Abstract
Bifidobacteria are common gut commensals with purported health-promoting effects. This has encouraged scientific research into bifidobacteria, though recalcitrance to genetic manipulation and scarcity of molecular tools has hampered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation. To overcome this problem and facilitate functional genomic analyses in bifidobacteria, we created a large Tn5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Transposon Directed Insertion Sequencing (TraDIS) approach. Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions. These essential genes encode functions involved in the so-called bifid-shunt, most enzymes related to nucleotide biosynthesis and a range of housekeeping functions. Comparison to the Bifidobacterium and B. breve core genomes highlights a high degree of conservation of essential genes at the species and genus level, while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria. This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria.
Highlights
Bifidobacteria are Gram-positive commensal microorganisms, widely encountered within the gut ecosystem of mammals and insects
Microarray-based approaches are used for mutant identification in TRAnsposon Site Hybridization (TraSH); while massive parallel sequencing is utilized in High-throughput Insertion Tracking Sequencing (HITS), Insertion Sequencing (In-Seq), Transposon Sequencing (Tn-seq) and Transposon Directed Insertion-Site Sequencing (TraDIS) technologies[17,18,19,20,21,22,23]
In a previous publication by our research group Tn5 transposon-mediated mutagenesis was accomplished in B. breve UCC2003, enabling relatively high insertion frequencies and generating single and stable transposon insertions per cell[16]
Summary
Bifidobacteria are Gram-positive commensal microorganisms, widely encountered within the gut ecosystem of mammals and (certain social) insects. The genome-wide identification of essential genes, defined as those that cannot be mutated in vitro, or those in which a mutation reduces growth or viability under particular conditions, has been performed in other microorganisms, where screenings of mutant collections following growth under specific conditions are coupled to en mass genome-wide location of genomic disruptions. Such an approach allows the identification of the complete set of genes essential for a certain phenotype or bacterial attribute in a single experiment. Comparative genomic analysis between the predicted essential genes and the Bifidobacterium core genome revealed a high level of conservation of essential genes across the genus Bifidobacterium and B. breve species, highlighting a set of highly conserved core metabolic functions, as well as a set of still uncharacterized essential functions in bifidobacteria
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