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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

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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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fullrecord <?xml version="1.0"?><rfc1807><datestamp>2018-08-29T14:37:18.7345314</datestamp><bib-version>v2</bib-version><id>43201</id><entry>2018-08-02</entry><title>Sporulation septation in Streptomyces as a model to investigate bacterial cell division.</title><swanseaauthors><author><sid>9a8a1b4b1baceea923153233c2f61171</sid><ORCID>NULL</ORCID><firstname>Suliman Ali</firstname><surname>Elgadi</surname><name>Suliman Ali Elgadi</name><active>true</active><ethesisStudent>true</ethesisStudent></author></swanseaauthors><date>2018-08-02</date><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.</abstract><type>E-Thesis</type><journal/><journalNumber></journalNumber><paginationStart/><paginationEnd/><publisher/><placeOfPublication/><isbnPrint/><issnPrint/><issnElectronic/><keywords>Streptomyces, bacteria</keywords><publishedDay>31</publishedDay><publishedMonth>12</publishedMonth><publishedYear>2012</publishedYear><publishedDate>2012-12-31</publishedDate><doi/><url/><notes/><college>COLLEGE NANME</college><department>Swansea University Medical School</department><CollegeCode>COLLEGE CODE</CollegeCode><institution>Swansea University</institution><degreelevel>Doctoral</degreelevel><degreename>Ph.D</degreename><apcterm/><lastEdited>2018-08-29T14:37:18.7345314</lastEdited><Created>2018-08-02T16:24:31.5998109</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Medicine</level></path><authors><author><firstname>Suliman Ali</firstname><surname>Elgadi</surname><orcid>NULL</orcid><order>1</order></author></authors><documents><document><filename>0043201-02082018162554.pdf</filename><originalFilename>10821593.pdf</originalFilename><uploaded>2018-08-02T16:25:54.3900000</uploaded><type>Output</type><contentLength>14901747</contentLength><contentType>application/pdf</contentType><version>E-Thesis</version><cronfaStatus>true</cronfaStatus><embargoDate>2018-08-02T16:25:54.3900000</embargoDate><copyrightCorrect>false</copyrightCorrect></document></documents><OutputDurs/></rfc1807>
spelling 2018-08-29T14:37:18.7345314 v2 43201 2018-08-02 Sporulation septation in Streptomyces as a model to investigate bacterial cell division. 9a8a1b4b1baceea923153233c2f61171 NULL Suliman Ali Elgadi Suliman Ali Elgadi true true 2018-08-02 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. E-Thesis Streptomyces, bacteria 31 12 2012 2012-12-31 COLLEGE NANME Swansea University Medical School COLLEGE CODE Swansea University Doctoral Ph.D 2018-08-29T14:37:18.7345314 2018-08-02T16:24:31.5998109 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Suliman Ali Elgadi NULL 1 0043201-02082018162554.pdf 10821593.pdf 2018-08-02T16:25:54.3900000 Output 14901747 application/pdf E-Thesis true 2018-08-02T16:25:54.3900000 false
title Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
spellingShingle Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
Suliman Ali Elgadi
title_short Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
title_full Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
title_fullStr Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
title_full_unstemmed Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
title_sort Sporulation septation in Streptomyces as a model to investigate bacterial cell division.
author_id_str_mv 9a8a1b4b1baceea923153233c2f61171
author_id_fullname_str_mv 9a8a1b4b1baceea923153233c2f61171_***_Suliman Ali Elgadi
author Suliman Ali Elgadi
author2 Suliman Ali Elgadi
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publishDate 2012
institution Swansea University
college_str Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description 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.
published_date 2012-12-31T03:54:27Z
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