[Introduction]
Of particular interest to this project is a predominant group of until recently unknown soil microorganisms of Bacillus benzoevorans- related bacteria, further referred to as the BACREX cluster . These microorganisms for the first time were detected in DrentseA grassland soils and contributed to 1/3 of the total bacterial community. As they are so abundant in this area, this project aims to cultivate them and to investigate their role in their natural environment. Furthermore the project aims to initiate the biotechnological exploitation of novel and predominant soil bacteria and to reveal their evolution in the environment. The main problem to solve is the demand to culture these microbes in the laboratory, since apparently less than 1% of present soil bacterial cells were cultivated successfully. Sequence analysis of directly isolated 16S rDNA molecules from soil indicated that the majority of environmental bacteria constitute of hitherto unknown species. [Research]
The cultivation strategy was applied to generate a comprehensive culture collection of strains, which were rapidly screened for the presence of novel groups via a multiplex Polymerase Chain Reaction (PCR) assay. This multiplex PCR also contains probes detecting other yet uncultured bacterial clusters, i.e. Acidobacter and Verucomicrobiales. PCR-positive strains were forwarded to the subsequent studies. Since less than 1% of present soil bacteria were recently successfully cultivated we developed a culture independent method for detection and monitoring of the BACREX community in different soil samples. We developed two 16S rRNA gene-targeted primer sets that can be used in a nested PCR approach, and can consecutively exclude non-BACREX species. These primers are used in combination with Denaturing Gradient Gel Electrophoresis (DGGE). The DGGE fingerprints give us clear view of BACREX diversity in the studied soil. This method allows fast monitoring of the community without being dependent on the cell conditions in the soil sample, which is another advantage.
A scientifically innovative aspect is to study soil bacteria dissemination in geographic scale (biogeography). In previous studies members of the BACREX cluster were detected in several areas in extraordinary abundance and diversity. From there, we will explore the extension of dissemination of the cluster, since the surrounding landscapes are of similar geological and agricultural history. The numerous samples were and still will be rapidly screened by the specific molecular and cultivation methods. Outcome will be a map of the BACREX Empire showing bacterial density of the cluster. The dissemination of these bacteria might be followed quite far, even over different EU member states. [Future Research]
For quantification of the BACREX cluster in different soil samples, a dot blot hybridization technique will be developed. This method will be based on 16S rDNA PCR products, which allows its application on soils where actively dividing can not be expected.
The evolutionary relationships among the BACREX cluster isolates will be investigated by the identification of different genes via representational difference analysis (RDA) for closely related strains, detection of gene-specific genomic diversity via Bacillus subtilis and/or Bacillus cereus DNA chips, genome fingerprinting (AFLP) and other physiological data appearing during taxonomic description. With respect to the geographical distribution of the BACREX cluster, a comprehensive evolutionary tree will be constructed. |
