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Elucidation of the Molecular Mechanisms Underlying the Production of Cyanobacterial Extracellular Polymeric Substances (EPS).

紀錄類型:	書目-電子資源 : Monograph/item
Title/Author:	Elucidation of the Molecular Mechanisms Underlying the Production of Cyanobacterial Extracellular Polymeric Substances (EPS)./
Author:	Povkhanych, Maksym.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2021,
Description:	48 p.
Notes:	Source: Dissertations Abstracts International, Volume: 84-01, Section: B.
Contained By:	Dissertations Abstracts International84-01B.
Subject:	DNA methylation. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29150595
ISBN:	9798835567515

Elucidation of the Molecular Mechanisms Underlying the Production of Cyanobacterial Extracellular Polymeric Substances (EPS).
Povkhanych, Maksym.

Elucidation of the Molecular Mechanisms Underlying the Production of Cyanobacterial Extracellular Polymeric Substances (EPS). - Ann Arbor : ProQuest Dissertations & Theses, 2021 - 48 p.

Source: Dissertations Abstracts International, Volume: 84-01, Section: B.

Thesis (M.Sc.)--Universidade do Porto (Portugal), 2021.

This item must not be sold to any third party vendors.

Many bacteria produce extracellular polymeric substances (EPS), mainly composed of heteropolysaccharides, that fulfil different biological roles depending on the organism and its environment. Due to their distinctive properties, the EPS produced by cyanobacteria are a promising resource for biotechnological and biomedical applications. However, it is necessary to unveil their biosynthetic pathway(s) to optimize their production. Previously, it was shown that cyanobacteria harbour genes encoding proteins related to the three main bacterial pathways of assembly and export of EPS, but often the complete set of proteins from a single pathway is absent. Mutational analysis using the model cyanobacterium Synechocystis sp. PCC 6803 confirmed the involvement of proteins from different pathways in the production of EPS, including the ABC transporter component Slr0977 (KpsM) and the polysaccharide copolymerase Sll0923 (Wzc). Together, these results suggest that the production of cyanobacterial EPS may not strictly follow one of the pathways previously characterized for other bacteria and raises the hypothesis of a crosstalk between different pathways. To clarify this hypothesis, it is important to continue elucidating the mechanisms of EPS production by identifying the functional partners of proteins related to different pathways.The aim of this work is to generate the molecular tools (plasmids) and develop the methodology to further characterize Synechocystis Wzc and KpsM, including the identification of their functional partners and/or in vivo characterization. For that, and moving from previous results, pull-down assays were performed using purified His6-Wzc as bait and Synechocystis protein extracts as target. The isolated proteins are being identified by mass spectrometry. In addition, to elucidate the proteins that interact with KpsM, an in vivo chemical crosslink followed by pull-down assays will be performed. For that, the sequences encoding the tagged version of the protein were cloned in a replicative vector under the regulation of different promoters. For one of the plasmids, the sequence was confirmed, and the plasmid will be used to transform the Synechocystis kpsM knockout mutant. In addition, the generation of plasmids to complement an Escherichia coli acapsular mutant with Synechocystis Wzc or its truncated version (without the C-terminal domain where its phosphorylation/dephosphorylation occurs) and evaluate its ability to restore capsule formation was initiated, with the final goal of clarifying the role of Wzc in an in vivo system.

ISBN: 9798835567515Subjects--Topical Terms:

3560639
DNA methylation.

Elucidation of the Molecular Mechanisms Underlying the Production of Cyanobacterial Extracellular Polymeric Substances (EPS).
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100 1 $a Povkhanych, Maksym. $3 3688035
245 1 0 $a Elucidation of the Molecular Mechanisms Underlying the Production of Cyanobacterial Extracellular Polymeric Substances (EPS).
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2021
300 $a 48 p.
500 $a Source: Dissertations Abstracts International, Volume: 84-01, Section: B.
500 $a Advisor: Pereira, Sara Bernardes;Tamagnini, Paula.
502 $a Thesis (M.Sc.)--Universidade do Porto (Portugal), 2021.
506 $a This item must not be sold to any third party vendors.
520 $a Many bacteria produce extracellular polymeric substances (EPS), mainly composed of heteropolysaccharides, that fulfil different biological roles depending on the organism and its environment. Due to their distinctive properties, the EPS produced by cyanobacteria are a promising resource for biotechnological and biomedical applications. However, it is necessary to unveil their biosynthetic pathway(s) to optimize their production. Previously, it was shown that cyanobacteria harbour genes encoding proteins related to the three main bacterial pathways of assembly and export of EPS, but often the complete set of proteins from a single pathway is absent. Mutational analysis using the model cyanobacterium Synechocystis sp. PCC 6803 confirmed the involvement of proteins from different pathways in the production of EPS, including the ABC transporter component Slr0977 (KpsM) and the polysaccharide copolymerase Sll0923 (Wzc). Together, these results suggest that the production of cyanobacterial EPS may not strictly follow one of the pathways previously characterized for other bacteria and raises the hypothesis of a crosstalk between different pathways. To clarify this hypothesis, it is important to continue elucidating the mechanisms of EPS production by identifying the functional partners of proteins related to different pathways.The aim of this work is to generate the molecular tools (plasmids) and develop the methodology to further characterize Synechocystis Wzc and KpsM, including the identification of their functional partners and/or in vivo characterization. For that, and moving from previous results, pull-down assays were performed using purified His6-Wzc as bait and Synechocystis protein extracts as target. The isolated proteins are being identified by mass spectrometry. In addition, to elucidate the proteins that interact with KpsM, an in vivo chemical crosslink followed by pull-down assays will be performed. For that, the sequences encoding the tagged version of the protein were cloned in a replicative vector under the regulation of different promoters. For one of the plasmids, the sequence was confirmed, and the plasmid will be used to transform the Synechocystis kpsM knockout mutant. In addition, the generation of plasmids to complement an Escherichia coli acapsular mutant with Synechocystis Wzc or its truncated version (without the C-terminal domain where its phosphorylation/dephosphorylation occurs) and evaluate its ability to restore capsule formation was initiated, with the final goal of clarifying the role of Wzc in an in vivo system.
520 $a Varias bacterias produzem substancias polimericas extracelulares (EPS), compostas principalmente por heteropolissacarideos que cumprem diferentes funcoes biologicas dependendo do organismo e do seu meio ambiente. Devido as suas propriedades distintas, os EPS produzidos por cianobacterias sao um recurso promissor para aplicacoes biotecnologicas e biomedicas. No entanto, e necessario elucidar a(s) sua(s) via(s) biossintetica(s) para otimizar a sua producao. Foi demonstrado previamente que as cianobacterias contem genes que codificam proteinas relacionadas com as tres principais vias bacterianas de montagem e exportacao de EPS; contudo, o conjunto completo de proteinas de uma unica via esta frequentemente ausente. A geracao de mutantes utilizando a cianobacteria modelo Synechocystis sp. PCC 6803 confirmou o envolvimento de proteinas de diferentes vias na producao de EPS, incluindo o componente transportador ABC Slr0977 (KpsM) e a proteina da familia "polysaccharide co-polimerase" Sll0923 (Wzc). Em conjunto, estes resultados sugerem que a producao de EPS em cianobacterias pode nao seguir estritamente uma das vias previamente caracterizadas para outras bacterias e levanta a hipotese de haver um cruzamento (ou crosstalk) entre diferentes vias. Para esclarecer esta hipotese, e importante continuar a elucidar os mecanismos de producao de EPS, identificando os parceiros funcionais das proteinas relacionadas com as diferentes vias.O objetivo deste trabalho e gerar as ferramentas moleculares (plasmideos) e desenvolver a metodologia para posterior caracterizacao do Wzc e KpsM de Synechocystis, incluindo a identificacao dos seus parceiros funcionais. Para tal, e partindo de resultados anteriores, foram realizados ensaios de "pull-down" utilizando a proteina His6-Wzc purificado como "bait" e extratos proteicos de Synechocystis como "target". As proteinas isoladas estao a ser identificadas por espetrometria de massa. Para elucidar as proteinas que interagem com o KpsM, serao realizados ensaios de "crosslink" in vivo seguidos de "pull-down". Para tal, as sequencias que codificam a versao da proteina com uma cauda de poli-histidina foram clonadas num vetor replicativo sob a regulacao de diferentes promotores. Para um dos plasmideos, a sequencia foi confirmada e o plasmideo sera utilizado para transformar o mutante Synechocystis kpsM. Alem desta metodologia, foi iniciada tambem a geracao de plasmideos para complementar um mutante acapsular de E. coli com o Wzc de Synechocystis ou a sua versao truncada (sem o dominio C-terminal onde ocorre a sua fosforilacao/desfosforilacao) e avaliada a capacidade do mutante em restaurar a formacao da capsula, clarificando assim o papel do Wzc num sistema in vivo.
590 $a School code: 5896.
650 4 $a DNA methylation. $3 3560639
650 4 $a Polymers. $3 535398
650 4 $a Plasmids. $3 3319070
650 4 $a ABC transporters. $3 3688036
650 4 $a Phosphatase. $3 3561803
650 4 $a Biosynthesis. $3 516407
650 4 $a Carbon. $3 604057
650 4 $a Cloning. $3 571606
650 4 $a Binding sites. $3 3560349
650 4 $a Bacteria. $3 550366
650 4 $a Genetic engineering. $3 530509
650 4 $a DNA polymerase. $3 3561751
650 4 $a Molecular weight. $3 3683783
650 4 $a Genomes. $3 592593
650 4 $a E coli. $3 3556834
650 4 $a Cyanobacteria. $3 860872
650 4 $a Chromatography. $3 1073639
650 4 $a Phosphorylation. $3 816803
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650 4 $a Kinases. $3 3558077
650 4 $a Biochemistry. $3 518028
650 4 $a Bioengineering. $3 657580
650 4 $a Chemistry. $3 516420
650 4 $a Engineering. $3 586835
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650 4 $a Polymer chemistry. $3 3173488
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