ItemMetagenomic approaches for the discovery of pollutant-remediating enzymes: Recent trends and challenges(Springer Nature Ltd., 2022-09-27) Mukherjee, Arghya; Cotter, Paul D.Metagenomic studies in diverse environments have generated petabytes of sequencing data, allowing biologists to peer into the uncultivated microbial majority with unprecedented clarity. Such advances have heralded a new era of enzyme discovery where proteins of interest can be directly extracted from metagenomic sequencing data, although this remains a challenging task. Traditionally, metagenomic enzyme discovery, including that involved in xenobiotic degradation, has largely relied on functional insights derived from activity-guided or PCR-based metagenomics. Due to its untargeted and holistic nature, metagenomics allows us to probe the unknown and underexplored microbial diversity, which represents a key resource for novel biocatalyst discovery. Metagenomic shotgun sequencing-based enzyme discovery, additionally, avoids common biases introduced through PCR-based or activity-guided functional genomic methods. In this chapter, we have provided an overview of metagenomics in novel enzyme discovery with discussions on both the experimental and computational aspects of the same. We discuss in detail computational strategies for identifying possible enzyme candidates from shotgun sequencing data and experimental strategies for characterizing candidate enzymes once they have been identified. Finally, we review emerging methods of metagenomic enzyme discovery as well as future goals and challenges with an emphasis on metagenomic-based approaches. ItemDetermination of bifidobacterial carbohydrate utilization abilities and associated metabolic end products(Springer Science+Business Media, LLC, 2021-03-03) Solopova, Ana; van Sinderen, Douwe; van Sinderen, Douwe; Ventura, MarcoBifidobacteria are able to utilize a diverse range of host-derived and dietary carbohydrates, the latter of which include many plant-derived oligo- and polysaccharides. Different bifidobacterial strains may possess different carbohydrate utilization abilities. These metabolic abilities can be studied using classical bacterial growth assessment methods, such as measurement of changes in optical density or acidity of the culture in the presence of the particular carbohydrate to generate growth and acidification curves, respectively. Scientists may also be interested in the growth rate during the exponential growth phase, and the maximum OD that is reached on a particular sugar, or the length of the lag phase. Furthermore, high-performance liquid chromatography (HPLC) and high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) are extensively used in carbohydrate and metabolic end-product analysis due to their versatility and separation capabilities. ItemPhageome analysis of bifidobacteria-rich samples(Springer Science+Business Media, LLC, 2021-03-03) McDonnell, Brian; Casey, Eoghan; Milani, Christian; Lugli, Gabriele Andrea; Viappiani, Alice; Mahony, Jennifer; Ventura, Marco; van Sinderen, Douwe; van Sinderen, Douwe; Ventura, MarcoBifidobacteria are important early colonizers of the human intestinal tract. The relative abundance of bifidobacterial species may be modulated, in part, by bacteriophage activity. Metagenomic studies of these populations is a crucial step in understanding this important interaction. This chapter outlines the technical instructions required to analyze the virome of a bifidobacteria-rich sample, for example, an infant fecal sample. ItemBifidobacterium transformation(Springer Science+Business Media, LLC, 2021-03-03) Hoedt, Emily C.; Bongers, Roger S.; Bottacini, Francesca; Knol, Jan; MacSharry, John; van Sinderen, Douwe; van Sinderen, Douwe; Ventura, Marco; Nutricia Research Foundation; Science Foundation IrelandThe protocol presented in this chapter describes a generic method for electrotransformation of Bifidobacterium spp., outlining a technique that is ideal for conferring selective properties onto strains as well as allowing the user to introduce or knock out/in selected genes for phenotypic characterization purposes. We have generalized on the plasmid chosen for transformation and antibiotic selection marker, but the protocol is versatile in this respect and we are able to achieve transformation efficiencies up to 107 transformants/μg of DNA. ItemProtocol to select bifidobacteria from fecal and environmental samples(Springer Science+Business Media, LLC, 2021-03-03) Alessandri, Giulia; Ossiprandi, Maria Cristina; Ventura, Marco; van Sinderen, Douwe; van Sinderen, Douwe; Ventura, MarcoBifidobacteria are commensal microorganisms able to colonize several ecological niches. Since their discovery, culture-dependent methods combined with the most modern next-generation sequencing techniques have contributed to shed light on the ecological, functional and genomic features of bifidobacteria, purporting them as microorganisms with probiotic traits. Thanks to their acclaimed health-promoting effects, several members of the Bifidobacterium genus have been included in a variety of functional foods and drugs. In this context, the functional relevance of bifidobacteria in the gut explains ongoing efforts to isolate novel and potentially beneficial strains. For this purpose, development of effective and selective isolation protocols in concert with knowledge on the physiological characteristics of bifidobacterial are fundamental requirements for their recovery and discovery from their natural environments, in particular from fecal samples.