Analysis of LMW RNA Profiles of Frankia Strains by Staircase Electrophoresis

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SYSTEMATIC AND APPUED MICROBIOLOGY

System. App!. Microbio!. 21, 539-545 (1998) g _©_G_u_sta_v_F_isc_h_er_V_er_Ia_

Analysis of LMW RNA Profiles of Frankia Strains by Staircase Electrophoresis ENCARNA VELAZQUEZ l , EMILIO CERVANTES 2 , JOSE MARIANO IGUAL2, ALVARO PEIX 2 , PEDRO F. MATEOS l , SAAD BENAMAR3 , ANDRE MOIROUD 4 , CHRIS T. WHEELERs, JEFF DAwso~, DAVID LABEDA 7 , CLAUDINO RODRfGUEZ-BARRUEC0 2 , and EUSTOQUIO MARTINEZ-MOLINA l Departamento de Microbiologia y Genetica, Edificio Departamental, Salamanca, Spain IRNA-CSIC, Salamanca, Spain 3 Laboratoire de Biologie et Physiologie Vegetales et Forestieres, Ecole Normale Superieure, Bensouda, Fes. Maroc 4 Universite Lyon 1. Ecologie Microbienne, Villeurbanne Cedex, France 5 Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Scotland 6 Deparrment of Natural Ressources and Environmental Sciences, University of Illinois, l005a Plant Sciences Lab, Urbana, IL 7 National Center for Agricultural Urilization Research, 1815 North University, Peoria, IL USA I

2

Received July 21, 1998

Summary An optimized technique of polyacrylamide gel electrophoresis, Staircase Electrophoresis (SCE), was applied to determine the stable Low Molecular Weight RNA (LMW RNA) profiles of 25 Frankia strains from diverse geographic origins and host specificity groups as well as species from other actinomycete genera. Application of the technique permits the rapid identification of Frankia strains and their differentiation from other actinomycetes. The isolates used in this study were grouped in eight clusters, each comprising strains with identical LMW RNA profiles. Comparison of these results with others obtained from DNA sequences or DNA hybridization methods suggest a high degree of complexity in the genus Frankia. Application of SCE to profile LMW RNA should in rhe future facilitate biodiversity studies of Frankia and discrimination of new species.

Key words: Frankia - LMW RNA - Nitrogen fixation - Bacterial taxonomy

Introduction Bacteria belonging to the genus Frankia are slow growing actinomycetes characterized by their capacity to fix atmospheric nitrogen in symbiotic associations resulting in the formation of nodule structures in the roots of many perennial woody dicotyledoneous plants. Since the first reproducible report of isolation of a Frankia strain in 1978 (CALLAHAN et aI., 1978) several hundred of isolates have been obtained for study in diverse laboratories throughout the world and considerable progress has been made in understanding many aspects of the Biology of these polymorphic bacteria (BENSON and SYLVESTER, 1993). As for Rhizobium, the first attempts to establish a classification of the genus Frankia were based on their host range (BECKING, 1970) and diverse phenotypic characteristics (LALONDE et aI., 1988). More recently, various techniques, based on molecular biology protocols, have been used for this purpose.

Studies of DNA relatedness, based on DNA-DNA reassociation kinetics, revealed the existence of at least nine genomic species (FERNANDEZ et aI., 1989). Among these, three contained strains compatible with the Alnus specificity group, five with the Elaeagnaceae and one with Casuarina. Genomic species 1 was proposed to be Frankia alnii, the type species of the genus. These studies are hampered by the difficulty to isolate and grow some strains in laboratory media as well as by the low efficiency of DNA extraction from Frankia cultures. Other approaches to allocating Frankia strains to taxonomic groups were based on the comparative analysis of PCR amplified sequences, an approach which also fa-

Abbreviations: LMW RNA - Low Molecular Weight RNA; SCE - Staircase Electrophoresis

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cilitated the characterization of uncultured Frankia strains, for which template DNA was directly obtained from the nodules. NAZARET et al. (1991) developed a protocol based on DNA amplification and sequencing of the 268 base pairs corresponding to partial ribosomal DNA sequences among eight of the previously described genomic species. Their work included the elaboration of a phylogenetic tree, showing a close relatedness among strains belonging to the Casuarina and Alnus infectivity groups, with both groups well separated from the Elaeagnus infectivity group. Atypical strains. ORS020602 (D11) and 55005 (DBB02060510) isolated from, but unable to reinfect Casuarina plants, were grouped close to Elaeagnus infective strains. In general, strains from Elaeagnus groups showed less diversity than those in the Alnus groups. From an analysis of PCR amplified sequences corresponding to 23S rRNA, HONNERLAGE et al. (1994) described seven groups, four related to the Alnus compatibility group, one including strains related to Casuarina compatibility group, another including Elaeagnus related strains and finally, a group consisting exclusively of the uncultured endophyte of Coriaria nepalensis. These results do not match exactly the results of the DNA-DNA reassociation experiments. Thus, different strains separated by a short distance in the phylogenetic tree of HONNERLAGE et a!. (1994) are sometimes included in different genomic species by FERNANDEZ et a!. (1989), whereas strains separated by longer distances are sometimes placed together. However, a similar grouping of strains as related to their host compatibilities is observed. Based on the complete nucleotide sequences of the 16S rRNA of eight Frankia strains, others in databases and DNA sequences amplified from nodules, NORMAND et a!. (1996) described the clustering of Frankia into four groups: Cluster 1 included strains that infect Alnus and Casuarina species, Cluster 2 included the unisolated symbiont of Dryas, related to the unisolated strains from Coriaria and Datisca, Cluster 3 included Elaeagnus infective Frankia strains and Cluster 4 included unclassified strains Ptl1 and atypical strains Cn7, Dc2 and AgBl-9. Given the present status of Frankia taxonomy, alternative methods may make an important contribution to the classification of the genus and determination of the phylogenetic relationships among diverse strains. BEYAZOVA and LECHEVAUER (1992) used LFRA to analyze the phylogenetic relationship among more than 100 Frankia strains. This technique is not particularly easy to master in order to obtain satisfactory and reproducible analysis and efforts to optimize other, simpler and faster methods, may results in significant advances in these studies. HOFLE (1988) proposed to use the profiles of Low Molecular Weight, stable RNA (LMW RNA) in studies of bacterial taxonomy. The LMW RNAs include the 5S rRNA and class 1 and 2 of tRNA (HOFLE, 1988). Recently, a new electrophoretic technique, Staircase Electrophoresis (SCE), has been developed that allows optimal separation of these molecules and their utilisation in bacterial taxonomy (CRuz-SANcHEZ et aI., 1997). This new technique has been applied to the differentiation of

species in the family Rhizobiaceae, where different LMW RNA profiles have been found to correspond well with the described genera and species (VELAzQUEZ et aI., 1998). In the present work, the LMW RNA profiles of Frankia isolates from diverse host plants and from diverse geographic provenances have been analysed.

Materials and Methods Bacterial strains and media: A description of the Frankia strains used in this study including the host plant from which they were derived and their geographic origin is shown in Table 1. Isolation of new strains for the present work was carried our by published protocols (LECHEVAUER and LECHEVALlER, 1990). Other aetinomvcetes obtained from culture colections are Rhodococcu~ rhodochrous CECf3046 (ATCC4273), Dacn'· lospoTangium aurantiacum CECf3288 (ATCC2391), Strept~­ myces cinammoneus (former Streptoverticillium cinammoneus) CECf3258 (ATCCI11874), Streptomyces kentuckense (former Streptoverticillium kentuckense) CECf3262 (ATCC12691), Micromonospora melanosporea CECf3087 (CBS 270.62), Streptomyces halstedii NRRL-2381 and Streptomyces lividans J11326. The Frankia strains used in this study were cultivated in Qmod medium (LALONDE and CALVERT, 1979) without glucose and with pyruvate at 0,5%. The cultures were kept at 25 ·C for three weeks. The other actinomycete strains were grown in YEG medium (Yeast Extract 0,7%, Glucose 1%) at 25 ·C for a week. RNA extraction and LMW RNA profile analysis: The RNA of the strains studied was extracted as described by HOFLE (1988). LMW RNA profiles were obtained using Staircase Elec· ttophoresis in 14% polyacrylamide gels under denaturing conditions in steps of 10 min, rising through a constant ramp with 50 V increases from 100 V to 2300 Vas reported earlier (CRUZSANCHEZ et aI., 1997). The foHowing commercial molecules from Boehringer Manheim (Manheim, Germany) and Sigma (St. Louis, MO, USA) were used as reference: 5S rRNA from Escherichia coli MRE 600 (120 and 115 nucleotides) (BIDLE and FLETCHER, 1995), tRNA specific for tyrosine from E. coli (85 nucleotides) and tRNA specific for valine from E. coli (77 nucleotides) (SPRlNZL et a!., 1985). Samples were prepared as reported elsewere (CRuz-SANCHEZ et a!., 1997). After electrophoresis, the gels were silver-stained as described by HAAS et a!. (1994). Data analysis and construction of dendrograms: The bands present in each profile were coded for input into a data base that included all the strains studied and Jaccard's similarity coefficient was calculated to construct the distance matrix. A dendrogram was constructed from the distance matrix using the Unweighted pair Group Arithmetic Mean (UPGMA).

Results RNA extraction and LMW RNA profile analysis The LMW RNA profiles of the strains used in this study are shown in Figure 1. All contain the three expected zones: 5S RNA, class 2 tRNA and class 1 tRNA (CRuz-SANcHEZ et a!', 1997). The number of bands present in the tRNA zone was as expected according to previous results obtained for the E. coli strain CECT99 (ATCC9637), using Staircase Electrophoresis (CRUZ-

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Fig. 1. LMW RNA profiles of the strains of actnomycetes used in the present work. (A) All lanes correspond to Frankia. Lane 1: Cn3 from Coriaria nepalensis. Lane 2: Cn7 from Coriaria nepalensis. Lane 3: Hr 114.2 from Hippophae rhamnoides. Lane 4: Hr 77.3 from Hippophae rhamnoides. Lane 5: Ag 67.5, Ag 95.2, AI 112.2, AcN14A, ENS010712, ENS010714, UGL011301, UGL013103, UGL013104, UGL010701, NRRLB-16510, all strains from different species of Almls, NRRLB-16510 (HrI1) from Hippophae rhamnoides. Lane 6: CSI020602, CSI020604, CSI020620, CSSI020621, UGL020603, all strains from Casuarina equisetifolia. Lane 7: Strains 55005, ORS020602, atypical from Casuarina and Strain Ea 32.1 isolated from Elaeagnus. Lane 8: Strains NRRLB-16422 (MgI8) and NRRLB-16423 (PtIl) isolated respectively from Myrica gale and PlIrshia tridentata. (B) All lanes correspond to other actinomycetes: Lane 1: RhodococclIs rhodochrolls CECT3046 (ATCC4273). Lane 2: Dactylosporangillm allrantiacllm CECT3288 (ATCC23491). Lane 3: Micromonospora melanosporea CECT3087 (CBS270.62). Lane 4: Streptomyces cinammonells CECT3258 (ATCCl1874). Lane 5: Streptoverticillium kentuckense CECT3262 (ATCC12691). Lane 6: Streptomyces halstedii NRRLB-2381. Lane 7: Streptomyces lividans JIl326.

SANCHEZ et aI., 1997) and for the members of the Rhizobiaceae (VELAzQUEZ et aI., 1998). The Frankia strains used in this study are distributed in eight groups, each showing a different LMW RNA profile (Figure lA). Groups 1 and 2 include strains isolated from Coriaria nepalensis: strain Cn3 (Fig. lA, lane 1) and Cn7 (Fig. lA, lane 2). Groups 3 and 4 include strains isolated from Hippophae rhamnoides: Hrl14.2 (Fig. lA, lane 3) and Hrn.3 (Fig. lA, lane 4), Group 5 (Fig. lA, lane 5) includes strains UGLOI0701, UGL013104, UGL013103, UGL011301, ENSOI0712, ENSOI0714, AcN14A, Ag67.5, Ag95.2 and Ar112.2, all of them isolated from diverse species of Alnus. Group 6 (Fig. lA, lane 6) includes atypical Frankia strains isolat-

ed from Casuarina that infect Elaeagnus (ORS010601 and 55005) as well as strains Ea31.1 isolated from Elaeagnus angustifolia. Group 7 (Fig. lA, lane 7) includes strains CSI010601, CSI010604, CSI010610, CSI010611 and UGL010603 isolated from Casuarina equisetifolia. Group 8 (Fig. lA, lane 8) includes strains NRRL-B16411 (MgI8) isolated from Myrica gale and NRRLB-16413 (PtIl) isolated from Purshia tridentata. The LMW RNA profiles of other actinomycetes are shown in figure 1B: Rhodococcus rhodochrous CECT3046 (Lane 1). Dactylosporangium aurantiacum CECT3288 (Lane 2), Micromonospora monosporea CECT3087 (Lane 3), Streptoverticillium cinammoneum CECT3258 (Lane 4), Streptoverticillium kentuckense

E. VELUQUEZ et al.

542

CECT3262 (Lane 5), Streptomyces halstedii NRRLB2382 (Lane 6) and Streptomyces lividans J11326 (Lane 7).

The Frankia strains used in this study are separated in two groups with a similarity coefficient of 0.4. One group includes strains NRRLB-l6422 (isolated from Myrica gale) and NRRLB-l6423 (isolated from Purshia tridentata), both strains with the same LMW RNA profile (Fig. lA, lane 8). The second group is subdivided in two with a coefficient of 0.5. One of these subgroups includes two strains isolated from Coriaria, CN3 and CN7 (Figure lA, lanes land 2). The remaining Frankia strains are divided in two groups with a similarity coefficient of 0.6. One of them comprises the two strains obtained from Hippophae rhamnoides (Hr 114.2 and Hr 77.3), the other is further subdivided into two groups with a similarity coefficient of 0.8. One of these comprises sev-

Data analysis and construction of dendrograms Construction of a dendrogram (Figure 2) revealed two major groups with a similarity coefficient of only 0.24. One contains Micromonospora and Dactylosporangium and the other group contains the rest of the actinomycetes (including Frankia strains) studied in this work. This second group is further divided in two clusters with a similarity coefficient of 0.3, one containing all the Frankia strains and the other the remaining actinomycetes.

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LMW RNA from Frankia

eral strains isolated from Casuarina equisetifolia (UGL020603) and the isolates obtained in Salamanca CSI020602, CSI020620 and CSI020621). The remaining strains are divided between a group that includes all the tested strains from Alnus and a final group with two atypical strains from Casuarina and strain Ea32.1 from Elaeagnus (similarity coefficient 0.86).

Discussion In recent years new techniques based on molecular biology protocols have been applied to the resolution of traditional questions in microbial taxonomy. The well known difficulties for isolating and growing in culture Frankia strains have led to the application of techniques which do not require previous steps of isolation and culture of microbial strains in the laboratory (HONNERLAGE et aI., 1994; NAZARET et aI., 1991; NORMAND et aI., 1996). Taxonomic studies of this genus have shown in general the relatedness among strains isolated from the same host plant, but there are controversial results concerning the relative taxonomic position in dendrograms of strains isolated from diverse host plants. As for other symbiotic, nitrogen-fixing bacteria, namely those in the family Rhizobiaceae, attempts were made initially to make the definition of species on the bases of the capacity to nodulate a particular host plant (BECKING, 1970). This character in itself is not a criterium robust enough for classification (TORREY, 1990) because particular plants may be nodulated by strains that were derived from different host species or even genera.

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In this sense, promiscuous hosts are Elaeagnus angustifolia and Alnus glutinosa. Also, most Frankia strains are able to reinfect individuals of the host plant species from which they were isolated. Nevertheless there are reports of so called atypical strains, i. e. strains unable to infect the host plant from which they are derived but able to infect other actinorhizal genera (BAKER, 1987). These results emphasize the need for the application of new taxonomic techniques, that in the Rhizobiaceae were decisive for the description of new genera and species. The application of the technique of Staircase electrophoresis to obtain the LMW-RNA profiles in the Rhizobiaceae resulted in the reported observations that different genera show differences in their 5S RNA whereas different species of the same genus show differences in the tRNAs and overall, the LMW-RNA profiles are characteristic of each species. Moreover, the analysis of the corresponding data by UPGMA resulted in similar dendrograms as those obtained by 16S rRNA sequencing and analysis in these bacteria (VELAZQUEZ et aI., 1998). Based on these considerations, the LMW RNA profiles of Frankia strains from diverse host plants and geographic provenances were analysed by Staircase Electrophoresis. The technique was simultaneously applied to other actinomycetes, showing different LMW RNA profiles among the diverse species and when compared to Frankia isolates (Figure 1). The clustering of actinomycetal species was similar to the obtained with 16S rRNA sequence analysis (GOODFELLOW, 1989), thus Dactylosporangium was more related to Micromonospora, being both separated from the other groups. Rhodococcus remained isolated from the species of the genus

Table 1. Characteristics of Frankia strains used in this study. Designation

Host source

Location

Source

Reference

NRRLB-16422 (MGI8) NRRLB-16423 (Pd1) ENS010712 (S12) ENS010714 (S14) UGLOl{)701 UGL013103 UGL013104 UGL011301 UGL020603 Ea32.1 Hr114.2 Hr77.3 Ag 67.5 Ag 97.2 Ar 112.2 ACN 14a ORS020602 (D11) Cn3 Cn7 55005 (DBB02060510) CS1020602 CS1020604 CS1020620 CS1020621

Myrica gale Purshia tridentata Alnus glutinosa Alnus glutinosa Alnus glutinosa Alnus ntbra Alnus rubra Alnus inokumai Casuarina equisetifolia Eleagnus angustifolia Hyppophae rhamnoides Hyppophae rhamnoides Alnus glutinosa Alnus glutinosa Alnus ntbra Alnus cordata Casuarina equisetifolia Coriaria nepalensis Coriaria nepalensis Casuarina equisetifolia

USA USA Morocco Morocco Scotland Scotland Scotland S. Korea Egypt Francia Francia Francia Francia Francia Francia Canada Senegal Pakistan Pakistan USA Spain Spain Spain Spain

D. Labeda D. Labeda S. Benamar S. Benamar C. T. Wheeler C. T. Wheeler C. T. Wheeler C. T. Wheeler C. T. Wheeler A. Moirud A. Moirud A. Moirud A.Moirud A. Moirud A. Moirud P. Normand H. RamIrez H. Ramirez H. Ramirez M.lgual this study this study this study this study

BAKER, 1987 BAKER, 1987 (Unpublished data) (Unpublished data) WHEELER et al., 1986 HOOKER and WHEELER, 1987 HOOKER and WHEELER, 1987 SAYED et al., 1997 (Unpublished data) (Unpublisehd data) (Unpublished data) (Unpublished data) (Unpublished data) (Unpublished data) (Unpublisehd data) NORMAND and LALONDE, 1982 GAUTHIER et aL, 1981 MIRZA et aL, 1992 MIRZA et aL, 1992 BAKER, 1987

Casuarinaequ~etifolia

Casuarina equisetifolia Casuarina equisetifolia Casuarina equisetifolia

544

E. VELAzQUEZ et al.

Streptomyces that clustered toghether confirming the finding of Wm and STACKEBRANDT (1990). Our results support the unification of the genera Streptoverticillium and Streptomyces as proposed by these authors because their 55 rRNA show an identical profile. Among the Frankia strains, eight LMW RNA profiles were observed that, in general, correspond to groups of strains isolated from different host plants, although strains with different LMW RNA profiles may be obtained from the same host plant (Figure lA, lanes 1 to 4) and strains with the same LMW RNA may be obtained from different hosts (Figure lA, lane 8). Strains isolated from Alnus consistently showed the same LMW RNA profile independently of the host species and their geographic origin (Table 1). Strain ACN14a was in cluster 1 of NORMAND et ai. (1996) and closely associated to CpIl, the strain designated as the rype strain for Frankia alnii, in the analysis of HONNERLAGE et ai. (1994). The uniformiry in the LMW RNA profiles obtained for the Alnus strains, including ACN14a, supports the designation of Frankia alnii as the species rype of the genus and this technique provides a rapid and efficient method to identify strains belonging to this species. The rypical strains isolated from Casuarina that were used in this study have an identical LMW RNA profile and constitute the same species. Arypical strains, isolated but unable to re-infect Casuarina, but able to infect Elaeagnus (BAKER, 1987; NAZARET et ai., 1991) 55005 and ORS020602 share their LMW RNA profile with strain Ea32.1 isolated from Elaeagnus and may thus be a separate species. The two Frankia species comprising the Alnus isolates and the rypical Casuarina isolates are close to each other phylogenetically, in agreement with the results of other authors (HONNERLAGE et ai., 1990; NAZARET et ai., 1991; NORMAND et ai., 1996). The low similariry coefficient among many clusters of Frankia strains makes it likely to consider the existence of different bacterial species (or even genera) in the genus Frankia. This possibiliry is supported by the observed varia.biliry in the region corresponding to 55 rRNA. Several aUthors have shown before that differences in the 55 rRNA reflect differences among bacterial genera (HOFLE, 1988; HOFLE, 1990; VELAzQUEZ et ai., 1998). Strains Hr114.2 and Hrn.3 isolated from Hippophae grouped in the same cluster but the coefficient of similariry (0,8) indicates that each of the strains may belong to a different species. Strains Cn3 and Cn7, isolated from Coriaria, grouped separately from the other Frankia strains. Similar results were obtained for strain Cn7 by RAMIREZ-SAAD et ai. (1998) using sequence data comparisons. Our results, showing a low coefficient of similariry between Cn3 and Cn7 (0.6), probably indicate that both strains may belong to different species. In summary, our results show that the grouping of Frankia isolates in taxonomic groups broadly reflects the host plants from which they were derived. This conclusion confirms previous analyses based on diverse tech-

niques (BEYAZOVA and LECHEVALlER, 1992; FERNANDEZ et ai., 1989; LALONDE et ai., 1988; NAZARET et ai., 1991; NORMAND et ai., 1996) but presents interesting exceptions that may be grouped in two classes: 1) Strains from very divergent host plants that share a very similar or identical LMW RNA profile, as for example MgI8 and PtI1 and 2) Strains isolated from the same host plant that have very divergent LMW RNA profiles, as several isolates obtained from Coriaria and Hippophae in our study. The results presented indicate the usefulness of LMW RNA profiles to differentiate species in the genus Frankia and to establish taxonomic groups among strains isolated from diverse host plants and from diverse geographic origins. The technique will allow in the future easy and rapid identification of Frankia strains and their allocation to the described species of the genus. This will improve significantly knowledge of the taxonomy of this bacterial group and help in studies of the biodiversity of these ecologically and economically important bacteria. Acknowledgements We thank PHILIPPE NORMAND, HUGO RAMIREZ SAAD and EMETERIO IGLESIAS JIMENEZ for suppling Frankia strains and their commentaries to this work. This work was supported by Grant CSIOl-97 of Junta de Castilla y Leon and the DGICYT (Direccion General de Investigacion Cientifica Tecnica) Grant PB921097.

References BAKER, D. D.: Relationships among pure-cultured strains of Frankia based on host specificity. Physiol. Plant. 70, 245-248 (1987). BECKING, J. H.: Frankiaceae fam. nov. (Actinomycelates) with one new combination and six new species of the genus Frankia Brunchorst 1886. Int. J. Syst. Bact. 20, 201-220 (1970). BENSON, D. R. and SILVESTER, W. B.: Biology of Frankia strains, Actinomycete symbionts of actinorhizal Plants. Microbiol. Rev. 57, 293-319 (1993). BEYAZOVA, M. and LECHEVALlER, M. P.: Low-frequency restriction fragment analysis of Frankia strains (Actinomycetales). Int. J. Syst. Bacteriol. 42, 422-433 (1992). BIDLE, K. D. and FLETCHER, M.: Comparison of free-living and Particle-associated bacterial communities in the Chesapeake bay by stable Low Molecular Weight RNA analysis. Appl. Environm. Microbiol. 61, 944-952 (1995). CALLAHAN, D., DEL TRIDIO, P., TORREY, J. G.: Isolation and cultivation in vitro of the actinomyete causing root nodulation in Comptonia. Science 199, 899-902 (1978). CRuZ-SANCHEZ, J. M., VELAzQUEZ, E. MATEOS, P. E, MARTlNEZMOLINA, E.: Enhancement of resolution of Low Molecular Weight RNA profiles by Staircase Electrophoresis. Electrophoresis 18, 1909-1911 (1977). FERNANDEZ, M. P., MEUGNIER, H., GRI/l.lONT, P. A. D., BARDIN, R.: Deoxyribonucleic acid relatedness among members of the genus Frankia. Int. J. Syst. Bacteriol. 39, 424-429 (1989). GAUTHIER, D., DIEM, H. G., DOMMERGUES, Y. R.: In vitro nitrogen fixation by two actinomycetous strains isolated from Casuarina nodules. Appl. Environ. Microbiol. 41, 306-308 (1981).

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Corresponding author: EMILIO CERVANTES, IRNA-CSIC, Apanado 257, 37080 Salamanca, Spain Phone: 011-34-923-219606, Fax: 011-34-923-219609

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