PCR primers based on different portions of insertion elements can assist genetic relatedness studies, strain fingerprinting and species identification in rhizobia

• Published in: FEMS microbiology ecology Vol. 54; no. 3; pp. 445 - 453
• Main Authors: Muresu, Rosella, Sulas, Leonardo, Polone, Elisa, Squartini, Andrea
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 01.11.2005; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Bacteria; Bacteriology; Base Sequence; Biological and medical sciences; Classification - methods; Cluster Analysis; Clustering; Computational Biology; DNA fingerprinting; DNA Fingerprinting - methods; DNA Primers - genetics; DNA Transposable Elements - genetics; Ecology; Fingerprinting; Fundamental and applied biological sciences. Psychology; Glutamate-ammonia ligase; Glutamine; Hedysarum coronarium; Heredity; Image Processing, Computer-Assisted; Insertion; IS element; Legumes; Microbiology; Miscellaneous; Molecular Sequence Data; Nitrogen fixation; Phylogeny; Polymerase Chain Reaction - methods; Primers; Rhizobial genetic diversity; Rhizobium; Rhizobium - genetics; Rhizobium sullae; rRNA 16S; Sequence Analysis, DNA; Species; Species Specificity; Strain fingerprinting; Taxonomy; Transposase; Transposase gene; Rhizobium; Strain fingerprinting; Rhizobial genetic diversity; IS element; Taxonomy; Identification; Transposable element; Symbiont; Strain; Polymerase chain reaction; Leguminosae; Ribosomal RNA; Dicotyledones; Fingerprint method; Angiospermae; Nitrogen fixation; 16S-RNA; Bacteria; Genetics; Spermatophyta; Fodder crop; Rhizobiaceae; Detection; Glutamine
• ISSN: 0168-6496; 1574-6941
• DOI: 10.1016/j.femsec.2005.05.008

Using the sequence of an insertion element originally found in Rhizobium sullae, the nitrogen-fixing bacterial symbiont of the legume Hedysarum coronarium, we devised three primer pairs ( inbound, outbound and internal primers) for the following applications: (a) tracing genetic relatedness within rhizobia using a method independent of ribosomal inheritance, based on the presence and conservation of IS elements; (b) achieve sensitive and reproducible bacterial fingerprinting; (c) enable a fast and unambiguous detection of rhizobia at the species level. In terms of taxonomy, while in line with part of the 16S rRNA gene- and glutamine synthetase I-based clustering, the tools appeared nonetheless more coherent with the actual geographical ranges of origin of rhizobial species, strengthening the European–Mediterranean connections and discerning them from the asian and american taxa. The fingerprinting performance of the outward-pointing primers, designed upon the inverted repeats, was shown to be at least as sensitive as BOX PCR, and to be functional on a universal basis with all 13 bacterial species tested. The primers designed on the internal part of the transposase gene instead proved highly species-specific for R. sullae, enabling selective distinction from its most related species, and testing positive on every R. sullae strain examined, fulfilling the need of PCR-mediated species identification. A general use of other IS elements for a combined approach to rhizobial taxonomy and ecology is proposed.