Diversity of Frankia strains nodulating HippÖphae salicifolia D. Don using FAME profiling as Chemotaxonomic markers

318 Journal of Basic Microbiology 2010, 50, 318–324

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Diversity of Frankia strains nodulating HippÖphae salicifolia D. Don using FAME profiling as chemotaxonomic markers

Arun Kumar Mishra, Anju Singh and Satya Shila Singh

Laboratory of Microbial genetics, Department of Botany, Banaras Hindu University, Varanasi-221005, India

Twelve Frankia strains isolated from HippÖphae salicifolia D. Don or Alnus glutinosa or Comptonia peregrine, showed the significant variation in fatty acid composition viz. palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidic acid (20:0) and erucic acid (22:1) suggesting the strain specific variability among the Frankia strains. Presence of Erucic acid (22:1), a major component of the oil obtained from the seeds of Brassica sp., albeit in lesser amount in the few studied frankial strains, is the first report. Cluster analysis on the basis of fatty acid composition suggests the presence of two distinct clusters with similitude coefficient ranging from 0.75 to 1.00. Cluster I with HsIi2 showed great divergence from other 11 frankial strains (Cluster II). The two sub groups were distinguished in cluster II: IIa contained five strains isolated from H. salicifolia and these strains are distantly related to the strains of cluster IIb isolated from different host. There is high degree of similarity among the frankial strains of Cluster IIb which suggests that the frankial strains might be evolved from the same ancestor. FAME profiling might be useful tool in the study of polyphasic approach based taxonomy and phylogenetic relationship.

Keywords: Frankia strains / Fatty acids / Taxonomy / Erucic acid

Received: September 26, 2009; accepted: December 20, 2009

DOI 10.1002/jobm.200900313

Introduction*

Frankia is widely distributed and has ability to nodulate with actinorhizal trees and fix atmospheric nitrogen [1] which makes the tree species potentially important for fuel-wood production, agroforestry and reclamation of infertile soils [2–4]. During the last few decades, num-bers of Frankia strains have been isolated from the root nodules of several actinorhizal plants [3–6] and their morphological, physiological, biochemical and molecu-lar characterization have been done [7–13]. Different types of lipids are found in frankial cells i.e. neutral lipids, glycolipids and polar lipids [14]. Myristic, palmi-tic, oleic, linoleic and linolenic acids were found to be the major fatty acids that have been identified in CpI1 and Casuarina-Frankia isolates [3, 14]. These fatty acids Correspondence: Dr. Arun Kumar Mishra, Laboratory of Microbial Genetics, Department of Botany, Banaras Hindu University, Varanasi-221005, India E-mail: [email protected]; [email protected] Phone: 0091-542-6701103(Office); 0091-9335474142(Mobile) Fax: 0091-542-2368174

play major role in the life of human beings and animals and are becoming increasingly important in the phar-maceutical industry as therapeutic agent for numerous health disorders [15–17]. Lipids are esters of fatty acids and the fatty acids are metabolites of acetyl CoA pathway, evolutionary very old and conservative. Besides morphological characteri-zation, fatty acid composition analyses are also helpful in taxonomic studies along with genotypic characteris-tics [17–20]. The concept of using a fatty acid profile in chemotaxonomy was initiated in cyanobacteria [21, 22] and thereafter in many environmentally relevant and biotechnologically important species [23]. Although diversity was studied among the number of Frankia strains nodulating various actinorhizal plants [3, 6, 12, 13, 24] but no reports are available that are related to the chemotaxanomical studies of the Frankia strains nodulating H. salicifolia. Hence, the present in-vestigation’s aim to study the fatty acid composition in the unexplored Frankia strains nodulating H. salicifolia growing along the higher altitude regions of the East-ern Himalayas and its application in the taxonomy.

Journal of Basic Microbiology 2010, 50, 318–324 FAME profiling of Frankia strains 319


Materials and methods

Organisms and culture conditions Ten different Frankia strains (HsIi2, HsIi4, HsIi5, HsIi8, HsIi9, HsIi10, HsIi11, HsIi12, HsIi13, HsIi14) were iso-lated from root nodules of H. salicifolia (Seabuckthorn) growing at high altitude areas in North Sikkim, India [6] and two reference strains (Ag4b and CpI2) that were isolated from A. glutinosa [25] and C. peregrina [26] re-spectively were received from Dr. Johannes Pasi Haan-suu, Department of Biosciences, University of Helsinki, Finland. All the strains were maintained in the steril-ized liquid BAP medium [27] pH 6.9, containing filter (0.45 μm) sterilized sodium pyruvate (10 mM), auto-claved ammonium chloride (5 mM) as a sole carbon and nitrogen source respectively. Antibiotics cyclohexemide (50 μg ml–1) and nalidixic acid (10 μg ml–1) were added to the autoclaved medium after filter (0.45 μm) sterili-zation to prevent any bacterial and fungal contamina-tion. Phosphate buffer (10 mM) and Fe-EDTA were auto-claved separately and added to the cooled medium. Strains were grown in properly washed and sterlized 250 ml Erlenmeyer flask containing 150 ml BAP me-dium with a 1000 μl suspension of Frankia cells growing at a concentration of 35 μg protein ml–1. All the strains were incubated in dark at 29 ± 0.5 °C in BOD (Biological Oxygen Demand) throughout the experimental setup.

Fatty acid analysis Fatty Acid Methyl Ester (FAME) analysis was done as per the protocol of Sasser [28; http://www.midi-inc.com] with slight modifications. Cultivated frankial cells were harvested by the centrifugation at 13,000 rpm for 10 min. The obtained pellets (100 mg) were saponified with reagent 1 (1 ml) [45 g NaOH in methanol (150 ml) and distilled water (150 ml)] by briefly vortexing and then placing it in hot water bath (100 °C) for 5 min. Again it was vortexed and further incubated in hot water bath for 25 min. Tubes were cooled and then reagent 2 (2 ml) [6N H2SO4 (325 ml) and methanol (275 ml)] was added. It was refluxed at 80 °C for 4 h. Excess of solvent (methanol) was removed under vac-cum using rotary evaporator. The residue was extracted with reagent 3 (10 ml) [Hexane:methyl tert-butyl ether (1 :1)] and the process was repeated thrice to achieve maximum extraction. The extract was left as such for 12 h at room temperature for complete evaporation. The residue was finally dissolved in 1.25 ml of reagent 3. A sample clean up was done by reagent 4 (3 ml) [10.8 g NaOH in 900 ml distilled water] and tumbled for 5 min. About 2/3 of the organic phase was pipetted into GC vial. Extracted methyl ester of fatty acids (3 μl) was

injected directly into the Gas Chromatograph (Perkin Elmer CLARUS_500) fitted with a flame ionization de-tector (FID) and Elite1 column (stainless steel column, 15 m long). The column temperature was initially kept at 150 °C for 1 min followed by a rise of 10 °C min–1 upto 270 °C for 2 min. The detector temperature was kept at 300 °C. Nitrogen was used as the carrier gas, with flow rate of 26 ml min–1 (Split mod). Mixture of fatty acids was kept as standard. Retention time of the samples was compared with the retention time of their respective standard so as to know the composition of methyl esters of fatty acids present. Further, amount of the fatty acid was calculated with the help of peak are by using the following formula in terms of percentage:

Amount of fatty acid (%) =

Peak area of the fatty acid X100(Total peak area Peak area of Hexane)−

where, Total peak area = A Peak area of Hexane = B Total fatty acid present (C) = (A – B).

Phylogenetic analysis The fatty acids data was numerically analyzed by using the NTSYS – pc 2.1 version software (Applied Biostatis-tics, Inc, Setanket, NY), and results were presented as dendrogram generated by UPGMA clustering method.

Results

Fatty acid analysis of the twelve Frankia strains isolated either from H. salicifolia or A. glutinosa or C. peregrina was based on the peak area of hexane and fatty acids (Fig. 1 A–L). All the strains showed the presence of ester linked fatty acid ranging from 16–22 carbon atoms (Table). Four major polar fatty acids viz. palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3) were found to be present in all the frankial strains whether isolated from H. salicifolia or A. glutinosa or C. peregrine. Exception was only observed in HsIi2 where oleic acid was absent. Arachidic (20:0) and erucic acid (22:1) were found only amongst few strains. Ara-chidic acid was present in HsIi9, HsIi10, HsIi11, HsIi12, HsIi13 and CpI2. Erucic acid was highest in HsIi14 fol-lowed by HsIi13, Ag4b, HsIi12, HsIi10 and CpI2. The highest percentage of linolenic acid (18:3) was observed amongst all the experimental frankial strains (49.78–69.52%). Linoleic (9.15–25.68%), palmitic (0.96–21.45%) and oleic acid (4.12–15.38%) were present in

320 A. K. Mishra et al. Journal of Basic Microbiology 2010, 50, 318–324


Figure 1. Gas chromatograms of twelve Frankia strains (A) HsIi2, (B) HsIi4, (C) HsIi5, (D) HsIi8, (E) HsIi9, (F) HsIi10, (G) HsIi11, (H) HsIi12, (I) HsIi13, (J) HsIi14, (K) Ag4b, (L) CpI2 isolated from different actinorhizal plants. Each peak area is representing specific fatty acid at specific retention time and is coded by a number: 1 – Hexane; 2 – Palmitic acid; 3 – Oleic acid; 4 – Linoleic acid; 5 – Linolenic acid; 6 – Arachidic acid; 7 – Erucic acid.



Table. Fatty acid composition of different Frankia strains isolated from different actinorhizal plants.

Host Frankia Total Hexane Total Palmitic Oleic Linoleic Linolenic Arachidic Erucic plant strains peak peak cellular acid acid acid acid acid acid area area fatty acid (16 :0) (18 :0) (18 :2) (18 :3) (20 :0) (22 :1)

[A] [B] [A–B] = [C] Peak % Peak % Peak % Peak % Peak % Peak % area area area area area area

Hippophae HsIi2 100 94.7 5.3 0.9 16.8 – – 1.2 22.8 3.2 60.0 – – – – Salicifolia HsIi4 100 89.6 10.4 1.6 1.0 0.4 15.4 2.5 24.2 5.8 55.5 – – – – HsIi5 100 93.2 6.8 1.2 16.8 0.4 5.1 1.5 22.5 3.8 55.4 – – – – HsIi8 100 84.4 15.5 2.5 15.8 0.7 4.8 3.8 24.5 8.6 55.0 – – – – HsIi9 100 80.7 19.7 3.4 17.4 0.9 4.7 4.4 22.7 10.5 55.5 0.1 0.6 – – HsIi10 100 89.6 10.4 1.7 15.9 0.4 4.1 2.5 24.2 5.4 52.2 0.2 1.6 0.2 1.7 HsIi11 100 92.9 7.1 1.1 15.1 0.3 3.8 1.6 22.4 3.6 49.8 0.1 2.0 – – HsIi12 100 90.5 9.5 1.6 16.3 0.4 4.5 2.4 25.7 4.8 50.0 0.2 1.7 0.2 1.9 HsIi13 100 91.5 8.5 1.8 20.7 0.6 7.2 1.0 11.6 4.8 56.0 0.2 1.7 0.2 2.7 HsIi14 100 95.7 4.3 0.8 19.2 0.3 6.5 0.4 9.7 2.6 60.2 – – 0.2 4.2 Alnus glutinosa

Ag4b 100 92.1 7.8 1.7 21.5 0.6 7.2 0.9 11.4 4.5 57.2 – – 0.2 2.7

Comptonia peregrina

CpI2 100 83.0 16.9 2.6 15.3 0.8 4.8 1.6 9.2 11.8 69.5 0.1 0.5 0.1 0.5

Fatty acid percentage was calculated according to the formula explained in Materials and methods section.

lesser amount in comparison to linolenic acid. Both arachidic and erucic acid were present in smaller quan-tity. Variation in the total cellular fatty acid, the per-centage composition of fatty acids and fatty acid profil-ing suggests strain specificity amongst the Frankia strains. A dendrogram was constructed which was based on the generated fatty acid profiling of each of the frankial

strains isolated from different hosts (Fig. 2). Cluster analysis revealed two major clusters (Cluster I and Clus-ter II) representing the twelve strains. The Cluster II was further divided into two subgroups i.e. subcluster IIa and IIb. Cluster I had only HsI2 genotype. The geno-types of Cluster IIa were HsIi4, HsIi5, HsIi8, HsIi9 and HsIi11 whereas the genotypes of cluster II b contained HsIi10, CpI2, HsIi13, HsIi12, Ag4b and HsIi14.

Figure 2. Dendrogram generated with NTSYS 2.1 version software on the basis of fatty acid profiling of twelve Frankia strains using UPGMA cluster analysis. Coefficient (X-axis) refers to a similitude coefficient.



Discussion

Studies on Frankia strains are very important as they nodulate H. salicifolia during actinorhizal symbiotic association and fix tremendous amount of atmospheric nitrogen which makes the tree as a pioneers of ecologi-cal succession. In spite of their ecological importance and environmental concern, their taxonomy and iden-tification are still problematic and often uncertain be-cause they are based on current morphological and physiological approaches which creates blurred image and are usually varied at different conditions [11, 29–31]. Therefore, a biochemical approach has gained an interest for the characterization of the frankial strains on the basis of fatty acid composition in the last few decades [3, 31–33]. It is true that pharmaceutical im-portance of the fatty acid is increasing day by day [16, 17]. In addition to this, it has also been proved useful for the chemotaxonomic studies of many environmen-tally relevant and biotechnologically important species at the family, genus and species level [31–36]. Our find-ings show the presence of fatty acids ranging from 16 to 22 carbon atoms in tested Frankia strains (Table1). However, 16 to 18 carbon fatty acids (Palmitic, Lino-lenic, Linoleic and oleic acid) were present in all the strains except HsIi2 where oleic acid was absent. Pres-ence and absence of fatty acids appear to be the charac-teristic of the frankial strains isolated from different host (Fig. 1). It is in accordance with the fatty acids found in the strain CpI1 [14] and the Casuarina-Frankia isolates where 12–18 carbon polar fatty acids were found [3]. In actinomycetes, fatty acids are the impor-tant component of the lipid laminae of the vesicles where nitrogen fixation is occurred [8, 14, 37–39]. These fatty acids prevent the diffusion of oxygen into the vesicles and hence play a major role in protecting the nitrogenase enzyme [14]. The lipid content is varied according to the high partial pressure of oxygen (pO2) [39]. The enzyme nitrogenase is essential for nitrogen fixation, is irreversibly inactivated on the exposure to atmospheric levels of oxygen [40]. Other frankial strains isolated from different hosts also have these fatty acids [31–33]. According to Tunlid et al [14] and Mirza et al. [32], the composition of fatty acids of several frankial strains showed significant similarity whereas the frankial isolates of Casuarina showed significant varia-tion in fatty acids composition [3]. All these fatty acids ranging from 15–20 carbon atoms are also found in nitrogen fixing symbiotic bacteria i.e. Agrobacterium and Bradyrhizobium [41]. Erucic acid (22:1) is monosaturated fatty acid and forms one of the major components of the oils obtained from the seeds of Brassica sp. and Hip-

pÖphae sp. First time, it has been reported in our ex-perimental Frankia strains though it was lesser in amount as compared to Brassica sp. and HippÖphae sp. The actinomycete Frankia is symbiotically associated with the root nodules of HippÖphae sp. shows the pres-ence of erucic acid as one of its fatty components so it may be possible that the gene responsible for the bio-synthesis of erucic acid might have been found and expressed in Frankia due to adaptive evolution [42] through infrequent horizontal gene transfer from plants to bacteria as reported earlier [43]. This result is consistent with the earlier observations of bacterial species [44–46]. From our experimental findings, it was clear that all the twelve strains of Frankia have a different percentage of various fatty acids as was observed among the other strains of Frankia isolated from various sources [3, 47]. Results proved that qualitative analysis of fatty acid composition showed significant variation while quanti-tative analysis showed minor differences in fatty acid composition. But when total cellular fatty acids were considered (Table 1), there were significant variation was observed among the strains whether they were isolated from the same host or from the other. This clarifies the specificity of the fatty acid composition in each and every Frankia strain. It was earlier reported that fatty acid profiling is characteristics feature of the genus Frankia and is useful in taxonomical study [32]. The constructed dendrogram (Fig. 2) through cluster analysis using UPGMA showed intrastrains variation among the strains at the biochemical level. Twelve frankial isolates were grouped in two major clusters based on fatty acid profiling. HsIi2 of cluster I was dis-tantly related to all other 11 strains of Cluster II. The strains of subcluster II a (HsIi4, HsIi5, HsIi8, HsIi9 and HsIi11) were most similar strains which were phyloge-netically distantly related to the closely related strains of subcluster IIb (HsIi10, CpI2, HsIi13, HsIi12, Ag4b and HsIi14). There is high degree of similarity among the frankial strains of Cluster IIb, even though isolated from different hosts, suggested that frankial strains might be evolved from the same ancestor. In other words, we can say that the fatty acids profile of these frankial strains appears to be conserved at the genus-level as reported by others [4, 32, 33]. In view of the above findings, it was concluded that a more realistic approaches i.e. the polyphasic appro-aches including genotypic, chemotaxonomic and phe-notypic methods is more reliable for the taxonomical study and diversity. Thus, such biochemistry-based taxonomies should be combined with other bacterio-logical approaches in order to gain an overall picture of



the target species, which would consequently allow each to be differentiated. The authors noted that more appropriate experimental conditions need to be created for further research to investigate horizontal gene transfer.

Acknowledgements

We are thankful to DBT (Project Ref No. BT/PR9145/ AGR/21/233), CSIR and DST, New Delhi for their finan-cial support. The authors are grateful to Dr. G. Thomas, Dr. S. C. Tiwari and Dr. H. K. Sarma for their valuable suggestions. The Head, Department of Botany, BHU, Varanasi, India is gratefully acknowledged for provid-ing laboratory facilities. Department of Forest, Envi-ronment and Wildlife Management, Sikkim is grate-fully acknowledged.

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((funded by • DBT, CSIR and DST, New Delhi; grant numbers: BT/PR9145/AGR/21/233))

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