Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

Tachon, Sybille; Chambellon, Émilie; Yvon, Mireille

doi:10.1128/jb.01466-10

Cited by 18 publications

(14 citation statements)

References 39 publications

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“…1C and 1D). It has been shown that a general NADH oxidase activity, which efficiently consumes dissolved oxygen, is not important for L. lactis as a protection against oxidative stress under normal growth conditions (57). Indeed, this was confirmed in our study, since the residual O 2 did not affect exponential growth at 30°C.…”

Section: Discussionsupporting

confidence: 77%

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Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Chen

Shen

Solem

et al. 2013

Appl Environ Microbiol

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Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen. Lactococcus lactis is a mesophilic bacterium with an optimum growth temperature around 30°C (1) that is widely used in the dairy industry for production of cheese and buttermilk, where it plays a crucial role in flavor and texture formation. During cheese production, L. lactis is frequently exposed to a variety of stresses, such as heat stress (2). Superoptimal temperatures cause the denaturation of macromolecules and induce chaperone proteins, such as DnaK, GroEL, and GroES, which then help proteins to fold correctly (2). The chaperones are essential for growth at elevated temperatures, and mutants with defects in chaperone activity display a pronounced temperature-sensitive phenotype (3). The opposite effect is sometimes seen when overexpressing chaperones, and in L. lactis this results in improved fitness and lactic acid production at high temperatures (4, 5). The viability of some L. lactis strains at high temperatures is also improved by an incorporation of high concentrations of NaCl into the medium or a preadaptation prior to inoculation (6), and this phenomenon was demonstrated to be correlated with an induction of several chaperones (7). Meanwhile, metabolic responses are regarded as important factors in handling heat stress. It has been demonstrated that genes in several metabolic pathways are important for handling heat stress (8), and a recently recognized c-di-AMP-specific phosphodiesterase in L. lactis was also found to be important for heat tole...

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Section: Discussionsupporting

confidence: 77%

“…5). NADH oxidase, which converts O 2 into H 2 O using NADH, is the main enzyme involved in oxygen consumption in L. lactis (57). The higher FAD and NoxE activity in the presence of riboflavin at 37°C could apparently provide the explanation for the difference in oxygen consumption ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Chen

Shen

Solem

et al. 2013

Appl Environ Microbiol

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“…Our results showed that most of pyruvate produced during glucose consumption was used to produce lactate in aerobic culture (Figure 1a), indicating that pyruvate oxidase apparently removed little pyruvate from this pathway. NADH oxidase is the most common enzyme responsible for the production of H 2 O 2 from oxygen and is highly-active in LAB (Condon 1987; Higuchi et al 2000; Tachon et al 2011). LAB are known to possess either a NADH: H 2 O 2 or a NADH: H 2 O oxidase, or sometimes both (Condon 1987; Higuchi et al 2000).…”

Section: Discussionmentioning

confidence: 99%

Influence of oxygen on NADH recycling and oxidative stress resistance systems in Lactobacillus panis PM1

2013

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Lactobacillus panis strain PM1 is an obligatory heterofermentative and aerotolerant microorganism that also produces 1,3-propanediol from glycerol. This study investigated the metabolic responses of L. panis PM1 to oxidative stress under aerobic conditions. Growth under aerobic culture triggered an early entrance of L. panis PM1 into the stationary phase along with marked changes in end-product profiles. A ten-fold higher concentration of hydrogen peroxide was accumulated during aerobic culture compared to microaerobic culture. This H2O2 level was sufficient for the complete inhibition of L. panis PM1 cell growth, along with a significant reduction in end-products typically found during anaerobic growth. In silico analysis revealed that L. panis possessed two genes for NADH oxidase and NADH peroxidase, but their expression levels were not significantly affected by the presence of oxygen. Specific activities for these two enzymes were observed in crude extracts from L. panis PM1. Enzyme assays demonstrated that the majority of the H2O2 in the culture media was the product of NADH: H2O2 oxidase which was constitutively-active under both aerobic and microaerobic conditions; whereas, NADH peroxidase was positively-activated by the presence of oxygen and had a long induction time in contrast to NADH oxidase. These observations indicated that a coupled NADH oxidase - NADH peroxidase system was the main oxidative stress resistance mechanism in L. panis PM1, and was regulated by oxygen availability. Under aerobic conditions, NADH is mainly reoxidized by the NADH oxidase - peroxidase system rather than through the production of ethanol (or 1,3-propanediol or succinic acid production if glycerol or citric acid is available). This system helped L. panis PM1 directly use oxygen in its energy metabolism by producing extra ATP in contrast to homofermentative lactobacilli.

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“…Due to the fact that oxygen generates NADH oxidase activity, these enzymes may regenerate NADH. Another possibility for NADH regeneration in presence of oxygen is NAD cytoplasmic oxidase NOX_E (Tachon et al 2011). Together with the production of lactate, ethanol, diacetyl and acetoin also regenerate NADH (Salminen et al 2004).…”

Section: Discussionmentioning

confidence: 98%

Effect of pH and dilution rate on specific production rate of extra cellular metabolites by Lactobacillus salivarius UCO_979C in continuous culture

Valenzuela

Pinuer

Cancino

2015

Appl Microbiol Biotechnol

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The effect of pH and dilution rate on the production of extracellular metabolites of Lactobacillus salivarius UCO_979 was studied. The experiments were carried out in continuous mode, with chemically defined culture medium at a temperature of 37 °C, 200 rpm agitation and synthetic air flow of 100 ml/min. Ethanol, acetic acid, formic acid, lactic acid and glucose were quantified through HPLC, while exopolysaccharide (EPS) was extracted with ethanol and quantified through the Dubois method. The results showed no linear trends for the specific production of lactic acid, EPS, acetic acid and ethanol, while the specific glucose consumption and ATP production rates showed linear trends. There was a metabolic change of the strain for dilution rates below 0.3 h(-1). The pH had a significant effect on the metabolism of the strain, which was evidenced by a higher specific glucose consumption and increased production of ATP at pH 6 compared with that obtained at pH 7. This work shows not only the metabolic capabilities of L. salivarius UCO_979C, but also shows that it is possible to quantify some molecules associated with its current use as gastrointestinal probiotic, especially regarding the production of organic acids and EPS.

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Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

Cited by 18 publications

References 39 publications

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Influence of oxygen on NADH recycling and oxidative stress resistance systems in Lactobacillus panis PM1

Effect of pH and dilution rate on specific production rate of extra cellular metabolites by Lactobacillus salivarius UCO_979C in continuous culture

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