Bacterial filamentation as a survival strategy : a goldmine for the discovery of new cell division regulators
- Publication Type:
- Thesis
- Issue Date:
- 2016
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Mycobacterium tuberculosis the causative organism of tuberculosis has been plaguing humanity for centuries. The number of effective antibiotics is dwindling due to the rise of multi-drug resistance within the species and new drugs need to be developed that target essential components of the bacterial life cycle. Bacterial cell division is an essential and highly conserved process across bacteria and new drugs that target this process could have broad-spectrum implications.
Bacilli can survive changes in their environment by forming filamentous cells, where 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, including Escherichia coli and Mycobacterium tuberculosis where it has been proposed to be required for replication and persistence within the human host. The process by which filamentation occurs in bacteria is not well understood. However, understanding filamentation can aid in identifying opportunities for new therapeutics and in addition, explore cell division in Mycobacteria as they are missing many of the key cell division genes present in model organisms like Escherichia coli and Bacillus subtilis.
The overall aim of this work was to use flow cytometry-based cell sorting to identify and characterize novel proteins that regulate cell division in Mycobacteria and allow persistence in mycobacterial disease. This was done by screening expression libraries of Mycobacterium bovis BCG genomic DNA (gDNA) hosted in E. coli and later Mycobacterium smegmatis, to identify clones expressing cell division proteins and regulatory genes via a filamentous phenotype.
The method for flow cytometry screening had to first be verified through the completion of a screen of a library of environmental DNA collected from the marine algae Ulva australis. Large environmental DNA inserts were sub-cloned and re-screened using flow cytometry-based cell sorting to identify genes causing filamentation when expressed. One reproducibly filamentous clone contained the Periplasmic Binding Protein Type-1 Superfamily conserved domain and we found that the overexpression of this gene caused a filamentous phenotype, which in turn showed that a single gene causing a filamentous phenotype could be identified with the flow cytometry based cell sorting method.
A library of M. bovis BCG gDNA was constructed and hosted in E. coli. This library was screened using flow cytometry-based cell sorting but no filamentous clones were found. The host species was then changed to M. smegmatis for better expression of heterologous genes and a modified expression vector utilizing the TET-ON/OFF inducible expression system was shown to work for the expression of cloned genes. Unfortunately after repeated attempts, a library of M. bovis BCG gDNA was unable to be constructed and screened for mycobacterial cell division genes and regulators.
Bacterial filamentation and cell division are important areas of investigation for clinically relevant bacteria. The information that can be gleaned from these investigations may lead to the next generation of antimicrobials.
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