Characterisation and analysis of polar growth proteins within rod-shaped bacterial species

Hutchinson, Alec Jack Cameron (2023) Characterisation and analysis of polar growth proteins within rod-shaped bacterial species. Masters thesis, University of East Anglia.

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Polar growth is the main mechanism used by most rod-shaped bacteria to drive growth within their life cycle. Known phylum that use polar growth include the Gram-positive Actinobacteria and the Gram-negative Pseudomonadota. Many of the Gram-positive bacteria utilise a homologue of the large DivIVA family as a vital protein in their growth mechanism due to its innate ability to localise to the pole of the cell. Whilst DivIVA naturally accumulates at the pole, the partner proteins of DivIVA that build up the growth mechanism are recruited and regulated to drive functional polar growth, though the range of partner proteins of DivIVA vary by species. A well-documented example of this the Tip Organising Centre (TIPOC) in the Actinobacteria Streptomyces coelicolor. S. coelicolor is a GC-rich, soil-dwelling filamentous bacteria thoroughly researched for its vast capacity for secondary metabolite production. S. coelicolor grows through polar growth at the tip of its filamentous hyphae utilising the TIPOC: a large multi-protein complex. This complex is constructed from three smaller protein complexes (DivIVA, Scy and FilP) and is known for its ability to interact with other important cellular mechanisms such as the ParAB system (used for chromosome segregation). However, unlike Gram-positive bacteria, many of the Gram-negative bacteria lack a DivIVA homologue and a common mechanism across these species is yet to be found. Though a new protein called Growth Pole Ring (GPR) has been identified recently in Agrobacterium tumefaciens which affects polar growth.

In this study, we have characterised a new potential partner protein of the TIPOC encoded by the previously researched gene SCO5569. SCO5569 has been designated dia due to its ability to affect the determined hyphal diameter during active polar growth. We used: computational analysis of Dia and other homologs to infer structural properties and functions of the protein, in vivo BACTH assays to test for direct protein:protein interactions and in vitro SDS-PAGE to analyse the oligomerisation of Dia. The computational analysis revealed Dia is highly prevalent in many Grampositive Actinobacteria species and a few Gram-negative phyla. Dia and its homologs all share a conserved region between residues 35-144 which was inferred to possess the function of DivIVA and ATP synthase subunit B domains. We indicated that Dia is capable of self-interaction along with direct interaction with DivIVA, Scy, FilP, ParB and ParH. These interactions suggest that Dia may be novel components of both the TIPOC and ParAB system of S. coelicolor. The isolated and overexpressed oligomerised Dia likely forms a dimer which was resistant to high temperature (95°C) and ί-mercaptoethanol. Alongside investigating dia, we investigated possible homologues of GPR as alternatives to DivIVA for Gram-negative rod-shaped bacteria that possess polar growth. We identified an extensive range of GPR homologs throughout Gram-negative bacteria. We analysed and localised a protein in the a-proteobacteia Labrenzia aggregata which showed signs of similarity, though very distantly related. The L. aggregata GPR homologs appeared to retain a conserved region of GPR between residues 278-1815 which possessed domains of GPR, some linked to its role in polar growth. We designated this protein Lcy and used in vivo fluorescent microscopy to find that Lcy localises at the pole of L. aggregata throughout its life cycle including during cell division. This study has helped to reveal a new potential TIPOC component which may directly interact with the ParAB system. This suggests a new link between the mechanisms that drive polar growth and chromosome segregation in bacteria. Finally, we have demonstrated that, as an alternative to DivIVA, homologs of GPR are more prevalent in Gram-negative bacteria, even distantly related species. This distantly related homolog appeared to retain the function for polar growth.

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 14 Dec 2023 14:19
Last Modified: 14 Dec 2023 14:19

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