H ₂ O ₂ Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

Abstract

e Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H 2 O 2 , inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H 2 O 2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The K m for FMN is 30 ؎ 8 M, in accordance with its proposed in vivo role in H 2 O 2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H 2 O 2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H 2 O 2 production in L. johnsonii.

Genome Sequence and Transcriptome Analysis of Meat-Spoilage-Associated Lactic Acid Bacterium Lactococcus piscium MKFS47

“…We also found several enzymes that could be involved in hydrogen peroxide production: the superoxide dismutase SodA (LACPI_0465), the alpha-glycerophosphate oxidase GlpO (LACPI_1161), and two NADPH-dependent flavin mononucleotide (FMN) reductases (LACPI_0686 and LACPI_0687) showing homology to the corresponding enzymes from L. johnsonii involved in hydrogen peroxide production (46). According to data from the hydrogen peroxide production assay, L. piscium produced hydrogen peroxide only from glycerol under aerobic conditions but not from glucose both aerobically and anaerobically.…”

Altered Microbiota Composition Mediates Depressive Behavior During Chronic Stress

“…Both our and previous data show that Lactobacillus can produce high amounts of ROS 166,176,178 . Previous evidence also shows that that the molecule can inhibit IDO1 activity 166 .…”

“…One approach to understand this process would be to genetically manipulate the Lactobacilli to silence or boost their ROS production. Such an experiment would be technically challenging, given that Lactobacilli evolved several genes involved in ROS secretion 161,162,176,178 . Nevertheless, it would definitely be informative about the importance of Lactobacillus-derived ROS for regulating kynurenine metabolism.…”

“…IDO1 is the main enzyme responsible for conversion of tryptophan to kynurenine and its expression and activity are inhibited by reactive oxygen species (ROS) 166 . Members of the Lactobacillus family have the capacity to produce high levels of ROS, as a way of fending off other bacteria and maintaining their niche 177,178 . Though difficult to show, it is conceivable that ROS molecules can diffuse from the lumen to signal to the intestinal epithelial cells.…”

“…Perhaps best studied in the vagina, Lactobacilli protect against infection by producing a diversity of antimicrobial factors, including lactic acid, peroxide, bacteriocins, as well as by resource competition [176][177][178] . Although in a few contexts increased levels of Lactobacilli are associated with pathology, e.g.…”