Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems.

BACKGROUND: Bifidobacterial genome analysis has provided insights as to how these gut commensals adapt to and persist in the human GIT, while also revealing genetic diversity among members of a given bifidobacterial (sub)species. Bifidobacteria are notoriously recalcitrant to genetic modification, which prevents exploration of their genomic functions, including those that convey (human) health benefits. METHODS: PacBio SMRT sequencing was used to determine the whole genome seqeunces of two B. longum subsp. longum strains. The B. longum pan-genome was computed using PGAP v1.2 and the core B. longum phylogenetic tree was constructed using a maximum-likelihood based approach in PhyML v3.0. M.blmNCII was cloned in E. coli and an internal fragment if arfBarfB was cloned into pORI19 for insertion mutagenesis. RESULTS: In this study we present the complete genome sequences of two Bifidobacterium longum subsp. longum strains. Comparative analysis with thirty one publicly available B. longum genomes allowed the definition of the B. longum core and dispensable genomes. This analysis also highlighted differences in particular metabolic abilities between members of the B. longum subspecies infantis, longum and suis. Furthermore, phylogenetic analysis of the B. longum core genome indicated the existence of a novel subspecies. Methylome data, coupled to the analysis of restriction-modification systems, allowed us to substantially increase the genetic accessibility of B. longum subsp. longum NCIMB 8809 to a level that was shown to permit site-directed mutagenesis. CONCLUSIONS: Comparative genomic analysis of thirty three B. longum representatives revealed a closed pan-genome for this bifidobacterial species. Phylogenetic analysis of the B. longum core genome also provides evidence for a novel fifth B. longum subspecies. Finally, we improved genetic accessibility for the strain B. longum subsp. longum NCIMB 8809, which allowed the generation of a mutant of this strain.

Selective carbohydrate utilization by lactobacilli and bifidobacteria.

AIM: To evaluate the ability of specific carbohydrates, including commercially available products, to support the growth of representatives of two well-known groups of gut commensals, namely lactobacilli and bifidobacteria. METHODS AND RESULTS: Sixty-eight bacterial strains, representing 29 human-derived lactobacilli and 39 bifidobacteria (both human- and animal-derived), were tested for their ability to metabolize 10 different carbohydrates. Analysis of growth and metabolic activity was performed using a combination of diagnostic parameters, such as final OD600 , final pH, fermentation end products and growth rate. CONCLUSIONS: The data assembled in this study provide significant complementary and comparative information on the growth-promoting properties of a range of carbohydrates, while also investigating interspecies differences between lactobacilli and/or bifidobacteria with regard to their carbohydrate utilization abilities. Galacto-oligosaccharides (GOS) and lactulose were shown to support the most favourable growth characteristics, whereas relatively poor growth of lactobacilli and bifidobacteria was observed on inulin, maltodextrin and polydextrose. GOS/inulin (9 : 1) and fructo-oligosaccharides (FOS)/inulin mixtures supported mostly similar growth abilities to those obtained for GOS and FOS, respectively. Microbial consumption of GOS, as determined by high-performance anion-exchange chromatography with pulsed amperometric detection, was evident for both lactobacilli and bifidobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may allow for the rational prediction of lactobacilli and/or bifidobacteria to be used in conjunction with prebiotics, such as GOS, as synbiotics.

Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis.

So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus Bifidobacterium. In this report we analyzed three prophage-like elements that are present in the genomes of Bifidobacterium breve UCC 2003, Bifidobacterium longum NCC 2705, and Bifidobacterium longum DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNA(Met) gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the attB sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a B. longum DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among B. breve strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.

Cloning and sequence analysis of putative histidine protein kinases isolated from Lactococcus lactis MG1363.

Eight recombinant plasmids harboring chromosomal fragments of Lactococcus lactis MG1363 were shown to phenotypically suppress a histidine protein kinase (HPK) deficiency in either of two different E. coli strains. Sequence analysis of the plasmid inserts revealed five different complete or partial open reading frames (ORFs) specifying proteins with high similarity to HPKs. One of the plasmids also harbored an additional ORF, unrelated to HPKs, with suppressing activity.