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Soil Biology & Biochemistry 39 (2007) 372–377

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Short communication

Genetic diversity among Elaeagnus compatible Frankia strains andsympatric-related nitrogen-fixing actinobacteria revealed by nifH

sequence analysis

Maher Gtaria,�, Lorenzo Brusettib, Abdenaceur Hassenc, Diego Morab,Daniele Daffonchiob, Abdellatif Boudabousa

aDepartement de Biologie, Laboratoire Microorganismes et Biomolecules Actives, Faculte des Sciences de Tunis, Campus Universitaire, 2092 Tunis, TunisiabDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita degli Studi, via Celoria 2, 20133 Milano, Italy

cLaboratoire Eau et Environnement, Institut National de Recherche Scientifique et Technique, BP24-1082, Cite Mahrajene, Tunis, Tunisia

Received 26 April 2006; received in revised form 27 June 2006; accepted 4 July 2006

Available online 14 August 2006

Abstract

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and

Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and

Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like

actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional

microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root

nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic

trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from

Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying

intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open

network investigations on the natural history of nitrogen-fixing gene among actinobacteria.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Frankia; Nitrogen-fixing actinobacteria; NifH; Phylogeny; Tunisia

Frankia genus was made up of actinobacteria distin-guished by their ability to establish nitrogen-fixingactinorhizes in diverse woody dicotyledonous roots (Ben-son and Silvester, 1993). Phylogenetic studies of Frankia

strains based on 16S rRNA gene, glutamine synthetase I(GlnA) and glutamine synthetase II (GlnII) sequences havegenerally revealed four major clades (Normand et al., 1996;Clawson et al., 2004; Cournoyer and Lavire, 1999; Gtari etal., 2004). Frankia strains in these clades can be distin-guished on the basis of cultivability, morphology, andplant root infectivity (Benson and Silvester, 1993). Strainsfrom clade I have not been isolated in culture, while typicalFrankia isolates have been obtained from clades II and III.

e front matter r 2006 Elsevier Ltd. All rights reserved.

ilbio.2006.07.005

ing author. Tel./fax: +216 70 860 553.

ess: maher.gtari@fst.rnu.tn (M. Gtari).

Clade II strains grow preferentially on organic acids,whereas Clade III strains grow on both organic acids andsimple sugars (Benson and Silvester, 1993). Althoughrelatively few plants have been studied, Clade II strainsappear to infect their hosts by root hair infection and CladeIII strains infect by either intercellular penetration or roothair infection depending on the plant (Berry et al., 1986;Bosco et al., 1992; Cournoyer et al., 1993; Miller andBaker, 1985; Racette and Torrey, 1989). Atypical (non-infective and/or non-nitrogen-fixing) Frankia strains ob-tained from actinorhizal plants such as Ceanothus,Coriaria, Datisca, and Purshia species, form a broad groupand were associated to clade IV. This group was describedon the basis of 16S rRNA gene sequence (Normand et al.,1996; Huguet et al., 2001). Due to the lack of solidphenotypic features of typical Frankia strains, the inclusion

ARTICLE IN PRESSM. Gtari et al. / Soil Biology & Biochemistry 39 (2007) 372–377 373

of this clade as member of the Frankia assemblage isquestionable and merits further investigations. Nitrogenfixation, one of the two enigmatic points of suchactinorhizal isolates, being the second infectivity, couldbe elucidated by using genes encoding nitrogenase complexenzymes, important feature of Frankia within actinobac-teria. The intergenic spacer between nifD and nifK genes(IGS nifD-K) has provided a powerful approach to assessthe diversity of Frankia strains in culture and directlywithin root nodules (Jamann et al., 1993; Rouvier et al.,1996; Lumini and Bosco, 1996; Lumini and Bosco, 1999).The nitrogenase iron protein gene, nifH, has been alsoreported to be in agreement with the phylogeny inferredfrom 16S rRNA gene sequences for several nitrogen-fixingmicroorganisms (Hennecke et al., 1985; Young, 1992; Zehret al., 1995; Ueda et al., 1995; Borneman et al., 1996;Ohkuma et al., 1996; Achouak et al., 1999; Raymond et al.,2003). However, phylogenetic studies of Frankia, based onnifH sequences, are rare and non-exhaustive (Jeong et al.,1999; Nick et al., 1992).

The objective of this study was to obtain informationabout the diversity and phylogenetic relationship ofFrankia nifH gene sequences and related nitrogen-fixingactinobacteria isolated from root nodule of Elaeagnus fromTunisian soils. When this study was started, no nifH

sequences from clade IV Frankia of taxa had beendeposited in Database (Gtari et al., 2004, EMBL accessionnumber AJ545036).

Isolation, subcultivation and purification procedures ofstrains: BMG5.2, BMG5.3, BMG5.4, BMG5.5, BMG5.10,BMG5.11, BMG5.12 and BMG5.6 used in this study, weredetailed elsewhere (Gtari et al., 2004). Infectivity tests ofisolates were carried out on Elaeagnus angustifolia, Alnus

glutinosa and Casuarina equisetifolia seedlings as describedby Bosco et al. (1992). NifH gene amplification wasperformed on DNA extracted from cultured strains andnodule lobes (Gtari et al., 2002, 2004) by using primersdescribed by Normand et al. (1988) and an innerset ATGGC(G/T)GCCATGGCCGAG and GTTGAT(G/A)CGGCGGCGA designed in this study to perform nestedPCR. For RFLP analysis PCR products were digested withAluI, RsaI, ThaI and HaeIII and electrophoresed in vertical6% polyacrylamide gel. Clustering of restriction patterns wasperformed with MVSP software (version 3.131, KovackComputing Service, Pentraeth, UK). Sequences were deter-mined by cycle sequencing (Urzı et al., 2001). Partialnucleotide and predicted aminoacid sequences of nifH genewere analyzed using Clustalw (http://clustalw.genome.ad.jp)and phylogenetic trees were achieved using PHYLIP(Felsenstein, 1993) and TREE-PUZZLE (Strimmer andHaeseler, 1996) utilities. Bootstrap values were determinedfrom 1000 replicates (Felstein, 1985). The nifH sequenceswere deposited in the EMBL nucleotide sequence database(GenBank/EMBL/DDBJ) under accession numbers fromAJ545030 to AJ545037.

All seven Frankia strains BMG5.2, BMG5.3, BMG5.4,BMG5.5, BMG5.10, BMG5.11 and BMG5.12 were found

to effectively nodulate 100% of Elaeagnus plantlets. StrainsBMG5.2 and BMG5.12 were also found able to induce 1–5small root nodules in some A. glutinosa plantlets with meanpercentages of nodulated plants ranging from 20% to 30%,respectively. However, nodulated Alnus plantlets alwaysremained small and yellowish. Despite initial re-nodulationexperiments of Elaeagnus axenic seedlings with strainBMG5.6 gave nodules (Gtari et al., 2004), after repeatedstrain subculturing the nodulation capacity was lost. StrainBMG5.6 never showed the capacity of nodulating Alnus

and Casuarina plantlets.Using primers set designed by Normand et al. (1988),

700 bp bands were amplified from Tunisian isolates using anifH specific PCR assay. For nifH RFLP, four restrictionenzymes, AluI, RsaI, HaeIII and ThaI, were selected on thebasis of in silico digestion using DNAMAN version 5.2.2software of Frankia nifH sequences available in Genbankand those of Tunisian isolates. A considerable diversity wasobserved among analyzed strains from Elaeagnus andAlnus compatible groups.Similar nifH RFLP profiles were found with the

restriction enzymes used between Alnus and Elaeagnus

specificity group. The Casuarina infective strain CcI3showed more divergent band patterns (Fig. 1). However,the UPGMA dendrogram obtained by PCR–RFLPanalysis permitted the discrimination between the twohost-specificity groups. The analysis allowed concludingthat strain BMG5.6, a closely related strain to Frankia

assemblage (Gtari et al., 2004), was associated withElaeagnus compatible strains.A nested PCR assay for the characterization of Frankia

and related nifH in nodules was developed by using primersof Normand et al. (1988) as outer primers and two primers,designed in this study as inner primers. The inner primerswere tested with several diazotrophic bacteria, and PCRrevealed that these primers are specific for PCR amplifica-tion of nifH gene from Alnus, Elaeagnus compatible strainsas well as the actinobacterium BMG5.6. We hence usedthis approach to analyze Frankia nifH in Elaeagnus rootnodules developed by plant-trapping assay with soilsampled in the region of Sfax. From the soil we previouslyisolated strain BMG5.6 (Gtari et al., 2004). PCR amplifi-cation was performed from peeled and non-peeled rootnodules (Fig. 2). Screening of 16 lobe nodules permitted thedetection of PCR–RFLP band patterns typical of strainBMG5.6 in one unpeeled and three peeled lobe nodules.Neighbor-Joining algorithm tree based on nifH gene

nucleotide sequences showed shown four main clades(Fig. 3). A deeper clade I included uncultured endosym-bionts from Ceanothus caeruleus, Coriaria nepalensis andDatisca cannabina with sequence identity from 81% to95%. Clade II included two infective isolates fromCasuarina CcI3 and INPCe16 (97.3% of identity) and thenitrogen-fixing actinobacterial isolate 7501. Strains in cladeIII share 93.8–98.9% of identity and were represented bythe Alnus–Myrica infective group strains; ArI3 ACN14aand FaC1 (Alnus infective) and Mrp128 (Myrica infective).

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ThaIAluI RsaI HaeIII2030405060708090100

FaCI

Mrp128

ACN14a

ArI3

HRN18a

BMG5.12

BMG5.2

BMG5.10

BMG5.4

BMG5.11

BMG5.3

BMG5.5

BMG5.6

EANpec

EuIK1

CcI3

Percentage of similarity

Fig. 1. In silico RFLP analysis of nifH from Frankia and nitrogen-fixing actinobacteria (strain BMG5.6) and the derived UPGMA-based genetic

relationship. The RFLP analysis was performed with the enzymes AluI, RsaI, HaeIII and ThaI. For the construction of the dendrogram the Jaccard’s

coefficient was used.

Fig. 2. Restriction patterns of amplified nifH on DNA extracted directly from lobe nodules collected from Elaeagnus capturing bioassay using Sfax soil.

Restriction fragments were separated on a 6% polyacrylamide gel. Lanes N1–N4 represent different nodules. The asterisk indicates RFLP pattern

identical to those of strain BMG5.6.

M. Gtari et al. / Soil Biology & Biochemistry 39 (2007) 372–377374

Clade IV, with a sequence identity ranging from 93.8% to99.3%, included Elaeagnus-infective strains HRN18a,EuIK1, EANpec, BMG5.2, BMG5.3, BMG5.4, BMG5.5,BMG5.10, BMG5.11 and BMG5.12 and the nitrogen-fixing actinobacterial strain BMG5.6.

PCR–RFLP approach gene has been routinely used inother regions of Frankia genome such as 16S rRNA gene(Huguet et al., 2001), IGS 16S–23S (Rouvier et al., 1996),IGS nifD-K (Jamann et al., 1993, Lumini and Bosco,1996). On nifH gene this approach was revealed to be anadditional powerful tool to assess Frankia diversity.Furthermore, PCR–RFLP on nifH, permitted the detection

of other nitrogen-fixing actinobacteria with restrictionpatterns identical to strain BMG5.6. The DNA wasextracted from lobe nodules sampled from Elaeagnus

plantlets grown, as Frankia-trapping bioassay, on soil(rendzine in sabkha soil, pH 9) sampled from Sfax, (351840

Northern latitude, 101360 Eastern longitude, 50m altitude),the site where strain BMG5.6 has been isolated. Thisindicated that the presence of Frankia related strains withinElaeagnus nodules is not sporadic. In addition, BMG5.6PCR–RFLP patterns were detected in DNA extractedfrom peeled lobe nodules, indicating that beside Frankia,the internal nodule tissues could be an appropriate

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Fig. 3. Neighbour-joining phylogenetic tree based on nifH nucleotide sequences, of Frankia and related nitrogen fixing actinobacteria. Genbank accession

numbers are given in parenthesis. Numbers on the tree branches are bootstrap values. Only bootstrap values higher than 400 positive replicates are shown.

M. Gtari et al. / Soil Biology & Biochemistry 39 (2007) 372–377 375

ecological niche for other nitrogen-fixing actinobacteria.Strain BMG5.6 showed important phenotypic featureswith typical Frankia isolates including cultural behavior onDPM medium and nitrogen fixation (as proved byacetylene reduction assay) (Gtari et al., 2004). Isolationof other nitrogen-fixing actinobacteria from root nodule ofCasuarina has been reported (Valdes et al., 2005) demon-strating that they are widespread and often associated withactinorhizal plants. By their ability to fix nitrogen, theycould exert beneficial effects to the host plant. However,the ecological interaction between these actinobacteria andFrankia is not yet clear.

Phylogenetic analysis of the partial nifH sequences fromFrankia revealed sufficient variation at the DNA sequencelevel enough to distinguish between different strains andgroups from Frankia genus and from other nitrogen-fixingbacteria. Several aspects of the nifH phylogenetic trees,proposed here were consistent with phylogenies based onthe 16S rRNA, GlnA and GlnII genes (Normand et al.,1996; Huguet et al., 2001; Clawson et al., 2004; Cournoyerand Lavire, 1999; Gtari et al., 2004), except two mainpoints. The first point was represented by the segregationof Alnus–Myrica–Casuarina clade in two clades, oneincluding strains from Alnus–Myrica-infecting group and

a second consisting of Casuarina-infective strains. Inaddition, and contrary to phylogenetic trees based on 16SrRNA gene and GlnII, strain BMG5.6 clustered withtypical Elaeagnus compatible Frankia strains. Likewise,strain 7501, the Mexican isolate from Casuarina nodule, isclustered with Casuarina-infective Frankia. When compar-ing the nifH DNA sequences of strains BMG5.6 and 7501with sequences of other Frankia strains, they were morerelated to sequences of Frankia, from clades II–IV than tosequences of Frankia associated to Coriaria, Datisca andCeanothus. For protein-coding genes such as nifH,phylogenetic relationship is influenced by the position ofthe nucleotide substitution. In particular, nucleotidesubstitutions in the third nucleotide positions of codonscan have low evolutionary implication. Thus, phylogenetictrees lack liability, mainly when closely related taxa orspecies with higher G+C content are considered (Gupta,1998). Hence a tree has been constructed from predictedaminoacid sequences (result not shown). Despite thedecrease of informative positions (Normand et al., 1988),Frankia the nifH-based phylogenetic tree was consistentwith 16S rRNA and GlnII phylogenies (Gtari et al., 2004).Nevertheless, a higher conservation of nifH sequences hasbeen detected between Frankia strains and the actinobac-

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Fig. 4. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of Frankia, Micromonospora genera and the intermediate nitrogen-fixing

actinobacteria. Numbers between parentheses are accession numbers. Only bootstrap values higher than 400 positive replicates are shown.

M. Gtari et al. / Soil Biology & Biochemistry 39 (2007) 372–377376

terial isolates BMG5.6 and 7501, which remain in aberranttree branch positions. This result suggests a horizontaltransfer of nifH among the different Frankia groups.According to Smith et al. (1992), the most crucialcomponent in establishing horizontal transfer is that thephylogeny of the sequences thought to be transferredshould conform to a conventional phylogeny except for theradically aberrant position of one member or group. UsingRibosomal Database Project II database (Cole et al., 2003),strain BMG5.6 has a 16S rRNA gene sequence identityintermediate between Frankia (96.1–96.4%) and Micro-

monospora species (95.7–96.3% by considering Micromo-

nospora rhodorangea, M. echinospora, M. globosa, M.

purpurea, M. melanospora, M. halophytica, and Micro-

monospora citrea species). In the 16S rRNA gene phyloge-netic tree, strain BMG5.6 occupied a position between theatypical group of Frankia strains and Micromonospora andthe assemblage of Eleagnaceae–Rhamnaceae/Alnus–Myri-

ca–Casuarina-infective strains and a series of unculturednodule microsymbionts (Fig. 4).

These results demonstrate that in the root noduleenvironment Frankia genus is not the unique actinobacter-ial taxon that harbors nitrogenase gene. In fact strains

BMG5.6 and 7501 living in the same environmental nicheof Frankia, the actinorhizal root nodules, constituteanother actinobacterial group closely related to Frankia

and carrying a similar nitrogenase enzyme. Whether nifH

was present in an actinobacterial ancestor or washorizontally transferred between Frankia and close-relatedactinobacterial strains BMG5.6 and 7501 merits furtherinvestigation.

Maher Gtari was supported by a grant from the DirectionGenerale de Recherche Scientifique et Technologique(DGRST) of the Ministere de l’Enseignement Superieureof Tunisia and the Comite Mixte de CooperationUniversitaire (CMCU 03/50908).

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