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Screening and optimization of a nitrate reductase-producing Staphylococcus simulans UV-11 and its application

ABSTRACT: A strain of Staphylococcus simulans D14 (S. simulans D14) showed the highest nitrate reductase activity (NRA) of 4.52 mM NO2?/mg dry weight by the spectrophotometric method, which was screened from traditional Chinese sausage. When the UV mutagenesis time was 25 s, the positive mutation rate was the highest at 26.60%. The NRA of the obtained positive mutant UV-11 was 9.21 mM NO2?/mg dry weight, and the activity was found to be 1.04-fold higher than that of the original strain S. simulans D14. A Plackett–Burman design (PBD) was employed to screen the significant variables pH, KNO3 (%) and incubation time (h), and response surface methodology (RSM) was used to optimize the significant variables using a Box–Behnken design (BBD). The results showed that the NRA of S. simulans UV-11 was 15.22 mM NO2?/mg dry weight under optimum conditions of 37 °C, pH 6.5, incubation time 15 h, KNO3 0.045%, NaCl 5%, NaNO2 0.015%, peptone 1%, and D–mannitol 1%, which increased by 65.2% compared with the unoptimized medium. Natural curing agents (containing 107 CFU/g S. simulans UV-11 under optimal conditions and 1.40% celery powder, T2) were added to the cured meat model. T2 produced significantly lighter and redder signals than the control group (C) and the addition of 150 ppm NaNO2 group (T1). The thiobarbituric acid reactive substance (TBARS) of T2 was 2.30 mg malonaldehyde/kg product and residual nitrite of T2 was 7.1 ppm after 14 days of storage,which were lower than those groups of C and T1. Taking into account the results of cured meat models, S. simulans UV-11 could be selected as a potential starter culture for the processing of natural meat products.

SUBMITTER: Sun M

PROVIDER: S-EPMC7875452 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Project description:In fungi, transcriptional activation of genes involved in NO3(-) assimilation requires the presence of an inducer (nitrate or nitrite) and low intracellular concentrations of the pathway products ammonium or glutamine. In Aspergillus nidulans, the two transcription factors NirA and AreA act synergistically to mediate nitrate/nitrite induction and nitrogen metabolite derepression, respectively. In all studied fungi and in plants, mutants lacking nitrate reductase (NR) activity express nitrate-metabolizing enzymes constitutively without the addition of inducer molecules. Based on their work in A. nidulans, Cove and Pateman proposed an "autoregulation control" model for the synthesis of nitrate metabolizing enzymes in which the functional nitrate reductase molecule would act as co-repressor in the absence and as co-inducer in the presence of nitrate. However, NR mutants could simply show "pseudo-constitutivity" due to induction by nitrate which accumulates over time in NR-deficient strains. Here we examined this possibility using strains which lack flavohemoglobins (fhbs), and are thus unable to generate nitrate internally, in combination with nitrate transporter mutations (nrtA, nrtB) and a GFP-labeled NirA protein. Using different combinations of genotypes we demonstrate that nitrate transporters are functional also in NR null mutants and show that the constitutive phenotype of NR mutants is not due to nitrate accumulation from intracellular sources but depends on the activity of nitrate transporters. However, these transporters are not required for nitrate signaling because addition of external nitrate (10 mM) leads to standard induction of nitrate assimilatory genes in the nitrate transporter double mutants. We finally show that NR does not regulate NirA localization and activity, and thus the autoregulation model, in which NR would act as a co-repressor of NirA in the absence of nitrate, is unlikely to be correct. Results from this study instead suggest that transporter-mediated NO?? accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed "pseudo-constitutive".

Project description:Nitrogen is one of the most important macro-nutrients for plant growth and crop productivity. The amount of synthetic nitrogen fertilizers supplied to crops has dramatically increased, leading to a notable rise in crop yields. However, excessive nitrogen use has an enormous negative impact on ecosystems and human health through the emission of intense greenhouse gases, such as nitric oxide derived from the nitrate (NO3 -) assimilation cascade. Additionally, owing to the development of extensive irrigation in agriculture, crops are known to suffer from high salt stress. The effect of excessive nitrogen fertilizer application has been studied in some crops, but the effect of high nitrate level and salt stress on plant stress tolerance has not been studied in detail. Therefore, in this study we aimed to study the effects of high concentrations of NO3 - on salt stress tolerance in Arabidopsis. In addition, since anthocyanin functions as a reactive oxygen species (ROS) scavenger under abiotic stress conditions, we investigated whether enhanced anthocyanin content helps Arabidopsis to withstand higher salt stress levels under high NO3 - concentrations by using pap1-D/fls1ko double mutant plants, which accumulate excessive amount of anthocyanin. We found that Col-0 plants are more sensitive to salt stress under high NO3 - concentrations. Although both the pap1-D/fls1ko and fls1ko plants accumulated higher anthocyanin levels and radical scavenging activities than Col-0 plants under both normal and salt stress conditions, the fls1ko plants exhibited much better growth than the pap1-D/fls1ko plants. It appears that the enhanced NR activities and transcript levels of NIA1 and NIA2 in pap1-D/fls1ko and fls1ko plants led to an increase in the synthesis of proteins and proline, which increases osmolytes against salt stress. Our results demonstrate that optimal levels of anthocyanin accumulation can enhance growth performance of plants under high NO3 - and salt stress conditions.

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Screening and optimization of a nitrate reductase-producing Staphylococcus simulans UV-11 and its application

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