The project proposes to advance technology development on several fronts in order to solve the critical problems of microbial identification, classification, detection and quantitation. The aims are to develop technologies that can become the basis of new automated procedures that can rapidly, reliably and inexpensively identify microorganisms, and to compare and validate them with particularly problematic bacterial taxa. The specific goals are:
1.The development of new technologies exhibiting good potential for rapid, accurate and automated identification of known and, more importantly, unknown microbes.
2.The establishment of new/expand existing data bases appropriate to the new technologies.
3.The assessment of developing/developed technologies with respect to taxonomy, cost and convenience, automation, applicability to relevant environmental samples.
4.to expand our microbial taxonomy base, particularly with respect to new environmental organisms and particularly with respect to non-culturables.
5.The development of automated technologies.
Attainment of the goals detailed in this proposal has resulted in an increase in the utility of microbial taxonomy, particularly with respect to the identification of new isolates with biotechnological and environmental relevance. This foundation of an information base has been utilized in assessing the impacts of anthropogenic influences, including genetically engineered microorganisms, on the environment and microbial diversity.
A model polyphasic study of the pseudomonads has been completed with the analysis of more than 300 strains belonging to more than 60 species of the genus Pseudomonas (sensu stricto), as well as species classified at one time as Pseudomonas but which belong to genetically-distinct phyla, comprise a phylogenetic framework for the comparison of biotechnologically-relevant organisms. Sequence data of the 16S and portions of 23S rRNA genes (GBF,MHH,IFR,TUM), ribotypes (PAS), small molecular-weight stable RNA (5S and tRNA) profiles (GBF), polar lipid and fatty acid characterization (GBF,RUG), total protein profiles (RUG) and DNA fingerprinting (RUG) have been correlated. Nested sets of gene probes have been developed for genus and species identification (TUM). The sequence database update, compiled by UIA, of the small subunit rRNA (16S rRNA) and the large subunit rRNA (23S rRNA) now includes more than 7,000 and 430, respectively, nearly complete sequences.
The model polyphasic analyses of coryneform bacterial reference species and environmental isolates have been started and are on-going, in collaboration with the Hans-Knöll Institute (HKI) for Natural Products, and now have advanced to the completion of the distribution of more than 450 selected reference strains to specified partners (GBF, IFR, TUM, PAS). The analyses, for the most part, have been completed and are reported in this final report.
MAJOR SCIENTIFIC BREAKTHROUGHS:
* The project has seen the introduction to the scientific community and the updating of the ARB program package, developed at the Technical University of Munich (TUM), for the analysis of rRNA sequence data.
* The development of software for the enhanced automation and sensitivity of profiling methods.
* An updated rRNA probe data base is publicly available via WWW.
* Improvement of the accessibility and ease of downloading of sequence data via the WWW.
* Accumulation of extensive chemotaxonomic and phenotypic data bases.
* A Molecular Microbial Ecology Manual has been published in which at least 7 chapters are written by HRAMI partners.
* The taxonomic analyses of the pseudomonads carried out by the individual partners have been com-pleted and compiled in a special issue of Systematic and Applied Microbiology (December, 1996).
* The 16S rRNA gene sequences of reference strains of the following genera have been completed: Actinomyces, Arcanobacterium, Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, Oerskovia and Sanguibacter.
* A comprehensive 16S rRNA gene sequence data base has been established for coryneforms and related actinomycete taxa, facilitating the recognition of new genera and species.
* A PCR sequencing strategy has been developed and evaluated for the rapid characterisation of the gene encoding the S' subunit of DNA-dependent RNA polymerase.
* Methods for bacterial cell extractions and DNA extractions from different soil types were modified and optimised. A rapid method for the isolation of total DNA from soils was developed. The method gives approximately 3 times more DNA compared to DNA extraction based previous methods, and the DNA can be used directly for PCR amplification.
* A highly sensitive method was developed for DNA quantification, allowing the DNA concentration in crude soil extracts to be measured. The method facilitates monitoring of DNA yields and, thereby, optimising the DNA extraction methods.
* A method has been developed to isolate ribosomes from peaty grassland soil and to quantify different bacterial groups by RT-PCR and TGGE profiling.
* A non-radioactive micro-method for DNA-DNA hybridizations have been developed. It is useful for Gram-negative, Gram-positive Bacteria and Archaebacteria.
* Establishment of a nonradioactive format for the detection of amplified nucleic acids allowing automated high-output analyses.
* Cy3-labeled, rRNA-targeted oligonucleotide probes yield high in situ detection rates in various environments.
* The determination of the resolution capacity of the intergenic spacer region: an identical sequence of the intergenic spacer region most probably indicates species identity; the rRNA intergenic spacer region provides some useful phylogenetic information mainly at a subspecies level; the level of sequence heterogeneity within the spacer region does not allow to define the taxonomic level of separation, i.e. species or subspecies.
* The phylogenetic position of the endophyte and co-symbionts in Ceanothus caeruleus root nodules have been determined on the basis of 16S rRNA sequences.
* The in situ identification of Candidatus Parachlamydia Acanthamoebae and Polynucleobacter necessarius.
* Nucleic acid-based techniques have been developed for the identification of P. stutzeri and for its detection in environmental samples.
* An epitope, widely distributed within the Bacteria and Archaea domains, could be mapped to the most N-terminal hexamer SKEKFE of the EF-Tu molecule.
* The monoclonal antibody, mAB 900, was used to design a dot-blot assay with a detection limit of 103 bacteria / ml.
* A scFv-fragment of mAB 900 was generated in E.coli for the direct examination of bacterial cells in environmental and clinical samples.
KEYWORDS:Microbial taxonomy, Phylogeny, Identification, Detection, Diversity, Pseudo-monads, Pseudomonas (sensu stricto), Pseudomonas stutzeri, Coryneform bacteria, Uncultured bacteria, Ribosomal RNA (rRNA), 16S rRNA, 23S rRNA, Gene probes, Cy3, In Situ hybridisation probing, Whole-cell hybridization, Oligonucleotide, Flow cytometry, Ribotyping, Chemotaxonomy, Fatty-Acid profiling, Protein fingerprinting, DNA sequencing, Sequence alignment, Databases, Secondary structure, Software, Fingerprinting, AFLPTM, Nonradioactive DNA-DNA hybridization, Nucleic acid extraction, DNA extraction, DNA quantification, Reassociation kinetics, Melting profile, PCR, Competitive RT-PCR, Q-PCR system 5000, Ribosome isolation, Root nodules, TGGE, Intergenic spacer, DNA-dependent RNA polymerase, Molecular chronometer, Soil, Bulking sludge, Sediments, Aquatic, Aquifer.
Funding SchemeCSC - Cost-sharing contracts
RG6 2EF Reading
07071 Palma De Mallorca
6703 CT Wageningen