Bacterial filamentation as a survival strategy: searching for novel cell division regulators in Mycobacteria. — The Association Specialists
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Bacterial filamentation as a survival strategy: searching for novel cell division regulators in Mycobacteria. (348)

Samuel J Burns 1 , Catherine Burke 1 , Warwick Britton 2 3 , James A Triccas 2 3 , Torsten Thomas 4 , Elizabeth J Harry 1
  1. ithree institute, University of Technology, Sydney, Sydney, NSW, Australia
  2. Centenary Institute of Cancer Medicine and Cell Biology, Sydney, NSW, Australia
  3. Department of Infectious Diseases and Immunology, The University of Sydney, Sydney, NSW, Australia
  4. School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, Sydney, NSW, Australia

Background

Bacilli can survive changes in their environment by forming filaments; cell division is inhibited while growth and DNA replication continue, giving rise to very long cells (up to 40 µm). Filamentation has been observed in both non-pathogenic and pathogenic bacteria. In Mycobacterium tuberculosis (the cause of tuberculosis which killed 1.4 million people in 2011) filamentation has been proposed to be required for replication and persistence within macrophages. The process by which filamentation occurs is not well understood. However, understanding filamentation can aid in identifying opportunities for new therapeutics. In addition, this work will explore cell division in Mycobacteria as they are missing many of the key genes present in model organisms like Escherichia coli and Bacillus subtilis.

Aim

Identify and characterise novel proteins that regulate cell division in Mycobacteria and allow its persistence in disease.

Method

Screening of expression libraries of Mycobacterium bovis BCG gDNA in Mycobacterium smegmatis and screening the clones for a filamentous phenotype in a novel approach using flow cytometry. Filamentation is confirmed by microscopy, genes sequenced and investigated.

Results

The method for flow cytometry screening was verified through the screen of a library of environmental DNA. A preliminary screen has been done on a BCG gDNA library in E. coli which will be followed by a screen of BCG gDNA in Mycobacterium smegmatis.To achieve this it has been shown M. smegmatis forms filaments (up to 30 µm) under conditions which prevent division, making it suitable for flow cytometry screening of filamentous cells.

Conclusion

The use of flow cytometry for sorting bacterial populations based on the filamentous phenotype is effective. M. smegmatis can filament extensively, enabling flow-cytometry based identification of these cells in screening the BCG expression library. Subsequent library ‘hits’ will allow identification and characterization of novel cell division genes and regulators in Mycobacteria.