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

Abstract

In Staphylococcus carnosus, the nreABC (for nitrogen regulation) genes were identified and shown to link the nitrate reductase operon (narGHJI) and the putative nitrate transporter gene narT. An nreABC deletion mutant, m1, was dramatically affected in nitrate and nitrite reduction and growth. Transcription of narT, narGHJI, and the nitrite reductase (nir) operon was severely reduced even when cells were cultivated anaerobically without nitrate or nitrite. nreABC transcripts were detected when cells were grown aerobically or anaerobically with or without nitrate or nitrite. NreA is a GAF domain-containing protein of unknown function. In vivo and in vitro studies showed that NreC is phosphorylated by NreB and that phospho-NreC specifically binds to a GC-rich palindromic sequence to enhance transcription initiation. This binding motif was found at the narGHJI, nir, and narT promoters but not at the moeB promoter. NreB is a cytosolic protein with four N-terminal cysteine residues. The second cysteine residue was shown to be important for NreB function. In vitro autophosphorylation of NreB was not affected by nitrate, nitrite, or molybdate. The nir promoter activity was iron dependent. The data provide evidence for a global regulatory system important for aerobic and anaerobic metabolism, with NreB and NreC forming a classical two-component system and NreB acting as a sensor protein with oxygen as the effector molecule.Staphylococcus carnosus, traditionally used as a starter culture in the production of raw fermented sausages, reduces nitrate to ammonia in two steps: (i) nitrate is taken up and reduced by a dissimilatory nitrate reductase to nitrite, which is subsequently excreted, and (ii) after depletion of nitrate, the accumulated nitrite is imported and reduced by an NADHdependent nitrite reductase to ammonia, which then accumulates in the medium. Nitrate reductase is a membrane-bound enzyme involved in energy conservation, whereas nitrite reductase is a cytosolic enzyme involved in NADH reoxidation. 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-mediat...

Characterization of the Oxygen-Responsive NreABC Regulon of Staphylococcus aureus

“…The response regulator NreC of S. carnosus has been shown to bind upstream of the nitrate reductase and nitrite reductase operons and the narT gene, and the consensus sequence TAGGGN 4 CCCTA was identified as the binding site (9). Inspection of the upstream regions of nirR, narG, and narK of S. aureus revealed conserved sequences with a high degree of similarity to the S. carnosus consensus motif (Fig.…”

“…In S. carnosus, the nreABC transcript has been reported to be present during aerobic and anaerobic growth, a requirement expected for an oxygen-sensing system (9). The putative S. aureus nreABC promoter resembles a typical A -dependent promoter (data not shown), which presumably leads to constitutive expression.…”

“…Upon oxygen depletion, autophosphorylation activity of the cytoplasmic histidine kinase NreB increases, which depends on the formation of an oxygen-labile iron-sulfur cluster (16). The response regulator NreC is phosphorylated by NreB and specifically binds to the promoters of the nitrate reductase and nitrite reductase operons and of the narT gene to enhance transcription (9). NarT shares 75% identity with the S. aureus nitrite extrusion protein NarK and is likely to be involved in nitrate and nitrite transport (6,8).…”

“…In S. aureus, the two-component system SrrAB (for staphylococcal respiratory response AB; synonym, SrhSR), homologous to Bacillus subtilis ResDE (37), has been described as one major regulatory system involved in anaerobic gene regulation (28,38,39). Another direct staphylococcal oxygen sensing system, NreABC (for nitrogen regulation), has been identified and characterized in S. carnosus (9). Upon oxygen depletion, autophosphorylation activity of the cytoplasmic histidine kinase NreB increases, which depends on the formation of an oxygen-labile iron-sulfur cluster (16).…”

At the Crossroads of Bacterial Metabolism and Virulence Factor Synthesis in Staphylococci

“…Under anaerobic conditions with Fe 2ϩ , the iron-sulfur cluster is intact, facilitating the autophosphorylation of NreB, which transfers the phosphate to NreC. In Staphylococcus carnosus, the transfer of the NreB phosphate to NreC activates the transcription of the nitrite reductase (annotated as nasDE or nirBD) and respiratory nitrate reductase (narGHJI) operons and the nitrite and/or nitrate transporter (narT) (59). Similarly, S. aureus NreC activates the transcription of the nitrate and nitrite reductase operons and the nitrite extrusion protein (NarK) and differentially regulates several nitrogen metabolism genes (e.g., arcABDC, ureBCEF, and glnRA) (192).…”