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

Abstract

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.

Keywords: Staphylococcus xylosus; coagulase-negative Staphylococcus; nitric oxide; nitric oxide synthase; nitrosoheme; oxidative stress.

FIGURE 1

Growth of Staphylococcus xylosus strains in TSB under aerobic conditions. (A) S. xylosus C2a and Δnos. (B) ΔnospRBnos and ΔnospRB473 with chloramphenicol. Data represent means ± SD from n = 3 independent biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Growth of S. xylosus strains in chemically defined minimal medium in the Bioscreen Assay. (A) Growth of S. xylosus C2a, Δnos, and ΔnospRBnos, (B) Growth of C2a, Δnos and ΔnospRBnos with phenylalanine (phe), (C) Growth of C2apRB473 (identical growth as C2apRB474), ΔnospRB473 (identical growth as ΔnospRB474), ΔnospRBpdt and ΔnospRBnospdt. Data represent means ± SD from n = 3 independent biological replicates. ***p < 0.001.

FIGURE 3

Formation of red myoglobin derivatives. S. xylosus C2apRB473, ΔnospRB473 and ΔnospRBnos were incubated in TSB supplemented with metmyoglobin (2 mg/mL) under (A) limited oxygenation (no stirring and reaction mixture covered with mineral oil) and (B) aerobic conditions (150 rpm). Formation of red pigment was evaluated by measuring absorbance. Data represent means ± SD from n = 3 independent biological replicates. **p < 0.01; ***p < 0.001 at λ_{548 nm} and λ_{581 nm.}

FIGURE 4

Evidence of nitrosoheme. Nitrosoheme was extracted from nitrosomyoglobin with 80% acetone from supernatants of strains grown in TSB supplemented with metmyoglobin and inoculated under (A) limited oxygenation (no stirring and reaction mixture covered with mineral oil) and (B) aerobic conditions (150 rpm). Curves show spectra representative of three biological replicates.

FIGURE 5

Colony pigment production by S. xylosus strains. (A) S. xylosus C2apRB473, ΔnospRB473, ΔnospRBnos, ΔnospRBpdt and ΔnospRBnospdt streaked on Tryptic Soy Agar (TSA) plates containing chloramphenicol for three days. (B) Measurement of pigmentation after methanol extraction at λ_{460 nm}. The relative optical density is normalized to C2a, which was set at 100. Data represent means ± SD from n = 3 independent biological replicates. **p < 0.01.

FIGURE 6

Effect of nos deletion on sensitivity to oxidative stress. S. xylosus C2a, Δnos, and ΔnospRBnos strains were grown under aerobic conditions up to OD = 1 and treated for 1 h with 150 mM hydrogen peroxide (H₂O₂). Cells, before and after treatment, were enumerated. Data represent means ± SD from n = 2 independent biological replicates. *p < 0.05.

FIGURE 7

Expression of nos and kat genes. S. xylosus C2a, Δnos, and ΔnospRBnos strains were grown in TSB under aerobic conditions for 6 and 24 h. Expression of nos and kat genes was evaluated by qRT-PCR. The Livak method (2^-ΔΔCt) was applied to analyze expression, using sod as reference gene. Results are expressed as means ± SD from n = 3 independent biological replicates.