Nitric oxide synthase: What is its potential role in the physiology of staphylococci in meat products?

Coagulase-negative staphylococci are frequently isolated from meat products and two species are used as starter cultures in dry fermented sausages. In these products, they face various environmental conditions such as variation of redox potential and oxygen levels that can lead to oxidative stress. Furthermore, when nitrate and nitrite are added as curing salts, staphylococci also experience nitrosative stress. A nos gene encoding a nitric oxide synthase (NOS) is present in the genome of all staphylococci. NOS produces nitric oxide (NO) and citrulline from arginine, but its activity is still poorly characterized, particularly in coagulase-negative staphylococci. NO is highly reactive with a broad spectrum of activity resulting from targeting metal centres (heme and non-heme) and protein thiols. At low concentration, NO acts as a signalling molecule, while at higher concentration it generates stress. Thus, it was initially suggested that staphylococcal NOS counteract oxidative stress in relation to PerR and Fur regulators. In the physiology of staphylococci, it has recently been highlighted that NO controls the rate of aerobic respiration and regulates the transition from aerobic to nitrate respiration and also helps maintain the membrane potential in relation to the two-component systems SrrAB and AirRS. As NO interacts with heme centres, it binds the heme iron atom of myoglobin to form nitrosomyglobin, which is the typical red pigment of cured meat. However, the contribution of NOS to this reaction in meat products has yet to be evaluated.

Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives.

As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored.

Insight into the Genome of Staphylococcus xylosus, a Ubiquitous Species Well Adapted to Meat Products.

Staphylococcus xylosus belongs to the vast group of coagulase-negative staphylococci. It is frequently isolated from meat products, either fermented or salted and dried, and is commonly used as starter cultures in sausage manufacturing. Analysis of the S. xylosus genome together with expression in situ in a meat model revealed that this bacterium is well adapted to meat substrates, being able to use diverse substrates as sources of carbon and energy and different sources of nitrogen. It is well-equipped with genes involved in osmotic, oxidative/nitrosative, and acidic stress responses. It is responsible for the development of the typical colour of cured meat products via its nitrate reductase activity. It contributes to sensorial properties, mainly by the the catabolism of pyruvate and amino acids resulting in odorous compounds and by the limiting of the oxidation of fatty acids, thereby avoiding rancidity.

Evidence for Nitric Oxide Synthase Activity in Staphylococcus xylosus Mediating Nitrosoheme Formation.

Staphylococcus xylosus is used as a starter culture in fermented meat products and contributes to color formation by the reduction of nitrate to nitrite. Nitrite is a food additive that is chemically turned to nitric oxide (NO) in meat but its safety has been questioned. The objective of this study was to determine the ability of NO synthase (NOS) of S. xylosus C2a to produce NO. For this purpose, a nos deletion mutant (Δnos) in S. xylosus was constructed and NO production was evaluated in a test based on its ability to form nitrosomyoglobin and nitrosoheme. Production of NO was abrogated in the Δnos mutant under aerobic conditions and reduced about 35-40% comparing to the wild type C2a under limited oxygenation. This mutant was sensitive to oxidative stress. The expression of genes encoding catalase was modulated in the mutant with an up-regulation of katA and a down-regulation of katB and katC. The Δnos mutant displayed high colony pigmentation after prolonged growth on agar medium. Finally, the Δnos mutant showed no growth in minimal medium. Growth was not restored in the minimal medium by complementation with nos, but was restored by either addition of phenylalanine or complementation with pdt, a gene that encodes a prephenate dehydratase involved in phenylalanine biosynthesis and co-transcribed with nos. Our findings clearly demonstrate NOS-mediated NO production in S. xylosus, a meat-associated coagulase-negative Staphylococcus.