Laboratoire dEcologie Microbienne, UMR CNRS 5557, Université Lyon 1, 43 Bld du 11 Novembre 1918, 69622 Villeurbanne Cedex, France1
Université Louis Pasteur/CNRS, Institut Le Bel, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France2
Department of Environmental Horticulture, University of California, Davis, CA 95616, USA3
Author for correspondence: Anne-Marie Domenach. Tel: +33 472431379. Fax: +33 43721223. e-mail: domenach{at}biomserv.univ-lyon1.fr
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ABSTRACT |
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Keywords: Frankia strains, lipids, hopanoids, nitrogen
The GenBank/EMBL/DDBJ accession numbers for the sequences in this paper are AJ25138891 and AJ2513934.
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INTRODUCTION |
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Frankia is a nitrogen-fixing actinomycete which can form root nodules with woody plant hosts in eight angiosperm families. It is also able to fix nitrogen as a free-living organism over a wide range of oxygen concentrations (Silvester et al., 1990 ). Biological nitrogen fixation constitutes a paradox for aerobic organisms as it relies on the oxygen-labile nitrogenase enzyme while, at the same time, the process is an energy-demanding function which implies aerobic respiration. Aerobic nitrogen-fixing micro-organisms have developed strategies to regulate their intracellular oxygen concentration (for a review, see Gallon, 1992
). Because Frankia can fix nitrogen under aerobic conditions, adaptive mechanisms for oxygen protection must exist.
In Frankia, the nitrogenase is localized in specialized structures called vesicles (Meesters et al., 1987 ). The vesicles differentiate mainly under nitrogen-limiting conditions. Each vesicle is surrounded by an external multilamellate lipid envelope which presumably functions as an oxygen diffusion barrier (Lamont et al., 1988
; Harriott et al., 1991
). Structural studies have shown that in cultured Frankia, the thickness of vesicle envelopes and the number of lipid layers increased in response to an increased oxygen concentration (Parsons et al., 1987
). Four hopanoids, two bacteriohopanetetrol isomers, (Ia) and (Ib), and two bacteriohopanetetrol phenylacetyl monoesters, (IIa) and (IIb), represented 84% of the dry weight of purified vesicle envelope preparations (Berry et al., 1993
), and 80% of the total lipids of vesicle clusters (Kleemann et al., 1994
). Thus the bacteriohopanetetrols (Ia) and (Ib) together with their phenylacetic acid esters (IIa) and (IIb) were proposed to form the major physical barrier to protect nitrogenase against oxygen (Berry et al., 1993
). It is not yet known whether these lipids might play an additional role in other stages of the Frankia life cycle. In Frankia strains HFPArI3 (Berry & Torrey, 1979
) and HFPCcI3 (Zhang & Torrey, 1985
), the two bacteriohopanetetrols represented a large fraction (2050%) of total lipids under nitrogen-replete conditions (Berry et al., 1991
), but a comparison of hopanoids in different genomic species of Frankia, in cells grown under nitrogen-replete versus nitrogen-depleted conditions, has not yet been carried out.
In this report, we have quantified the ratio of hopanoids/total lipids, including the identification of new hopanoid structures in Frankia cells. This was realized in several Elaeagnus-compatible Frankia strains characterized by the nifDK region, and in two reference strains from the two other main infectivity groups, cultivated with and without nitrogen.
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METHODS |
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Sequencing of PCR-amplified fragments.
Elaeagnus-infective strains were characterized based on the PCR-RFLP HaeIII profile of the nifDK region (Nalin et al., 1997 ). One representative strain of each nif-HaeIII profile was used for the nifDK intergenic region identification. PCR amplifications were performed in a total volume of 50 µl, in 0·2 ml Eppendorf tubes, using a thermocycler (9600; Perkin-Elmer).
Amplification of the nifDK region directly from cells in liquid cultures was performed with primers FGPD807 (5'-CACTGCTACCGGTCGATGAA-3') and FGPK700' (5'-CGAGGTAGGTCTCGAAACCGG-3') as described by Jamann et al. (1993 ). Five microlitres of liquid culture was added to the reaction buffer [10 mM Tris/HCl (pH 8·3), 1·5 mM MgCl2, 50 mM KCl, 0·01% (w/v) gelatin], 200 µM each dNTP, 1 µM each primer and 2·5 units Taq polymerase (Gibco-BRL). The following program was used: initial denaturation for 2 min at 94 °C, and 35 cycles of denaturation (45 s at 95 °C), annealing (45 s at 55 °C) and extension (45 s at 72 °C), the final extension being longer (2 min at 72 °C). Before sequencing, the amplification reaction mix was purified with Centricon-30 concentrators (Amicon-Grace). The amplicons were sequenced using the Amplicycle Sequencing Kit (Perkin-Elmer) by the direct sequencing method of Winship (1989
). The two amplification primers were used as well as FGP-D169 [5'-ATGGACATCGC(CG)ATCAA-3'], FGP-K333' (5'-CCGGGCGAAGTGGCT-3') and FGP-K1 (5'-GTGACGACGACTCCC-3'), FGP-K64' (5'-CCTCGTCCTTGAACA-3') described by Navarro et al. (1997
). The sequences were determined on both strands (GenBank accession nos AJ25138891 and AJ2513934).
DNA sequence analysis.
The phylogenetic analyses were based on the sequences of an approximately 420 bp DNA fragment of the nifDK intergenic region according to Nalin et al. (1999 ). Sequences were aligned and compared with the alignment of 14 closely related Frankia strains described by Navarro et al. (1997
), using the multiple-alignment CLUSTAL X algorithm (Thompson et al., 1997
), with manual refinements in the non-coding regions. Distances were calculated by pairwise comparison according to Kimuras two-parameter model (Kimura, 1980
), and phylogenetic analyses were made using the neighbour-joining distance method of Saitou & Nei (1987
). The topology of the tree was tested by performing 1000 bootstraps (Felsenstein, 1985
) and parsimony analysis (Swofford, 1993
).
Lipid extraction and analysis.
Total cultures were sonicated for 3 min at 60% of the maximal power and 50% active cycles (Sonifier 250; Branson UltraSonics) for each strain and each treatment and for three replicates. Lipids from the sonicated samples were extracted according to Bligh & Dyer (1959 ) (CHCl3/CH3OH/H2O, 2:2:1·8, by vol., 38 ml). Total lipids extracted from each sample were concentrated under nitrogen to a volume of 50 µl immediately prior to the HPLC injection.
Separation and detection of total lipids by HPLC was performed according to Moreau et al. (1990 ) on a silica gel Si-60 Lichrosorb column of 10 cm with an internal diameter of 5x10-4 cm (Chrompack) at a flow rate of 0·5 ml min-1. Quantifications were done with an evaporative light-scattering detector (ELSD IIA model; Varex) by integration of peak areas in the chromatogram (Kleemann et al., 1994
).
HPLC peaks containing hopanoids were collected. Compounds were acetylated and identified by direct inlet impact MS according to the characteristic fragmentation ions of the triterpenoid pentacyclic skeleton, as described earlier (Berry et al., 1991 ). A large-scale culture of Frankia afforded enough material for the spectroscopic identification of the hopanoids. The structures of the novel hopanoids moretanol (IV) and bacteriohopanetetrol propionate (IV) and the stereochemistry of bacteriohopanetetrol (Ia) and (Ib) side chains were determined after isolation of the compounds by spectroscopic methods (1H- and 13C-NMR, MS), by derivatization and finally by comparison of the data with those obtained from synthetic reference compounds (Rosa Putra, 1998
). The two bacteriohopanetetrols (Ia) and (Ib) only differed by the configuration at C-34 as shown by the comparison of the 1H-NMR spectra of their tetra-acetates with those of reference compounds (Bisseret & Rohmer, 1989
; Rosa Putra, 1998
).
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RESULTS |
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DISCUSSION |
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The nifDK region, which has been widely used for the characterization of new Frankia isolates (Navarro et al., 1997 ), permits the discrimination between two closely related Alnus-infective strains (Nalin et al., 1995
). The nifDK intergenic spacer sequences have been used to characterize newly detected Gymnostoma-infective Frankia strains, which were found to be clustered with the Elaeagnus-infectivity group confirming the plant nodulation bioassay (Navarro et al., 1997
). This region allows for discrimination at the infraspecific level and thus is very useful for strain characterization in the Frankia genus.
In Elaeagnus-infective strains, we confirm and extend earlier observations on Frankia hopanoids (Berry et al., 1991 , 1993
): bacteriohopanetetrols (Ia) and (Ib) and their phenylacetates (IIa) and (IIb) belong to the main triterpenoids found in these bacteria. In addition, two novel hopanoids were identified: a bacteriohopanetetrol propionate (III) as a minor compound and, surprisingly, moretan-29-ol (IV), which was one of the major hopanoids (Rosa Putra, 1998
). Detailed structure determinations will be reported elsewhere. This is the first report of a (17 ß-H, 21
-H) hopanoid from a prokaryote. The presence of such a C30 triterpene is a striking feature, and its significance is not clear. Over 30 hopanoid structures have been described from bacteria (Ourisson & Rohmer, 1992
; Rohmer, 1993
). They primarily differ by their side chain structures. Hopanoids have already been found in nitrogen-fixing bacteria, such as Azotobacter vinelandii, Beijerinckia indica and Beijerinckia mobilis (Vilchèze et al., 1994
), and in the Bradyrhizobium species (Kannenberg et al., 1996
), but not in all of them. No common structural features were found, however, for the hopanoid side chains in nitrogen-fixing bacteria. The significance of such structural diversity is not obvious. Hopanoids were assumed to act as membrane stabilizers, in a fashion similar to that of sterols in eukaryotic membranes (Ourisson & Rohmer, 1992
). Such a structural role is compatible with molecular diversity, provided the triterpenoids fulfil all structural features required for membrane stabilizers (Rohmer et al., 1979
; Simonin et al., 1992
). Our findings of additional hopanoids in Frankia strains, especially the moretan-29-ol, under nitrogen-replete conditions are highly suggestive that hopanoids might be implicated in other processes besides the oxygen protection of nitrogenase.
The total amount of hopanoids has been estimated at approximately 35 mg (g lyophilized cells) -1 (data not shown). All Frankia strains tested from three infectivity groups show a high hopanoids/lipids ratio, expanding the results obtained by Berry et al. (1991 ) on strains HFPArI3 and HFPCcI3. This ratio was found to be much higher than those of other hopanoid-producing bacteria, even higher than those of Z. mobilis (Hermans et al., 1991
) and Al. acidocaldarius (Poralla et al., 1984
), which were previously known to produce the highest amounts of hopanoids. This suggests that the large amount of hopanoids (2087% of total lipids) is a general feature of the Frankia genus and could be used as a phenotypic characteristic of this genus among actinomycetes (Marechal et al., 2000
). Such high concentrations are probably not compatible with their commonly accepted role as membrane stabilizers, at least in the way described for sterols interacting with the phospholipid side chains.
The moretan-29-ol (IV) and the bacteriohopanetetrols (Ia) and (Ib), the most abundant hopanoids in Frankia cells, have been found in different ratios among the Frankia strains tested. Quantitative variations under the alternative nitrogen conditions were much less important in our experiments, since such variation represented only up to 20%. A similar result was found in another nitrogen-fixing bacterium, Az. vinelandii, where production of hopanoids was not stimulated under nitrogen-fixing conditions (Vilchèze et al., 1994 ).
The consistently high proportion of hopanoids observed, even under nitrogen-replete conditions, indicates that in all tested Frankia strains, most of the hopanoids are synthesized independently of the nitrogen source of the culture. Nevertheless, hopanoids are the main lipid component of the vesicles (Kleemann et al., 1994 ), which are essentially produced under nitrogen-fixing conditions. This suggests that hopanoids could be remobilized rather than neosynthesized under nitrogen-free culture conditions in order to build up the multilamellate envelope of the vesicles.
The structural diversity of hopanoids and their high proportion in all Frankia populations over different infectivity groups suggest that these compounds play important roles in Frankia cells. Their roles could be expressed in symbiotic interactions, i.e. in root nodules, where hopanoids were first reported (Berry et al., 1991 ), and also under in vitro culture conditions, where they represent up to 87% of the total lipids detected. Hopanoids might have a fundamental function in Frankia ecology, and the variations observed in the hopanoid concentration between strains would be the expression of different adaptive capacities. This hypothesis might point to new functions for hopanoids that are currently being tested.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Berry, A. M., Moreau, R. A. & Jones, A. D. (1991). Bacteriohopanetetrol: abundant lipid in Frankia cells and in nitrogen-fixing nodule tissue. Plant Physiol 95, 111-115.
Berry, A. M., Harriott, O. T., Moreau, R. A., Osman, S. F., Benson, D. R. & Jones, A. D. (1993). Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase. Proc Natl Acad Sci USA 90, 6091-6094.[Abstract]
Bisseret, P. & Rohmer, M. (1989). Bacterial sterol surrogates. Determination of the absolute configuration of bacteriohopanetetrol side chain by hemisynthesis of its diastereoisomers. J Org Chem 54, 2958-2964.
Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911-917.
Felsenstein, J. (1985). Confidence limits on phylogenies: approach using bootstrap. Evolution 39, 783-791.
Fernandez, M. P., Meugnier, H., Grimont, P. A. D. & Bardin, R. (1989). Deoxyribonucleic acid relatedness among members of the genus Frankia. Int J Syst Bacteriol 39, 424-429.
Fontaine, M. S., Lancelle, S. A. & Torrey, J. G. (1984). Initiation and ontogeny of vesicles in cultured Frankia sp. strain HFPArI3. J Bacteriol 160, 921-927.[Medline]
Gallon, J. R. (1992). Reconciling the incompatible: N2 fixation and O2. New Phytol 122, 571-609.
Harriott, O. T., Khairallah, L. & Benson, D. R. (1991). Isolation and structure of the lipid envelopes from the nitrogen-fixing vesicles of Frankia sp. strain CpI1. J Bacteriol 173, 2061-2067.[Medline]
Hermans, M. A. F., Neuss, B. & Sahm, H. (1991). Content and composition of hopanoids in Zymomonas mobilis under various growth conditions. J Bacteriol 173, 5592-5595.[Medline]
Hirsh, A., McKhann, H., Reddy, A., Liao, J., Fang, Y. & Marshall, C. (1995). Assessing horizontal transfer of nif HDK genes in eubacteria: nucleotide sequence of nif K from Frankia strain HFPCcI3. Mol Biol Evol 12, 16-27.[Abstract]
Jamann, S., Fernandez, M. P. & Normand, P. (1993). Typing method for N2-fixing bacteria based on PCR/RFLP application to the characterization of Frankia strains. Mol Ecol 2, 17-26.[Medline]
Kannenberg, E. L., Perzi, M., Muller, P., Hartner, T. & Poralla, K. (1996). Hopanoid lipids in Bradyrhizobium and other plant-associated bacteria and cloning of the Bradyrhizobium squalene-hopene cyclase. Plant Soil 186, 107-112.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111-120.[Medline]
Kleemann, G., Alskog, G., Berry, A. M. & Huss-Danell, K. (1994). Lipid composition and nitrogenase activity of the symbiotic Frankia (Alnus incana) in response to different oxygen concentrations. Protoplasma 183, 107-115.
Lamont, L. H., Silvester, B. & Torrey, J. G. (1988). Nile red fluorescence demonstrates lipid in the envelope of vesicles from N2-fixing cultures of Frankia. Can J Microbiol 34, 656-660.
Marechal, J., Clement, B., Nalin, R., Gandon, C., Orso, S., Cvejic, H., Bruneteau, M., Berry, A. M. & Normand, P. (2000). A recA gene analysis confirms the close proximity of Frankia to Acidothermus. Int J Syst Evol Microbiol 50, 781-785.[Abstract]
Meesters, T., van Vliet, M. & Akkermans, A. (1987). Nitrogenase is restricted to the vesicles in Frankia strain EAN1pec. Physiol Plant 70, 267-271.
Moreau, R. A., Asmann, P. T. & Norman, H. A. (1990). Quantitative analysis of the major classes of plant lipids by high performance liquid chromatography and flame ionization detection (HPLC-FID). Phytochemistry 29, 2461-2466.
Murry, M., Fontaine, M. S. & Torrey, J. G. (1984). Growth kinetics and nitrogenase induction in Frankia sp. HFP ArI3 grown in batch culture. Plant Soil 78, 61-78.
Nalin, R., Domenach, A. M. & Normand, P. (1995). Molecular structure of the Frankia spp. nifD-K intergenic spacer and design of Frankia genus compatible primer. Mol Ecol 4, 483-491.[Medline]
Nalin, R., Normand, P. & Domenach, A. M. (1997). Distribution and N2-fixing activity of Frankia strains in relation with soil depth. Physiol Plant 99, 732-738.
Nalin, R., Normand, P., Simonet, P. & Domenach, A. M. (1999). Polymerase chain reaction and hybridization on DNA extracted from soil as a tool for Frankia spp. population distribution studies in soil. Can J Bot 77, 1239-1247.
Navarro, E., Nalin, R., Gauthier, D. & Normand, P. (1997). The nodular microsymbionts of Gymnostoma spp. are Elaeagnus-infective Frankia strains. Appl Environ Microbiol 63, 1610-1616.[Abstract]
Oh, B., Twiggs, P., Hong, J., Mullin, B. & An, C. (1997). nif V is contiguous to nif HDK in Frankia strain FaC1. Physiol Plant 99, 707-713.
Ourisson, G. & Rohmer, M. (1992). The hopanoids. Part 2: the biohopanoids, a novel class of bacterial lipids. Accounts Chem Res 25, 403-407.
Parsons, R., Silvester, W., Harris, S., Gruijters, W. & Bullivant, S. (1987). Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase. Plant Physiol 83, 728-731.
Poralla, K., Härtner, T. & Kannenberg, E. (1984). Effect of temperature and pH on the hopanoid content of Bacillus acidocaldarius. FEMS Microbiol Lett 23, 253-256.
Rohmer, M. (1993). The biosynthesis of triterpenoids by the hopane series in Eubacteria: a mine of new enzyme reactions. Pure Appl Chem 65, 1293-1298.
Rohmer, M., Bouvier, P. & Ourisson, G. (1979). Molecular evolution of biomembranes: structural equivalents and phylogenetic precursors of sterols. Proc Natl Acad Sci USA 76, 847-851.[Abstract]
Rohmer, M., Bouvier-Nave, P. & Ourisson, G. (1984). Distribution of hopanoid triterpenes in prokaryotes. J Gen Microbiol 130, 1137-1150.
Rosa Putra, S. (1998). Rle du 1-désoxy-D-xylulose dans la biosynthèse des isoprénoides. PhD thesis, Université Louis Pasteur, Strasbourg, France.
Saitou, R. R. & Nei, M. (1987). A neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.[Abstract]
Schulenberg-Schell, H., Neuss, B. & Sahm, H. (1989). Quantitative determination of various hopanoids in microorganisms. Anal Biochem 181, 120-124.[Medline]
Shine, J. & Dalgarno, L. (1974). The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71, 1342-1346.[Abstract]
Silvester, W. B., Harris, S. L. & Tjepkema, J. D. (1990). Oxygen regulation and hemoglobin. In The Biology of Frankia and Actinorhizal Plants, pp. 157-173. Edited by C. R. Schwintzer & J. D. Tjepkema. New York: Academic Press.
Simonin, P., Jürgens, U. J. & Rohmer, M. (1992). 350-ß-6-Amino-6-deoxyglucopyranosyl bacteriohopanetetrol, a novel triterpenoid of the hopane series from the cyanobacterium Synechocystis sp. PCC 6714. Tetrahedron Lett 33, 3629-3632.
Swofford, D. L. (1993). PAUP phylogenetic analysis using parsimony, version 3.1. Illinois Natural History Survey, Champaign.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
Vilchèze, C., Llopiz, P., Neunlist, S., Poralla, K. & Rohmer, M. (1994). Prokaryotic triterpenoids: new hopanoids from the nitrogen-fixing bacteria Azotobacter vinelandii, Beijerinckia indica and Beijerinckia mobilis. Microbiology 140, 2749-2753.
Winship, P. R. (1989). An improved method for directly sequencing PCR amplified material using dimethyl sulfoxide. Nucleic Acids Res 17, 1266.[Medline]
Zhang, Z. & Torrey, J. G. (1985). Studies of an effective strain of Frankia from Allocasuarina lehmanniana of the Casuarinaceae. Plant Soil 87, 1-16.
Received 14 April 2000;
revised 4 July 2000;
accepted 18 July 2000.