Periodic Reporting for period 1 - MBD (Mapping bacterial division in Bacillus subtilis)
Periodo di rendicontazione: 2016-09-01 al 2018-08-31
The bacterial cell cycle is of fundamental importance and an important potential antibiotic target; however, many details of the molecular mechanisms involved remain elusive. Sporulation in Bacillus subtilis is an important model system for cell cycle studies. Here, I applied the next generation sequencing techniques, including ribosome profiling method to search for novel factors involved in the of B. subtilis cell cycle. Results were further investigated using advanced genetic methods and microscopic techniques to characterize the functions of the novel factors, gaining insights into their role in the cell cycle.
The cell cycle of the Gram-positive model bacterium B. subtilis is one of the most studied and best understood, though many facets of the process remain to be worked out. B. subtilis sporulation serves as an excellent model with which to study various cell cycle processes. Unlike canonical ("vegetative") division, in which the cells divide precisely in the middle to generate identical daughter cells, during sporulation the cells divide in a highly asymmetrical manner, leading to the formation of a small spore and a large mother cell. The major factors involved in sporulation division are the same as in vegetative division, but the overall process is drastically reorganized to achieve specific changes not only in the position of the chromosomes and division septum but also their ultrastructure. Sporulation is a non-essential process but it can be induced conveniently and relatively synchronously. These attractive properties, and the exceptionally well-developed methods to investigate the molecular cell biology of B. subtilis, make sporulation an extremely powerful experimental system. There are also important practical reasons for studying sporulation. First, cell cycle events such as replication, segregation and division are underexploited potential targets for antibacterials with novel modes of action, which are desperately needed for the fight against antibacterial resistance. Second, for a number of important bacterial pathogens, including Clostridium difficile, B. anthracis and B. cereus, endospore formation is an important factor in pathogenesis.
Although the transcriptional regulation of sporulation has been extensively studied and most of the key factors have been identified and characterized, almost nothing was known about regulation at the translational level. In the MBD project, I studied the role of translation regulation on the gene expression regulation of the factors involved in the process of spore formation in a Bacillus subtilis.
The project is primarily basic research but as described above, the results have potential impact in at least three areas. The first will be a better understanding of the basic physiology and regulatory process in bacteria. Although a non-pathogenic "model" bacterium, B. subtilis is closely related to various important pathogens, including B. anthracis, B. cereus, various clostridia, Listeria monocytogenes, and various Gram-positive cocci (Staphylococcus, Streptococcus, etc). New basic understanding of B. subtilis will inform and enhance our ability to predict the physiology and regulatory processes of many pathogens. Second, the cell cycle provides an important set of potential targets for novel antibiotic agents. Identifying these targets and understanding their function could facilitate the discovery and development of novel antibiotic leads. Third, B. subtilis and close relatives are important industrial organisms, responsible for the commercial production of secreted industrial enzymes, particularly various hydrolases (proteases, amylases, etc), and some small high value molecules (e.g. riboflavin). A better understanding of B. subtilis physiology and translational regulation could impact on our ability to manipulate this organism to improve the yield of these and other potential commercial products.
Objectives of the project may be summarized as follows:
Use of ribosomal profiling to elucidate new factors and regulatory processes operating during the early stages of sporulation. The main focus was on:
Switch from vegetative growth to the starvation / sporulation response
Characterise the properties of novel cell cycle and sporulation factors in terms of:
Mutant phenotype
Localization of the protein (fluorescence imaging)
Regulation relative to the well-known sporulation pathway
Identify and characterise factors involved in ribosomal reprogramming.
The cell cycle of the Gram-positive model bacterium B. subtilis is one of the most studied and best understood, though many facets of the process remain to be worked out. B. subtilis sporulation serves as an excellent model with which to study various cell cycle processes. Unlike canonical ("vegetative") division, in which the cells divide precisely in the middle to generate identical daughter cells, during sporulation the cells divide in a highly asymmetrical manner, leading to the formation of a small spore and a large mother cell. The major factors involved in sporulation division are the same as in vegetative division, but the overall process is drastically reorganized to achieve specific changes not only in the position of the chromosomes and division septum but also their ultrastructure. Sporulation is a non-essential process but it can be induced conveniently and relatively synchronously. These attractive properties, and the exceptionally well-developed methods to investigate the molecular cell biology of B. subtilis, make sporulation an extremely powerful experimental system. There are also important practical reasons for studying sporulation. First, cell cycle events such as replication, segregation and division are underexploited potential targets for antibacterials with novel modes of action, which are desperately needed for the fight against antibacterial resistance. Second, for a number of important bacterial pathogens, including Clostridium difficile, B. anthracis and B. cereus, endospore formation is an important factor in pathogenesis.
Although the transcriptional regulation of sporulation has been extensively studied and most of the key factors have been identified and characterized, almost nothing was known about regulation at the translational level. In the MBD project, I studied the role of translation regulation on the gene expression regulation of the factors involved in the process of spore formation in a Bacillus subtilis.
The project is primarily basic research but as described above, the results have potential impact in at least three areas. The first will be a better understanding of the basic physiology and regulatory process in bacteria. Although a non-pathogenic "model" bacterium, B. subtilis is closely related to various important pathogens, including B. anthracis, B. cereus, various clostridia, Listeria monocytogenes, and various Gram-positive cocci (Staphylococcus, Streptococcus, etc). New basic understanding of B. subtilis will inform and enhance our ability to predict the physiology and regulatory processes of many pathogens. Second, the cell cycle provides an important set of potential targets for novel antibiotic agents. Identifying these targets and understanding their function could facilitate the discovery and development of novel antibiotic leads. Third, B. subtilis and close relatives are important industrial organisms, responsible for the commercial production of secreted industrial enzymes, particularly various hydrolases (proteases, amylases, etc), and some small high value molecules (e.g. riboflavin). A better understanding of B. subtilis physiology and translational regulation could impact on our ability to manipulate this organism to improve the yield of these and other potential commercial products.
Objectives of the project may be summarized as follows:
Use of ribosomal profiling to elucidate new factors and regulatory processes operating during the early stages of sporulation. The main focus was on:
Switch from vegetative growth to the starvation / sporulation response
Characterise the properties of novel cell cycle and sporulation factors in terms of:
Mutant phenotype
Localization of the protein (fluorescence imaging)
Regulation relative to the well-known sporulation pathway
Identify and characterise factors involved in ribosomal reprogramming.
Work packages
Ribosome profiling
The method of ribosome profiling (RP) was set up in the laboratory and detailed protocol for RP in B. subtilis was obtained. Method was successfully applied to sporulating cells. I have carried on bioinformatical analyses on collected data and created a list of major translational changes during sporulation.
Characterisation of novel sporulation factors
From the list of changes, I have selected factors for further analyses. After training in B. subtilis genetics, I constructed gene knock-outs and reporter gene fusions for further analyses. Training in fluorescence imaging methods allowed me to carry on phenotypic and localization studies.
Transfer of knowledge
I applied RP to other projects in Errington lab. Moreover, I trained PhD student from Errington lab in the method so it can be used routinely by members of the lab.
Training
I have attended: Realize you potential - workshop for women in science; and Grant writing workshop.
Public engagement
I wrote an article for Gettyscience and volunteered in a Soapbox science initiative to promote STEM in public.
Dissemination
- Seminar presentation - I presented twice at Superlab - a weekly scientific meeting of the Centre for Bacterial Cell Biology, Newcastle University, and at AwayDay - annual meeting of the Institute for Cell and Molecular Biosciences - Newcastle University
- Conference presentation
09/2017 EMBO conference in Heidelberg, Germany. Protein Synthesis and Translational Control
06/2017 EMBO conference in Heidelberg, Germany. New Approaches and Concepts in Microbiology. Poster presentation: Quick ribosome profiling in sporulating Bacillus subtilis.
07/2016 EMBO conference in Strasbourgu, France. Ribosome Structure and Function.
- Invited lectures -
11/2017 Science Polish Perspective - Cambridge - Undercover translation. Hidden secrets of the ribosomes.
10/2017 Science Polish Perspective - meet up Berlin - Panel discussion: Science across borders: Polish-German perspectives for scientific excellence
11/2016 Institute of Bioorganic Chemistry, Polish Academy of Science in Poznan "Above and Beyond RNAseq. The lesson to be learned from the Ribosome".
Ribosome profiling
The method of ribosome profiling (RP) was set up in the laboratory and detailed protocol for RP in B. subtilis was obtained. Method was successfully applied to sporulating cells. I have carried on bioinformatical analyses on collected data and created a list of major translational changes during sporulation.
Characterisation of novel sporulation factors
From the list of changes, I have selected factors for further analyses. After training in B. subtilis genetics, I constructed gene knock-outs and reporter gene fusions for further analyses. Training in fluorescence imaging methods allowed me to carry on phenotypic and localization studies.
Transfer of knowledge
I applied RP to other projects in Errington lab. Moreover, I trained PhD student from Errington lab in the method so it can be used routinely by members of the lab.
Training
I have attended: Realize you potential - workshop for women in science; and Grant writing workshop.
Public engagement
I wrote an article for Gettyscience and volunteered in a Soapbox science initiative to promote STEM in public.
Dissemination
- Seminar presentation - I presented twice at Superlab - a weekly scientific meeting of the Centre for Bacterial Cell Biology, Newcastle University, and at AwayDay - annual meeting of the Institute for Cell and Molecular Biosciences - Newcastle University
- Conference presentation
09/2017 EMBO conference in Heidelberg, Germany. Protein Synthesis and Translational Control
06/2017 EMBO conference in Heidelberg, Germany. New Approaches and Concepts in Microbiology. Poster presentation: Quick ribosome profiling in sporulating Bacillus subtilis.
07/2016 EMBO conference in Strasbourgu, France. Ribosome Structure and Function.
- Invited lectures -
11/2017 Science Polish Perspective - Cambridge - Undercover translation. Hidden secrets of the ribosomes.
10/2017 Science Polish Perspective - meet up Berlin - Panel discussion: Science across borders: Polish-German perspectives for scientific excellence
11/2016 Institute of Bioorganic Chemistry, Polish Academy of Science in Poznan "Above and Beyond RNAseq. The lesson to be learned from the Ribosome".
From the list of factors identified in the ribosome profiling experiment, I have selected several candidate which are investigated using various approaches, including genetics (classical gene deletions and CRISPRinterference - which allows to study depletion of the essential genes). I am observing interesting phenotypes which may implicate ribosome remodelling.