Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

Abstract

ABSTRACT Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef -A), membrane pump Ef -B, biotin acceptor protein Ef -D, and the novel subunit Ef -H. Our results show that in E. faecalis , subunits Ef -A, Ef -D, and Ef -H form a cytoplasmic soluble complex (termed Ef -AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef -H subunit, coexpression of oad genes was performed in Escherichia coli , showing that this subunit is vital for Ef -A and Ef -D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB -deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef -AHD complex is functional in E. faecalis. These results suggest that the Ef -AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef -B subunit to achieve its maximal alkalinization capacity during citrate fermentation.