The major objectives of the proposed research are:
to develop high resolution molecular methods for the rapid identification of microorganisms;
to assess the utility and to compare the efficiency of the different methods developed;
to exploit them to expand our microbial taxonomy base, in particular to microorganisms from natural environments;
to automate the developed methods.
Quantitative analysis of microbial populations in the environment have been based mainly on conventional cultivation methods, neglecting those taxa that prove resistant to cultivation. Recently new methods became available to identify organisms at the ribosomal ribonucleic acid (rRNA) level. 16S rRNA contains large amounts of phylogenetic information and is a relevant target for probe designing.
Studies are concerned with the construction and use of oligonucleotide probes in the environment, concentrating on the identification of Nitrosospira and Frankia in soil and on syntrophic propionate oxidizing bacteria in anaerobic granular sludge. Part of the 16S rRNA from different strains of these bacteria have been sequenced and a specific oligonucleotide probes have been constructed for them.
Application of these probes to environmental samples necessitates adaptation of the deoxyribonucleic acid (DNA) RNA extraction procedures. So far there are no methods available that garantuee quantification of nucleic acids in complex ecosystems (eg soil). Special attention has been given to the standardization of the extraction procedure from different ecosystems. Data will be presented on the efficiency of different extraction procedures from soil and actinorhizal root nodules.
Ribosomal ribonucleic acid (RNA) (or genes) act as reliable molecular chronometers and are currently the most powerful molecules for measuring the evolutionary and genealogical relationships of bacteria. In addition to being valuable phylogenetic markers the enormous potential of these molecules for the design of highly specific oligonucleotide probes for microbial identification detection has recently become apparent. To date most probe development has been based on smallsubunit ribosomal RNA (rRNA) (16S). The widespread applicability of large subunit probes to problems of bacterial identification and detection is however seriously hampered by the lack of comprehensive sequence databases which in turn is due to current limitations in the rapidity of sequencing methodologies for these molecules. Primary sequences of large subunit rRNAs are invariably generated using gene cloning (which is time consuming and not practicable for large scale database construction). It is now recognized that polymerase chain reaction (PCR) methodology, an enzymatic method for in vitro deoxyribonucleic acid (DNA) amplification, can provide a rapid means of obtaining rRNA sequences. A set of conserved and semiconserved oligonucleotide primers have been developed to facilitate the direct sequencing of the large subunit rRNA genes of the economically important mastitic pathogen Streptococcus uberis. Diagnostic signatures in the large subunit rRNA have been identified and used for the design of highly specific gene probes to differentiate Streptococcus uberis from Streptococcus parauberis and other pyrogenic streptococci.
Rapid identification of culturable and nonculturable microorganisms requires new tools such as nucleic acid probes.
Published ribosomal ribonucleic acid (rRNA) targeted oilgonucleotide probes and their optimal hybridization parameters are compiled in a database. Software for automated probe design based on aligned rRNA sequences is under development. Full length 16S rRNA sequence of Nitrobacter species strain 255, Nitrobacter species strain K34, Nitrospira species strain 295, Ilyobacter tataricus, I polytropus, Pelobacter propionicus, P acetylenicus, Ruminococcus pasteurii, Paracoccus denitrificans, Thiosphaera pantotropha, Thiobacillus cuprinus and 23 S rRNA sequences of Gram-positive bacteria with a low deoxyribonucleic acid (DNA) guanine and cytosine content, Paracoccus denitrificans, Pseudomonas diminuta, P stutzeri, Rhodopseudomonas palustris, Bradyrhizobium lupinii, B japonicum, Thiobacillus cuprinus, Vibrio vulnificus, Nannocystis exedens, Stigmatella aurantiaca have been determined.
Whole cell hybridization with fluorescent oligonucleotides allows the in situ identification of individual cells. Alternative, more sensitive probe labels have been evaluated. Identification of single microbial cells has been achieved with horseradish peroxidase labelled oligonucleotide. The combination of whole cell hybridization with fluorescent rRNA targeted oligonucleotide probes and flow cytometry has been optimized. A set of 16S and 23S based probes specific for the alpha-subclasses, beta-subclasses and gamma-subclasses of proteobacteria has been developed, and is currently used to enumerate bacteria in samples from aquatic environments. A combination of polymerase chain reaction (PCR) assisted 16S rRNA sequence retrieval and fluorescent oligonucleotide probing was applied to reveal the phylogenetic status of Sarcobium lyticum, a hitherto uncultured intracellular parasite of small amoebae.
A multidisciplinary research consortium (9 research laboratories and 1 software company) will develop technology in the fields of molecular sequence analysis, immunology, analytical chemistry, instrumentation and separation science, and apply this technology to the rapid and accurate identification of microorganisms. In a first step, ribosomal RNA sequences, chemical "biomarkers", macromolecular "profiles" and stable antigens, will be used to characterize a standard set of commercially and environmentally relevant groups of microorganisms.
Within these different analytical approaches determination and alignment of rRNA sequences (16S and 23S) enable definition of higher taxonomic relationships (genus-level and above) and thereby generate the backbone of a microbial taxonomy based on the phylogeny of microorganisms. Analysis of specific rRNA sequences will allow construction of species-specific oligonucleotide probes for bulk and single cell detection in environmental samples.
The chemotaxonomic approach will emphasise the search for qualitatively new microbial compounds (biomarkers) using high resolution analytical instrumentation like pyrolysis mass spectrometry and tandem mass spectrometry on whole microbial cells. All major macromolecules of the microbial cell (DNA, RNA and proteins) will be used to obtain fingerprints (profiles) by high resolution electrophoresis that enable a rapid overview of large sets of individual strains for their taxonomic grouping. The study of stable antigens will lead to strain-specific monoclonal antibodies used in automated bulk detection and single cell detection via epifluorescence microscopy.
In a second step, the results of this polyphasic approach will be compared and evaluated with respect to identification utility and analytical facility. Those analytical systems found to be useful will be used to expand taxonomic data bases (ie, MINE) and will be evaluated in terms of their applicability to the identification, quantitation, and sensitive detection of microbes in environmental samples. Additionally, the probes (nucleic acids, antibodies, etc.) generated for rapid identification and taxonomic purposes will be used for the development of highly sensitive procedures for the detection of specific organisms in environmental samples either in situ or following extraction from complex environmental matrices.
Finally, these new methods and probes are to be automated for the analysis of large numbers of environmental samples in order to study pressing environmental issues.
Funding SchemeCSC - Cost-sharing contracts
RG6 2EF Reading
6703 CT Wageningen