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

Hederstedt, Lars LU (2023) In Microorganisms 11(1).
Abstract

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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cytochrome bd, EetB, Enterococcus faecalis, ferric reductase, Ferrozine, heme, Npr
in
Microorganisms
volume
11
issue
1
article number
106
publisher
MDPI AG
external identifiers
  • scopus:85146638283
  • pmid:36677398
ISSN
2076-2607
DOI
10.3390/microorganisms11010106
language
English
LU publication?
yes
id
13176cf1-b2b1-40f4-b1f1-bb88f5ccbaea
date added to LUP
2023-02-13 15:04:29
date last changed
2024-09-20 08:57:57
@article{13176cf1-b2b1-40f4-b1f1-bb88f5ccbaea,
  abstract     = {{<p>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.</p>}},
  author       = {{Hederstedt, Lars}},
  issn         = {{2076-2607}},
  keywords     = {{cytochrome bd; EetB; Enterococcus faecalis; ferric reductase; Ferrozine; heme; Npr}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{MDPI AG}},
  series       = {{Microorganisms}},
  title        = {{Enterococcus faecalis NADH Peroxidase-Defective Mutants Stain Falsely in Colony Zymogram Assay for Extracellular Electron Transfer to Ferric Ions}},
  url          = {{http://dx.doi.org/10.3390/microorganisms11010106}},
  doi          = {{10.3390/microorganisms11010106}},
  volume       = {{11}},
  year         = {{2023}},
}