Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase Implications for NADPH recognition and structural stability

AbstractPhe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Å is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Å revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH

Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10(5) cells ml(−1). The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products

MicrobiomeSupport Mapping Data

This dataset is the result of a mapping exercise of the online database containing relevant information on microbiome-related policies and strategies, funding programmes, research projects and organisations. This included collecting research programmes and projects from (1) the full H2020 database on Cordis, (2) a database with research projects funded via ERA-NETs and Joint Programming Initiatives, (3) COST Action research networks, (4) European Molecular Biology Organization projects, (5) the Horizon Frontier Science Programme, (6) the Bill and Melinda Gates Foundation, and (7) the Gordon and Betty Moore Foundation as well as relevant new policies, research strategies and infrastructures and knowledge platforms. The database is accessible on the project website: LIN

Rapid Fluorescence Assessment of the Viability of Stressed Lactococcus lactis

The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using Lactococcus lactis subsp. lactis ML3 exposed to different stress treatments. The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70°C very well. However, after treatment up to 60°C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50°C and was completely lost for those treated at 60°C. In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared. The acidification capacity corresponded with the number of CFU. The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters. The cF labeling depended on membrane integrity, as was confirmed by PI exclusion. The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration). When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment. This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed L. lactis and has potential for physiological research and dairy applications related to lactic acid bacteria

Flow Cytometric Assessment of Viability of Lactic Acid Bacteria

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70°C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications

Metadata harmonization–Standards are the key for a better usage of omics data for integrative microbiome analysis

International audienceAbstract Background Tremendous amounts of data generated from microbiome research studies during the last decades require not only standards for sampling and preparation of omics data but also clear concepts of how the metadata is prepared to ensure re-use for integrative and interdisciplinary microbiome analysis. Results In this Commentary, we present our views on the key issues related to the current system for metadata submission in omics research, and propose the development of a global metadata system. Such a system should be easy to use, clearly structured in a hierarchical way, and should be compatible with all existing microbiome data repositories, following common standards for minimal required information and common ontology. Although minimum metadata requirements are essential for microbiome datasets, the immense technological progress requires a flexible system, which will have to be constantly improved and re-thought. While FAIR principles (Findable, Accessible, Interoperable, and Reusable) are already considered, international legal issues on genetic resource and sequence sharing provided by the Convention on Biological Diversity need more awareness and engagement of the scientific community. Conclusions The suggested approach for metadata entries would strongly improve retrieving and re-using data as demonstrated in several representative use cases. These integrative analyses, in turn, would further advance the potential of microbiome research for novel scientific discoveries and the development of microbiome-derived products