Publications by authors named "Douwe Molenaar"

83 Publications

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

Proteome constraints reveal targets for improving microbial fitness in nutrient-rich environments.

Mol Syst Biol 2021 04;17(4):e10093

TiFN, Wageningen, the Netherlands.

Cells adapt to different conditions via gene expression that tunes metabolism for maximal fitness. Constraints on cellular proteome may limit such expression strategies and introduce trade-offs. Resource allocation under proteome constraints has explained regulatory strategies in bacteria. It is unclear, however, to what extent these constraints can predict evolutionary changes, especially for microorganisms that evolved under nutrient-rich conditions, i.e., multiple available nitrogen sources, such as Lactococcus lactis. Here, we present a proteome-constrained genome-scale metabolic model of L. lactis (pcLactis) to interpret growth on multiple nutrients. Through integration of proteomics and flux data, in glucose-limited chemostats, the model predicted glucose and arginine uptake as dominant constraints at low growth rates. Indeed, glucose and arginine catabolism were found upregulated in evolved mutants. At high growth rates, pcLactis correctly predicted the observed shutdown of arginine catabolism because limited proteome availability favored lactate for ATP production. Thus, our model-based analysis is able to identify and explain the proteome constraints that limit growth rate in nutrient-rich environments and thus form targets of fitness improvement.
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http://dx.doi.org/10.15252/msb.202010093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022198PMC
April 2021

Macrophage ATP citrate lyase deficiency stabilizes atherosclerotic plaques.

Nat Commun 2020 12 8;11(1):6296. Epub 2020 Dec 8.

Department of Medical Biochemistry, Experimental Vascular Biology, Amsterdam Infection and Immunity, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Macrophages represent a major immune cell population in atherosclerotic plaques and play central role in the progression of this lipid-driven chronic inflammatory disease. Targeting immunometabolism is proposed as a strategy to revert aberrant macrophage activation to improve disease outcome. Here, we show ATP citrate lyase (Acly) to be activated in inflammatory macrophages and human atherosclerotic plaques. We demonstrate that myeloid Acly deficiency induces a stable plaque phenotype characterized by increased collagen deposition and fibrous cap thickness, along with a smaller necrotic core. In-depth functional, lipidomic, and transcriptional characterization indicate deregulated fatty acid and cholesterol biosynthesis and reduced liver X receptor activation within the macrophages in vitro. This results in macrophages that are more prone to undergo apoptosis, whilst maintaining their capacity to phagocytose apoptotic cells. Together, our results indicate that targeting macrophage metabolism improves atherosclerosis outcome and we reveal Acly as a promising therapeutic target to stabilize atherosclerotic plaques.
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http://dx.doi.org/10.1038/s41467-020-20141-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722882PMC
December 2020

Searching for principles of microbial physiology.

FEMS Microbiol Rev 2020 11;44(6):821-844

Systems Biology Lab, AIMMS, De Boelelaan 1108, 1081 HZ, VU University, Amsterdam, The Netherlands.

Why do evolutionarily distinct microorganisms display similar physiological behaviours? Why are transitions from high-ATP yield to low(er)-ATP yield metabolisms so widespread across species? Why is fast growth generally accompanied with low stress tolerance? Do these regularities occur because most microbial species are subject to the same selective pressures and physicochemical constraints? If so, a broadly-applicable theory might be developed that predicts common microbiological behaviours. Microbial systems biologists have been working out the contours of this theory for the last two decades, guided by experimental data. At its foundations lie basic principles from evolutionary biology, enzyme biochemistry, metabolism, cell composition and steady-state growth. The theory makes predictions about fitness costs and benefits of protein expression, physicochemical constraints on cell growth and characteristics of optimal metabolisms that maximise growth rate. Comparisons of the theory with experimental data indicates that microorganisms often aim for maximisation of growth rate, also in the presence of stresses; they often express optimal metabolisms and metabolic proteins at optimal concentrations. This review explains the current status of the theory for microbiologists; its roots, predictions, experimental evidence and future directions.
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http://dx.doi.org/10.1093/femsre/fuaa034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685786PMC
November 2020

Transthyretin-Binding Activity of Complex Mixtures Representing the Composition of Thyroid-Hormone Disrupting Contaminants in House Dust and Human Serum.

Environ Health Perspect 2020 01 31;128(1):17015. Epub 2020 Jan 31.

Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden.

Background: House dust contains many organic contaminants that can compete with the thyroid hormone (TH) thyroxine () for binding to transthyretin (TTR). How these contaminants work together at levels found in humans and how displacement from TTR relates to -TTR binding is unknown.

Objectives: Our aims were to determine the TTR-binding potency for contaminant mixtures as found in house dust, maternal serum, and infant serum; to study whether the TTR-binding potency of the mixtures follows the principle of concentration addition; and to extrapolate the TTR-binding potency to inhibition levels of -TTR binding in maternal and infant serum.

Methods: Twenty-five contaminants were tested for their capacity to compete for TTR-binding with a fluorescent FITC- probe. Three mixtures were reconstituted proportionally to median concentrations for these chemicals in house dust, maternal serum, or infant serum from Nordic countries. Measured concentration-response curves were compared with concentration-response curves predicted by concentration addition. For each reconstituted serum mixture, its inhibitor-TTR dissociation constant () was used to estimate inhibition levels of -TTR binding in human blood.

Results: The TTR-binding potency of the mixtures was well predicted by concentration addition. The inhibition in FITC- binding observed for the mixtures reflecting median concentrations in maternal and infant serum was extrapolated to 1.3% inhibition of -TTR binding in maternal and 1.5% in infant blood. For nontested mixtures reflecting high-end serum concentrations, these estimates were 6.2% and 4.9%, respectively.

Discussion: The relatively low estimated inhibition levels at median exposure levels may explain why no relationship between exposure to TTR-binding compounds and circulating levels in humans has been reported, so far. We hypothesize, however, that 1.3% inhibition of -TTR binding may ultimately be decisive for reaching a status of maternal hypothyroidism or hypothyroxinemia associated with impaired neurodevelopment in children. https://doi.org/10.1289/EHP5911.
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http://dx.doi.org/10.1289/EHP5911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015555PMC
January 2020

New Insights Into Cinnamoyl Esterase Activity of .

Front Microbiol 2019 8;10:2597. Epub 2019 Nov 8.

Escola Superior de Biotecnologia, Centro de Biotecnologia e Química Fina, Universidade Católica Portuguesa, Porto, Portugal.

Some strains of possess cinnamoyl esterase activity that can be relevant in the malolactic stage of wine production liberating hydroxycinnamic acids that are precursors of volatile phenols responsible for sensory faults. The objective of this study was to better understand the basis of the differential activity between strains. After initial screening, five commercial strains of were selected, three were found to exhibit cinnamoyl esterase activity (CE+) and two not (CE-). Although the use of functional annotation of genes revealed genotypic variations between the strains, no specific genes common only to the three CE+ strains could explain the different activities. Pasteurized wine was used as a natural source of tartrate esters in growth and metabolism experiments conducted in MRS medium, whilst commercial -caftaric acid was used as substrate for enzyme assays. Detoxification did not seem to be the main biological mechanism involved in the activity since unlike its phenolic cleavage products and their immediate metabolites (caffeic acid and 4-ethylcatechol), -caftaric acid was not toxic toward . In the case of the two CE+ strains Oenos and CiNe, wine-exposed samples showed a more rapid degradation of -caftaric acid than the unexposed ones. The CE activity was present in all cell-free extracts of both wine-exposed and unexposed strains, except in the cell-free extracts of the CE- strain CH11. This activity may be constitutive rather than induced by exposure to tartrate esters. -caftaric acid was totally cleaved to -caffeic acid by cell-free extracts of the three CE+ strains, whilst cell-free extracts of the CE- strain CH16 showed significantly lower activity, although higher for the strains in experiments with no prior wine exposure. The EstB28 esterase gene, found in the genomes of the 5 strains, did not reveal any difference on the upstream regulation and transport functionality between the strains. This study highlights the complexity of the basis of this activity in wine related population. Variable cinnamoyl esterases or/and membrane transport activities in the strains analyzed and a possible implication of wine molecules could explain this phenomenon.
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http://dx.doi.org/10.3389/fmicb.2019.02597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857119PMC
November 2019

Vanishing white matter: deregulated integrated stress response as therapy target.

Ann Clin Transl Neurol 2019 08 18;6(8):1407-1422. Epub 2019 Jul 18.

Child Neurology, Emma Children's Hospital, Amsterdam University Medical Centers, Vrije Universiteit and Amsterdam Neuroscience, Amsterdam, The Netherlands.

Objective: Vanishing white matter (VWM) is a fatal, stress-sensitive leukodystrophy that mainly affects children and is currently without treatment. VWM is caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B) that is crucial for initiation of mRNA translation and its regulation during the integrated stress response (ISR). Mutations reduce eIF2B activity. VWM pathomechanisms remain unclear. In contrast with the housekeeping function of eIF2B, astrocytes are selectively affected in VWM. One study objective was to test our hypothesis that in the brain translation of specific mRNAs is altered by eIF2B mutations, impacting primarily astrocytes. The second objective was to investigate whether modulation of eIF2B activity could ameliorate this altered translation and improve the disease.

Methods: Mice with biallelic missense mutations in eIF2B that recapitulate human VWM were used to screen for mRNAs with altered translation in brain using polysomal profiling. Findings were verified in brain tissue from VWM patients using qPCR and immunohistochemistry. The compound ISRIB (for "ISR inhibitor") was administered to VWM mice to increase eIF2B activity. Its effect on translation, neuropathology, and clinical signs was assessed.

Results: In brains of VWM compared to wild-type mice we observed the most prominent changes in translation concerning ISR mRNAs; their expression levels correlated with disease severity. We substantiated these findings in VWM patients' brains. ISRIB normalized expression of mRNA markers, ameliorated brain white matter pathology and improved motor skills in VWM mice.

Interpretation: The present findings show that ISR deregulation is central in VWM pathomechanisms and a viable target for therapy.
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http://dx.doi.org/10.1002/acn3.50826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689685PMC
August 2019

A systematic assessment of current genome-scale metabolic reconstruction tools.

Genome Biol 2019 08 7;20(1):158. Epub 2019 Aug 7.

Systems Bioinformatics, AIMMS, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

Background: Several genome-scale metabolic reconstruction software platforms have been developed and are being continuously updated. These tools have been widely applied to reconstruct metabolic models for hundreds of microorganisms ranging from important human pathogens to species of industrial relevance. However, these platforms, as yet, have not been systematically evaluated with respect to software quality, best potential uses and intrinsic capacity to generate high-quality, genome-scale metabolic models. It is therefore unclear for potential users which tool best fits the purpose of their research.

Results: In this work, we perform a systematic assessment of current genome-scale reconstruction software platforms. To meet our goal, we first define a list of features for assessing software quality related to genome-scale reconstruction. Subsequently, we use the feature list to evaluate the performance of each tool. To assess the similarity of the draft reconstructions to high-quality models, we compare each tool's output networks with that of the high-quality, manually curated, models of Lactobacillus plantarum and Bordetella pertussis, representatives of gram-positive and gram-negative bacteria, respectively. We additionally compare draft reconstructions with a model of Pseudomonas putida to further confirm our findings. We show that none of the tools outperforms the others in all the defined features.

Conclusions: Model builders should carefully choose a tool (or combinations of tools) depending on the intended use of the metabolic model. They can use this benchmark study as a guide to select the best tool for their research. Finally, developers can also benefit from this evaluation by getting feedback to improve their software.
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http://dx.doi.org/10.1186/s13059-019-1769-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685185PMC
August 2019

Finding Functional Differences Between Species in a Microbial Community: Case Studies in Wine Fermentation and Kefir Culture.

Front Microbiol 2019 25;10:1347. Epub 2019 Jun 25.

Systems Bioinformatics, VU University Amsterdam, Amsterdam, Netherlands.

Microbial life usually takes place in a community where individuals interact, by competition for nutrients, cross-feeding, inhibition by end-products, but also by their spatial distribution. Lactic acid bacteria are prominent members of microbial communities responsible for food fermentations. Their niche in a community depends on their own properties as well as those of the other species. Here, we apply a computational approach, which uses only genomic and metagenomic information and functional annotation of genes, to find properties that distinguish a species from others in the community, as well as to follow individual species in a community. We analyzed isolated and sequenced strains from a kefir community, and metagenomes from wine fermentations. We demonstrate how the distinguishing properties of an organism lead to experimentally testable hypotheses concerning the niche and the interactions with other species. We observe, for example, that , a dominant organism in kefir, stands out among the because it potentially has more amino acid auxotrophies. Using metagenomic analysis of industrial wine fermentations we investigate the role of an inoculated in malolactic fermentation. We observed that thrives better on white than on red wine fermentations and has the largest number of phosphotransferase system among the bacteria observed in the wine communities. Also, together with , and genera had the highest number of genes involved in biosynthesis of amino acids.
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http://dx.doi.org/10.3389/fmicb.2019.01347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603220PMC
June 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

Taxonomic and Functional Characterization of the Microbial Community During Spontaneous Fermentation of Riesling Must.

Front Microbiol 2019 9;10:697. Epub 2019 Apr 9.

Section for Evolutionary Genomics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

Although there is an extensive tradition of research into the microbes that underlie the winemaking process, much remains to be learnt. We combined the high-throughput sequencing (HTS) tools of metabarcoding and metagenomics, to characterize how microbial communities of Riesling musts sampled at four different vineyards, and their subsequent spontaneously fermented derivatives, vary. We specifically explored community variation relating to three points: (i) how microbial communities vary by vineyard; (ii) how community biodiversity changes during alcoholic fermentation; and (iii) how microbial community varies between musts that successfully complete alcoholic fermentation and those that become 'stuck' in the process. Our metabarcoding data showed a general influence of microbial composition at the vineyard level. Two of the vineyards (4 and 5) had strikingly a change in the differential abundance of . We therefore additionally performed shotgun metagenomic sequencing on a subset of the samples to provide preliminary insights into the potential relevance of this observation, and used the data to both investigate functional potential and reconstruct draft genomes (bins). At these two vineyards, we also observed an increase in non-Saccharomycetaceae fungal functions, and a decrease in bacterial functions during the early fermentation stage. The binning results yielded 11 coherent bins, with both vineyards sharing the yeast bins and . Read recruitment and functional analysis of this data revealed that during fermentation, a high abundance of might serve as a biocontrol agent against bacteria, via a putative iron depletion pathway, and this in turn could help dominate the fermentation. During alcoholic fermentation, we observed a general decrease in biodiversity in both the metabarcoding and metagenomic data. Unexpected behavior was observed in vineyard 4 according to metagenomic analyses based on reference-based read mapping. Analysis of open reading frames using these data showed an increase of functions assigned to class Actinobacteria in the end of fermentation. Therefore, we hypothesize that bacteria might sit-and-wait until activity slows down. Complementary approaches to annotation instead of relying a single database provide more coherent information true species. Lastly, our metabarcoding data enabled us to identify a relationship between stuck fermentations and abundance. Given that robust chemical analysis indicated that although the stuck samples contained residual glucose, all fructose had been consumed, we hypothesize that this was because fructophilic , rather than , dominated these fermentations. Overall, our results showcase the different ways in which metagenomic analyses can improve our understanding of the wine alcoholic fermentation process.
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http://dx.doi.org/10.3389/fmicb.2019.00697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465770PMC
April 2019

Comparative genomics of human Lactobacillus crispatus isolates reveals genes for glycosylation and glycogen degradation: implications for in vivo dominance of the vaginal microbiota.

Microbiome 2019 03 29;7(1):49. Epub 2019 Mar 29.

Department of Molecular Cell Biology, Faculty of Science, O|2 Lab Building, VU University, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands.

Background: A vaginal microbiota dominated by lactobacilli (particularly Lactobacillus crispatus) is associated with vaginal health, whereas a vaginal microbiota not dominated by lactobacilli is considered dysbiotic. Here we investigated whether L. crispatus strains isolated from the vaginal tract of women with Lactobacillus-dominated vaginal microbiota (LVM) are pheno- or genotypically distinct from L. crispatus strains isolated from vaginal samples with dysbiotic vaginal microbiota (DVM).

Results: We studied 33 L. crispatus strains (n = 16 from LVM; n = 17 from DVM). Comparison of these two groups of strains showed that, although strain differences existed, both groups degraded various carbohydrates, produced similar amounts of organic acids, inhibited Neisseria gonorrhoeae growth, and did not produce biofilms. Comparative genomics analyses of 28 strains (n = 12 LVM; n = 16 DVM) revealed a novel, 3-fragmented glycosyltransferase gene that was more prevalent among strains isolated from DVM. Most L. crispatus strains showed growth on glycogen-supplemented growth media. Strains that showed less-efficient (n = 6) or no (n = 1) growth on glycogen all carried N-terminal deletions (respectively, 29 and 37 amino acid deletions) in a putative pullulanase type I protein.

Discussion: L. crispatus strains isolated from LVM were not phenotypically distinct from L. crispatus strains isolated from DVM; however, the finding that the latter were more likely to carry a 3-fragmented glycosyltransferase gene may indicate a role for cell surface glycoconjugates, which may shape vaginal microbiota-host interactions. Furthermore, the observation that variation in the pullulanase type I gene is associated with growth on glycogen discourages previous claims that L. crispatus cannot directly utilize glycogen.
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http://dx.doi.org/10.1186/s40168-019-0667-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441167PMC
March 2019

Adaption to glucose limitation is modulated by the pleotropic regulator CcpA, independent of selection pressure strength.

BMC Evol Biol 2019 01 10;19(1):15. Epub 2019 Jan 10.

Molecular Genetics Group, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.

Background: A central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution experiments are ideally suited to approach these questions. For example, prolonged chemostats provide a constant environment in which the growth rate can be set, and the adaptive process of the organism to such environment can be subsequently characterized.

Results: We performed parallel laboratory evolution of Lactococcus lactis in chemostats varying the quantitative value of the selective pressure by imposing two different growth rates. A mutation in one specific amino acid residue of the global transcriptional regulator of carbon metabolism, CcpA, was selected in all of the evolution experiments performed. We subsequently showed that this mutation confers predictable fitness improvements at other glucose-limited growth rates as well. In silico protein structural analysis of wild type and evolved CcpA, as well as biochemical and phenotypic assays, provided the underpinning molecular mechanisms that resulted in the specific reprogramming favored in constant environments.

Conclusion: This study provides a comprehensive understanding of a case of microbial evolution and hints at the wide dynamic range that a single fitness-enhancing mutation may display. It demonstrates how the modulation of a pleiotropic regulator can be used by cells to improve one trait while simultaneously work around other limiting constraints, by fine-tuning the expression of a wide range of cellular processes.
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http://dx.doi.org/10.1186/s12862-018-1331-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327505PMC
January 2019

Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With .

Front Microbiol 2018 21;9:2218. Epub 2018 Sep 21.

Microbiology and Systems Biology, Netherlands Organization for Applied Scientific Research (TNO), Amsterdam, Netherlands.

A variety of foods fermented with lactic acid bacteria serve as dietary staples in many African communities; yet, their bacterial profiles are poorly characterized. The integration of health-promoting probiotics into naturally fermented milk products could make a profound impact on human health. Here, we characterize the bacterial community composition of a naturally fermented milk product () from northern Senegal, prepared in wooden bowls (lahals) with a bacterial biofilm to steer the fermentation process. We incorporated a probiotic starter culture containing the most documented probiotic strain GG (generic strain name yoba 2012) into the local fermentation process. Bar-coded 16S rRNA amplicon sequencing of samples indicated that the bacterial community of has high species richness with over 100 bacterial genera; however, few have high abundance. In contrast to the diverse bacterial compositions of other characterized naturally fermented milk products, the composition of predominantly consists of the lactic acid bacteria and , resembling the bacterial composition in regular yogurt. The bacterial community composition of varies geographically based on the presence of some genera, including , and , but this trend is not consistent within production communities. The diversity of bacterial communities is much higher in the biofilm than in the naturally fermented milk products, which is in turn greater than in commercial yogurts. Addition of a starter culture with yoba 2012 to milk in led to substantial growth of this probiotic bacterium during the fermentation process. Two independent quantitative PCR-analyses specific for yoba 2012 indicated a 20- to 60-fold increase in the total number of probiotic bacteria in the first batch after inoculation. A similar increase of the probiotic was observed in a variation of prepared with carbohydrate-rich millet granules () added prior to fermentation. This study shows the feasibility of integrating health-promoting probiotic strains into naturally fermented foods produced in regions with a high prevalence of malnutrition.
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http://dx.doi.org/10.3389/fmicb.2018.02218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160551PMC
September 2018

Training for translation between disciplines: a philosophy for life and data sciences curricula.

Bioinformatics 2018 07;34(13):i4-i12

Department of Computer Science, IBIVU Centre for Integrative Bioinformatics Vrije Universiteit Amsterdam, HV Amsterdam, Netherlands.

Motivation: Our society has become data-rich to the extent that research in many areas has become impossible without computational approaches. Educational programmes seem to be lagging behind this development. At the same time, there is a growing need not only for strong data science skills, but foremost for the ability to both translate between tools and methods on the one hand, and application and problems on the other.

Results: Here we present our experiences with shaping and running a masters' programme in bioinformatics and systems biology in Amsterdam. From this, we have developed a comprehensive philosophy on how translation in training may be achieved in a dynamic and multidisciplinary research area, which is described here. We furthermore describe two requirements that enable translation, which we have found to be crucial: sufficient depth and focus on multidisciplinary topic areas, coupled with a balanced breadth from adjacent disciplines. Finally, we present concrete suggestions on how this may be implemented in practice, which may be relevant for the effectiveness of life science and data science curricula in general, and of particular interest to those who are in the process of setting up such curricula.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022589PMC
July 2018

A benzene-degrading nitrate-reducing microbial consortium displays aerobic and anaerobic benzene degradation pathways.

Sci Rep 2018 03 14;8(1):4490. Epub 2018 Mar 14.

Wageningen University & Research, Laboratory of Microbiology, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.

In this study, we report transcription of genes involved in aerobic and anaerobic benzene degradation pathways in a benzene-degrading denitrifying continuous culture. Transcripts associated with the family Peptococcaceae dominated all samples (21-36% relative abundance) indicating their key role in the community. We found a highly transcribed gene cluster encoding a presumed anaerobic benzene carboxylase (AbcA and AbcD) and a benzoate-coenzyme A ligase (BzlA). Predicted gene products showed >96% amino acid identity and similar gene order to the corresponding benzene degradation gene cluster described previously, providing further evidence for anaerobic benzene activation via carboxylation. For subsequent benzoyl-CoA dearomatization, bam-like genes analogous to the ones found in other strict anaerobes were transcribed, whereas gene transcripts involved in downstream benzoyl-CoA degradation were mostly analogous to the ones described in facultative anaerobes. The concurrent transcription of genes encoding enzymes involved in oxygenase-mediated aerobic benzene degradation suggested oxygen presence in the culture, possibly formed via a recently identified nitric oxide dismutase (Nod). Although we were unable to detect transcription of Nod-encoding genes, addition of nitrite and formate to the continuous culture showed indication for oxygen production. Such an oxygen production would enable aerobic microbes to thrive in oxygen-depleted and nitrate-containing subsurface environments contaminated with hydrocarbons.
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http://dx.doi.org/10.1038/s41598-018-22617-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852087PMC
March 2018

Systems biology of lactic acid bacteria: For food and thought.

Curr Opin Syst Biol 2017 Dec;6:7-13

Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, O|2 Building, Section Systems Bioinformatics, Location Code 2E51, De Boelelaan 1085, NL-1081HV Amsterdam, The Netherlands.

Lactic acid bacteria (LAB) ferment plants, fish, meats and milk and turn them into tasty food products with increased shelf life; other LAB help digesting food and create a healthy environment in the intestine. The economic and societal importance of these relatively simple and small bacteria is immense. In this review we hope to show that their adaptations to nutrient-rich environments provides fascinating and often puzzling behaviours that give rise to many fundamental evolutionary biological questions in need of a systems biology approach. We will provide examples of such questions, compare the (metabolic) behaviour of LAB to that of other model organisms, and provide the latest insights, if available.
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http://dx.doi.org/10.1016/j.coisb.2017.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489361PMC
December 2017

Experimental evolution and the adjustment of metabolic strategies in lactic acid bacteria.

FEMS Microbiol Rev 2017 08;41(Supp_1):S201-S219

Systems Bioinformatics, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.

Experimental evolution of microbes has gained lots of interest in recent years, mainly due to the ease of strain characterisation through next-generation sequencing. While evolutionary and systems biologists use experimental evolution to address fundamental questions in their respective fields, studies with lactic acid bacteria are often more directed by applied questions. Insight into population and genome dynamics are valuable for experimental design and data interpretation, and it is becoming increasingly apparent how different constraints limit and govern the outcome of microbial adaptation to a selective environment. Examples for such constraints are the finite membrane and cellular space which can lead to trade-offs between cellular strategies. A powerful perspective is that of resource allocation, which allows cells to maximise fitness. This impacts on metabolic strategies that have different protein/resource demands. This review focuses on parameters and forces that shape cellular optimisation processes and that are determining for the outcome of laboratory evolution experiments. Phenotypic changes of experimentally evolved lactic acid bacteria will be discussed in the light of the selection conditions and the prevailing constraints.
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http://dx.doi.org/10.1093/femsre/fux024DOI Listing
August 2017

Nonhierarchical Flux Regulation Exposes the Fitness Burden Associated with Lactate Production in Synechocystis sp. PCC6803.

ACS Synth Biol 2017 03 12;6(3):395-401. Epub 2016 Dec 12.

Molecular Microbial Physiology Group, Faculty of Science, Swammerdam Institute for Life Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.

Cyanobacteria are mostly engineered to be sustainable cell-factories by genetic manipulations alone. Here, by modulating the concentration of allosteric effectors, we focus on increasing product formation without further burdening the cells with increased expression of enzymes. Resorting to a novel 96-well microplate cultivation system for cyanobacteria, and using lactate-producing strains of Synechocystis PCC6803 expressing different l-lactate dehydrogenases (LDH), we titrated the effect of 2,5-anhydro-mannitol supplementation. The latter acts in cells as a nonmetabolizable analogue of fructose 1,6-bisphosphate, a known allosteric regulator of one of the tested LDHs. In this strain (SAA023), we achieved over 2-fold increase of lactate productivity. Furthermore, we observed that as carbon is increasingly deviated during growth toward product formation, there is an increased fixation rate in the population of spontaneous mutants harboring an impaired production pathway. This is a challenge in the development of green cell factories, which may be countered by the incorporation in biotechnological processes of strategies such as the one pioneered here.
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http://dx.doi.org/10.1021/acssynbio.6b00235DOI Listing
March 2017

Molecular assessment of bacterial vaginosis by Lactobacillus abundance and species diversity.

BMC Infect Dis 2016 Apr 23;16:180. Epub 2016 Apr 23.

Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University, Amsterdam, The Netherlands.

Background: To date, women are most often diagnosed with bacterial vaginosis (BV) using microscopy based Nugent scoring or Amsel criteria. However, the accuracy is less than optimal. The aim of the present study was to confirm the identity of known BV-associated composition profiles and evaluate indicators for BV using three molecular methods.

Methods: Evaluation of indicators for BV was carried out by 16S rRNA amplicon sequencing of the V5-V7 region, a tailor-made 16S rRNA oligonucleotide-based microarray, and a PCR-based profiling technique termed IS-profiling, which is based on fragment variability of the 16S-23S rRNA intergenic spacer region. An inventory of vaginal bacterial species was obtained from 40 females attending a Dutch sexually transmitted infection outpatient clinic, of which 20 diagnosed with BV (Nugent score 7-10), and 20 BV negative (Nugent score 0-3).

Results: Analysis of the bacterial communities by 16S rRNA amplicon sequencing revealed two clusters in the BV negative women, dominated by either Lactobacillus iners or Lactobacillus crispatus and three distinct clusters in the BV positive women. In the former, there was a virtually complete, negative correlation between L. crispatus and L. iners. BV positive subjects showed cluster profiles that were relatively high in bacterial species diversity and dominated by anaerobic species, including Gardnerella vaginalis, and those belonging to the Families of Lachnospiraceae and Leptotrichiaceae. Accordingly, the Gini-Simpson index of species diversity, and the relative abundance Lactobacillus species appeared consistent indicators for BV. Under the conditions used, only the 16S rRNA amplicon sequencing method was suitable to assess species diversity, while all three molecular composition profiling methods were able to indicate Lactobacillus abundance in the vaginal microbiota.

Conclusion: An affordable and simple molecular test showing a depletion of the genus Lactobacillus in combination with an increased species diversity of vaginal microbiota could serve as an alternative and practical diagnostic method for the assessment of BV.
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http://dx.doi.org/10.1186/s12879-016-1513-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841971PMC
April 2016

Public goods and metabolic strategies.

Curr Opin Microbiol 2016 06 4;31:109-115. Epub 2016 Apr 4.

Department of Systems Bioinformatics, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.

Microbial growth can be characterized by a limited set of macroscopic parameters such as growth rate, biomass yield and substrate affinity. Different culturing protocols for laboratory evolution have been developed to select mutant strains that have one specific macroscopic growth parameter improved. Some of those mutant strains display tradeoffs between growth parameters and changed metabolic strategies, for example, a shift from respiration to fermentation. Here we discuss recent studies suggesting that metabolic strategies and growth parameter tradeoffs originate from a common set of physicochemical and cellular constraints, associated with the allocation of intracellular resources over biosynthetic processes, mostly protein synthesis. This knowledge will give insight in ecological and biological concepts and can be used for metabolic and evolutionary engineering strategies.
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http://dx.doi.org/10.1016/j.mib.2016.03.007DOI Listing
June 2016

Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

FEBS J 2015 Jun 24;282(12):2394-407. Epub 2015 Apr 24.

Systems Bioinformatics, VU University, Amsterdam, The Netherlands.

Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them.
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http://dx.doi.org/10.1111/febs.13289DOI Listing
June 2015

Protein costs do not explain evolution of metabolic strategies and regulation of ribosomal content: does protein investment explain an anaerobic bacterial Crabtree effect?

Mol Microbiol 2015 Jul 9;97(1):77-92. Epub 2015 May 9.

Systems Bioinformatics IBIVU, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.

Protein investment costs are considered a major driver for the choice of alternative metabolic strategies. We tested this premise in Lactococcus lactis, a bacterium that exhibits a distinct, anaerobic version of the bacterial Crabtree/Warburg effect; with increasing growth rates it shifts from a high yield metabolic mode [mixed-acid fermentation; 3 adenosine triphosphate (ATP) per glucose] to a low yield metabolic mode (homolactic fermentation; 2 ATP per glucose). We studied growth rate-dependent relative transcription and protein ratios, enzyme activities, and fluxes of L. lactis in glucose-limited chemostats, providing a high-quality and comprehensive data set. A three- to fourfold higher growth rate rerouted metabolism from acetate to lactate as the main fermentation product. However, we observed hardly any changes in transcription, protein levels and enzyme activities. Even levels of ribosomal proteins, constituting a major investment in cellular machinery, changed only slightly. Thus, contrary to the original hypothesis, central metabolism in this organism appears to be hardly regulated at the level of gene expression, but rather at the metabolic level. We conclude that L. lactis is either poorly adapted to growth at low and constant glucose concentrations, or that protein costs play a less important role in fitness than hitherto assumed.
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http://dx.doi.org/10.1111/mmi.13012DOI Listing
July 2015

How fast-growing bacteria robustly tune their ribosome concentration to approximate growth-rate maximization.

FEBS J 2015 May 26;282(10):2029-44. Epub 2015 Mar 26.

Systems Bioinformatics, VU University, Amsterdam, The Netherlands.

Maximization of growth rate is an important fitness strategy for bacteria. Bacteria can achieve this by expressing proteins at optimal concentrations, such that resources are not wasted. This is exemplified for Escherichia coli by the increase of its ribosomal protein-fraction with growth rate, which precisely matches the increased protein synthesis demand. These findings and others have led to the hypothesis that E. coli aims to maximize its growth rate in environments that support growth. However, what kind of regulatory strategy is required for a robust, optimal adjustment of the ribosome concentration to the prevailing condition is still an open question. In the present study, we analyze the ppGpp-controlled mechanism of ribosome expression used by E. coli and show that this mechanism maintains the ribosomes saturated with its substrates. In this manner, overexpression of the highly abundant ribosomal proteins is prevented, and limited resources can be redirected to the synthesis of other growth-promoting enzymes. It turns out that the kinetic conditions for robust, optimal protein-partitioning, which are required for growth rate maximization across conditions, can be achieved with basic biochemical interactions. We show that inactive ribosomes are the most suitable 'signal' for tracking the intracellular nutritional state and for adjusting gene expression accordingly, as small deviations from optimal ribosome concentration cause a huge fractional change in ribosome inactivity. We expect to find this control logic implemented across fast-growing microbial species because growth rate maximization is a common selective pressure, ribosomes are typically highly abundant and thus costly, and the required control can be implemented by a small, simple network.
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http://dx.doi.org/10.1111/febs.13258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672707PMC
May 2015

Genome-wide transposon mutagenesis indicates that Mycobacterium marinum customizes its virulence mechanisms for survival and replication in different hosts.

Infect Immun 2015 May 17;83(5):1778-88. Epub 2015 Feb 17.

Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands

The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.
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http://dx.doi.org/10.1128/IAI.03050-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399070PMC
May 2015

Availability of public goods shapes the evolution of competing metabolic strategies.

Proc Natl Acad Sci U S A 2013 Aug 12;110(35):14302-7. Epub 2013 Aug 12.

Systems Bioinformatics/Center for Integrative Bioinformatics Vrije Universiteit, Amsterdam Institute for Molecules Medicines and Systems, Vrije Universiteit Amsterdam, 1081HV Amsterdam, The Netherlands.

Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli, yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell-cell interactions, and the relationships between environmental and metabolic constraints.
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http://dx.doi.org/10.1073/pnas.1308523110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3761572PMC
August 2013

Bioinformatics and systems biology: bridging the gap between heterogeneous student backgrounds.

Brief Bioinform 2013 Sep 19;14(5):589-98. Epub 2013 Apr 19.

Centre for Integrative Bioinformatics VU (IBIVU), VU University Amsterdam, Amsterdam, The Netherlands. Tel.: +31 20 59 87816; Fax: +31 20 59 87653;

Teaching students with very diverse backgrounds can be extremely challenging. This article uses the Bioinformatics and Systems Biology MSc in Amsterdam as a case study to describe how the knowledge gap for students with heterogeneous backgrounds can be bridged. We show that a mix in backgrounds can be turned into an advantage by creating a stimulating learning environment for the students. In the MSc Programme, conversion classes help to bridge differences between students, by mending initial knowledge and skill gaps. Mixing students from different backgrounds in a group to solve a complex task creates an opportunity for the students to reflect on their own abilities. We explain how a truly interdisciplinary approach to teaching helps students of all backgrounds to achieve the MSc end terms. Moreover, transferable skills obtained by the students in such a mixed study environment are invaluable for their later careers.
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http://dx.doi.org/10.1093/bib/bbt023DOI Listing
September 2013

How biochemical constraints of cellular growth shape evolutionary adaptations in metabolism.

Genetics 2013 Jun 27;194(2):505-12. Epub 2013 Mar 27.

Systems Bioinformatics, The Centre for Integrative Bioinformatics VU, VU University, 1081 HV, Amsterdam, The Netherlands.

Evolutionary adaptations in metabolic networks are fundamental to evolution of microbial growth. Studies on unneeded-protein synthesis indicate reductions in fitness upon nonfunctional protein synthesis, showing that cell growth is limited by constraints acting on cellular protein content. Here, we present a theory for optimal metabolic enzyme activity when cells are selected for maximal growth rate given such growth-limiting biochemical constraints. We show how optimal enzyme levels can be understood to result from an enzyme benefit minus cost optimization. The constraints we consider originate from different biochemical aspects of microbial growth, such as competition for limiting amounts of ribosomes or RNA polymerases, or limitations in available energy. Enzyme benefit is related to its kinetics and its importance for fitness, while enzyme cost expresses to what extent resource consumption reduces fitness through constraint-induced reductions of other enzyme levels. A metabolic fitness landscape is introduced to define the fitness potential of an enzyme. This concept is related to the selection coefficient of the enzyme and can be expressed in terms of its fitness benefit and cost.
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http://dx.doi.org/10.1534/genetics.113.150631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664859PMC
June 2013

Genome instability in Lactobacillus rhamnosus GG.

Appl Environ Microbiol 2013 Apr 25;79(7):2233-9. Epub 2013 Jan 25.

Yoba for Life foundation, Amsterdam, The Netherlands.

We describe here a comparative genome analysis of three dairy product isolates of Lactobacillus rhamnosus GG (LGG) and the ATCC 53103 reference strain to the published genome sequence of L. rhamnosus GG. The analysis showed that in two of three isolates, major DNA segments were missing from the genomic islands LGGISL1,2. The deleted DNA segments consist of 34 genes in one isolate and 84 genes in the other and are flanked by identical insertion elements. Among the missing genes are the spaCBA genes, which encode pilin subunits involved in adhesion to mucus and persistence of the strains in the human intestinal tract. Subsequent quantitative PCR analyses of six commercial probiotic products confirmed that two more products contain a heterogeneous population of L. rhamnosus GG variants, including genotypes with or without spaC. These results underline the relevance for quality assurance and control measures targeting genome stability in probiotic strains and justify research assessing the effect of genetic rearrangements in probiotics on the outcome of in vitro and in vivo efficacy studies.
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http://dx.doi.org/10.1128/AEM.03566-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623246PMC
April 2013

Vesicle trafficking via the Spitzenkörper during hyphal tip growth in Rhizoctonia solani.

Antonie Van Leeuwenhoek 2013 Apr 20;103(4):921-31. Epub 2013 Jan 20.

Fungal Cell Biology Group, University of Edinburgh, Daniel Rutherford Building, Edinburgh, EH9 3JH, UK.

Growing hyphae of Rhizoctonia solani were stained with the endocytic marker dye FM4-64 and imaged by confocal microscopy. Staining of the plasma membrane was followed by labeling of organelles in the cytoplasm (after ~1 min) and of the Spitzenkörper (Spk; after ~2 min). Fluorescence recovery after photobleaching (FRAP) of the stained Spk demonstrated the vectorial flow of secretory vesicles from the apical cytoplasm to the Spk. This flux was modelled in a two-compartment model. The turnover time of the vesicles of the Spk was estimated to be 1.3-2.5 min. These results are roughly consistent with the expected flux of vesicles through the Spk based on the number of secretory vesicles within the Spk and the number of secretory vesicles that would be necessary to fuse with the apical plasma membrane to maintain hyphal extension rates. These results suggest that membrane retrieval via endocytosis is not as significant as previously suggested.
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http://dx.doi.org/10.1007/s10482-012-9873-1DOI Listing
April 2013