Enterococcus faecalis NADH Peroxidase-Defective Mutants Stain Falsely in Colony Zymogram Assay for Extracellular Electron Transfer to Ferric Ions

Enterococcus faecalis cells can reduce ferric ions and other electron acceptors by extracellular electron transfer (EET). To find mutants with enhanced or defective EET, strain OG1RF with random transposon insertions in the chromosome was screened for ferric reductase activity by colony zymogram staining using the chromogenic ferrous-chelating compound Ferrozine. The screen revealed npr, eetB, and ndh3 mutants. The aberrant ferric reductase phenotype of Npr (NADH peroxidase)-defective mutants was found to be a property of colonies and not apparent with washed cells grown in liquid culture. EetB- and Ndh3-defective mutants, in contrast, consistently showed low ferric reductase activity. It is concluded that colony zymogram staining for ferric reductase activity using Ferrozine can be misleading, especially through false negative results. It is suggested that hydrogen peroxide produced in the colony quenches the zymogram staining. In addition, it is demonstrated that the negative effect of heme on EET to ferric ion in E. faecalis is relieved by cytochrome bd deficiency. The findings can help to identify bacteria with EET ability and contribute to our understanding of EET in Gram-positive bacteria and the physiology of E. faecalis.