Two putative Enterococcus faecalis fabG genes do not encode β-ketoacyl-acyl carrier protein reductases

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Enterococcus faecalis possesses three putative beta-ketoacyl-acyl carrier protein (ACP) reductases (FabG).
• FabG1 is part of the main fatty acid synthesis operon, while FabG2 and FabG3 are located elsewhere.
• Understanding the functional roles of these FabG enzymes is crucial for comprehending fatty acid biosynthesis in E. faecalis.

Purpose of the Study:

• To determine the functional significance of the three putative FabG enzymes in Enterococcus faecalis.
• To identify which FabG enzyme is responsible for the essential beta-ketoacyl-ACP reductase activity.
• To investigate the regulation of fabG2 expression.

Main Methods:

• Complementation assays using an Escherichia coli fabG(Ts) strain.
• Gene deletion analysis of E. faecalis fabG1.
• Growth studies of mutant strains with and without oleic acid supplementation.
• Analysis of fabG2 expression using a beta-galactosidase transcriptional fusion and investigating the role of FabT.

Main Results:

• Only Enterococcus faecalis FabG1 could complement the growth of the E. coli fabG(Ts) strain at the non-permissive temperature.
• Deletion of E. faecalis fabG1 resulted in an auxotrophic strain requiring oleic acid for growth.
• Plasmid-borne fabG1 restored growth, while fabG2 and fabG3 could not.
• FabG2 expression was not affected by fabT deletion or fatty acid supplementation, despite a putative FabT binding site.

Conclusions:

• Enterococcus faecalis FabG1 is the only functional beta-ketoacyl-ACP reductase essential for fatty acid synthesis.
• FabG2 and FabG3 are likely non-functional or have redundant roles not essential for growth under tested conditions.
• The regulation of fabG2 by FabT appears minimal or absent in E. faecalis.