Abstract

Novel anti-microbial agents are needed to overcome problems of resistance and provide protection against infection. The control of microbial growth is also an essential part of product preservation in the cosmeceutical industry. Despite high through put screening the discovery rate of new antimicrobial compounds has declined. We aim to reverse this trend by exploiting biodiversity within the uncultured microbial populations in the environment and clone habitat metagenomes using bacterial artificial chromosomes.

To evaluate the cloning procedure, habitat diversity will be analysed at the functional and taxonomic level. Targeted assays will identify antimicrobial activities of clones for use in control of infections, as transdermal applications and as preservatives for pharmaceuticals. We aim to combine chemical novelty with novelty in application.

Objectives

The problems of selective culture will be solved whilst simultaneously evaluating alternative approaches to exploiting bacterial diversity in natural environments, thus obtaining novel antimicrobial agents. This will be achieved by using a systematic approach to produce genetic diversity from the soil both by cultivation and other means. The availability of the streptomycete artificial chromosome provides the opportunity to clone high molecular weight DNA from total community extracts. The biosynthetic potential of uncultured actinomycete groups will be evaluated. Cloned pathways provide considerable advantages because of their ease of detection and the project will also use hosts such as Streptomyces lividans. New molecules will be identified and tested for diverse uses in systemic, dermal and transdermal applications and as biocides for preservation of pharmaceutical products.

Description of the work

Total community DNA will be extracted from environmental samples, which have been chosen because of their taxonomic and functional diversity within the actinomycete populations. High molecular weight DNA will be cloned into specialised vectors for expression in a S. lividans host. Environmental gene libraries will be evaluated for diversity in target genes such as 16S rRNA and recA, biological activity and metabolite profiles. In parallel, new methods will be applied to isolate a diverse range of actinomycetes from the same samples. The culturable groups will be screened in the same way, as both will be subjected to metabolite analysis using HPLC-DAD and MALD-MS for rapid detection of new activities. The ultimate objective is to detect new antimicrobial agents using targeted screens.

WP1 aims to produce a molecular toolkit for screening actinomycete diversity in soils, isolates and clone libraries.

WP2 will involve the screening of soil samples using the toolkit and select 4 samples for detailed analysis.

WP3 involves the selective isolation of actinomycetes from the selected environments

WP4 involves the provision of four environmental clone libraries which together will provide some 3000 isolates and 60,000 clones.

WPs 5 and 6 will determine metabolite profiles.

WP7 will determine biological activity. Highly specific targeted assays will include in vitro and in vivo activity with a focus on the control of targeted microbial species. The precise chemical structure of new molecules will be established in WP7 using Atmospheric Pressure Chemical Ionisation mass spectra and NMR.

WP8. will involve further analysis of stability and toxicology that will enable new applications for drug delivery and the use as preservatives to be established.

The main goal of the project is the provision and testing of an alternative strategy to selective culture.

Deliverables

• Development of a molecular toolkit for evaluating diversity within actinomycete populations.
• A strategy for cloning high molecular weight DNA from soil and stable integration into the chromosome of S .lividans host.
• Rapid methods for detection of altered fermentation profiles of clone libraries using MALD-MS.
• Provision of molecular structure and activity profiles and development of novel applications for use of antimicrobial agents as anti-infectives, dermal patch treatments and biocidal agents.

Contacts

Coordinator

• E M H WELLINGTON / University of Warwick Department of Biological Sciences / UNITED KINGDOM

Participant

• Biosearch Italia SpA Microbial Fermentation / ITALY
• Spiros POTINOS / Institution Lavipharm S A GREECE
• National & Kapodistrian University of Athens Institute of General Botany, Dept of Biology / GREECE
• Joachim VATER / Technische Universität Berlin Max-Volmer-Institut für Biophysikalische Chemie / GERMANY
• Lubbert DIJKHUIZEN / University of Groningen Microbial Physiology / NETHERLANDS
• Alan WARD / University of Newcastle upon Tyne Department of Agricultural and Environmental Science / UNITED KINGDOM
• Hans-Peter FIEDLER / Universität Tübingen Mikrobiologisches Institut / GERMANY