Publications by authors named "Rob J M van Spanning"

28 Publications

  • Page 1 of 1

High biodiversity in a benzene-degrading nitrate-reducing culture is sustained by a few primary consumers.

Commun Biol 2021 May 5;4(1):530. Epub 2021 May 5.

Department of Molecular Cell Biology, AIMMS, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.
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http://dx.doi.org/10.1038/s42003-021-01948-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099898PMC
May 2021

Effects of DNA preservation solution and DNA extraction methods on microbial community profiling of soil.

Folia Microbiol (Praha) 2021 Apr 9. Epub 2021 Apr 9.

Systems Biology Lab, Department of Molecular Cell Biology, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands.

Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.
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http://dx.doi.org/10.1007/s12223-021-00866-0DOI Listing
April 2021

Long-term exposure of activated sludge in chemostats leads to changes in microbial communities composition and enhanced biodegradation of 4-chloroaniline and N-methylpiperazine.

Chemosphere 2020 Mar 22;242:125102. Epub 2019 Oct 22.

Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, the Netherlands.

Exposure history and adaptation of the inoculum to chemicals have been shown to influence the outcome of ready biodegradability tests. However, there is a lack of information about the mechanisms involved in microbial adaptation and the implication thereof for the tests. In the present study, we investigated the impact of a long-term exposure to N-methylpiperazine (NMP) and 4-chloroaniline (4CA) of an activated sludge microbial community using chemostat systems. The objective was to characterize the influence of adaptation to the chemicals on an enhanced biodegradation testing, following the OECD 310 guideline. Cultures were used to inoculate the enhanced biodegradability tests, in batch, before and after exposure to each chemical independently in chemostat culture. Composition and diversity of the microbial communities were characterised by 16s rRNA gene amplicon sequencing. Using freshly sampled activated sludge, NMP was not degraded within the 28 d frame of the test while 4CA was completely eliminated. However, after one month of exposure, the community exposed to NMP was adapted and could completely degrade it. This result was in complete contrast with that from the culture exposed for 3 months to 4CA. Long term incubation in the chemostat system led to a progressive loss of the initial biodegradation capacity of the community, as a consequence of the loss of key degrading microorganisms. This study highlights the potential of chemostat systems to induce adaptation to a specific chemical, ultimately resulting in its biodegradation. At the same time, one should be critical of these observations as the dynamics of a microbial community are difficult to maintain in chemostat, as the loss of 4CA biodegradation capacity demonstrates.
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http://dx.doi.org/10.1016/j.chemosphere.2019.125102DOI Listing
March 2020

Circular Spectropolarimetric Sensing of Vegetation in the Field: Possibilities for the Remote Detection of Extraterrestrial Life.

Astrobiology 2019 10 27;19(10):1221-1229. Epub 2019 Aug 27.

Leiden Observatory, Leiden University, Leiden, The Netherlands.

Homochirality is a generic and unique property of all biochemical life, and the fractional circular polarization of light it induces therefore constitutes a potentially unambiguous biosignature. However, while high-quality circular polarimetric spectra can be easily and quickly obtained in the laboratory, accurate measurements in the field are much more challenging due to large changes in illumination and target movement. In this study, we measured various targets in the field, up to distances of a few kilometers, using the dedicated circular spectropolarimeter TreePol. We show how photosynthetic life can readily be distinguished from abiotic matter. We underline the potential of circular polarization signals as a remotely accessible means to characterize and monitor terrestrial vegetation, for example, for agriculture and forestry. In addition, we discuss the potential of circular polarization for the remote detection of extraterrestrial life.
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http://dx.doi.org/10.1089/ast.2019.2050DOI Listing
October 2019

Biodegradation of metformin and its transformation product, guanylurea, by natural and exposed microbial communities.

Ecotoxicol Environ Saf 2019 Oct 10;182:109414. Epub 2019 Jul 10.

Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098, XH Amsterdam, the Netherlands.

Metformin (MET) is a pharmaceutical product mostly biotransformed in the environment to a transformation product, guanylurea (GUA). In ready biodegradability tests (RBTs), however, contrasting results have been observed for metformin. The objective of this study was to measure the biodegradation of MET and GUA in RBTs, using activated sludge from the local wastewater treatment plant, either directly or after pre-exposure to MET, in a chemostat. The activated sludge community was cultivated in chemostats, in presence or absence of MET, for a period of nine months, and was used in RBT after one, three and nine months. The results of this study showed that the original activated sludge was able to completely remove MET (15 mg/l) and the newly produced GUA (50% of C) under the test conditions. Inoculation of the chemostat led to a rapid shift in the community composition and abundance. The community exposed to 1.5 mg/l of MET was still able to completely consume MET in the RBTs after one-month exposure, but three- and nine-months exposure resulted in reduced removal of MET in the RBTs. The ability of the activated sludge community to degrade MET and GUA is the result of environmental exposure to these chemicals as well as of conditions that could not be reproduced in the laboratory system. A MET-degrading strain belonging to the genus Aminobacter has been isolated from the chemostat community. This strain was able to completely consume 15 mg/l of MET within three days in the test. However, community analysis revealed that the fluctuation in relative abundance of this genus (<1%) could not be correlated to the fluctuation in biodegradation capacity of the chemostat community.
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http://dx.doi.org/10.1016/j.ecoenv.2019.109414DOI Listing
October 2019

Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans.

mBio 2019 07 9;10(4). Epub 2019 Jul 9.

Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism's central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism. Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable "operating system" of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.
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http://dx.doi.org/10.1128/mBio.00805-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747724PMC
July 2019

Microbial Communities in Sediments From Four Mildly Acidic Ephemeral Salt Lakes in the Yilgarn Craton (Australia) - Terrestrial Analogs to Ancient Mars.

Front Microbiol 2019 6;10:779. Epub 2019 May 6.

Centro de Química-Física Molecular-Institute of Nanoscience and Nanotechnology (CQFM-IN), Institute for Bioengineering and Biosciences (iBB), Departamento de Engenharia Química, Instituto Superior Técnico (IST), Universidade de Lisboa, Lisbon, Portugal.

The Yilgarn Craton in Australia has a large number of naturally occurring shallow ephemeral lakes underlain by a dendritic system of paleodrainage channels. Processes like evaporation, flooding, erosion, as well as inflow of saline, often acidic and ion-rich groundwater contribute to the (dynamic) nature of the lakes and the composition of the sediments. The region has previously been described as an analog environment for early Mars due to its geological and geophysical similarities. Here, we investigated sediment samples of four lake environments aimed at getting a fundamental understanding of the native microbial communities and the mineralogical and (bio)chemical composition of the sediments they are associated with. The dominant mineral phases in the sediments were quartz, feldspars and amphiboles, while halite and gypsum were the only evaporites detected. Element analysis revealed a rich and complex image, in which silicon, iron, and aluminum were the dominant ions, but relative high concentrations of trace elements such as strontium, chromium, zirconium, and barium were also found. The concentrations of organic carbon, nitrogen, and phosphorus were generally low. 16S amplicon sequencing on the Illumina platform showed the presence of diverse microbial communities in all four lake environments. We found that most of the communities were dominated by extremely halophilic Archaea of the family. The dynamic nature of these lakes appears to influence the biological, biochemical, and geological components of the ecosystem to a large effect. Inter- and intra-lake variations in the distributions of microbial communities were significant, and could only to a minor degree be explained by underlying environmental conditions. The communities are likely significantly influenced by small scale local effects caused by variations in geological settings and dynamic interactions caused by aeolian transport and flooding and evaporation events.
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http://dx.doi.org/10.3389/fmicb.2019.00779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512757PMC
May 2019

Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations.

Photosynth Res 2019 May 23;140(2):129-139. Epub 2018 Aug 23.

Leiden Observatory, Leiden University, P.O. Box 9513, 2300 RA, Leiden, The Netherlands.

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light-harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study, we have measured the circular polarizance of several multicellular green, red, and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life.
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http://dx.doi.org/10.1007/s11120-018-0572-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548066PMC
May 2019

Imaging linear and circular polarization features in leaves with complete Mueller matrix polarimetry.

Biochim Biophys Acta Gen Subj 2018 06 9;1862(6):1350-1363. Epub 2018 Mar 9.

Optical Sensing Lab, Department of Electrical and Computer Engineering, North Carolina State University, Raleigh, NC 27695, USA.

Spectropolarimetry of intact plant leaves allows to probe the molecular architecture of vegetation photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological information. In addition to the molecular signals due to the photosynthetic machinery, the cell structure and its arrangement within a leaf can create and modify polarization signals. Using Mueller matrix polarimetry with rotating retarder modulation, we have visualized spatial variations in polarization in transmission around the chlorophyll a absorbance band from 650 nm to 710 nm. We show linear and circular polarization measurements of maple leaves and cultivated maize leaves and discuss the corresponding Mueller matrices and the Mueller matrix decompositions, which show distinct features in diattenuation, polarizance, retardance and depolarization. Importantly, while normal leaf tissue shows a typical split signal with both a negative and a positive peak in the induced fractional circular polarization and circular dichroism, the signals close to the veins only display a negative band. The results are similar to the negative band as reported earlier for single macrodomains. We discuss the possible role of the chloroplast orientation around the veins as a cause of this phenomenon. Systematic artefacts are ruled out as three independent measurements by different instruments gave similar results. These results provide better insight into circular polarization measurements on whole leaves and options for vegetation remote sensing using circular polarization.
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http://dx.doi.org/10.1016/j.bbagen.2018.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970099PMC
June 2018

Regulation of nitrogen metabolism in the nitrate-ammonifying soil bacterium Bacillus vireti and evidence for its ability to grow using N2 O as electron acceptor.

Environ Microbiol 2016 09 18;18(9):2937-50. Epub 2016 Jan 18.

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Science, Ås, Norway.

Bacillus vireti is a nitrate-ammonifying bacterium and a partial denitrifier, reducing NO3 (-) , NO2 (-) , NO and N2 O with NarG, NrfA, CbaA and NosZ respectively. Growth is optimized through successive use of the electron acceptors O2 and NO3 (-) , followed by NO2 (-) , NO and N2 O. Fermentation takes place simultaneously with anaerobic respiration. When grown in batch culture with 5 mM initial NO3 (-) , transcription of nrfA was high and most NO3 (-) was reduced to NH4 (+) . With 20 mM initial NO3 (-) , nrfA transcription was lower and more than 50% of the nitrate was recovered as NO, N2 O and N2 . Analysis of gene transcription patterns and corresponding gas kinetics indicated that O2 and NO2 (-) or NO are main controllers of nrfA, nirB, cbaA and nosZ transcription. This was corroborated by analyses of putative binding regions for specific transcriptional regulators. Furthermore, we demonstrate that N2 O reduction in B. vireti supports growth. The high nosZ transcription but low N2 O production seen at 5 mM NO3 (-) implies that this organism can use N2 O reductase to scavenge N2 O from other organisms in the soil, thus possibly acting as a net sink for N2 O.
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http://dx.doi.org/10.1111/1462-2920.13124DOI Listing
September 2016

The nitrate-ammonifying and nosZ-carrying bacterium Bacillus vireti is a potent source and sink for nitric and nitrous oxide under high nitrate conditions.

Environ Microbiol 2014 Oct 15;16(10):3196-210. Epub 2014 May 15.

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Science, Ås, Norway.

Several Gram-positive bacteria carry genes for anaerobic reduction of NO3(-) via NO2(-) to NH4(+) or gaseous nitrogen compounds, but the processes are understudied for these organisms. Here, we present results from a whole-genome analysis of the soil bacterium Bacillus vireti and a phenotypic characterization of intermediate and end-products, formed under anoxic conditions in the presence of NO3(-). Bacillus vireti has a versatile metabolism. It produces acetate, formate, succinate and lactate from fermentation and performs dissimilatory nitrate reduction via NO2(-) to ammonium (DNRA) using NrfA, while NirB may detoxify NO2(-) in the cytoplasm. Moreover, it produces NO from an unknown source and reduces it via N2O to N2 using two enzymes connected to denitrification: an unusual NO reductase, qCuA Nor encoded by cbaA, and a z-type N2O reductase, encoded by nosZ. In batch cultures, B. vireti reduced all NO3(-) to NO2(-) before the NO2(-) was reduced further. The quantities of all products varied with the initial NO3(-) concentration. With 5 mM NO3(-) , 90% was reduced to NH4 (+) while with ≥ 20 mM NO3(-), 50% was reduced to NO, N2O and N2. This organism is thus an aggressive NO2(-) accumulator and may act as a net source and sink of NO and N2O.
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http://dx.doi.org/10.1111/1462-2920.12478DOI Listing
October 2014

Expression of nitrous oxide reductase in Paracoccus denitrificans is regulated by oxygen and nitric oxide through FnrP and NNR.

Microbiology (Reading) 2012 Mar 15;158(Pt 3):826-834. Epub 2011 Dec 15.

Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway.

The reductases performing the four steps of denitrification are controlled by a network of transcriptional regulators and ancillary factors responding to intra- and extracellular signals, amongst which are oxygen and N oxides (NO and NO2(-)). Although many components of the regulatory network have been identified, there are gaps in our understanding of their role(s) in controlling the expression of the various reductases, in particular the environmentally important N(2)O reductase (N(2)OR). We investigated denitrification phenotypes of Paracoccus denitrificans mutants deficient in: (i) regulatory proteins (three FNR-type transcriptional regulators, NarR, NNR and FnrP, and NirI, which is involved in transcription activation of the structural nir cluster); (ii) functional enzymes (NO reductase and N(2)OR); or (iii) ancillary factors involved in N(2)O reduction (NirX and NosX). A robotized incubation system allowed us to closely monitor changes in concentrations of oxygen and all gaseous products during the transition from oxic to anoxic respiration. Strains deficient in NO reductase were able to grow during denitrification, despite reaching micromolar concentrations of NO, but were unable to return to oxic respiration. The FnrP mutant showed linear anoxic growth in a medium with nitrate as the sole NO(x), but exponential growth was restored by replacing nitrate with nitrite. We interpret this as nitrite limitation, suggesting dual transcriptional control of respiratory nitrate reductase (NAR) by FnrP and NarR. Mutations in either NirX or NosX did not affect the phenotype, but the double mutant lacked the potential to reduce N(2)O. Finally, we found that FnrP and NNR are alternative and equally effective inducers of N(2)OR.
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http://dx.doi.org/10.1099/mic.0.054148-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541799PMC
March 2012

Ferric reductase A is essential for effective iron acquisition in Paracoccus denitrificans.

Microbiology (Reading) 2009 Apr;155(Pt 4):1294-1301

Department of Biochemistry, Faculty of Science, Masaryk University, Czech Republic, CZ-611 37 Brno, Czech Republic.

Based on N-terminal sequences obtained from the purified cytoplasmic ferric reductases FerA and FerB, their corresponding genes were identified in the published genome sequence of Paracoccus denitrificans Pd1222. The ferA and ferB genes were cloned and individually inactivated by insertion of a kanamycin resistance marker, and then returned to P. denitrificans for exchange with their wild-type copies. The resulting ferA and ferB mutant strains showed normal growth in brain heart infusion broth. Unlike the ferB mutant, the strain lacking FerA did not grow on succinate minimal medium with ferric 2,3-dihydroxybenzoate as the iron source, and grew only poorly in the presence of ferric sulfate, chloride, citrate, NTA, EDTA and EGTA. Moreover, the ferA mutant strain was unable to produce catechols, which are normally detectable in supernatants from iron-limited wild-type cultures. Complementation of the ferA mutation using a derivative of the conjugative broad-host-range plasmid pEG400 that contained the whole ferA gene and its putative promoter region largely restored the wild-type phenotype. Partial, though significant, restoration could also be achieved with 1 mM chorismate added to the growth medium. The purified FerA protein acted as an NADH : FMN oxidoreductase and catalysed the FMN-mediated reductive release of iron from the ferric complex of parabactin, the major catecholate siderophore of P. denitrificans. The deduced amino acid sequence of the FerA protein has closest similarity to flavin reductases that form part of the flavin-dependent two-component monooxygenases. Taken together, our results demonstrate an essential role of reduced flavins in the utilization of exogenous ferric iron. These flavins not only provide the electrons for Fe(III) reduction but most probably also affect the rate of siderophore production.
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http://dx.doi.org/10.1099/mic.0.022715-0DOI Listing
April 2009

Characterization of the quinone reductase activity of the ferric reductase B protein from Paracoccus denitrificans.

Arch Biochem Biophys 2009 Mar 30;483(1):29-36. Epub 2008 Dec 30.

Department of Biochemistry, Masaryk University, Brno, Czech Republic.

The ferric reductase B (FerB) protein of Paracoccus denitrificans exhibits activity of an NAD(P)H: Fe(III) chelate, chromate and quinone oxidoreductase. Sequence analysis places FerB in a family of soluble flavin-containing quinone reductases. The enzyme reduces a range of quinone substrates, including derivatives of 1,4-benzoquinone and 1,2- and 1,4-naphthoquinone, via a ping-pong kinetic mechanism. Dicoumarol and Cibacron Blue 3GA are competitive inhibitors of NADH oxidation. In the case of benzoquinones, FerB apparently acts through a two-electron transfer process, whereas in the case of naphthoquinones, one-electron reduction takes place resulting in the formation of semiquinone radicals. A ferB mutant strain exhibited an increased resistance to 1,4-naphthoquinone, attributable to the absence of the FerB-mediated redox cycling. The ferB promoter displayed a high basal activity throughout the growth of P. denitrificans, which could not be further enhanced by addition of different types of naphthoquinones. This indicates that the ferB gene is expressed constitutively.
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http://dx.doi.org/10.1016/j.abb.2008.12.016DOI Listing
March 2009

Effects of probiotic Lactobacillus salivarius W24 on the compositional stability of oral microbial communities.

Arch Oral Biol 2009 Feb 30;54(2):132-7. Epub 2008 Oct 30.

Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Louwesweg 1, 1066 EA Amsterdam, The Netherlands.

Probiotics are microorganisms beneficial to gastrointestinal health. Although some strains are also known to possess positive effects on oral health, the effects of most intestinal probiotics on the oral microflora remain unknown. We assessed the ability of the intestinal probiotic Lactobacillus salivarius W24 to incorporate into and to affect the compositional stability and cariogenicity of oral microbial communities. Microtiter plates with hydroxyapatite discs were incubated with W24 ("+W24") or without W24 ("-W24") and saliva from four individuals in plain ("-sucrose") or sucrose-supplemented ("+sucrose") medium. Biofilms were subjected to community profiling by 16S rRNA gene-based Denaturing Gradient Gel Electrophoresis (DGGE) after 72h growth. Diversity (Shannon-Weaver index) and similarities (Pearson correlation) between biofilm communities were calculated. Microcosms "+sucrose" were less diverse and more acidic than "-sucrose" microcosms (p<0.001). The effects of W24 on the community profiles were pH dependent: at pH 4 ("+sucrose"), the respective "+W24" and "-W24" microcosms differed significantly more from each other than if the pH was approximately 7 ("-sucrose"). The pH of "+W24/+sucrose" microcosms was lower (p<0.05) than the pH of the microcosms supplemented with sucrose alone ("-W24/+sucrose"). Although not able to form a monospecies biofilm, L. salivarius W24 established itself into the oral community if inoculated simultaneously with the microcosm. In the presence of sucrose and low pH, W24 further lowered the pH and changed the community profiles of these microcosms. Screening of probiotics for their effects on oral microbial communities allows selecting strains without a potential for oral health hazards.
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http://dx.doi.org/10.1016/j.archoralbio.2008.09.007DOI Listing
February 2009

The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems.

Biochim Biophys Acta 2008 Dec 30;1777(12):1480-90. Epub 2008 Sep 30.

Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany.

Respiration is fundamental to the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. In principle, the free energy of a redox reaction catalysed by a membrane-bound electron transport chain is transduced via the generation of an electrochemical ion (usually proton) gradient across a coupling membrane that drives ATP synthesis. The proton motive force (pmf) can be built up by different mechanisms like proton pumping, quinone/quinol cycling or by a redox loop. The latter couples electron transport to a net proton transfer across the membrane without proton pumping. Instead, charge separation is achieved by quinone-reactive enzymes or enzyme complexes whose active sites for substrates and quinones are situated on different sides of the coupling membrane. The necessary transmembrane electron transport is usually accomplished by the presence of two haem groups that face opposite sides of the membrane. There are many different enzyme complexes that are part of redox loops and their catalysed redox reactions can be either electrogenic, electroneutral (non-proton motive) or even pmf-consuming. This article gives conceptual classification of different operational organisations of redox loops and uses this as a platform from which to explore the biodiversity of quinone/quinol-cycling redox systems.
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http://dx.doi.org/10.1016/j.bbabio.2008.09.008DOI Listing
December 2008

MLPA diagnostics of complex microbial communities: relative quantification of bacterial species in oral biofilms.

J Microbiol Methods 2008 Dec 13;75(3):558-65. Epub 2008 Sep 13.

Department of Cariology, Endodontology, Pedodontology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, The Netherlands.

A multitude of molecular methods are currently used for identification and characterization of oral biofilms or for community profiling. However, multiplex PCR techniques that are able to routinely identify several species in a single assay are not available. Multiplex Ligation-dependent Probe Amplification (MLPA) identifies up to 45 unique fragments in a single tube PCR. Here we report a novel use of MLPA in the relative quantification of targeted microorganisms in a community of oral microbiota. We designed 9 species specific probes for: Actinomyces gerencseriae, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans, Lactobacillus acidophilus, Rothia dentocariosa, Streptococcus mutans, Streptococcus sanguinis and Veillonella parvula; and genus specific probes for selected oral Streptococci and Lactobacilli based on their 16S rDNA sequences. MLPA analysis of DNA pooled from the strains showed the expected specific MLPA products. Relative quantification of a serial dilution of equimolar DNA showed that as little as 10 pg templates can be detected with clearly discernible signals. Moreover, a 2 to 7% divergence in relative signal ratio of amplified probes observed from normalized peak area values suggests MLPA can be a cheaper alternative to using qPCR for quantification. We observed 2 to 6 fold fluctuations in signal intensities of MLPA products in DNAs isolated from multispecies biofilms grown in various media for various culture times. Furthermore, MLPA analyses of DNA isolated from saliva obtained from different donors gave a varying number and intensity of signals. This clearly shows the usefulness of MLPA in a quantitative description of microbial shifts.
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http://dx.doi.org/10.1016/j.mimet.2008.08.012DOI Listing
December 2008

Novel nirK cluster genes in Nitrosomonas europaea are required for NirK-dependent tolerance to nitrite.

J Bacteriol 2005 Oct;187(19):6849-51

Evolutionary Genetics and Microbial Ecology Laboratory, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.

Nitrite reductase (NirK) of Nitrosomonas europaea confers tolerance to nitrite (NO2-). The nirK gene is clustered with three genes of unknown physiological function: ncgABC. At present, this organization is unique to nitrifying bacteria. Here we report that the ncgABC gene products facilitate NirK-dependent NO2- tolerance by reversing the negative physiological effect that is associated with the activity of NirK in their absence. We hypothesize that the ncg gene products are involved in the detoxification of nitric oxide that is produced by NirK.
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http://dx.doi.org/10.1128/JB.187.19.6849-6851.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251594PMC
October 2005

NosX function connects to nitrous oxide (N2O) reduction by affecting the Cu(Z) center of NosZ and its activity in vivo.

FEBS Lett 2005 Aug;579(21):4605-9

Institute of Applied Biosciences, Division of Molecular Microbiology, University of Karlsruhe, D-76128 Karlsruhe, Germany.

The effect of loss of the 34-kDa periplasmic NosX protein on the properties of N2O reductase was investigated with an N2O-respiration negative, double mutant of the paralogous genes nosX and nirX of Paracoccus denitrificans. In spite of absence of whole-cell N2O-reducing activity, the purified reductase was catalytically active, which attributes NosX a physiological role in sustaining the reaction cycle. N2O reductase exhibited the spectroscopic features of Cu(A) and the redox-inert, paramagnetic state, Cu(Z)*, of the catalytic center. Cu(Z)*, hitherto considered the result of spontaneous reaction of the reductase with dioxygen, attains cellular significance.
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http://dx.doi.org/10.1016/j.febslet.2005.07.023DOI Listing
August 2005

Denitrification and ammonia oxidation by Nitrosomonas europaea wild-type, and NirK- and NorB-deficient mutants.

Microbiology (Reading) 2004 Dec;150(Pt 12):4107-14

Department of Microbiology, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany.

The phenotypes of three different Nitrosomonas europaea strains--wild-type, nitrite reductase (NirK)-deficient and nitric oxide reductase (NorB)-deficient strains--were characterized in chemostat cell cultures, and the effect of nitric oxide (NO) on metabolic activities was evaluated. All strains revealed similar aerobic ammonia oxidation activities, but the growth rates and yields of the knock-out mutants were significantly reduced. Dinitrogen (N2) was the main gaseous product of the wild-type, produced via its denitrification activity. The mutants were unable to reduce nitrite to N2, but excreted more hydroxylamine leading to the formation of almost equal amounts of NO, nitrous oxide (N2O) and N2 by chemical auto-oxidation and chemodenitrification of hydroxylamine. Under anoxic conditions Nsm. europaea wild-type gains energy for growth via nitrogen dioxide (NO2)-dependent ammonia oxidation or hydrogen-dependent denitrification using nitrite as electron acceptor. The mutant strains were restricted to NO and/or N2O as electron acceptor and consequently their growth rates and yields were much lower compared with the wild-type. When cells were transferred from anoxic (denitrification) to oxic conditions, the wild-type strain endogenously produced NO and recovered ammonia oxidation within 8 h. In contrast, the mutant strains remained inactive. For recovery of ammonia oxidation activity the NO concentration had to be adjusted to about 10 p.p.m. in the aeration gas.
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http://dx.doi.org/10.1099/mic.0.27382-0DOI Listing
December 2004

Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor.

Mol Microbiol 2004 Oct;54(1):148-58

BioCentrum Amsterdam, Department of Molecular Cell Physiology, Vrije Universiteit, de Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands.

Production of nitric oxide (NO) and nitrous oxide (N(2)O) by ammonia (NH(3))-oxidizing bacteria in natural and man-made habitats is thought to contribute to the undesirable emission of NO and N(2)O into the earth's atmosphere. The NH(3)-oxidizing bacterium Nitrosomonas europaea expresses nitrite reductase (NirK), an enzyme that has so far been studied predominantly in heterotrophic denitrifying bacteria where it is involved in the production of these nitrogenous gases. The finding of nirK homologues in other NH(3)-oxidizing bacteria suggests that NirK is widespread among this group; however, its role in these nitrifying bacteria remains unresolved. We identified a gene, nsrR, which encodes a novel nitrite (NO(2) (-))-sensitive transcription repressor that plays a pivotal role in the regulation of NirK expression in N. europaea. NsrR is a member of the Rrf2 family of putative transcription regulators. NirK was expressed aerobically in response to increasing concentrations of NO(2) (-) and decreasing pH. Disruption of nsrR resulted in the constitutive expression of NirK. NsrR repressed transcription from the nirK gene cluster promoter (P(nir)), the activity of which correlated with NirK expression. Reconstruction of the NsrR-P(nir) system in Escherichia coli revealed that repression by NsrR was reversed by NO(2) (-) in a pH-dependent manner. The findings are consistent with the hypothesis that N. europaea expresses NirK as a defence against the toxic NO(2) (-) that is produced during nitrification.
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http://dx.doi.org/10.1111/j.1365-2958.2004.04248.xDOI Listing
October 2004

Nitric oxide oscillations in Paracoccus denitrificans: the effects of environmental factors and of segregating nitrite reductase and nitric oxide reductase into separate cells.

Arch Biochem Biophys 2004 Sep;429(2):237-43

Department of Biochemistry, Faculty of Science, Masaryk University, Kotlárská 2, CZ-61137 Brno, Czech Republic.

Nitric oxide is a denitrification intermediate which is produced from nitrite and then further converted via nitrous oxide to nitrogen. Here, the effect of low concentrations of the protonophore carbonylcyanide m-chlorophenylhydrazone on the time courses for dissolved gases was examined. While NO was found to oscillate, N(2)O only increased gradually as the reduction of nitrite progressed. The frequency and shape of protonophore-induced NO oscillations were influenced by temperature and the concentration of electron donor N,N,N',N'-tetramethyl-p-phenylene diamine (TMPD) in a manner compatible with the observed differential effects on the two involved enzyme activities. We demonstrated the existence of a pH interval, where [NO] oscillates even without uncoupler addition. Occurrence of nitric oxide oscillations in mixtures of a nitrite reductase mutant with a nitric oxide reductase mutant suggests that they cannot be due to a competition of the enzymes for redox equivalents from one common respiratory chain.
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http://dx.doi.org/10.1016/j.abb.2004.06.026DOI Listing
September 2004

Nitrosomonas europaea expresses a nitric oxide reductase during nitrification.

J Bacteriol 2004 Jul;186(13):4417-21

BioCentrum Amsterdam, Department of Molecular Cell Physiology, Vrije Universiteit, The Netherlands.

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.
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http://dx.doi.org/10.1128/JB.186.13.4417-4421.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC421603PMC
July 2004

Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans.

Microbiology (Reading) 2003 Dec;149(Pt 12):3405-3412

Department of Molecular Cell Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, NL-1081 HV Amsterdam, The Netherlands.

In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, beta-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of beta-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic beta-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N(2)O. Additions of nitrate or nitroprusside lowered beta-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.
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http://dx.doi.org/10.1099/mic.0.26546-0DOI Listing
December 2003

A mutant of Paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase.

J Bacteriol 2003 Nov;185(21):6308-15

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.

In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative electron carriers from the cytochrome bc(1) complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c(550). The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c(550) or both these proteins. We concluded that pseudoazurin and cytochrome c(550) are the alternative electron mediator proteins between the cytochrome bc(1) complex and the cytochrome cd(1)-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC219389PMC
http://dx.doi.org/10.1128/JB.185.21.6308-6315.2003DOI Listing
November 2003

DNA supercoiling by gyrase is linked to nucleoid compaction.

Mol Biol Rep 2002 ;29(1-2):79-82

Molecular Cell Physiology, Free University, Amsterdam, The Netherlands.

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 microm cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.
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http://dx.doi.org/10.1023/a:1020318705894DOI Listing
July 2003

Nitrite reductase of Nitrosomonas europaea is not essential for production of gaseous nitrogen oxides and confers tolerance to nitrite.

J Bacteriol 2002 May;184(9):2557-60

BioCentrum Amsterdam, Department of Molecular Cell Physiology, Vrije Universiteit, NL-1081 Amsterdam, The Netherlands.

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC134999PMC
http://dx.doi.org/10.1128/JB.184.9.2557-2560.2002DOI Listing
May 2002

Expression of the mau gene cluster of Paracoccus denitrificans is controlled by MauR and a second transcription regulator.

Microbiology (Reading) 1997 Mar;143 ( Pt 3):793-801

Departments of Microbial Physiology, Faculty of Biology, Biocentrum Amsterdam, Vrije Universiteit,De Boelelaan 1087, NL-1081 HV,The Netherlands.

The mau gene cluster of Paracoccus denitrificans constitutes 11 genes (10 are located in the transcriptional order mauFBEDACJGMN; the 11th, mauR, is located upstream and divergently transcribed from these genes) that encode a functional methylamine-oxidizing electron transport branch. The mauR gene encodes a LysR-type transcriptional activator essential for induction of the mau operon. In this study, the characteristics of that process were established. By using lacZ transcriptional fusions integrated into the genome of P. denitrificans, it was found that the expression of the mauR gene during growth on methylamine and/or succinate was not autoregulated, but proceeded at a low and constant level. The mauF promoter activity was shown to be controlled by MauR and a second transcriptional regulator. This activity was very high during growth on methylamine, low on succinate plus methylamine, and absent on succinate alone. MauR was overexpressed in Escherichia coli by using a T7 RNA polymerase expression system. Gel shift assays indicated that MauR binds to a 403 bp DNA fragment spanning the mauR-mauF promoter region. It is concluded from these results that the expression of the structural mau genes is dependent on MauR and its inducer, methylamine, as well as on another transcription factor. Both activators are required for high-level transcription from the mauF promoter. It is hypothesized that the two activators act synergistically to activate transcription: the effects of the two activators are not additive and either one alone activates the mauF promoter rather weakly.
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http://dx.doi.org/10.1099/00221287-143-3-793DOI Listing
March 1997