The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans.
ABSTRACT: Nitrite, in equilibrium with free nitrous acid (FNA), can inhibit both aerobic and anaerobic growth of microbial communities through bactericidal activities that have considerable potential for control of microbial growth in a range of water systems. There has been much focus on the effect of nitrite/FNA on anaerobic metabolism and so, to enhance understanding of the metabolic impact of nitrite/FNA on aerobic metabolism, a study was undertaken with a model denitrifying bacterium Paracoccus denitrificans PD1222. Extracellular nitrite inhibits aerobic growth of P. denitrificans in a pH-dependent manner that is likely to be a result of both nitrite and free nitrous acid (pKa = 3.25) and subsequent reactive nitrogen oxides generated from the intracellular passage of FNA into P. denitrificans. Increased expression of a gene encoding a flavohemoglobin protein (Fhp) (Pden_1689) was observed in response to extracellular nitrite. Construction and analysis of a deletion mutant established Fhp to be involved in endowing nitrite/FNA resistance at high extracellular nitrite concentrations. Global transcriptional analysis confirmed nitrite-dependent expression of fhp and indicated that P. denitrificans expressed a number of stress response systems associated with protein, DNA and lipid repair. It is therefore suggested that nitrite causes a pH-dependent stress response that is due to the production of associated reactive nitrogen species, such as nitric oxide from the internalisation of FNA.
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 ?M. 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 pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P. denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the cobK and cobJ genes with their orthologues suggests that P. denitrificans uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions, P. denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that P. denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.
Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type incubated in continuous culture (continuous culture (CSTR)) in minimal media with aerobic or anaerobic conditions. The goal was to define the core respiratory genes. Overall design: Two growth conditions, three biological replicates of each condition. Each sample hybridised in a two-channel hybridization against Paracoccus denitrificans genomic DNA as the comparator/reference, which also acted as a control for spot quality.
Project description:Free nitrous acid (FNA), which is the protonated form of nitrite and inevitably produced during biological nitrogen removal, has been demonstrated to strongly inhibit the activity of polyphosphate accumulating organisms (PAOs). Herein we reported an efficient process for wastewater treatment, i.e., the oxic/anoxic/oxic/extended-idle process to mitigate the generation of FNA and its inhibition on PAOs. The results showed that this new process enriched more PAOs which thereby achieved higher phosphorus removal efficiency than the conventional four-step (i.e., anaerobic/oxic/anoxic/oxic) biological nutrient removal process (41 ± 7% versus 30 ± 5% in abundance of PAOs and 97 ± 0.73% versus 82 ± 1.2% in efficiency of phosphorus removal). It was found that this new process increased pH value but decreased nitrite accumulation, resulting in the decreased FNA generation. Further experiments showed that the new process could alleviate the inhibition of FNA on the metabolisms of PAOs even under the same FNA concentration.
Project description:Hydrogen sulfide produced by sulfate-reducing bacteria (SRB) in sewers causes odor problems and asset deterioration due to the sulfide-induced concrete corrosion. Free nitrous acid (FNA) was recently demonstrated as a promising antimicrobial agent to alleviate hydrogen sulfide production in sewers. However, details of the antimicrobial mechanisms of FNA are largely unknown. Here, we report the multiple-targeted antimicrobial effects of FNA on the SRB Desulfovibrio vulgaris Hildenborough by determining the growth, physiological, and gene expression responses to FNA exposure. The activities of growth, respiration, and ATP generation were inhibited when exposed to FNA. These changes were reflected in the transcript levels detected during exposure. The removal of FNA was evident by nitrite reduction that likely involved nitrite reductase and the poorly characterized hybrid cluster protein, and the genes coding for these proteins were highly expressed. During FNA exposure, lowered ribosome activity and protein production were detected. Additionally, conditions within the cells were more oxidizing, and there was evidence of oxidative stress. Based on an interpretation of the measured responses, we present a model depicting the antimicrobial effects of FNA on D. vulgaris These findings provide new insight for understanding the responses of D. vulgaris to FNA and will provide a foundation for optimal application of this antimicrobial agent for improved control of sewer corrosion and odor management.IMPORTANCE Hydrogen sulfide produced by SRB in sewers causes odor problems and results in serious deterioration of sewer assets that requires very costly and demanding rehabilitation. Currently, there is successful application of the antimicrobial agent free nitrous acid (FNA), the protonated form of nitrite, for the control of sulfide levels in sewers (G. Jiang et al., Water Res 47:4331-4339, 2013, http://dx.doi.org/10.1016/j.watres.2013.05.024). However, the details of the antimicrobial mechanisms of FNA are largely unknown. In this study, we identified the key responses (decreased anaerobic respiration, reducing FNA, combating oxidative stress, and shutting down protein synthesis) of Desulfovibrio vulgaris Hildenborough, a model sewer corrosion bacterium, to FNA exposure by examining the growth, physiological, and gene expression changes. These findings provide new insight and underpinning knowledge for understanding the responses of D. vulgaris to FNA exposure, thereby benefiting the practical application of FNA for improved control of sewer corrosion and odor.
Project description:The characterization of the hydroxylamine oxidase from the heterotrophic nitrifier Paracoccus denitrificans GB17 indicates the enzyme to be entirely distinct from the hydroxylamine oxidase from the autotrophic nitrifier Nitrosomonas europaea. Hydroxylamine oxidase from P. denitrificans contains three to five non-haem, non-iron-sulphur iron atoms as prosthetic groups, predominantly co-ordinated by carboxylate ligands. The interaction of the enzyme with the electron-accepting proteins cytochrome C556 and pseudoazurin is mainly hydrophobic. The catalytic mechanism of hydroxylamine oxidase from P. denitrificans is different from the enzyme from N. europaea because the production of nitrite by the former requires molecular oxygen. Under anaerobic conditions the enzyme makes nitrous oxide as a sole product.
Project description:Ergothioneine (ESH) is a low-molecular-mass thiol present in millimolar concentrations in a limited number of tissues, including erythrocytes, kidney, seminal fluid and liver; however, its biological function is still unclear. In the present study we investigated the role of ESH in the catabolism of S-nitrosoglutathione (GSNO). The results show that: (1) GSNO decomposition is strongly influenced by ESH (k"=0.178+/-0.032 M(-1) x s(-1)); (2) ammonia is the main nitrogen-containing compound generated by the reaction; and (3) nitrite is practically absent under both aerobic and anaerobic conditions. These findings are markedly different from those reported for the GSH-induced decomposition of GSNO, in which the nitrogen-containing end products are nitrite, ammonia and nitrous oxide (N(2)O) under aerobic conditions but nitrite, ammonia, nitric oxide (NO) and small quantities of hydroxylamine under anaerobic conditions. Considering the high concentration of ESH in specific cells, the reaction with GSNO should be considered as an important molecular event occurring in the cell.
Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type grown to mid-exponential phase in minimal media with either 13 uM (Cu-H) or 0.5 uM (Cu-L) Cu regimes. The goal was to define the effects of Cu-limitation on denitrification genes Two growth conditions, three biological replicates of each condition. Each sample hybridised in a two-channel hybridization against Paracoccus denitrificans genomic DNA as the comparator/reference, which also acted as a control for spot quality. Cu-concentration 13 uM (Cu-H) versus 0.5 uM Cu (Cu-L) in anaerobic growth conditions.
Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type grown to mid-exponential phase in minimal media with either 13 uM (Cu-H) or 0.5 uM (Cu-L) Cu regimes. The goal was to define the effects of Cu-limitation on denitrification genes Overall design: Two growth conditions, three biological replicates of each condition. Each sample hybridised in a two-channel hybridization against Paracoccus denitrificans genomic DNA as the comparator/reference, which also acted as a control for spot quality. Cu-concentration 13 uM (Cu-H) versus 0.5 uM Cu (Cu-L) in anaerobic growth conditions.