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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image High-resolution automated microbial identification (HRAMI) and applications to biotechnologically-relevant ecosystems

Background and objectives (1)

This project aimed to develop technology to solve the problems of microbial identification, classification, detection and quantification. We aimed to develop new automated procedures to rapidly, reliably and inexpensively identify micro-organisms and for the comparison and validation of problematic bacterial taxa. It was hoped that the new methods would enable the expansion of microbial taxonomy databases to include unknown microbes.

(1) This project was a direct follow-on of EC project “High resolution of automated microbial identification: improvement of nucleic acid probe techniques” (BIOT-CT91-0294).



Approach and methodology

A model polyphasic study of the pseudomonads was used to analyse several hundred strains belonging to over 60 species of the genus Pseudomonas (sensu stricto) and species formerly classified as Pseudomonas, which are now known to belong to genetically distinct phyla. Sequence data of the 16S and portions of the 23S rRNA genes, ribotypes, small molecular weight stable RNA (5S and tRNA) profiles, polar lipid and fatty acid characterisation, total protein profiles and DNA fingerprinting were correlated. Nested sets of gene probes were developed for genus and species identification.


Main findings and outcome

A database of microbial taxonomy was established and used to assess the impact of anthropogenic influences, including genetically engineered micro-organisms, on the environment and microbial diversity. The database now includes over 430 small subunit rRNA sequences and over 7000 large subunit rRNA sequences.

The model polyphasic analyses were completed for over 450 selected reference strains.

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Conclusions

An automated and highly sensitive software programme for the analysis of rRNA sequence data was developed and used to compile a database of rRNA probes which is publicly available. We also compiled extensive chemotaxonomic and phenotypic databases as well as a taxonomic database of the pseudomonads.

The 16S rRNA gene sequences of reference strains was completed for Actinomyces, Arcanobacterium, Arthobacter, Brevibacterium, Cellulomonas, Corynebacterium, Oerskovia and Sanguibacter. Establishment of comprehensive 16S rRNA gene sequence databases for coryneforms and actinomycetes facilitated the recognition of new genera and species.

We developed and evaluated a PCR sequencing strategy for the rapid characterisation of the gene encoding the ß’ sub-unit of DNA-dependent RNA polymerase.

Modification and optimisation of methods for bacterial cell and DNA extraction from different soil types gave approximately three times more DNA than previous methods and the resultant DNA could be used directly for PCR amplification. A highly sensitive method was developed for DNA quantification that could measure the DNA concentration in crude soil extracts. Similarly we developed a method to isolate ribosomes from peaty grassland soil and to quantify different bacterial groups by RT-PCR and TGGE profiling.

We developed non-radioactive methods for DNA-DNA hybridisation for Gram-negative bacteria, Gram-positive bacteria and Archaebacteria and for the detection of amplified nucleic acids. We showed that Cy3-labeled, rRNA-targeted oligonucleotide probes yield high in situ detection rates in a variety of environments.

Nucleic acid-based techniques were utilised to identify and to detect Pseudomonas stutzeri in environmental samples. We also successfully identified Candidatus Parachlamydia acanthamoebae and Polynucleobacter necessarius in situ.

We designed a dot-blot assay using the monoclonal antibody, mAB 900, which had a detection limit of 103 bacteria/ml and a scFv-fragment of mAB 900 was generated in E. coli which allowed direct examination of bacteria in environmental and clinical samples.

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Major publications

Glöckner F.O., Amann R., Alfreider A., Pernthaler J., Psenner R., Trebesius K., Schleifer K.-H., “An optimized in situ hybridization protocol for planktonic bacteria”.
Syst. Appl. Microbiol.,
19, 1996, pp. 403-406.

Moore E.R.B., Mau M., Arnscheidt A., Böttger E.C., Hutson R.A., Collins M.D., Van de Peer Y., De Wachter R., Timmis K.N., “The determination and comparison of the 16S rRNA gene sequence of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships”.
System. Appl. Microbiol.,
19, 1996, pp. 478-492.

Pascual C., Lawson P.A., Farrow J.A.E., Gimenez M.N., Collins M.D., “Genealogical relationships within the genus Corynebacterium as revealed by 16S ribosomal ribonucleic acid gene sequences”.
Int. J. System. Bacteriol.,
45, 1995, pp. 724-725.

Strunk O., Gross O., Reichel B., May M., Hermann S., Stuckmann N., Nonhoff B., Ginhart T., Vibig A., Lenke M., Ludwig T., Bode A, Schleifer K.-H., Ludwig W., ARB: a software environment for sequence data http:/www.mikro.biologie.tu-uenchen.de, Department of Microbiology, Technical University of Munich, Munich, Germany, 1997.

Tesar M., Hoch C., Moore E.R.B., Timmis K.N., “Westprinting: development of a rapid immunochemical identification for species within the genus Pseudomonas (sensu stricto)”.
System. Appl. Microbiol., 19, 1996, pp. 577-588.
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imageResearch project
 

Contract number
BIO2-CT94-3098

Period
January 1995 – July 1997

Coordinator
K.N. Timmis
National Research Centre for Biotechnology (GBF)
Braunschweig (DE)

 
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Partners


A. Akkermans
Agricultural University of Wageningen (NL)

D. Bitter-Suermann,
E. Böttger

Med. Univ. Hannover (DE)

D. Collins
AFRC Institute of Food Research
Reading (UK)

K. Schleifer
Technical University of Munich (DE)

P. Grimont
Institut Pasteur
Paris (FR)

K. Kesters
Rijksuniversiteit Gent (BE)

J. Lalucat
Instituto de Estudios Avanzados de las Islas Baleares
Palma de Mallorca (ES)

R. de Wachter
University of Antwerp (BE)

J. Zeyer
Swiss Federal Institute of Technology (ETH)
Zurich (CH)

V. Torsvik
University of Bergen (NO)

H. Roth
Herolab GmbH Laborgeräte
Wiesloch (DE)

 
 
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