TAXONOMIC DIVERSITY OF PSEUDOMONADS REVEALED BY COMPUTER-INTERPRETATION OF RIBOTYPING DATA

Citation
R. Brosch et al., TAXONOMIC DIVERSITY OF PSEUDOMONADS REVEALED BY COMPUTER-INTERPRETATION OF RIBOTYPING DATA, Systematic and applied microbiology, 19(4), 1996, pp. 541-555
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology,"Biothechnology & Applied Migrobiology
ISSN journal
0723-2020
Volume
19
Issue
4
Year of publication
1996
Pages
541 - 555
Database
ISI
SICI code
0723-2020(1996)19:4<541:TDOPRB>2.0.ZU;2-1
Abstract
In the framework of a European project entitled ''High Resolution Auto mated Microbial Identification'', the ability of rRNA gene restriction pattern determination (ribotyping) to uncover the taxonomic diversity of a collection of bacteria was tested on 226 strains of Pseudomonas sensu late. Improved ribotyping methodology included automated DNA ext raction and purification, low voltage electrophoresis, vacuum transfer of restricted DNA fragments to nylon membranes, hybridization with a chemically labeled probe in a hybridization incubator, computer captur e of restriction patterns with automatic migration measurements, fragm ent size interpolation, and comparison of patterns using programs of t he Taxotron package (Institut Pasteur, Paris, France). Two endonucleas es were used (SmaI and HincII). The strains, belonging to more than 40 nomenspecies, displayed 169 unique rRNA gene restriction patterns wit h SmaI, and 159 unique rRNA gene restriction patterns with HincII. A c ombined analysis of both restriction data yielded 79 ribogroups or iso lated ribotypes. Most (92.7%) ribogroups were homogeneous with respect s to nomenspecies. Some nomenspecies were split in several ribogroups (e.g. P. putida, P. fluorescens, P. marginalis, P. pseudoalcaligenes). Nomenspecies represented by single strains gave isolated ribotypes. B iovars of P. fluorescens and P. putida formed distinct ribogroups. How ever, P. marginalis pathovars were not separated by ribotyping. When t he ribotyping data (ribogroups) were compared to published phenotypic data (phenons) obtained with Biotype-100 strips (BioMerieux, La Balme- les-Grottes, France) and Biolog GN Microplates (Biolog, Hayward, CA), an exclusive phenon-ribogroup correspondence was observed for 13 Bioty pe-100 phenons/ribogroups and for 14 Biolog phenons/ribogroups. Other Biotype-100 or Biolog phenons showed partial correspondence with ribog roups or were split into several ribogroups. Most unclustered strains in phenotypic analyses were also unclustered in the ribotyping study. DNA-DNA hybridization was used to verify some species delineation. Rib ogroup R38 which contained most of P. fluorescens biovar I strains was found to constitute a DNA hybridization group distinct from P. fluore scens strains in other ribogroups or biovars. The type strain of P. fl uorescens, which was unclustered by ribotyping, was found isolated by DNA relatedness. Ribogroup R1 which contained most of P. putida biovar A strains was found to constitute a DNA relatedness group distinct fr om biovar B strains. A total of 30 isolates from polluted environment were included in the study and 9 of them were identified to six known taxons. Thirteen other isolates constituted five ribogroups not repres ented by a reference strain and seven isolates gave isolated ribotypes . This work showed the identification potential of ribotyping and the need to extend the ribotype database. It is now clear that the ribotyp e carries taxonomic information in addition to typing information.