automated microbial identification (HRAMI) and applications to biotechnologically-relevant
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
The model polyphasic analyses were completed for over 450 selected reference
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.
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.
January 1995 July 1997
National Research Centre for Biotechnology (GBF)
Agricultural University of Wageningen (NL)
Med. Univ. Hannover (DE)
AFRC Institute of Food Research
Technical University of Munich (DE)
Rijksuniversiteit Gent (BE)
Instituto de Estudios Avanzados de las Islas Baleares
Palma de Mallorca (ES)
R. de Wachter
University of Antwerp (BE)
Swiss Federal Institute of Technology (ETH)
University of Bergen (NO)
Herolab GmbH Laborgeräte