Project description:Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated.
Project description:Paracoccus denitrificans is a well studied model organism with respect to its aerobic and anaerobic respiratory enzymes. However, until now, the growth medium for this organism has not been optimized for anaerobic growth. In particular, the requirements of P. denitrificans for trace elements (TEs) are not well known. In the present study we aimed to improve growth rates of P. denitrificans Pd1222 on a defined medium under anoxic conditions. We designed media containing different combinations of TEs at various concentrations, and tested their performance against previously reported media. Our results suggest that growth rate and yield depend on the availability and concentration of TEs in the medium. A chelated TE solution was more suitable than an acidified TE solution. Highest growth rates were achieved with medium comprising the TEs iron, manganese, molybdenum, copper and zinc ranging from 0.1 to 9 μM. On this medium, P. denitrificans Pd1222 grew with a generation time of 4.4 h under anoxic conditions and 2.8 h under oxic conditions. Diauxic growth was clearly shown with respect to nitrate and nitrite reduction under anoxic conditions.
Project description:A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO3- is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCEParacoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans, which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates.
Project description:Denitrification is a respiratory process by which nitrate is reduced to dinitrogen. Incomplete denitrification results in the emission of the greenhouse gas nitrous oxide and this is potentiated in acidic soils, which display reduced denitrification rates and high N2O/N2 ratios compared to alkaline soils. In this work, impact of pH on the proteome of the soil denitrifying bacterium Paracoccus denitrificans PD1222 was analysed with nitrate as sole energy and nitrogen source under anaerobic conditions at pH ranging from 6.5 to 7.5. Quantitative proteomic analysis revealed that the highest difference in protein representation was observed when the proteome at pH 6.5 was compared to the reference proteome at pH 7.2. However, this difference in the extracellular pH was not enough to produce modification of intracellular pH, which was maintained at 6.5 ± 0.1. The biosynthetic pathways of several cofactors relevant for denitrification and nitrogen assimilation like cobalamin, riboflavin, molybdopterin and nicotinamide were negatively affected at pH 6.5. In addition, peptide representation of reductases involved in nitrate assimilation and denitrification were reduced at pH 6.5. Data highlight the strong negative impact of pH on NosZ synthesis and intracellular copper content, thus impairing active NosZ assembly and, in turn, leading to elevated nitrous oxide emissions.