Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA1
Author for correspondence: Jerry D. Johnson. Tel: +1 307 766 3300. Fax: +1 307 766 5098. e-mail: jdjohn{at}uwyo.edu
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ABSTRACT |
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Keywords: Frankia, promoter sequences, in vitro transcription
Abbreviations: TSP, transcription start point
The GenBank accession number for the sequence reported in this paper is AY008259.
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INTRODUCTION |
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Comparatively little is known of Frankia molecular biology and genetics. Only a few Frankia genes that encode proteins have been cloned and completely sequenced. These include genes from the nif operon of several strains (Harriott et al., 1995 ; Nalin et al., 1995
; Normand et al., 1988
), ferredoxin subunits from strain EUIK1 (Yoo et al., 1999
) and glnA (Hosted et al., 1993
) plus glnII (Rochefort & Benson, 1990
) from strain CpI1. The rRNA operon of strain ORS020606 (Normand et al., 1992
) and partial 16S rRNA sequences have been characterized from many strains (Nazaret et al., 1991
; Normand et al., 1996
). The control of gene expression has not previously been addressed in Frankia although Cournoyer & Normand (1994)
examined promoter activity of Frankia DNA in Streptomyces and Escherichia coli. Also, numerous attempts to develop a transformation system for Frankia have failed perhaps, in part, due to a lack of basic information about gene expression.
The transcriptional regulation of genes involved in nitrogen fixation (nif) and other nitrogen-regulated processes (ntr) has been investigated in rhizobia and enteric bacteria. Both sets of genes are transcribed by an RNA polymerase holoenzyme containing a sigma factor, N, encoded by the ntrA gene (Gussin et al., 1986
). The recognition sequence for this holoenzyme is distinct from that of the standard E. coli promoter and is referred to here as the nif/ntr promoter. Maximal gene expression is also dependent upon sequences far upstream of the transcriptional start site (Alvarez-Morales et al., 1986
; Buck et al., 1986
; Reitzer & Magasanik, 1986
) that act as prokaryotic enhancer elements (Hunt & Magasanik, 1985
; Wedel et al., 1990
). Enhancer-binding proteins are different for the two sets of genes but similar in primary structure and function. The transcriptional activator for the ntr genes is encoded by the ntrC gene and the nif activator is the product of the nifA gene (Ditta, 1989
). The results presented herein suggest that Frankia may use mechanisms similar to those of the enteric bacteria and rhizobia in the expression of nitrogen-regulated genes. We have identified a cloned Frankia DNA that directs initiation of transcription in a Frankia cell-free extract from two promoter regions. The upstream promoter, TSP-1, contains sequences similar to the canonical E. coli promoter and a Streptomyces promoter element. The downstream promoter, TSP-2, is tentatively identified as an ntr promoter by the presence of sequences closely related to the consensus NtrC-binding site and the nif/ntr promoter. Using RT-PCR, a mRNA was identified in extracts from Frankia ArI3 that represents a large ORF just downstream of the TSP-1 and 2 promoter elements. This corroborates the biological significance of the in vitro studies.
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METHODS |
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E. coli cultures were grown at 37 °C in LB broth containing the appropriate antibiotic (Sambrook et al., 1989 ). Plasmid constructs were transformed into E. coli cells made competent for DNA uptake by CaCl2 treatment (Sambrook et al., 1989
).
Transcription extracts and assays.
Transcription extracts were prepared essentially according to the procedure of Westpheling et al. (1985) . All manipulations were done at 04 °C. Two grams (wet wt) of Frankia strain CpI1 cells were collected by centrifugation at 6000 g for 10 min and washed twice in 80 ml aliquots of buffer A (0·01 M Tris/HCl, pH 8·0, 1 mM EDTA, 0·3 mM DTT, 0·3 mg PMSF ml-1, 10%, v/v, glycerol) containing 0·1 M KCl, then resuspended in 40 ml of the same buffer. The cells were subjected to two passages through a French pressure cell at 3040 kPa. The lysate was centrifuged at 100000 g for 1 h to remove cell debris. The supernatant was collected and loaded onto a 2 ml column of heparin-agarose at a flow rate of 1015 ml h-1. The column was washed with 8·0 ml buffer A containing 0·1 M KCl and the bound material was eluted by raising the KCl concentration to 0·5 M. Fractions of 1·0 ml were collected and aliquots were assayed for protein concentration and RNA polymerase activity. Active fractions were divided into aliquots and stored at -80 °C.
Transcription reactions were performed in a volume of 50 µl containing 0·5 or 1·0 µg plasmid DNA, 20 µg extract protein and 1·0 µM [-32P]UTP (7·4 TBq mmol-1) in transcription buffer (40 mM Tris/HCl, pH 8·0, 10 mM MgCl2, 10 mM DTT, 50 µg BSA ml-1, 150 µM each of ATP, CTP and GTP). The final KCl concentration was adjusted to 200 mM for the Frankia extract, an amount that had been shown to produce maximal transcription activity (data not shown). The reactions were incubated at 37 °C for 10 or 15 min then placed on ice and quenched with an equal volume of a stop solution (100 mM sodium acetate, pH 5·2, 0·4% SDS, 1 mg yeast tRNA ml-1). Incorporation of 32P into TCA precipitable material was determined by Cerenkov counting.
Construction of luciferase promoter-probe clones.
Plasmid pFIT001 containing the Vibrio harveyi luxAB genes on a 4·0 kbp insert (Legocki et al., 1986 ) was the generous gift of Dr R. P. Legocki (Boyce Thompson Institute, Cornell University, USA). A 0·6 kbp SalI fragment (Baldwin et al., 1984
) was deleted to form
P, the promoterless luciferase cassette. Total DNA from Frankia strain ArI5 was partially digested with SalI and ligated into SalI-digested
P.
Primer extension analysis of RNA.
RNA templates for primer extension were produced according to the procedure for in vitro transcription except that unlabelled UTP was used at 150 µM and 1 U RNase Block II (Stratagene) or 20 U RNasin (Promega) RNase inhibitor was included. RNA templates were also produced from the same plasmid DNAs using 1 U E. coli RNA polymerase (US Biochemical) and reaction conditions as recommended by the supplier. Transcription reactions were incubated at 37 °C for 15 min then placed on ice and quenched with an equal volume of 3 M sodium acetate (pH 4·6) containing 1 mg yeast tRNA ml-1. Duplicate reactions were sometimes performed and combined at this stage to increase the amount of RNA available for primer extension. Reaction mixtures were extracted sequentially with phenol, phenol/chloroform/isoamyl alcohol (50:49:1) and chloroform prior to the addition of a two- to tenfold molar excess of 32P-labelled oligonucleotide primer (90180 GBq mmol-1). Primers used were either P, 5'-CGTACGGCTTTCAAT-3', or T3 (Stratagene) for the GLO and SKG7 transcripts, respectively. The template/primer mixture was precipitated with ethanol, resuspended in 30 µl 40 mM PIPES (pH 6·4), 1 mM EDTA, 0·4 M NaCl, 80% formamide, heated to 70 °C for 10 min and annealed for 1824 h at 30 °C. Nucleic acids were precipitated by the addition of 170 µl H2O and 400 µl absolute ethanol and incubation at 0 °C for 1 h. Pellets were collected by centrifugation and gently resuspended in 20 µl 50 mM Tris/HCl (pH 8·3), 75 mM KCl, 10 mM MgCl2, 2 mM each dATP, dGTP, dCTP and dTTP, 10 mM DTT, 20 U RNasin. Primers were extended with 200 U MMLV reverse transcriptase (Bethesda Research Labs) at 37 °C for 2 h. Un-annealed RNA was removed by addition of 1 µl 0·5 M EDTA and 1 µl 5 mg DNase-free RNase A ml-1 followed by incubation at 37 °C for 30 min. The mixture was extracted with an equal volume of phenol/chloroform/isoamyl alcohol and nucleic acids precipitated with 2 vols ethanol. Pellets were resuspended in 3 µl 80% formamide, 10 mM EDTA (pH 8·0), 1 mg xylene cyanol ml-1, 1 mg bromophenol blue ml-1 and heated at 95 °C for 5 min before electrophoresis on a 7% polyacrylamide gel containing 8 M urea (Sambrook et al., 1989
).
General molecular biology techniques.
Restriction enzyme digestions were performed according to suppliers directions. Single-stranded oligodeoxynucleotides were labelled using T4 polynucleotide kinase and [-32P]ATP (Sambrook et al., 1989
). Unincorporated ATP was removed using DuPont Nensorb columns according to the manufacturers directions. Plasmids to be used in restriction analysis, subcloning and sequencing were prepared by the alkaline lysis method and further purified by polyethylene glycol precipitation (Sambrook et al., 1989
). In the preparation of templates for transcription, RNase digestion was avoided and contaminating RNA was removed by either Sephacryl S-1000 chromatography (Raymond et al., 1988
) or equilibrium centrifugation in CsCl containing ethidium bromide (Sambrook et al., 1989
). DNA sequencing was performed with Taq DNA polymerase according to suppliers instructions (Promega). For sequencing stretches with high G+C content, the reactions were performed at 75 °C and gel compressions minimized by lyophilizing the reaction mixtures before addition of 80% formamide gel loading buffer. Sequencing reactions were heated in boiling water and analysed on 4% or 7% polyacrylamide gels containing 8 M urea (Sambrook et al., 1989
).
Southern blotting.
DNA from agarose gels was transferred to Zeta-Probe nylon hybridization membrane (Bio-Rad Laboratories) using either capillary transfer or pressure blotting. DNA was fixed to the membrane by baking at 80 °C for 20 min in vacuo. Membranes were prehybridized in 510 ml 50% formamide, 5xSSC, 5xDenhardts solution, 5 mM sodium pyrophosphate, 250 µg denatured and sheared herring sperm DNA ml-1, 0·5% SDS for 112 h at 42 °C. Hybridization was performed in a solution of 50% formamide, 5xSSC, 1xDenhardts solution, 5 mM sodium pyrophosphate, 100 µg denatured and sheared herring sperm DNA ml-1, 0·5% SDS and 32P-labelled RNA probe for 1821 h at 42 °C. Blots were washed twice for 10 min each at 25 °C in 1xSSC, 0·1% SDS followed by a 20 min wash at 65 °C in 0·1xSSC, 0·1% SDS.
Construction of deletion subclones.
The 1·9 kb insert of GLO7 was subcloned into the SalI site of Bluescript SK+ (Stratagene) and a clone, SKG7, was selected in which transcription from the Frankia promoter was oriented in the opposite direction to that of the vector ß-galactosidase gene. SKG7 DNA was digested with XhoI/KpnI and nested deletions were created using exonuclease III with S1 nuclease.
RNA extraction and RT-PCR.
These procedures were done as described (T. John, J. Rice & J. Johnson, unpublished) except strain ArI5 cells grown as described in bacterial cultures were used. Primers for PCR were 5'-CACCGTCAATCGCTTCTTC-3' and 5'-GAAAACTGACCCCCTGCTGG-3', which anneal to positions 10001018 and 13201301 from the GTG start codon (see Fig. 5a).
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RESULTS |
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The inserts in GLO3 and GLO7 exhibited significantly higher lux transcription in the Frankia extract than pFIT001 (Fig. 2). All GLO clones produced more luxAB transcript than the promoter-deleted clone,
P. In addition, GLO3, GLO4 and GLO7 produced RNAs that hybridized to the insert bands, suggesting that a transcription start site was present within the insert.
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Comparison of GLO7 with other bacterial promoter elements
The sequence of the luxAB proximal region of the GLO7 insert was determined from GLO7 and SKG73
6. An overlapping clone, produced by BclI digestion of Frankia ArI5 DNA, was isolated using the SKG7
6 fragment as a probe. The combined sequence is deposited in GenBank under accession no. AY008259. The locations of TSP-1 and 2 as well as the end points of SKG7 clones
5 and
6 are identified in Fig. 5(a)
. The GLO7 sequence was searched for similarity to promoter elements identified in other bacteria. A sequence similar to the NtrC consensus binding site identified in E. coli and Salmonella (Buck et al., 1986
) and to a similar element upstream of the Frankia CpI1 glnII gene (Rochefort & Benson, 1990
) was found beginning 101 bp upstream of TSP-1 (Fig. 5b
).
In addition, the immediate upstream regions of both TSP-1 and TSP-2 contained sequences with identity to E. coli canonical promoter elements (Fig. 5a). These similarities are apparently sufficient for recognition in E. coli as evidenced by primer extension of RNA transcribed in vitro by purified E. coli RNA polymerase from the GLO7 DNA as template. Two primer extension products corresponding exactly to TSP-1 and TSP-2 were present, but at much lower levels than those produced from RNA made by Frankia extracts (data not shown). A region with 5 of 7 identity to a Streptomyces -35 element (Taguchi et al., 1989
) is also present in an appropriate region upstream of TSP-1 (Fig. 5a
).
The TSP-1 region also has two sequence blocks, centred at -8 and -29 bp upstream, that have substantial identities at or near the 5' end of the Frankia CpI1 glnII gene (Fig. 5b). An element similar to the nif/ntr promoter consensus characterized in Rhizobium and Klebsiella (Ausubel, 1984
; Barrios et al., 1999
) is present between 2 and 17 bp upstream from TSP-2 (Fig. 5c
).
In vivo activity of the TSP-1 and 2 sequences
The GLO7 fragment terminated 136 bp downstream from TSP-1. A clone overlapping GLO7 by 853 bp was isolated from a BclI digest of Frankia ArI5 DNA. Sequence analysis of this clone revealed the presence of an ORF encoding 498 aa that initiated with a GTG codon 64 bp downstream from TSP-1 (Fig. 5a). A ShineDalgarno sequence, GGAGG, begins 12 bp upstream of the putative start codon, 3 bp beyond the usual range of spacing (Barrick et al., 1994
). The ORF has a high G+C content in the third codon position (76·4%) and is terminated by tandem UGA codons (data not shown). All of these features suggest that this ORF encodes a protein. However, a BLASTP comparison of the deduced amino acid sequence of the putative protein with the GenBank database does not identify significant identities with any known protein. The best matches, E=2x10-5, are with other hypothetical proteins rich in proline and arginine.
In vivo transcription of the ORF was verified by RT-PCR using total RNA extracted from Frankia ArI5 cells. The RNA preparation was used as template with random decanucleotide primers to produce a cDNA copy. This DNA was amplified by PCR using primers nested within the ORF. A fragment of the expected size, 321 bp, was produced in the reaction (data not shown). No such product was evident in a control reaction in which the reverse transcriptase step was omitted. The product is therefore dependent on RNA present in the cell extract. The DNA sequence of the 321 bp RT-PCR fragment corresponds perfectly to the region of the ORF bracketed by the primers identified in Methods. This result supports the conclusion that the promoter region(s) identified by in vitro analyses also function in vivo.
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DISCUSSION |
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Because the promoters described here are located at the extreme 3' end of the GLO7 insert, the identity of any corresponding gene was not evident. An overlapping fragment of Frankia ArI5 DNA was therefore cloned to determine whether the putative promoter region was associated with any functional gene(s). The proximal ATG downstream from GLO7p2 is in-frame with a stop codon. DNA sequence analysis of the overlapping fragment identified a 498 aa ORF with a potential GTG start codon 64 bp downstream from TSP-1. The ORF has a ShineDalgarno sequence (Gold, 1988 ) and bias in third codon position G+C content of 76·4%. Further, the ORF is terminated by tandem nonsense codons. These are all indicators that the ORF is a functional gene.
To determine whether this region of the Frankia DNA is transcribed in vivo, an RNA preparation from actively growing ArI5 cells was used as the template for RT-PCR. The PCR primers were nested within the ORF and produced a 321 bp fragment, as expected from the corresponding DNA sequence (data not shown). A control reaction in which the reverse transcription was not done showed no product, indicating that the PCR depends on an RNA template. The DNA sequence of the amplified fragment was identical to the ORF, confirming that the PCR product represents transcription from the ORF. The presence of the in vivo transcript provides strong support for the conclusion that the transcription analyses done in vitro reflect the in vivo activity of the Frankia RNA polymerase.
Most Streptomyces promoters are not recognized in E. coli, perhaps due to differences in G+C content (Bibb & Cohen, 1982 ; Buttner & Brown, 1987
). In this study, Frankia promoters that are operative in E. coli were selected by a promoter screening in that organism. The promoters GLO7p1 and GLO7p2 contain homologies to E. coli canonical promoter elements and the transcription start sites were shown to be identical using both Frankia and E. coli RNA polymerase in vitro. Whether or not recognition in E. coli is a general feature of Frankia promoters awaits further investigation.
The presence of sequences resembling NtrC binding sites (Fig. 5a) and
N-RNA polymerase recognition sequences (Fig. 5b
, c
) in Frankia suggests that the actinomycete may regulate ntr genes in a manner similar to other diazotrophs and that it may contain proteins that are functionally and structurally similar to NtrA and NtrC. However, this conclusion must be tempered by the lack of similarity of the downstream ORF to any known ntr gene.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the contributions of Ms Shawna Clark, Ms Holly Magna and Ms Katja Manninen to the success of this work.
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REFERENCES |
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Ausubel, F.(1984). Regulation of nitrogen fixation genes. Cell 37, 5-6.[Medline]
Baker, D. & Selig, E.(1984). Frankia: new light on an actinomycete symbiont. In Biological, Biochemical and Biomedical Aspects of Actinomycetes , pp. 563-574. Edited by L. Ortiz-Ortiz, L. Bojalil & V. Yakoleff. New York:Academic Press.
Baldwin, T., Berends, T., Bunch, T., Holzman, T., Rausch, S., Shamansky, L., Treat, M. & Ziegler, M.(1984). Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry 23, 3663-3667.[Medline]
Barrick, D., Villanueba, K., Childs, J., Kalil, R., Schneider, T. D., Lawrence, C. E., Gold, L. & Stormo, G. D.(1994). Quantitative analysis of ribosome binding sites in E. coli. Nucleic Acids Res 22, 1287-1295.[Abstract]
Barrios, H., Valderrama, B. & Morett, E.(1999). Compilation and analysis of 54-dependent promoter sequences. Nucleic Acids Res 27, 4305-4313.
Bibb, M. & Cohen, S.(1982). Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen Genet 187, 265-277.[Medline]
Buck, M., Miller, S., Drummond, M. & Dixon, R.(1986). Upstream activator sequences are present in the promoters of nitrogen fixation genes. Nature 320, 374-378.
Buck, M., Gallegos, M.-T., Studholme, D. J., Guo, Y. & Gralla, J. D.(2000). The bacterial enhancer-dependent 54 (
N) transcription factor. J Bacteriol 182, 4129-4136.
Buttner, M. & Brown, N.(1987). Two promoters from the Streptomyces plasmid pIJ101 and their expression in Escherichia coli. Gene 51, 179-186.[Medline]
Cournoyer, B. & Normand, P.(1994). Gene expression in Frankia: characterization of promoters. Microbios 78, 229-236.[Medline]
Ditta, G.(1989). Regulation of nif genes in Rhizobium. In PlantMicrobe Interactions , pp. 11-24. Edited by T. Kosuge & E. Nester. New York:McGraw-Hill.
Gold, L.(1988). Posttranscriptional regulatory mechanisms in Escherichia coli. Ann Rev Biochem 57, 199-233.[Medline]
Gussin, G. N., Ronson, C. W. & Ausubel, F. M.(1986). Regulation of nitrogen fixation genes. Ann Rev Genet 20, 567-591.[Medline]
Harriott, O. T., Hosted, T. J. & Benson, D. R.(1995). Sequences of nifX, nifW, nifZ, nifB and two ORF in the Frankia nitrogen fixation gene cluster. Gene 161, 63-67.[Medline]
Hosted, T. J., Rochefort, D. A. & Benson, D. R.(1993). Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1. J Bacteriol 175, 3679-3684.[Abstract]
Hunt, T. P. & Magasanik, B.(1985). Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL. Proc Natl Acad Sci USA 82, 8453-8457.[Abstract]
Legocki, R., Legocki, T., Baldwin, T. & Szalay, A.(1986). Bioluminescence in soybean root nodules: demonstration of a general approach to assay gene expression in vivo using bacterial luciferase. Proc Natl Acad Sci USA 83, 9080-9084.[Abstract]
Nalin, R., Domenach, A. M. & Normand, P.(1995). Molecular structure of the Frankia spp. nifDK intergenic spacer and design of a Frankia genus compatible primer. Mol Ecol 4, 483-491.[Medline]
Nazaret, S., Cournoyer, B., Normand, P. & Simonet, P.(1991). Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences. J Bacteriol 173, 4072-4078.[Medline]
Normand, P., Simonet, P. & Bardin, R.(1988). Conservation of nif sequences in Frankia. Mol Gen Genet 213, 238-246.[Medline]
Normand, P., Cournoyer, B., Simonet, P. & Nazaret, S.(1992). Analysis of a ribosomal RNA operon in the actinomycete Frankia. Gene 111, 119-124.[Medline]
Normand, P., Orso, S., Cournoyer, B., Jeannin, P., Chapelon, C., Dawson, J., Evtushenko, L. & Misra, A. K.(1996). Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae. Int J Syst Bacteriol 46, 1-9.[Abstract]
Obata, S., Taguchi, S., Kumagi, I. & Miura, K.(1989). Molecular cloning and nucleotide sequence determination of the gene encoding Streptomyces subtilisin inhibitor (ssi). J Biochem 105, 367-371.[Abstract]
Raymond, G., Bryant, P., Nelson, A. & Johnson, J.(1988). Large-scale isolation of covalently closed circular DNA using gel filtration chromatography. Anal Biochem 173, 125-133.[Medline]
Reitzer, L. & Magasanik, B.(1986). Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell 45, 785-792.[Medline]
Rochefort, D. A. & Benson, D. R.(1990). Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1. J Bacteriol 172, 5335-5342.[Medline]
Sambrook, J., Fritsch, E. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Press.
Stowers, M. D.(1987). Collection, isolation, cultivation, and maintenance of Frankia. In Symbiotic Nitrogen Fixation Techniques , pp. 29-53. Edited by G. H. Elkan. New York:Marcel Dekker.
Taguchi, S., Nishiyama, K., Kumagi, I. & Miura, K.(1989). Analysis of transcriptional control regions in the Streptomyces subtilisin-inhibitor-encoding gene. Gene 84, 279-286.[Medline]
Tjepkema, J., Ormerod, W. & Torrey, J.(1980). Vesicle formation and acetylene reduction activity in Frankia sp. CpI1 cultured in defined nutrient media. Nature 287, 633-635.
Tjepkema, J. D., Schwintzer, C. R. & Benson, D. R.(1986). Physiology of actinorhizal nodules. Ann Rev Plant Physiol 37, 209-232.
Wedel, A., Weiss, D., Popham, D., Droge, P. & Kustu, S.(1990). A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science 248, 486-490.[Medline]
Westpheling, J., Ranes, M. & Losick, R.(1985). RNA polymerase heterogeneity in Streptomyces coelicolor. Nature 313, 22-27.[Medline]
Yoo, W. Y., Sung, S. B. & An, C. S.(1999). Nucleotide sequences of the 2-oxoacid ferredoxin oxidoreductase and ferredoxin genes from Frankia strain EuIK1, a symbiont of Eleagnus umbellata root nodules. Can J Bot 72, 1279-1286.
Received 19 July 2000;
revised 3 October 2000;
accepted 16 October 2000.
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