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Cloning and purification of the two component system GraSR involved in glycopeptide antibiotic resistance in Staphylococcus aureus (Mu5o), and characterization of the Citrate/methylcitrate synthase from Bacillus subtilis strain 168

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Jeremiah Onyemachi (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Dansantila Golemi-Kotra

Abstract: Staphylococcus aureus is a Gram-positive bacterium that causes varieties of serious infections, and thus remains a significant threat to public health. The Mu50 glycopeptide-resistant S.aureus strain has reduced susceptibility to vancomycin, a glycopeptide antibiotic that is heavily relied on for the treatment of infections caused by S.aureus. The two component system GraSR genes from Mu50 glycopeptide-resistant S.aureus have been identified as a mediator in a rapid signal transduction cascade that occurs when the bacteria cell wall is stressed by antibiotics. Mu50 S.aureus, GraS and GraR were successfully cloned, expressed and purified using Glutathione Sepharose 4B for GraS which is designed for a single step purification of GST fusion protein, while GraR was purified using Fast Protein Liquid Chromatography with an anion exchanger resin DEAE Sepharose. The proteins are now ready for subsequent kinetics studies to investigate their role in cell wall stress by antibiotics, and regulation in cell wall biosynthesis. The Analysis of the Native Molecular Weight of the Citrate/Methylcitrate Synthase mmgD from strain 168" of Bacillus subtilis, a rod shape gram-positive bacterium, capable of forming endospore during inadequate supply of nutrients, and the mother cell metabolic gene (mmg) is one operon that is expressed during this sporulation. The mmgD protein is one of the six open reading frames (ORFs) of the mmg operon and was shown previously to be a combined citrate/methylcitrate synthase. The goal of this work was to determine the native molecular weight of this protein. Gel filtration chromatography is one of the methods use in determining the native state of a protein by comparing to known standard proteins molecular weights. The previously cloned mmgD gene was expressed, purified and subjected to gel filtration chromatography to establish its apparent native molecular weight. The analysis showed that a major fraction of the protein existed as a monomer while a very small fraction eluted from this column showed an aggregation of proteins.

Additional Information

Publication
Thesis
Language: English
Date: 2011
Keywords
Cloning, Two Component System GraSR, Purification, Staphylococcus aureus (Mu5o), Bacillus subtilis Strain 168, Citrate/Methylcitrate Synthase
Subjects
Staphylococcus aureus $x Molecular aspects
Molecular cloning
Bacillus subtilis $x Research