Microbiology - Journal Articles
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Item From raw milk cheese to the gut: investigating the colonization strategies of Bifidobacterium mongoliense(American Society for Microbiology, 2024-08-16) Longhi, Giulia; Lugli, Gabriele Andrea; Tarracchini, Chiara; Fontana, Federico; Bianchi, Massimiliano Giovanni; Carli, Elisa; Bussolati, Ovidio; van Sinderen, Douwe; Turroni, Francesca; Ventura, Marco; Dudley, Edward G.; Science Foundation Ireland; Ministero dell’Istruzione, dell’Università e della RicercaThe microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the Bifidobacterium mongoliense species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between B. mongoliense and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 B. mongoliense strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in B. mongoliense, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3’-sialyllactose (3′-SL) or 6’-sialyllactose (6′-SL). The B. mongoliense BMONG18 was chosen, due to its superior ability to utilize 3′-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.Item Functional and practical insights into three lactococcal antiphage systems(American Society for Microbiology, 2024-08-13) Grafakou, Andriana; Mosterd, Cas; de Waal, Paul P.; van Rijswijck, Irma M. H.; van Peij, Noël N. M. E.; Mahony, Jennifer; van Sinderen, Douwe; Ercolini, Danilo; Science Foundation IrelandThe persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.Item Development of a ribosome profiling protocol to study translation in Kluyveromyces marxianus(Oxford University Press, 2022) Fenton, Darren; Kiniry, Stephen J.; Yordanova, Martina M.; Baranov, Pavel V.; Morrissey, John P.; Horizon 2020 Framework Programme; Science Foundation Ireland; Health Research Board; Wellcome TrustKluyveromyces marxianus is an interesting and important yeast because of particular traits such as thermotolerance and rapid growth, and for applications in food and industrial biotechnology. For both understanding its biology and developing bioprocesses, it is important to understand how K. marxianus responds and adapts to changing environments. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. We report here the development of a ribosome profiling method for K. marxianus, which allows codon resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs. To aid in the analysis and sharing of ribosome profiling data, we added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. We also provide a set of step-by-step protocols for the experimental and bioinformatic methods that we developed.Item Reconstruction of a catalogue of genome-scale metabolic models with enzymatic constraints using GECKO 2.0(Springer Nature, 2022) Domenzain, Ivan; Sánchez, Benjamín; Anton, Mihail; Kerkhoven, Eduard J.; Millan-Oropeza, Aaron; Henry, Celine; Siewers, Verena; Morrissey, John P.; Sonnenschein, Nikolaus; Nielsen, Jens; Horizon 2020 Framework Programme; Knut och Alice Wallenbergs Stiftelse; Novo Nordisk FondenGenome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into such models was first enabled by the GECKO toolbox, allowing the study of phenotypes constrained by protein limitations. Here, we upgrade the toolbox in order to enhance models with enzyme and proteomics constraints for any organism with a compatible GEM reconstruction. With this, enzyme-constrained models for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus are generated to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions reveal that upregulation and high saturation of enzymes in amino acid metabolism are common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO is expanded with an automated framework for continuous and version-controlled update of enzyme-constrained GEMs, also producing such models for Escherichia coli and Homo sapiens. In this work, we facilitate the utilization of enzyme-constrained GEMs in basic science, metabolic engineering and synthetic biology purposes.Item Bacteriocin diversity, function, discovery and application as antimicrobials(Springer Nature, 2024-05-10) Sugrue, Ivan; Ross, R. Paul; Hill, ColinBacteriocins are potent antimicrobial peptides that are produced by bacteria. Since their discovery almost a century ago, diverse peptides have been discovered and described, and some are currently used as commercial food preservatives. Many bacteriocins exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. The molecular structures, mechanisms of action and resistance have been determined for a number of bacteriocins, but most remain incompletely characterized. These gene-encoded peptides are amenable to bioengineering strategies and heterologous expression, enabling metagenomic mining and modification of novel antimicrobials. The ongoing global antimicrobial resistance crisis demands that novel therapeutics be developed to combat infectious pathogens. New compounds that are target-specific and compatible with the resident microbiota would be valuable alternatives to current antimicrobials. As bacteriocins can be broad or narrow spectrum in nature, they are promising tools for this purpose. However, few bacteriocins have gone beyond preclinical trials and none is currently used therapeutically in humans. In this Review, we explore the broad diversity in bacteriocin structure and function, describe identification and optimization methods and discuss the reasons behind the lack of translation beyond the laboratory of these potentially valuable antimicrobials.