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Final Report Summary - AMBER (Advanced Multidisciplinary Training in Molecular Bacteriology)

The overall objective of the research programme Advance Multidisciplinary training in Molecular Bacteriology (AMBER) was to provide a comprehensive, timely and multidisciplinary training in microbiology for young European researchers. Within this Marie Curie International Training Network, we aimed at investigating an important and multifaceted bacterial problem: the intertwinement of central carbon metabolism and main cellular processes. The model system used was the bacterium B. subtilis. Two companies joined our consortium as active participants: Kemin and Puratos. The advancement in life sciences has created a growing number of disciplines that are required to elucidate the increasingly complicated biological questions. This has led to an unsurpassed level of knowledge, but it has also resulted in a continuous specialization in PhD trainings, which makes the students less prepared for complex present day biological questions. The programme aimed at providing a better comprehensive and multidisciplinary PhD training in microbiology.

The reason why relatively little is known about the role of carbon metabolism and other cellular processes is because of the complexity and intertwined at different levels. This research problem could therefore only be tackled by applying a comprehensive set of multidisciplinary (systems) approach. Within the AMBER consortium we have brought together 10 groups that are experts in the different relevant disciplines: Cell division, DNA replication, cell wall synthesis, RNA processing, metal homeostasis, metabolomics, stress management, non-ribosomal peptides, probiotics, and enzyme production. The consortium intended therefore to apply a wide variety of tools that span the complete gamut of molecular biological approaches.

The work was organised in 12 work packages (WP) leading to the following main results:

WP1: Training of ESRs and ERs.
Main achievements: 8 ESRs, 2 ERs and 8 short-term fellows received a comprehensive, timely and multidisciplinary training in microbiology, investigating how central carbon metabolism is linked to the different key cellular processes. All ESRs and ERs performed their secondments at other beneficiaries, including the industrial partners, acquiring a broad range of knowledge and skills, as well as profiting from extensive professional networks across Europe. Fellows benefitted from a high-quality and close supervision and co-supervision by the senior PIs of the network.

WP2: Carbon metabolism and cell division.
Main objective: Understand how carbon metabolism, and in particular WhiA, regulates cell division.
Main achievements: WhiA appeared to be also required for DNA replication and DNA damage repair. Interestingly, the absence of WhiA influences the metabolism of branched-chain amino acids.

WP3: Carbon metabolism and DNA replication.
Main objective: Unravel the regulatory interplay between carbon metabolism and chromosomal replication.
Main achievements: YabA negatively controls the initiation of chromosomal replication in Bacillus subtilis. We showed that YabA is phosphorylated on its Serine 71 by the Ser/Thr kinase YabT. This phosphorylation is not involved in replication initiation control but plays a role in sporulation and biofilm formation and modulates an atypical ATP/GTPase.

WP4: Carbon metabolism and cell wall synthesis.
Main objective: unravel the connections between central carbon metabolism and biosynthesis and cell wall structure in the Gram-positive model bacterium B. subtilis.
Main achievements: The deletion of ugtP caused higher peptidoglycan precursor levels and a higher activity of the LytE hydrolase.

WP5: Carbon metabolism and RNA processing.
Main objective: elucidate the regulatory function of glycolytic enzymes on mRNA processing in B. subtilis.
Main achievements: we solved the crystal structure of the mRNA processing enzyme PnpA in complex with ADP, and showed a dynamic localization of proteins involved in RNA-metabolism, including the paralogous RNases J1 and J2.

WP6: Carbon metabolism and metal homeostasis.
Main objective: understand at system's level how the global regulation of B. subtilis metabolism is integrated with the control of metal ion homeostasis.
Main achievements: we have determined the global metal homeostasis by means of proteomics and transcriptomics studies.

WP7: Carbon metabolism and the metabolome.
Main objective: analyse the B. subtilis ‘metabolome’ in different glycolytic mutants and under different growth conditions.
Main achievements: we were able to detect differences in the intra and extracellular metabolome profile of Bacillus subtilis BSB1 wild type and glycolytic mutants involved in teichoic acid pathway of the cell wall. Moreover, pyruvate kinase mutants was studied in several media conditions revealing also an impact in the cell wall precursors.

WP8: Carbon metabolism and stress management.
Main objective: understand the interplay between bacterial stress management and central carbon metabolism.
Main achievements: we examined noise in gene expression in single Bacillus subtilis cells under different growth conditions and constructed mathematical models to predict this.

WP9: Carbon metabolism and non-ribosomal peptide synthesis.
Main objective: understand (and control) the relationship between central carbon metabolism and non-ribosomal peptide synthesis.
Main achievements: mathematical models were constructed to predict the Branched-Chain Amino Acids (BCAAs) metabolic pathway of B. subtilis and verified this by constructing knockout mutants based on the model prediction. This was applied to study the production of surfactin production in relation with extracellular metabolites production.

WP10: Carbon metabolism and secondary metabolite production.
Main objective: investigate how production of active metabolites in B. subtilis is regulated under different growth conditions and developmental stages.
Main achievements: the lipopetide profile of B. subtilis strain PB6 was characterized using glucose as a carbon source, and the relation between carbon metabolism and differential synthesis of extra cellular polymeric substances was investigated.

WP11: Carbon metabolism and heterologous enzyme production.
Main objective: better understand the link between carbon metabolism and enzyme expression to improve B. subtilis strains and fermentation for optimal enzyme production.
Main achievements: through biochemistry and omics technologies, we observed that specific parameters of fermentation have a relevant impact in the cells, triggering a significant modification in the enzyme production.

WP12: Outreach activities.
Main achievements: The primary means to share the results of this project with the wider public was by publishing in peer reviewed journals: 8 peer-reviewed publications were achieved during the project, 3 are still under review and 7 are being written. In addition, AMBER fellows made 36 (oral and paper) presentations with their research results at national and international scientific conferences and workshops. The highly successful International symposium on “Central Carbon Metabolism and its Effect on Bacterial Processes” saw the participation of eminent international speakers and the attendance of more than 100 participants among students and researchers. A very successful outreach event “Tales of Bacillus subtilis: from origin of life on earth to delicacy” was organized in Micropia, the only museum of microbes, in the centre of Amsterdam. During the evening invited guests and the AMBER fellows revealed fascinating stories about the bacterium Bacillus subtilis. The audience learned how this bacterium travels through space and might have started life on this planet, colonizes our intestines, trains our immune system, spoils our food and at the same time is able to produce the most tasteful delicacies, which were served afterwards. Many local outreach activities were also carried out by the AMBER fellows, in particular with students. All dissemination events/news were advertised on the AMBER website, where all the related materials (programs, pictures, publications, etc.) are publicly available, also to a wider audience: www.amber-itn.eu.

The multidisciplinary trained fellows investigated, in key areas of microbial physiology, the intertwinement of central carbon metabolism and main cellular processes in Bacillus subtilis, a Gram positive model organism widely used in the biotechnology industry. They were provided a well-balanced and high-quality training and secondments opportunities and supervision arrangements, equipping them with all crucial skills required to pursue a successful career in academia and/or the private sector. ESRs at the end of AMBER are very much advanced in their PhD programmes, as half of them have already submitted or defended their thesis. The rest are expected to submit and defend in 2017. One ER was offered a contract at the end of her fellowship. One ESR of an academic beneficiary is employed in a start-up company of the university and one short-term fellow is now doing a PhD at the institute where she did her fellowship research.
Importantly, AMBER also developed exploitable foreground: a patent application is in the process of being submitted by an academic beneficiary with possible applications in the Agricultural and Cosmetics sectors and some internally exploitable foreground has also been developed by the industrial partners (Feed additives, Biotech & Agrofood sectors).

For further information you can contact:
Prof Leendert Hamoen, AMBER Project Coordinator
University of Amsterdam, Faculty of Science
Swammerdam Institute for Life Sciences (SILS)
Bacterial Cell Biology department
+31 (0) 20 525 5115
l.w.hamoen@uva.nl
Project website: www.amber-itn.eu

Reported by

UNIVERSITEIT VAN AMSTERDAM
Netherlands

Subjects

Life Sciences
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