Abstract

This project follows up the exploitation of novel predominant soil bacteria and the revelation of their evolution. The recently discovered BACREX group (related to Bacillus) represented 20% of all soil bacteria in a Dutch grassland soil and was at least in part culturable. Due to their apparent competitiveness among other bacteria they will be screened for antimicrobial activity. Active compounds will be identified and tested for health-related exploitation potential. The course of evolution of the BACREX group, as visible in its microdiversity, will be investigated by genome analysis. With DNA/RNA based techniques we will follow their spatial dissemination through the Netherlands and beyond. Different populations will be located and compared for evolutionary relationships by genome analysis. We will describe the most important soil bacteria hitherto cultured and track their adaptation to different niches and their dissemination through European landscapes.

Objectives

This project will link pioneer research on bacterial evolution, microdiversity and metabolic capabilities of yet unexplored bacteria abundant in agricultural soil with the exploitation of their activity potential. It is focused on the recently cultured BACREX cluster predominant in soils, especially Dutch grasslands.

The specific objectives include:

• Culture collection of BACREX strains, phenotypic characterisation and description of novel species.
• Dissemination of BACREX bacteria monitored by culture-independent 16S rRNA methods and long-distance sampling beyond the Dutch grasslands including other EU states.
• Novel methods to capture yet uncultured bacteria.
• Fermentation optimization and high-throughput screening for antibiotic/anti-tumour compounds.
• Identification of variable genes among BACREX genomes to reveal selective forces of microdiversity.
• Reconstruction of the evolution of BACREX strains in soil.

Description of the work

A comprehensive culture collection of BACREX strains will be generated. Genomic and phenotypic analyses will be performed to characterise the new species and to allow reliable identification. The geographical dissemination of BACREX strains will be monitored by 16S rRNA directly extracted from soil samples.

The present BACREX bacteria will be unveiled by cloning and sequencing of PCR-amplified 16S rDNA. Their species richness and abundance in microbial communities of different agricultural soils will be spatially monitored by 16S rDNA analysis. BACREX strains recalcitrant to cultivation will be enriched by cell sorting methods. bioLeads GmbH, specialised on high-throughput screening for novel drug compounds, will reveal secondary metabolite diversity by mass spectrometry. Automated tests for antibiotic, cytostatic and other compounds and spectroscopic structure resolution will identify novel drug candidates. BACREX microdiversity will be assessed on the genome level. The variable DNA parts among BACREX genomes will be selectively amplified by RDA. Cloning and sequencing of RDA fragments will identify the differing genes to reveal the impact of natural selection. The known genome sequence of the BACREX-relative Bacillus subtilis and other public databases will serve as a reference. The metabolic potential of BACREX strains will be shown by hybridisation of their DNA with DNA chips carrying the B. subtilis genome. All data on geographic dissemination, phenotypic and genomic diversity of BACREX strains will be combined to a comprehensive view on their evolution in European agricultural soils, that allows for the exploitation of BACREX specific genes and expression products.

Deliverables

• Culture collection of BACREX strains.
• Dissemination map of BACREX bacteria populations in European agricultural soils.
• Cell sorting enrichment method for uncultured bacteria.
• Fermentation protocols for BACREX strains.
• Isolation and characterisation of efficacy and molecular structure of novel drug candidates.
• Estimation of selective forces on soil bacteria microdiversity by analysis of genome diversity among the BACREX cluster populations.
• Construction of an evolutionary tree of the BACREX cluster.

Contacts

Coordinator

• Andreas FELSKE / National Research Centre for Biotechnology (GBF Division of Microbiology / GERMANY

Participant

• Paul de VOS / Universiteit Gent Laboratorium voor Microbiologie, Vakgroep Biochemie / BELGIUM
• Wageningen University Department of Biomolecular Sciences / NETHERLANDS
• bioLeads GmbH GERMANY