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- Title
- IN-FRAME MUTAGENESIS OF GENES ENCODING A SELENIUM-DEPENDENT MOLYBDENUM HYDROXYLASE AND PUTATIVE ACCESSORY PROTEINS IN ENTEROCOCCUS FAECALIS.
- Creator
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Mallard, Christopher, Self, William, University of Central Florida
- Abstract / Description
-
Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative selenium-dependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play...
Show moreEnterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative selenium-dependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.
Show less - Date Issued
- 2010
- Identifier
- CFE0003420, ucf:53152
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003420
- Title
- Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis.
- Creator
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Nelson, Samantha, Self, William, Moore, Sean, Rohde, Kyle, University of Central Florida
- Abstract / Description
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A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH),...
Show moreA previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.
Show less - Date Issued
- 2014
- Identifier
- CFE0005388, ucf:50455
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005388