Bacterial protein secretion is a fundamental biological process of the utmost relevance to human health. On one hand, this process can be exploited successfully to the benefit of human health through the biotechnological production of biopharmaceuticals. On the other hand, secreted bacterial toxins and virulence factors represent a major threat to human health. The Twin-arginine translocation (Tat) machinery represents a recently discovered, yetbut widely conserved system for bacterial protein secretion. This multi-subunit nanomachine can transport fully folded proteins and thus has a huge potential for biopharmaceutical production in bacterial species that are already used for this purpose, including Bacillus, Escherichia coli and Streptomyces. It has furthermore been demonstrated that critical virulence factors are secreted via Tat in important pathogens, such as Pseudomonas aeruginosa and E. coli O157. The Tat machine programme aims at the multidisciplinary functional genomic characterisation of the Tat nanomachine for both biotechnological and biomedical purposes.
The Tat machine project sought to eliminate existing bottlenecks in the Tat nanomachine that limit biopharmaceutical production in Bacillus, E. coli and Streptomyces, as well as to characterise the structure and function of Tat nanomachines from a few selected Gram-positive and Gram-negative bacteria, including major pathogens. To reach these goals, the full potential of bioinformatics, comparative and structural genomics, and proteomics will be exploited. The Tat machine partnership has a proven track record in the application of these cutting-edge technologies.
Problem:
This programme is focused on the twin-arginine translocation (Tat) protein transporter that is widespread in bacterial plasma membranes. This system assembles to form a circa 1 mDa nanomachine that is uniquely able to translocate a wide range of large, folded (even oligomeric) proteins across the tightly sealed bacterial plasma membrane. The system differs in both structural and mechanistic respects from all other known protein translocases and, although only identified in bacteria in 1998, it is already clear that the system has significant potential for biomedical and biotechnological research and exploitation.
Aim:
The Tat machine programme is aimed at (a) exploiting the unique abilities of the system for the production of biomedically important, heterologous proteins, (b) understanding the role of Tat in a range of pathogenic bacteria, and (c) solving the three-dimensional structure of representative Tat machines. The data acquired from (b) and (c) will underpin the future development of novel anti-infective compounds that specifically target the Tat nanomachine.
Expected and obtained results:
The deliverables of the Tat machine programme include knowledge of a fundamental biological system, the Tat nanomachine, that is of relevance to human health. Specifically, this includes:
- Detailed structure of Tat complexes from representative Gram-negative and Gram-positive species. This is an ambitious aim. The project has established a pipeline for the efficient delivery of a wide range of Tat complexes to partners with strong track records in the elucidation of membrane protein structures. First structural data has become available.
- Development of super-secreting strains of B. subtilis and Streptomyces coelicolor, capable of exporting heterologous proteins with high efficiency. These strains will fill major gaps in the present repertoire of bacterial vehicles for protein production. Especially, the Tat system of S. coelicolor has turned out to have a high application potential.
- Understanding of the overall role of Tat in a limited series of pathogenic bacteria, including the identification of specific virulence determinants that employ this export pathway. Our studies have so far addressed the Tat systems of pathogenic E. coli, Pseudomonas, Shigella, Staphylococcus and Vibrio species. The results obtained will be of biomedical interest and will serve to reinforce efforts to design anti-infectives.
- In-depth understanding of the Tat translocation mechanism has been achieved by a combined biochemical/genetic analysis of the Tat translocation process. The results are to the benefit of all the afore-mentioned elements of this project.
Potential applications:
The Tat machine programme focuses on the need to translate genome data into practical applications, both in the fields of medicine (controlling infectious disease) and biotechnology. The project has been formulated to provide innovative solutions to the industry. Furthermore, the partnership will create an important knowledge base needed to foster European competitiveness in the area of antibiotics and biotechnology research.
Coordinator:
Prof. Jan Maarten van Dijl
University Medical Center Groningen (UMCG)
Department of Medical Microbiology
Hanzeplein 1
P.O. Box 30 001
9700 RB Groningen, Netherlands
Tel. +31 503633079
Fax +31 503633528
E-mail:
j.m.van.dijl@med.umcg.nl
Management Assistant
Dr S. Bron
University of Groningen
Haren, Netherlands
Partners:
Prof. C. Robinson
University of Warwick
Warwick, England, UK
Prof. O.P. Kuipers
University of Groningen
Haren, Netherlands
Dr M. Kolkman
Genencor International BV
Leiden, Netherlands
Prof. Dr M. Müller
Universitätsklinikum Freiburg
Freiburg, Germany
Prof. T. Palmer
University of Dundee
Dundee, Scotland, UK
Dr L. F. Wu
Laboratoire de Chimie Bactérienne (LCB) UPR9043, CNRS
Marseille, France
Prof. Dr M. Hecker
Ernst-Moritz-Arndt-Universitaet Greifswald
Greifswald, Germany
Prof. Dr W. Kühlbrandt and Dr K. Model
Max-Planck Institute of Biophysics
Frankfurt am Main, Germany
Prof. S. Iwata and Dr L. Carpenter
Imperial College of Science, Technology and Medicine
London, England, UK
Prof. Dr R. Freudl
Forschungszentrum Jülich GmbH
Jülich, Germany
