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Multicopy suppression of the folA null mutation escherichia coli

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Multicopy suppression of the folA null mutation escherichia coli

Chirwa, Neema Tiyowoyechi (2004) Multicopy suppression of the folA null mutation escherichia coli. PhD thesis, Concordia University.

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Abstract

In Escherichia coli , Dihydrofolate reductase coded by the folA gene plays a central role. It is required for the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced by cells synthesizing thymidylate. Deletion of the folA gene in E. coli K-12 strains is not lethal but generates auxotrophies when glycine or methionine is omitted from a combination of supplements containing pantothenate, thymidine, adenine and histidine. We have isolated pSD6P, which contains part of the csgD sequence, as a multicopy suppressor of the glycine auxotrophy. Multicopy suppressors are genes that when overexpressed alleviates one or more growth requirements of a strain. CsgD codes for a transcriptional regulator required for the synthesis of curlin subunits that are used in the synthesis of extracellular matrix important for biofilm formation. Curli fibers are also thought to be important for infection of host cells and confer additional protection against damaging agents or predators in the environment. We have shown that increasing the expression of the glyA gene can alleviate the glycine auxotrophy of a folA null strain. Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is required for the conversion of serine to glycine. We have measured SHMT activity and shown that cells expressing csgD from multicopy plasmid have increased levels of activity. We have also monitored the effect of CsgD on transcription of genes involved in one-carbon metabolism using a lac-based reporter system. We have shown that the presence of CsgD increases the transcription of the glyA and purU genes. PurU codes for a formyltetrahydrofolate hydrolase and is important for balancing the cell's need for one-carbon units and glycine. The role of CsgD is not limited to curli or one-carbon metabolism. Our results suggest that expression of hmp , a gene adjacent to glyA , is slightly induced. Hmp codes for a flavohaemoglobin with denitrification properties. Interestingly, the csgD -mediated induction of the glyA gene but not of hmp requires the known regulators (MetR and PurR) of glyA transcription.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Chirwa, Neema Tiyowoyechi
Pagination:xi, 119 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Ph. D.
Program:Chemistry and Biochemistry
Date:2004
Thesis Supervisor(s):Herrington, M. B
ID Code:7830
Deposited By:Concordia University Libraries
Deposited On:18 Aug 2011 14:07
Last Modified:19 Aug 2011 04:10
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