Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus

Abstract

Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (>10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.Nitrate can be used by many bacteria as a source of assimilable nitrogen or as a terminal electron acceptor under anoxic conditions (nitrate respiration). In the assimilatory process, which may occur aerobically or anaerobically, nitrate is ultimately reduced to ammonia (NH 3 ) and subsequently incorporated into biomass. This pathway is performed by many bacteria, fungi, and plants. In respiration, nitrate is used as an alternative electron acceptor when oxygen is not available. The enzymes for this pathway are found only in bacteria. Two main forms have been described so far, and in both, nitrate reduction is coupled to the generation of a proton motive force (3, 17), which is directly utilized as a source of energy or transformed into ATP by a membrane-associated ATPase. In one form, reported for Escherichia coli and other members of the family Enterobacteriaceae, the organisms reduce nitrate to nitrite, which is then excreted or further reduced to NH 3 by a dissimilatory (e.g., in E. coli [25]) or assimilatory nitrite reductase. The other form of nitrate respiration, denitrification, is defined as the reduction of nitrate to the gaseous oxides nitric oxide and nitrous oxide, which then may be further reduced to nitrogen (18,27). This form, generally found in obligately respiring bacteria such as pseudomonads, is crucial to nitrogen cycling in nature (10).Certain bacteria, such as strict aerobes, are able to perform assimilatory nitrate reduction but cannot use nitrate as the terminal electron acceptor. Other bacteria, such as E. coli and Salmonella typhimurium, do not assimilate nitrogen through nitrate reduction during aer...

Characterization of the Oxygen-Responsive NreABC Regulon of Staphylococcus aureus

“…In the presence of 2 mM nitrate or 1 mM nitrite, growth of the ⌬nreABC strains was slightly decreased under reduced oxygen tension in BM broth (data not shown). In S. carnosus wild type, the presence of higher nitrate concentrations (25 mM) under anoxic conditions resulted in a higher growth yield (23). During anoxic growth on BM agar plates with 20 mM nitrate, S. aureus SA113 wild type and the complemented mutant formed significantly larger colonies than the nreABC mutant, which displayed a small-colony phenotype under these conditions (Fig.…”

“…Nitrate (NO 3 Ϫ ) and nitrite (NO 2 Ϫ ) can be used as terminal electron acceptors under anaerobic conditions. In the foodgrade species Staphylococcus carnosus, used as a starter culture in the production of raw fermented sausages, the membranebound respiratory nitrate reductase, NarGHI, generates nitrite, which is further reduced to ammonia by a cytoplasmic NADH-dependent nitrite reductase encoded by the nirBD genes (23,24,26). In contrast to nitrate reduction, nitrite dissimilation is not coupled to the generation of a proton motive force and, hence, is not a respiratory pathway.…”

The Nitrate Reductase and Nitrite Reductase Operons and the narT Gene of Staphylococcus carnosus Are Positively Controlled by the Novel Two-Component System NreBC

“…The absence of oxygen and the presence of nitrate and/or nitrite induce nitrate reductase and nitrite reductase activities. Nitrite reduction is inhibited by nitrate and by high concentrations of nitrite (Ն10 mM), whereas nitrate reduction is not influenced by nitrite and ammonia (19).…”

“…The absence of oxygen and the presence of nitrate and/or nitrite induce nitrate reductase and nitrite reductase activities. Nitrite reduction is inhibited by nitrate and by high concentrations of nitrite (Ն10 mM), whereas nitrate reduction is not influenced by nitrite and ammonia (19).Although the amino acid sequences of the S. carnosus nitrate reductase and nitrite reductase enzymes are similar to those of the corresponding Escherichia coli proteins, we have found evidence that the regulatory proteins and operator sequences differ (21,24).In E. coli, expression from the nir promoter (P nir ) is dependent both on FNR (fumarate and nitrate reductase regulation) and on NarL or NarP (36, 37). Recently, Browning et al (7) have shown that P nir is repressed by three DNA binding proteins, i.e., Fis (factor for inversion stimulation), integration host factor (IHF), and H-NS (histone-like nucleoid structuring protein), and that NarL and NarP can relieve IHF-and Fismediated repression but are unable to counteract H-NS-mediated repression.…”

Complex genomic and phenotypic characterization of the related species Staphylococcus carnosus and Staphylococcus piscifermentans

“…As an apathogenic organism, S. carnosus is also widely used as a Gram-positive recipient strain for molecular cloning (Gotz, 1990;Halfmann et al, 1993;Ayora & Gotz, 1994;Connolly et al, 1994;Wieland et al, 1994 ;Heilmann et al, 1996 ;Neubauer & Gotz, 1996), as well as for the production of heterologous proteins (Demleitner & Gotz, 1994;Pschorr et al, 1994;Samuelson et al, 1995;Strauss & Gotz, 1996;Robert et al, 1996;Thumm & Gotz, 1997;Liljeqvist et R. Pantiieek and others Rosenstein et al, 1992;Wieland et al, 1995;Bruckner & Gotz, 1996;Bruckner, 1997) were developed.…”