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Sporulation septation in Streptomyces as a model to investigate bacterial cell division. / Suliman Ali Elgadi

Swansea University Author: Suliman Ali Elgadi

Abstract

Streptomyces are interesting gram positive filamentous bacteria and have been studied mostly in the context of antibiotic production. This system is controlled by specific networks of genes through regulatory signals that combine to regulate both morphological and physiological differentiation in th...

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Published: 2012
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa43201
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Abstract: Streptomyces are interesting gram positive filamentous bacteria and have been studied mostly in the context of antibiotic production. This system is controlled by specific networks of genes through regulatory signals that combine to regulate both morphological and physiological differentiation in the organism. But it is in the context of growth and cell division that they are also fascinating. FtsW is one of four Shape, Elongation, Division and Sporulation (SEDS) family proteins encoded in the genome of S. coelicolor. In this study the effect of ftsW complementation, and its overexpression in S. coelicolor, and role of other related proteins was studied. Complementation of an ftsW mutant resulted in a sporulating phenotype confirming that the white phenotype was due to ftsW disruption, and ftsW function was restored by the complementing plasmid. Macroscopically, fts W or ^/com plem entation or overexpression in S. coelicolor does not show any difference in phenotype. In addition, the microscopic analysis revealed that there is no effect of ftsW or ftsl overexpression on the sporulation septation and chromosomal condensation of aerial hyphae. FtsW localization was investigated revealing a diffuse distribution of fluorescence in the aerial hyphae of an ftsWcomplemented strain, due to in vivo proteolytic cleavage of the FtsW-mCherry translational fusion protein, precluding any inference of FtsW localisation itself. Also mycobacterial orthologs of FtsW and Ftsl cannot replace the function of their related streptomycete proteins. By construction of triple mutants of ftsW, sfr and rodA2 SEDS genes in one strain of S.coelicolor, results showed that these genes are dispensable for growth and survival. No difference was observed between the triple mutant strain that exhibited a white non-sporulating phenotype, and the fts W single mutant phenotype. An absence of sporulation septa in both the triple mutant and ftsW single mutant aerial hyphae, while an sfrlrodA2diO\xb\Q mutant has normal sporulation septa, confirming that FtsW is required specifically for sporulation septation. In contrast and from the results of fluorescence microscopical analysis of the vegetative mycelium of the triple mutant strain and the wild type strain, a similar staining pattern of vegetative septa were observed, suggesting that these genes (ftsW, sfr and rodA2) are not required for vegetative septation in S. coelicolor. In addition, in order to understand the cell division mechanism in S. coelicolor more clearly, the Bacterial Adenylate Cyclase Two- Hybrid (BACTH) system used to study protein-protein interactions. Proteins tested were FtsW, Ftsl, FtsZ, FtsQ, CrgA, Sfr, MreB, RodA, RodA2, penicillin binding proteins PBP1, PBP2 and PBP3) in several combinations of protein pairs. Notably, the results showed a strong interaction between CrgA and FtsQ, and also with other cell division proteins, suggesting aivcentral role for CrgA in the cell division process. According to this significant results, alignment of the S', coelicolor CrgA sequence with orthologs from other Actinobacteria was carried out, revealing only four amino acids G4 0, W45, N65 and Ws3 that are well conserved. After site directed mutagenesis to modify S. coelicolor CrgA, this revealed that the amino acids N65 and Wg3 are required for interaction of the protein with itself and PBP2.
Keywords: Streptomyces, bacteria
College: Faculty of Medicine, Health and Life Sciences