi14 (188.3), HsIi12 (176.7), HsIi8 (153.7) and HsIi13
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Figure 6. Catecholate type siderophore production in twelve strains of Frankia cultivated in BAP medium (Fe) with different concentrations of EDTA after 10 days of incubation. Bars indicate the standard error of the six replicates (n = 6).

(147.5) in Fe + EDTA condition. A similar trend was also observed in the reference strains Ag4b and CpI2. Maximum siderophore production was observed (~8 times higher compare to the control) in HsIi2 and HsIi10 under EDTA replete condition whereas in others, slight increase was observed either in the reference strains or in the strains isolated from Hippophae salicifolia.

Discussion
All the strains, either reference strains or those isolated from the root nodules of Hippophae salicifolia, showed a better growth in the iron supplemented (20 M) BAP medium when compared to the iron deplete (Fe) BAP medium (Fig. 1). Results proved that the growth of Frankia strongly depends on the availability of iron. Furthermore, the fact is strongly supported by the result that the strains growing either in the iron deplete and EDTA replete (Fe + EDTA) or iron deplete and EDTA deplete (Fe EDTA) medium showed a lesser growth in comparison to the strains grown in iron supplemented BAP medium (Fig. 4). The results were consistent with the earlier observations [4, 6, 18]. When the growth was compared between the strains growing in EDTA replete and deplete condition, more enhancement in growth was observed in EDTA deplete condition (Fig. 4). Probably, under iron deficient condition, EDTA present in the medium might bind with other
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metals and slowed the availability of other metals to the organisms that causes inhibition in many metabolic activities and ultimately the growth was reduced [28 30]. Similar results were also reported in some strains of Rhizobium and Bradyrhizobium under the effects of EDTA and 2,2-dipyridyl which proved that the above strains were susceptible to these chelators [31]. From Table 1, it was clearly observed that all the strains were capable of producing siderophore which were confirmed on the basis of CAS assay in iron and EDTA deprived condition. This assay consists of the competition for the ferric ion between the CAS-Fe3+ and the excreted siderophore. The higher affinity constant of the siderophore for iron led to a colour change of CAS (usually from blue to orange ) after 6 hours of incubation as also reported in Frankia strains Cj and G2 isolated from Casuarina junghuhniana and C. equisetifolia respectively [6] and Saccharopolyspora erythraea [32]. All the strains were capable of producing both the hydroxamate and catecholate type siderophore in the presence or absence of iron-supplemented medium. Maximum siderophore production was shown in the control condition (Fe) (Fig. 2 and 3). The different iron concentrations (3 M to 20 M) were found to be inhibitory for the siderophore production (either hydroxamate or catecholate type) in all the tested strains. Whereas in the Frankia strain Cj isolated from Casaurina junghuhniana, siderophore production is twice even at 0.5 M iron concentration as compared to the control [6]. This iron repressibility of siderophore biosynthesis is a
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common characteristic of many microorganisms producing these compounds [6, 33, 34]. Siderophore production (either hydroxamate or catecholate) is strain specific and varied among all the strains (including reference strains) [6, 14, 19]. Maximum siderophore producing strains were HsIi2 and HsIi10 (hydroxamate type) while HsIi5 and HsIi12 produced maximum catecholate type siderophore in the presence or absence of iron supplemented medium (Figs. 2 and 3). Strains growing in the EDTA sufficient medium showed the maximum excretion of both type of siderophore (hydroxamate and catecholate) in comparison to the strains growing in EDTA deficient condition (Figs. 5 and Fig. 6). Maximum siderophore producing strains were HsIi2 and HsIi10 in the presence or absence of EDTA supplemented medium either hydroxamate or catecholate type. Frankia sp. strain CeS15 and Frankia sp. strain 52065 always produces hydroxamate type siderophore [4, 19]. Rhizobium trifolii produces a catechol siderophore [35, 36] contrast to R. leguminosarum that only produces a hydroxamate siderophore in iron limiting condition [37]. R. meliloti produces a siderophore, rhizobactin, which lacks both hydroxamate and catechol functional groups [38] whereas fungi produces only hydroxamate siderophore [14, 39 41]. These results suggest that under iron deficient condition, the EDTA present in the medium causes inhibition in many metabolic activities of the strains that may create a stress condition in which iron is required for the protection of the cells. So, in the presence of EDTA, siderophore production might be increased to fulfill the demand of iron by the frankial cells. The availability of metals through plants and aquatic organisms depends on the free metal activity. The presence of chelators in the solutions reduces the metal availability to the organisms due to transformation in the cationic metal [28 30]. Studies showed that siderophore production in the iron stress and EDTA flourish conditions was accumulated late in the stationary phase. This is due to the fact that siderophore was produced late in the exponential growth phase of Frankia suggesting that siderophore might be used for scavenging of iron and sustaining growth and survival of the microorganism [6]. Figs. 2, 3, 5 and 6 show the enhanced level of siderophore production under the influence of iron chelator rather than the iron supplemented conditions. In view of the above findings, it is suggested that (i) growth of the strains were greatly affected under the iron, EDTA stress suggesting the importance of iron on the various biological activities, (ii) All the strains were capable of producing both catecholate and hydroxamate type siderophore but strains being more sidero 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

phore producer under the influence of chelator (40 M) and (iii) The high affinity of iron uptake helps the Frankia strain to survive competitively in the rhizosphere.

Acknowledgements
We would like to acknowledge, Dr. Johannes Pasi Haansuu, Department of Biosciences, University of Helsinki, Finland for providing two reference strains used in this work. We thankfully acknowledge, Head, Department of Botany, Banaras Hindu University, Varanasi, India for providing the necessary facilities.

References
[1] Chincholkar, S.B., Chaudhari, B.L., Talegaonkar, S.K. and Kothari, R.M., 2000. Microbial iron chelators: a tool for sustainable agriculture. In: Upadhayay, R.K., Mukherji, K.G. and Chamola, B.P. (eds.), Biocontrol Potential on Their Exploration in Crop Disease Management. Kluwer Academic, New York, pp. 49 70. [2] Sayyed, R.Z. and Chincholkar, S.B., 2006. Purification of siderophores of Alcaligenes faecalis on Amberlite XAD. Bioresource Technol., 97, 1026 1029. [3] Burgess, B.K., 1984. Structure and reactivity of nitrogenase-an overeview. In: Veeger, C. and Newton, W.E. (eds.), Advances in Nitrogen Fixation Research. Nijhoff/Junk, Pudoc Wagenningen, pp. 103 114. [4] Boyer, G.L., Stacie, A.K., Alexander, J.A. and Aronson, D.B., 1999. Siderophore formation in iron-limited cultures of Frankia sp. strain 52065 and Frankia sp. Strain CeS15. Can. J. Bot., 77, 1316 1320. [5] Silvester, W.B., Harris, S.L. and Tjepkema, J.D., 1990. Oxygen regulation and haemoglobin. In: Schwintzer, C.R. and Tjepkema, J.D. (eds.), The Biology of Frankia and Actinorhizal Plants. Academic Press, San Diego, pp. 157 176. [6] Arahou, M., Diem, H.G. and Sasson, A., 1998. Influence of iron depletion on growth and production of catechol siderophore by different Frankia strains. W. J. Microbiol. Biotechnol., 14, 31 36. [7] Ishimaru, C.A., 1993. Biochemical and genetic analysis of siderophore produced by plant-associated Pseudomonas and Erwinia species. In: Barton, L.B. and Heming B.C. (eds.), Iron Chelation in Plants and Soil Microorganisms. Academic Press, Inc., pp. 27 73. [8] Neilands, J.B., 1981. Iron absorption and transport in microorganism. Ann. Rev. Nutr., 1, 27 46. [9] Meyer, J., Halle, F., Hohnadel, D., Lemanceau, P. and Ratefiarivel, H., 1987. Siderophore of Pseudomonas, Biological properties. In: Winkelmann, G., Vander Helm, D. and Neilands, J.B. (eds.), Iron Transport in Microbes, Plants and Animals. VCH Verlagsgesellschaft, Weinheim, pp. 189 205.
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