Researech Cooperability Program, Crossing Borders Grant, 2015

Project leader: Dr. Dušica Vujaklija

Project co-leader: Prof. Boris Maček

Project co-workers:

• Prof. Iain Hunter, University of Strathclyde, Glasgow, UK
• dr. Paul Herron, University of Strathclyde, Glasgow, UK
• dr. Saša Kazazić, Ruđer Bošković Institute, CRO
• dr.Tina Paradžik, Ruđer Bošković Institute, CRO
• dr. Senka Džidić, Ruđer Bošković Institute, CRO
• dr. Želimira Filić, Ruđer Bošković Institute, CRO
• dr. Marin Roje, Ruđer Bošković Institute, CRO
• dr. Gerry Quinn, Ruđer Bošković Institute, CRO
• Ela Šarić, PhD student, Ruđer Bošković Institute, CRO
• Maja Šemanjski, Proteome Center Tübingen, Germany

Partner Institutions:

• Institute for Cell Biology, Proteome Center Tuebingen, Germany
• Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, UK

Project duration: 24 months

Administering organization: Institut Ruđer Bošković

S. rimosus growing on solid and in liquid media

The aim of this project is to determine the role of protein phosphorylation in antibiotic production from industrially important bacterium. Over the past decade phosphoproteomic studies of distantly related bacterial species have demonstrated protein phosphorylation on serine, threonine and tyrosine (Ser/Thr/Tyr) as widely employed posttranslational modification (PTM) of proteins which participate in various cellular processes, from basic metabolism to signaling and development. Recent advances in mass spectrometry-based proteomics has had a significant impact on rapid development of this field. Although this type of PTM is extensively used in bacteria and that there is a fast growing list of Ser/Thr/Tyr phosphorylated proteins, the extent and biological function of this PTM, in most cases, is not well defined. Therefore, this project will investigate the comparative phosphoproteome of Streptomyces rimosus strains that vary in the amounts of the antibiotic oxytetracycline (OTC) produced, in order to uncover the impact of this PTM on the antibiotic biosynthesis.

The great majority of natural products are synthesized by bacteria from the genus Streptomyces. These natural products include clinically-important antibiotics (tetracyclines, streptomycine, & ß-lactams), immunosuppressants and anti-cancer drugs (doxorubicin). The tetracycines are one of the most successful classes of antibiotics that include OTC produced by S. rimosus.

To the best of our knowledge, phosphoproteome analysis was never performed with industrially important streptomycetes. As such, we will use high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from cell lysates collected at different growth stages of selected S. rimosus production strains in order to identify key proteins whose phosphorylation status is associated with increased antibiotic production. Identification of novel regulatory proteins can be also applied to elevated antibiotic production in other antibiotic producing streptomycetes. The scientists involved in project have great expertise and contributed to make S. rimosus one of the best genetically described industrial species with well-developed genetic system. Therefore it is likely that their effort and proposed technology will ensure smooth project execution.

In general, this study is expected to provide a better understanding of signaling network for complex prokaryotes during their cellular differentiation that always correlates with the production of specialized metabolites.

Work Plan

Figure 1. Streptomyces rimosus strains - Evaluation of the culturing conditions, OTC production and mycelia differentiation (photos taken from the Mid-term report).

Researchers listed in the project were engaged during the first year of the project. They were performing planned experiments. Detailed results of the project achivements are described in the Mid-term report and submitted to the UKF.

S. Kazazić spent one month (March 2016) in Prof. B. Maček’s laboratory undertaking proteome and phosphoproteome analyses, while D. Vujaklija initiated the evaluation of S. rimosus G7 culturing and determination of the Ser/Thr/Tyr pohosphorylation at the various stages of the G7 growth during her stay in Glasgow in February 2016. In February 2017., dr. Gerry Quinn spent 3 weeks in the Proteome Center Tuebingen in Prof. B. Maček’s laboratory performing phosphoproteome analysis of selected S. rimosus, high OTC producer strain.

Dissemination activities

During the first year UKF funds supported participarition and results presentation for: (i) Tina Paradžik participation at a conference „Power of microbes in industry and environment 2016“,  28.9 - 1.10. 2016, Krk, Croatia and (ii) Tina Paraždik and Dušica Vujaklija at a conference „The 2nd International Conference on Post-Translational Modifications in Bacteria, PTM, 18.10 - 21.10. 2016. Lyon, France.

Prof. dr. sc. Boris Maček has been to the Institute on a working visit from 18th to 22nd of May 2016, and on 30th of December 2016.  

Equipment

Freezer (- 80˚C) and shaker for large scale production of bacteria cultures are delivered, installed and put in usage.