[show abstract][hide abstract] ABSTRACT: We show that the nitrate storing vacuole of the sulfide-oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation.
[show abstract][hide abstract] ABSTRACT: Nitrous oxide (N(2)O) is an environmentally important atmospheric trace gas because it is an effective greenhouse gas and it leads to ozone depletion through photo-chemical nitric oxide (NO) production in the stratosphere. Mitigating its steady increase in atmospheric concentration requires an understanding of the mechanisms that lead to its formation in natural and engineered microbial communities. N(2)O is formed biologically from the oxidation of hydroxylamine (NH(2)OH) or the reduction of nitrite (NO(-) (2)) to NO and further to N(2)O. Our review of the biological pathways for N(2)O production shows that apparently all organisms and pathways known to be involved in the catabolic branch of microbial N-cycle have the potential to catalyze the reduction of NO(-) (2) to NO and the further reduction of NO to N(2)O, while N(2)O formation from NH(2)OH is only performed by ammonia oxidizing bacteria (AOB). In addition to biological pathways, we review important chemical reactions that can lead to NO and N(2)O formation due to the reactivity of NO(-) (2), NH(2)OH, and nitroxyl (HNO). Moreover, biological N(2)O formation is highly dynamic in response to N-imbalance imposed on a system. Thus, understanding NO formation and capturing the dynamics of NO and N(2)O build-up are key to understand mechanisms of N(2)O release. Here, we discuss novel technologies that allow experiments on NO and N(2)O formation at high temporal resolution, namely NO and N(2)O microelectrodes and the dynamic analysis of the isotopic signature of N(2)O with quantum cascade laser absorption spectroscopy (QCLAS). In addition, we introduce other techniques that use the isotopic composition of N(2)O to distinguish production pathways and findings that were made with emerging molecular techniques in complex environments. Finally, we discuss how a combination of the presented tools might help to address important open questions on pathways and controls of nitrogen flow through complex microbial communities that eventually lead to N(2)O build-up.
[show abstract][hide abstract] ABSTRACT: Bacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, B. subtilis encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in B. subtilis 3610.
We show that B. subtilis 3610 produces intracellular NO via NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL). We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the nos gene, nor the exogenous addition of NO with NO donors affected (i) biofilm development, (ii) mature biofilm structure, and (iii) swarming motility in a qualitative and quantitative manner. In contrast, the nos knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal.
The results suggest that biofilm formation and swarming motility in B. subtilis represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in B. subtilis might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of B. subtilis from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic) conditions in the biofilm or dispersal from the biofilm.
[show abstract][hide abstract] ABSTRACT: Permeable or sandy sediments cover the majority of the seafloor on continental shelves worldwide, but little is known about their role in the coastal nitrogen cycle. We investigated the rates and controls of nitrogen loss at a sand flat (Janssand) in the central German Wadden Sea using multiple experimental approaches, including the nitrogen isotope pairing technique in intact core incubations, slurry incubations, a flow-through stirred retention reactor and microsensor measurements. Results indicate that permeable Janssand sediments are characterized by some of the highest potential denitrification rates (0.19 mmol N m−2 h−1) in the marine environment. Moreover, several lines of evidence showed that denitrification occurred under oxic conditions. In intact cores, microsensor measurements showed that the zones of nitrate/nitrite and O2 consumption overlapped. In slurry incubations conducted with 15NO3− enrichment in gas-impermeable bags, denitrification assays revealed that N2 production occurred at initial O2 concentrations of up to ~90 μM. Initial denitrification rates were not substantially affected by O2 in surficial (0–4 cm) sediments, whereas rates increased by twofold with O2 depletion in the at 4–6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), 29N2 and 30N2 were produced and O2 was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O2 may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.
The ISME Journal 04/2011; 5(4):776. · 8.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microorganisms can degrade saturated hydrocarbons (alkanes) not only under oxic but also under anoxic conditions. Three denitrifying isolates (strains HxN1, OcN1, HdN1) able to grow under anoxic conditions by coupling alkane oxidation to CO(2) with NO(3) (-) reduction to N(2) were compared with respect to their alkane metabolism. Strains HxN1 and OcN1, which are both Betaproteobacteria, utilized n-alkanes from C(6) to C(8) and C(8) to C(12) respectively. Both activate alkanes anaerobically in a fumarate-dependent reaction yielding alkylsuccinates, as suggested by present and previous metabolite and gene analyses. However, strain HdN1 was unique in several respects. It belongs to the Gammaproteobacteria and was more versatile towards alkanes, utilizing the range from C(6) to C(30). Neither analysis of metabolites nor analysis of genes in the complete genome sequence of strain HdN1 hinted at fumarate-dependent alkane activation. Moreover, whereas strains HxN1 and OcN1 grew with alkanes and NO(3) (-), NO(2) (-) or N(2)O added to the medium, strain HdN1 oxidized alkanes only with NO(3) (-) or NO(2) (-) but not with added N(2)O; but N(2)O was readily used for growth with long-chain alcohols or fatty acids. Results suggest that NO(2) (-) or a subsequently formed nitrogen compound other than N(2)O is needed for alkane activation in strain HdN1. From an energetic point of view, nitrogen-oxygen species are generally rather strong oxidants. They may enable enzymatic mechanisms that are not possible under conditions of sulfate reduction or methanogenesis and thus allow a special mode of alkane activation.
[show abstract][hide abstract] ABSTRACT: Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO3-) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO3- to nitrite (NO2-).
We show that dental plaque mediates denitrification of NO3- to nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) using microsensor measurements, 15N isotopic labelling and molecular detection of denitrification genes. In vivo N2O accumulation rates in the mouth depended on the presence of dental plaque and on salivary NO3- concentrations. NO and N2O production by denitrification occurred under aerobic conditions and was regulated by plaque pH.
Increases of NO concentrations were in the range of effective concentrations for NO signalling to human host cells and, thus, may locally affect blood flow, signalling between nerves and inflammatory processes in the gum. This is specifically significant for the understanding of periodontal diseases, where NO has been shown to play a key role, but where gingival cells are believed to be the only source of NO. More generally, this study establishes denitrification by human-associated microbial communities as a significant metabolic pathway which, due to concurrent NO formation, provides a basis for symbiotic interactions.
[show abstract][hide abstract] ABSTRACT: Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.
[show abstract][hide abstract] ABSTRACT: Bacterial targeting of tumours is an important anti-cancer strategy. We previously showed that strain SL7838 of Salmonella typhimurium targets and kills cancer cells. Whether NO generation by the bacteria has a role in SL7838 lethality to cancer cells is explored. This bacterium has the mechanism for generating NO, but also for decomposing it.
Mechanism underlying Salmonella typhimurium tumour therapy was investigated through in vitro and in vivo studies. NO measurements were conducted either by chemical assays (in vitro) or using Biosensors (in vivo). Cancer cells cytotoxic assay were done by using MTS. Bacterial cell survival and tumour burden were determined using molecular imaging techniques.
SL7838 generated nitric oxide (NO) in anaerobic cell suspensions, inside infected cancer cells in vitro and in implanted 4T1 tumours in live mice, the last, as measured using microsensors. Thus, under these conditions, the NO generating pathway is more active than the decomposition pathway. The latter was eliminated, in strain SL7842, by the deletion of hmp- and norV genes, making SL7842 more proficient at generating NO than SL7838. SL7842 killed cancer cells more effectively than SL7838 in vitro, and this was dependent on nitrate availability. This strain was also ca. 100% more effective in treating implanted 4T1 mouse tumours than SL7838.
NO generation capability is important in the killing of cancer cells by Salmonella strains.
[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) and nitrous oxide (N(2)O) are formed during N-cycling in complex microbial communities in response to fluctuating molecular oxygen (O(2)) and nitrite (NO(2)(-)) concentrations. Until now, the formation of NO and N(2)O in microbial communities has been measured with low spatial and temporal resolution, which hampered elucidation of the turnover pathways and their regulation. In this study, we combined microsensor measurements with metabolic modeling to investigate the functional response of a complex biofilm with nitrifying and denitrifying activity to variations in O(2) and NO(2)(-). In steady state, NO and N(2)O formation was detected if ammonium (NH(4)(+)) was present under oxic conditions and if NO(2)(-) was present under anoxic conditions. Thus, NO and N(2)O are produced by ammonia-oxidizing bacteria (AOB) under oxic conditions and by heterotrophic denitrifiers under anoxic conditions. NO and N(2)O formation by AOB occurred at fully oxic conditions if NO(2)(-) concentrations were high. Modeling showed that steady-state NO concentrations are controlled by the affinity of NO-consuming processes to NO. Transient accumulation of NO and N(2)O occurred upon O(2) removal from, or NO(2)(-) addition to, the medium only if NH(4)(+) was present under oxic conditions or if NO(2)(-) was already present under anoxic conditions. This showed that AOB and heterotrophic denitrifiers need to be metabolically active to respond with instantaneous NO and N(2)O production upon perturbations. Transiently accumulated NO and N(2)O decreased rapidly after their formation, indicating a direct effect of NO on the metabolism. By fitting model results to measurements, the kinetic relationships in the model were extended with dynamic parameters to predict transient NO release from perturbed ecosystems.
The ISME Journal 07/2009; 3(11):1301-13. · 8.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sandy sediments dominate the intertidal region of the Wadden Sea but so
far little is known about their role in the coastal N-cycle. We
investigated the potential N-loss rates at a sandflat (Janssand) in the
central German Wadden Sea by using a modified version of the whole core
incubation technique used for fine-grained sediments. In view of the
high permeability and strong pore water advection in these sediments,
the percolation method better represents the in situ conditions than the
conventional diffusive flux technique. Denitrification assays with those
permeable sediments incubated with 15NO3- indicate immediate 29N2 and
30N2 production. In contrast to the conventional views, our preliminary
results show that permeable Janssand sediments are characterized by some
of the highest denitrification rates in the marine environment.
Moreover, our results from gas-tight bag incubations indicate that
denitrification immediately occurs even under aerobic conditions, with
rates of 2.03±0.06 at 0-2 cm and 2.30±0.09 mmol m-3 h-1 at
2-4 cm of the sediments with the starting O2concentrations of 90 and 30
mol L-1, respectively. Additional evidence for denitrification in the
presence of free oxygen was obtained by simultaneous O2 and NOx
measurements with microsensors in percolated cores and Membrane Inlet
Mass Spectrometer measurements. We speculate that the observed high
denitrification rates in the presence of free oxygen might be an
adaptation of the denitrifying bacteria to recurrent tidally-induced
oscillations in pore water oxygen concentrations in the permeable
sediments of Janssand.
[show abstract][hide abstract] ABSTRACT: Pharmaceuticals are abundant at low concentrations (i.e. ng L(-1); microg L(-1)) in natural aquatic systems. However, very little is known about their effects on microorganisms. This study investigated the influence of the pharmaceuticals phenazone, amoxicillin and erythromycin, at low, non-toxic concentrations (i.e. 0.5-50 microg L(-1)), on the initial adhesion of bacteria to uncoated and iron-coated polystyrene. The influence of the pharmaceuticals on the initial adhesion of bacterial pure cultures (Escherichia coli, Aquabacterium commune, Bacillus subtilis) isolated from natural aquatic systems, was investigated with a plate assay. Initial adhesion of the pure cultures depended on the selected pharmaceutical, its concentration, the bacterial strain and the adhesion surface. Different combinations of these parameters resulted in inhibition, enhancement or had no effect on initial adhesion. In addition, a continuous flow system was used to investigate the influence of the pharmaceuticals on the initial adhesion of a drinking water microbial community. The drinking water community showed decreased adhesion in the presence of the pharmaceuticals regardless of adhesion surface. The results show that pharmaceuticals at environmentally relevant concentrations can influence the initial adhesion of bacteria. Thus, pharmaceutical compounds that are introduced to natural aquatic systems are able to exert subtle effects on bacteria.
[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) is a ubiquitous biomolecule that is known as a signaling compound in eukaryotes and prokaryotes. In addition, NO is involved in all conversions of the biogeochemical nitrogen cycle: denitrification, nitrification, and the anaerobic oxidation of ammonium (Anammox). Until now, NO has not been measured with high spatial resolution within microbial communities, such as biofilms, sediments, aggregates, or microbial mats, because the available sensors are not robust enough and their spatial resolution is insufficient. Here we describe the fabrication and application of a novel Clark-type NO microsensor with an internal reference electrode and a guard anode. The NO microsensor has a spatial resolution of 60-80 microm, a sensitivity of 2 pA microM-1, and a detection limit of approximately 30 nM. Hydrogen sulfide (H2S) was found to be a major interfering compound for the electrochemical detection of NO. The application of the novel NO microsensor to nitrifying biofilms and marine sediments revealed dynamic NO concentration profiles with peaks in the oxic parts of the samples. The local concentrations suggested that NO may be an important bioactive compound in natural environments. The consumption and production of NO occurs in separate regions of stratified microbial communities and indicates that it is linked to distinct biogeochemical cycles.