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Bioinformatics pipeline for metaproteomics data analysis : investigation on microbial populations and their respective functional role in cheese
No full textThe investigating functional molecular pathways of an organism are at the basis of the understanding of complex microbial ecosystems. In the case of unicellular organisms, the description of protein pathways provides information on the functionality of a microbial consortium.
We recently developed a semi-automated pipeline for the analysis of metaproteomics data to depict the newborn mouse gut phylotypes.1 Here we present a refined bioinformatics tool to extend the analysis to a more interesting description of the metabolic functions correlated with the characterized microbial taxa, using a typical PDO Italian cheese as model.
Metaproteomics raw data are analyzed to obtain a representation of the bacterial population at different taxonomic levels on the bases of the taxon-specificity of a tryptic peptide list, comparing it with that coming from the already available metaproteomics and metagenomics applications such as Megan2. As a further development we managed for the automated association of taxa to metabolic pathways (KEGG database) and of proteins to groups of cluster orthologs (COG). We optimized parameters to have the maximum number of protein and minimum FDR, and then developed a series of python scripts to integrate and improve, the output of available application, and manipulate raw data. This investigation aims at providing a functional insight in metagenomic analysis, and at offer a direct evaluation of protein functional pathways which are actually controlling the consortia homeostasis
Database Comparison and Bioinformatic Tools for Metaproteomic Approaches in Investigations on Microbial Populations
No full textDetermination of the absolute configurations of chiral organometallic complexes via density functional theory calculations of their vibrational circular dichroism spectra: The chiral chromium tricarbonyl complex of N-pivaloyl-tetrahydroquinoline
No full textThe racemate of the chiral tricarbonyl-η6-arene-chromium(0) complex, tricarbonyl-η6-N-pivaloyl-tetrahydroquinoline-chromium(0), 1, has been synthesized and resolved using chromatography on a (R,R)-Whelk-O1 column. The Absolute Configuration (AC) of 1 has been determined using vibrational circular dichroism (VCD) spectroscopy. The VCD spectrum of 1 has been predicted using the Stephens equation for vibrational rotational strengths, implemented using density functional theory (DFT) in the gaussian program. Using the B3PW91 functional and the 6-311++G (2d,2p) basis set, the predicted VCD spectrum of S-1 is in excellent agreement with the experimental VCD spectrum of (+)-1, leading unambiguously to the AC S-(+). It is concluded that VCD is a useful technique for determining the ACs of chiral organometallic complexes, given the use of optimum functionals and basis sets
Microbiota dynamics in cheese production
No full textGrana Padano (GP) is a PDO cheese that is made from raw cow’s milk. One of the problem which more frequently affects the production of GP and also other type of cheese with similar production is the “late blowing” that depends from the butyric fermentation product by Clostridia spp. This phenomenon particularly occurred in the fermentation process of GP. Clostridia, in particular C. tyrobutyricum, are inhibited by the addition of lysozyme, a natural bacteriolytic enzyme derived from egg chicken.
Aim of this investigation is to characterize the microbiota dynamics in experimental caseification of GP products in controlled critical parameters in GP ripening like clostridial spore count and lysozyme treatment
Grana Padano cheeses were producted in a pilot plant. Milk was inoculated with whey starter mixture. Two different variables in ripening conditions were considered: low or high C. tyrobutyricum spore count and lysozyme treatment or not. Aqueous phases were extracted from cheese aliquots at different timepoints and proteins analysed by 1- and 2D-LC MS/MS. Taxonomic group were assigned using sequence homology search on bacterial peptide counts and MEGAN [1].
The characteristic microbial signature results in important differences from ‘natural’ products, suggest also the environment plays a pivotal role in selection of functional microbiota, and it gives new insights to improve characterization of PDO
Differential proteomics investigation of Pseudomonas fluorescens chromogenics trains : insights in blue mozzarella
No full textThe "Bridge" Game: Role of the Fourth Player in Chiral Recognition
No full textA new team player: The “three-point interaction” model, which is usually employed to rationalize chiral recognition, does not account for the amazing enantioselectivity measured for the receptors of many proteic acceptors. Gas-phase experiments have indicated that at least a fourth “player” must be considered: the rigidity that a receptor opposes to distortions of its cavity resulting from noncovalent interactions with a chiral molecule
Proteomics Investigation of Pseudomonas Fluorescens Chromogenic Strains: Insight in Blue-Mozzarella
Get PDFIntroduction and Objectives:
In the last years an high number of mozzarella cheese coming from Italian and German establishments
caused complaints in consumers due to unusual pigmentation of dairyproducts Many microbiological analysis have been performed revealing an ubiquitous non-pathogenic bacteria, Pseudomonas fluorescens, as a causative agents liable for this phenomenon. Some specific strains of this microorganism own gene sequences coding for enzymes, able to produce dyes that can induce anomalous coloration to food. It has been verified that the occurrence of blue pigmentation in mozzarella cheese samples is however not related to chemical conditions, bacterial concentration, or critical temperature level. Both ribotyping and PFGE methodologies have underlined differences in Pseudomonas fluorescens strains isolated in food coming from different geographical areas. Nevertheless the phylogenetic assessment of bacteria pointed out no correlation between the genetic profile of the microorganism and its chromogenic behavior. the aim of this study was a deeper proteomic investigation of P. fluorescens isolates in order to evaluate how these bacteria could represent contamination source.
Methods:
A shotgun proteomic approach has been performed on isolated Pseudomonas fluorescens cultures coming from samples of mozzarella cheese either showing anomalous pigmentation or not. LC-MSMS experiments have been carried on by nanoHPLC runs and auto MSn acquisitions on a Bruker amaZon-ETD Ion Trap instrument. Protein identification has been executed either versus Pseudomonas dedicated DBs or on the open-reviewed databases.
Results and Discussion:
Obtained data highlighted several differential
expressed proteins in two conditions. Among these, key proteins are, on one hand, the major cold shock protein that increases its level in the anomalous pigmentation, and, on the other, the phosphate starvation-inducible protein that decreases respect to normal condition.
Conclusion:
Proteomics analysis could allowed us to differentiate among
chromogenic and non chromogenic strains at the molecular level, widening the knowledge on this bacteria and the molecular mechanism underlying this contamination. (P09.23
Unravelling the effect of clostridia spores and lysozyme on microbiota dynamics in Grana Padano cheese: A metaproteomics approach
Get PDFGrana Padano is a typical Italian Protected Designation of Origin (PDO) hard cheese largely consumed all over the world. The major problem during its production is represented by late blowing. Clostridia are gasogen bacteria responsible of the swelling during ripening, and they are partially counteracted by the use of egg white lysozyme as additive. In this work was applied, for the first time in cheese, a metaproteomic approach that identified the functional dynamics of microbial consortia in relation to the number of clostridia(spores and lysozyme treatment using experimental samples of Grana Padano cheese. We used a combined custom BLAST+/MEGAN/STAMP approach to obtain a global taxonomic view associated to low and high clostridial spores cheese without and with lysozyme. Main differences were highlighted in the bacilli class. Functional analysis with SEED provided a deep view into several metabolic pathways, highlighting the subsystems "amino acid and derivatives" and "clustering-based subsystem" as the targeted subsystems during lysozyme treatment in the high spore group. In these subsystems, acetate kinase from clostridia was one of the main enzymes affected by the lysozyme treatment.
Biological significance: Metaproteomics is a very promising and useful technique in the control of food safety and quality, from fresh products until 'ready to eat' food. Tools able to identify at molecular level the dynamic fingerprinting of food microbiota could be of great help to improve food safety and quality
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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