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...

SummaryThe nreABC ( n itrogen re gulation) operon encodes a new staphylococcal two-component regulatory system that controls dissimilatory nitrate/nitrite reduction in response to oxygen. Unlike other twocomponent sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB-cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desulphurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron-sulphur cluster formation) developed. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no ironsulphur cluster was incorporated; both findings corroborated the importance of the cysteine residues. Comparison of the kinase activity of (i) the reconstituted (ii) the unreconstituted, and (iii) the unreconstituted and deferrated NreB-His indicated that NreB kinase activity depended on iron availability and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4-8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1-2 iron-sulphur [4Fe-4S] 2+ clusters of the FNR-type. Unlike FNR, NreB does not act directly as transcriptional activator, but transfers the phosphoryl group to the response regulator NreC.

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Five DNA probes directed against different regions of the gene that encodes the dermonecrotic toxin of Pasteurella multocida subsp. multocida were examined for their ability to identify toxigenic P. multocida subsp. multocida strains. The specificities of the probes were studied with 96 strains of P. multocida subsp. multocida and 22 strains of 11 other bacterial species. Results of colony hybridization assays using these probes indicated that two of the five probes have potential diagnostic value. Atrophic rhinitis, a major respiratory disease in pigs, is characterized by sneezing, nose bleeding, shortening or twisting of the snout, atrophy of the nasal turbinate bones, and, in severe cases, impaired growth. Pasteurella multocida subsp. multocida strains that produce a dermonecrotic toxin (DNT), which is also called osteolytic toxin, are associated with the severe progressive form of the disease and are therefore considered to be the major etiologic agents. P. multocida subsp. multocida strains that do not produce the toxin do not cause atrophic rhinitis (4, 10, 13;

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the LambdaGEM‐11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. Multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.

Gametes of opposite mating type (mt (+) and mt (-)) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using (35)S-labeled flagella and the isolated mt (-)agglutination factor. It is shown that not only isolated flagella, but also the mt (-)agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt (-)agglutination factor in determining the sexual agglutinability of mt (-)gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt (-)agglutination factor can be completely inactivated.

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.