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PHYSIOLOGICAL RELEVANCE OF A TRNA-DEPENDENT MECHANISM FOR MEMBRANE MODIFICATION IN ENTEROCOCCUS FAECIUM
Title
PHYSIOLOGICAL RELEVANCE OF A TRNA-DEPENDENT MECHANISM FOR MEMBRANE MODIFICATION IN ENTEROCOCCUS FAECIUM.
Creator
Harrison, Jesse, Roy, Herve, University of Central Florida
Abstract / Description
Enterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG)...
Show moreEnterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG) in the cellular envelope of bacteria. Addition of amino acids to PG confers resistance to various therapeutic antimicrobial agents, and contributes to evasion of the host immune response in a number of clinically relevant microorganisms. E. faecium possesses two distinct aaPGSs: aaPGS1 and aaPGS2. In addition, another gene coding for a putative hydrolase (pHyd) is located in the same operon as aaPGS2, and has no known function. To investigate the physiological relevance of aa-PG formation, and the function of aaPGS1, aaPGS2, and pHyd in E. faecium, we generated individual knockouts of these genes using a markerless deletion strategy. Deletion of aaPGS1 did not noticeably alter lipid aminoacylation, whereas deletion of aaPGS2 led to a loss of aa-PG synthesis. Deletion of pHyd also led to a loss of lipid aminoacylation; however, additional experiments are needed to verify that expression of aaPGS2 (which resides just downstream in the same operon) is unaffected in the pHyd-deletion strain. Development of the mutant strains described here will enable us to investigate additional phenotypes associated with these genes, and to determine whether aa-PG formation contributes to antibiotic resistance in E. faecium as in several other pathogenic microorganisms.
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Date Issued
2012
Identifier
CFH0004308, ucf:45054
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004308
Membrane topology of a broad-spectrum resistance factor responsible for lipid modification in Enterococcus faecium.
Title
Membrane topology of a broad-spectrum resistance factor responsible for lipid modification in Enterococcus faecium.
Creator
Harrison, Jesse, Roy, Herve, Teter, Kenneth, Phanstiel, Otto, University of Central Florida
Abstract / Description
Aminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against...
Show moreAminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against osmotic stress and acidic conditions. aaPGS homologs are found in a variety of clinically relevant microorganisms, including Enterococcus faecium, which is increasingly found to be the etiologic agent of antibiotic-resistant nosocomial infections. Although the broad distribution of these virulence factors across bacterial species makes them attractive targets for therapeutic strategies, little is known about the structure of aaPGSs. Two aaPGS paralogs are found in E. faecium, one of which exhibits relaxed substrate specificity and is responsible for the transfer of Arg (R), Ala (A), and Lys (K) to PG (RakPGS). The catalytic site of RakPGS is located in the hydrophilic C-terminal domain, which is localized in the cytoplasm. The N-terminus contains an integral membrane domain that is thought to harbor flippase activity that translocates the neosynthesized aa-PG from the inner to the outer leaflet of the membrane. We are currently developing the substituted cysteine accessibility method (SCAM) and a dual-reporter fusion system, which exploits alkaline phosphatase (Pho) and ?-galactosidase (LacZ) activities, for investigating the membrane topology of RakPGS in E. faecium.
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Date Issued
2015
Identifier
CFE0006318, ucf:51566
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006318