Skip to main content
SpringerLink
Log in

Rapid identification of Rhizobium strains by targeted PCR fingerprinting

  • Chapter
Molecular Microbial Ecology of the Soil

Part of the book series: Developments in Plant and Soil Sciences ((DPSS,volume 83))

Abstract

Numerous polymerase chain reaction (PCR) based procedures are routinely used to produce genomic fingerprints of prokaryotes. Many of them have drawbacks however such as sensitivity to experimental variation, lack of reproducibility, poor resolution and the inability to distinguish between closely related strains. To overcome these difficulties, we developed an alternative procedure, Targeted PCR Fingerprinting (TPF) which is based upon the amplification of few but carefully selected markers, followed by high resolution RFLP analysis of the amplified DNA fragments. In contrast to most fingerprinting protocols that use low resolution agarose gels, TPF patterns are produced on denaturing polyacrylamide gels which allow the precise recording of the genomic fingerprints. TPF analysis, which can simultaneously process 96 samples in less than 12 h and remains unaffected by slight experimental variations, is particularly adapted for the rapid identification of target strains amongst many field isolates. Using PCR primers specific for the nifH and recA genes, this procedure was also sufficiently sensitive to discriminate between Rhizobium species NGR234 and R. fredii USDA257, two closely related bacteria in which the symbiotic loci are 98% homologous. Interestingly, comparison of several of their symbiotic genes as well as the partial DNA sequences of their 16S rDNA and recA genes suggest that chromosomes and symbiotic plasmids did not co-evolve, but that symbiotic functions were acquired by lateral gene transfer long after NGR234 and USDA257 diverged from their common ancestors. In this respect, TPF fingerprints produced with distinct chromosomal and plasmid born markers, such as the recA and the nifH genes in NGR234 and USDA257, are probably more likely to detect lateral transfer of genes in bacterial field-populations than procedures relying on the amplification of numerous fragments of unknown genomic position and biological function.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  • Amabile-Cuevas C F and Chicure M E 1992 Bacterial plasmids and gene flux. Cell 70, 189–199.

    Article  CAS  Google Scholar 

  • Balatti P A, Kovacs L G, Krishnan H B and Pucppkc S G 1995 Rhizobium sp. NGR234 contains a functional copy of the soybean cultivar specificity locus, noiXWBTUV. Mol. Plant-Microbe Interact. 8, 693–699.

    Google Scholar 

  • Berg D E, Akopyants N S and Kersulyte D 1994 Fingerprinting microbial genomes using the RAPD or AP-PCR method. Methods Mol. Cellular Biol. 5, 13–24.

    Google Scholar 

  • Beringer J E 1974 R-factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84, 188–198.

    CAS  Google Scholar 

  • Broughton W J, Wong C-H, Lewin A, Samrey U, Myint H, Meyer z A H, Dowling D N and Simon R 1986 Identification of Rhizobium plasmid sequences involve in recognition of Psophocarpus, Ugna, and other legumes. J. Cell Biol. 102, 1173–1182.

    Article  CAS  Google Scholar 

  • Chen W P and Kuo T T 1993 A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucl. Acids Res. 21, 2260.

    Article  CAS  Google Scholar 

  • Chua K J, Pankhurst C E, MacDonald P E, Hoperoft D H, Jarvis B D W and Scott D B 1985 Isolation and characterization of Tn5induced symbiotic mutants of Rhizobium loti. J. Bacteriol. 162, 335–343.

    CAS  Google Scholar 

  • Dams E L, Hendricks Y, van de Peer Y, Neefs J M, Smits G, Vandenbempt I and de Wachter R 1988 Compilation of small ribosomal subunit RNA sequences. Nucl. Acids Res. 16, r87 — r173.

    Article  CAS  Google Scholar 

  • Dobert R C, Breil B T and Triplett E W 1994 DNA sequence of the common nodulation genes of Bradyrhizobium elkanii and their phylogenetic relationship to those of other nodulating bacteria. Mol. Plant-Microbe Interact. 5, 564–572.

    Google Scholar 

  • Dooley J J and Harrison S P 1993 Phylogenetic grouping and identification of Rhizobium isolates on the basis of random amplified polymorphic DNA profiles. Can. J. Microbiol. 39, 665–673.

    Article  CAS  Google Scholar 

  • Eardly B D, Young J P W and Selander R K 1992 Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes. Appl. Environ. Microbiol. 58, 1809–1815.

    Google Scholar 

  • Ellsworth B D, Rittenhouse K D and Honeycutt R L 1993 Artifactual variation in randomly amplified polymorphic DNA banding pattern. Biotechniques 14, 214–217.

    CAS  Google Scholar 

  • Fox G E, Wisotzkey J D and Jurtschuk P J 1992 How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Bacteriol. 42, 166–170.

    Google Scholar 

  • Freiberg C, Fellay R, Bairoch A, Broughton W J, Rosenthal A and Perret X 1997 Molecular basis of symbiosis between Rhizobium and legumes. Nature 387, 394–401.

    Article  CAS  Google Scholar 

  • Hanahan D 1983 Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580.

    Article  CAS  Google Scholar 

  • Heron D S and Pueppke S G 1984 Mode of infection, nodulation specificity, and indigenous plasmids of 11 fast-growing Rhizobium japonicum strains. J. Bacteriol. 160, 1061–1066.

    CAS  Google Scholar 

  • Jarvis B D W, Downer H L and Young J P W 1992 Phylogeny of fast-growing soybean-nodulating rhizobia supports synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii. Int. J. Syst. Bacteriol. 42, 93–96.

    Google Scholar 

  • Kay H E, Coutinho H L C, Fattori M, Manfio G P, Goodacre R, Nuti M P, Basiglia M and Beringer J E 1994 The identification of Bradyrhizobium japonicum strains isolated from Italian soils. Microbiology 140, 2333–2339.

    Article  CAS  Google Scholar 

  • Krishnan H B, Lewin A, Fellay R, Broughton W J and Pueppke S G 1992 Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp. Mol. Microbiol. 6, 33213330.

    Google Scholar 

  • Kündig C, Beck C, Hennecke H and Göttfert M 1995 A single rRNA gene region in Bradyrhizobium japonicum. J. Bact. 177, 51515154.

    Google Scholar 

  • Kuykendall L D and Saxena B 1992 Genetic diversity in Bradyrhizobium japonicum Jordan 1982 and a proposal for Bradyrhizobium elkanii sp. nov. Can. J. Microbiol. 38, 501–505.

    Article  CAS  Google Scholar 

  • Laguerre G, Fernandez M P, Edel V, Normand P and Amarger N 1993 Genomic heterogeneity among French Rhizobium strains isolated from Phaseolus vulgaris. L. Int. J. Syst. Bacteriol. 43, 761–767.

    Google Scholar 

  • Laguerre G, Allard M R, Revoy F and Amarger N 1994 Rapid identification of rhizobia by restriction length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 60, 56–63.

    Google Scholar 

  • Lloyd A T and Sharp P M 1993 Evolution of the recA gene and the molecular phylogeny of bacteria. J. Mol. Evol. 37, 399–407. Martinez E, Romero D and Palacios R 1990 The Rhizobium genome. Crit. Rev. Plant Sci. 9, 59–93.

    Google Scholar 

  • Massol-Deya A A, Odelson D A, Hickey R F and Tiedje J M 1995 Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA). In Mol. Microbial Ecology Manual 3.3. 2. pp 1–8.

    Google Scholar 

  • Mercado-Blanco J and Toro N 1996 Plasmids in rhizobia: the role of nonsymbiotic plasmids. Mol. Plant-Microbe Interact. 9, 535545.

    Google Scholar 

  • Nouer 1-I F 1984 Structure of ribosomal RNA. An nu. Rev. Biochem. 53, 119–162.

    Article  Google Scholar 

  • Ochman H and Wilson A C 1987 Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. J. Mol. Evol. 26, 74–86.

    Article  CAS  Google Scholar 

  • Oyaizu H S, Matsumoto S, Minamisawa K and Gamov T 1993 Distribution of rhizobia in leguminous plants surveyed by phylogenetic identification. J. Gen. Appl. Microbiol. 39, 339–354.

    Google Scholar 

  • Perret X, Broughton W J and Brenner S 1991 Canonical ordered cosmid library of the symbiotic plasmid of Rhizobium species NGR234. Proc. Natl. Acad. Sci. USA 88, 1923–1927.

    Google Scholar 

  • Perret X 1992 Cartographic physique et génétique du génome de Rhizobium species NGR234. Ph.D. Thesis # 2489, University of Geneva, Geneva, Switzerland.

    Google Scholar 

  • Perret X, Fellay R, Bjourson A J, Cooper J E, Brenner S and Broughton W J 1994 Subtraction hybridization and shot-gun sequencing: a new approach to identify symbiotic loci. Nucl. Acids Res. 22, 1335–1341.

    Google Scholar 

  • Perret X. 1997 Charactrization of prokaryotic genomes by Targeted PCR Fingerprinting: various applications. In Boehringer Mannheim PCR-Bibliographie. pp 58–60.

    Google Scholar 

  • Pillai S D, Josephson K L, Bailey R L and Pepper I 1992 Specific detection of rhizobia in root nodules and soil using the polymerase chain reaction. Soil. Biol. Biochem. 24, 885–891.

    Google Scholar 

  • Relié B, Perret X, Estrada-Garcia M T, Kopcinska J, Golinowski W, Krishnan H B, Pueppke S G and Broughton W J 1994 Nod-factors of Rhizobium are a key to the legume door. Mol. Microbiol. 13, 171–178.

    Google Scholar 

  • Rosenberg C, Boistard P, Dénarié J and Casse-Delbart F 1981 Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol. Gen. Genet. 184, 326333.

    Google Scholar 

  • Sambrook J, Fritsch E F and Maniatis T 1989 Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor University Press, Cold Spring Harbor.

    Google Scholar 

  • Sanger F, Nicklen S and Coulson R A 1977 DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 1757–1761.

    Google Scholar 

  • Segovia L, Young J P W and Martinez-Romero E 1993 Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etti sp. nov. Int. J. Syst. Bacteriol. 43, 374–377.

    Google Scholar 

  • Schmidt T M 1994 Fingerprinting bacterial genomes using ribosomal RNA genes and operons. Methods Mol. Cellular Biol. 5, 3–12.

    CAS  Google Scholar 

  • Sobral B W, Honeycutt R J, Atherly A G, McClelland M 1991 Electrophoretic separation of the three Rhizobium meliloti replicons. J. Bact. 173, 5173–5180.

    CAS  Google Scholar 

  • Stanley J, Dowling D N and Broughton W J 1988 Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera. Mol. Gen. Genet 215, 32–37.

    Google Scholar 

  • Ueda T. Suga Y, Yahiro N and Matsugushi T 1995 Phylogeny of Sym plasmids of rhizobia by PCR-based sequencing of a nodC segment. J. Bacteriol. 177, 468–472.

    Google Scholar 

  • Vargas C, Martinez L J, Megias M and Quinto C 1990 Identification and cloning of nodulation genes and host-specificity determinants of the broad host-range Rhizobium leguminosarum biovar phaseoli strain CIAT899. Mol. Microbiol. 4, 1899–1910.

    Google Scholar 

  • Versalovic J, Koeuth T and Lupski J R 1991 Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl. Acids Res. 19, 6823–6831.

    Google Scholar 

  • Versalovic J, Schneider M, de Bruijn F J and Lupski J R 1994 Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cellular Biol. 5, 25–40.

    Google Scholar 

  • Williams J G K, Kubelic A R, Livak K J, Rafalski J A and Tingey S V 1990 DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res. 18, 6531–6535.

    Google Scholar 

  • Willems A and Collins D 1993 Phylogenetic analysis of rhizobia and agrobacteria based on 16S rRNA gene sequence. Int. J. Syst. Bacteriol. 43, 305–313.

    Article  CAS  Google Scholar 

  • Yamamoto S and Harayama S 1995 PCR amplification and direct sequencing of gyrB genes with universal primers and their appli 34b cation to the detection and taxonomic analysis of Pseudomonas putida strains. Appl. Environ. Microbiol. 61, 1104–1109.

    CAS  Google Scholar 

  • Yanagi M and Yamasato K 1993 Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol. Lett. 107, 115–120.

    CAS  Google Scholar 

  • Young J P W, Downer H L and Eardly B D 1991 Phylogeny of the phototropic Rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J. Bacteriol. 173, 2271–2277.

    CAS  Google Scholar 

  • Young J P W 1993 Molecular phylogeny of rhizobia and their relatives. In New Horizons in Nitrogen Fixation. Eds. R Palacios, J Mora and W E Newton. pp 587–592. Kluwer Academic Publishers, London.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire des Plantes Supérieures, University of Geneva, 1 chemin de l’Impératrice, 1292, Chambésy, Geneva, Switzerland

    X. Perret & W. J. Broughton

Authors
  1. X. Perret
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. J. Broughton
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Joint FAO/IAEA Programme, Soil Science Unit, Agency’s Laboratories, A-2444, Seibersdorf, Austria

    G. Hardarson

  2. LBPMS, University of Geneva, 1292, Chambesy, Geneva, Switzerland

    W. J. Broughton

Rights and permissions

Reprints and permissions

Copyright information

© 1998 Springer Science+Business Media Dordrecht

About this chapter

Cite this chapter

Perret, X., Broughton, W.J. (1998). Rapid identification of Rhizobium strains by targeted PCR fingerprinting. In: Hardarson, G., Broughton, W.J. (eds) Molecular Microbial Ecology of the Soil. Developments in Plant and Soil Sciences, vol 83. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-2321-3_2

Download citation

  • DOI: https://doi.org/10.1007/978-94-017-2321-3_2

  • Publisher Name: Springer, Dordrecht

  • Print ISBN: 978-90-481-5099-1

  • Online ISBN: 978-94-017-2321-3

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation