Bacterial filamentation as a survival strategy : identification and characterisation of a novel cell division inhibitor in Escherichia coli

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Bacterial cell division is tightly regulated to ensure that division occurs at the correct time and position in order to create two viable, genetically identical, daughter cells. In addition to correct timing and positioning, the inhibition of division is also important for survival in certain conditions. This inhibition of division results in filamentous cells, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. This is an important survival mechanism utilised by several bacteria in response to an environmental stimuli, including during pathogenesis and exposure to antibiotics. However, the underlying mechanisms of filamentation and regulators of cell division that enable this unique morphology remain largely unknown. A recent high-throughput over-expression screen in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 identified several potential division inhibitors, including 𝘺𝘮𝘧𝘔, a gene encoded within the e14 prophage. The overall aim of this thesis was to understand the biological condition in which YmfM may be functioning, as well as how it may be acting to inhibit division. The initial aim of this thesis was to verify the genes from the original screen that are responsible for causing filamentation. From this the expression of 𝘺𝘮𝘧𝘔 was shown to cause a complete inhibition of cell division and became the primary focus of this work. The e14 prophage is thought to encode for an SOS inducible cell division inhibitor, SfiC. However the exact gene responsible for this is unknown. In this thesis, YmfM was identified to be SfiC and is up-regulated by the SOS response. The inhibition of cell division during SOS has traditionally been attributed to SulA, which inhibits FtsZ polymerization and is activated by the RecA pathway. However, we have identified the likely role of 𝘺𝘮𝘧𝘔 in inhibiting division during SOS, suggesting that alternative pathways exist during stress. Bioinformatics analysis identified the context in which 𝘺𝘮𝘧𝘔 functions; it is conserved to 𝘌. 𝘤𝘰𝘭𝘪 and closely related gram-negative bacteria. Further, it was shown to be likely that 𝘺𝘮𝘧𝘔 functions with two other genes within the e14 prophage, 𝘺𝘮𝘧𝘓 and 𝘰𝘸𝘦𝘌. Finally, the initial characterisation of the mechanism of action of YmfM indicates that it inhibits division at the level of FtsZ ring assembly (early stages of division) and is independent of known cell division inhibitors SulA, MinC and SlmA. While more work is needed to fully characterise YmfM, this work highlights that there are multiple pathways which may inhibit cell division during stress and raises the question of the beneficial role of prophage encoded inhibitors for bacterial survival. Having a greater understanding of filamentation will not only enable us to understand the intricacies of cell division inhibition, but also how bacteria are able to cope with stress.
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