Journal: PLoS ONE

Article Title: DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

doi: 10.1371/journal.pone.0148887

Figure Lengend Snippet: (A) SEM was used to compare OMV formation. The IVp-II-regulated gene cluster was entirely (Δ3397–3403) or partially (only BF3403, the last gene of the cluster) deleted in the ON/ON genetic background of B . fragilis YCH46. Bar: 1 μm. (B) Comparison of OMV production. OMV production was quantified by measuring the protein content of culture supernatants. The graph indicates OMV production relative to that of the ON/ON (Δ3397–3403) mutant. The data are expressed as means ± standard deviation. The bars labeled with different letters indicate significant differences at p < 0.05.

Article Snippet: The B . fragilis YCH46 DNA microarray from NimbleGen Systems, which includes 4,527 target genes with at least 8 unique 60-mer synthetic oligonucleotide probes for each gene, was used for the comparative transcriptomic analysis of the mutant strains.

Techniques: Comparison, Mutagenesis, Standard Deviation, Labeling

Journal: PLoS ONE

Article Title: DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

doi: 10.1371/journal.pone.0148887

Figure Lengend Snippet: (A) Growth of B . fragilis YCH46 in GAM supplemented with the indicated bile concentrations. (B) qPCR was used to determine the relative abundance of the IVp-I-ON or IVp-II-ON genotypes after exposure to the indicated bile concentration. (C) Effect of bile exposure on the expression of an IVp-I-regulated EPS production gene (BF2767) and IVp-II-regulated membrane protein genes (BF3397 and BF3403). (D) OMV production of B . fragilis increased 7-fold after exposure to 5% bile. (E) Schematic of the 3 x FLAG-tag fusion to the C-terminus of the BF3397-encoded protein. (F) Enhancement of the BF3397-encoded membrane protein in response to 5% bile. A B . fragilis YCH46 derivative with a 3 x FLAG-tag sequence fused to BF3397, as shown in panel E, was exposed to 5% bile: the production of BF3397 was assessed by Western blotting with an anti-FLAG antibody. Upper, middle, and lower bands indicate the anti-FLAG tag antibody used for immunoprecipitation, BF3397 (unprocessed form), and N-terminally processed BF3397 (indicated by arrowheads), respectively.

Article Snippet: The B . fragilis YCH46 DNA microarray from NimbleGen Systems, which includes 4,527 target genes with at least 8 unique 60-mer synthetic oligonucleotide probes for each gene, was used for the comparative transcriptomic analysis of the mutant strains.

Techniques: Concentration Assay, Expressing, Membrane, FLAG-tag, Sequencing, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

doi: 10.1371/journal.pone.0148887

Figure Lengend Snippet: (A) Competitive growth assay of ON/ON B . fragilis mutant and its parental YCH46 stain in vitro . Equivalent cell mixture of wild and mutant B . fragilis cells was inoculated into GAM broth. Closed circles indicate the periodical change of total viable cell number in GAM broth. Columns indicate the relative abundance of the wild and mutant B . fragilis cell in GAM broth. (B) IVp-I/IVp-II ON/OFF prevalence during in vivo gut colonization IVp-I/IVp-II under in vivo gut colonization by B . fragilis . A cell suspension (7.0 x10 7 CFU in total) of wild-type B . fragilis was inoculated into three 8-week-old male BALB/cA germ-free mice via gavage. At 3, 7, 10, and 14 days after inoculation, feces samples were collected. DNA was extracted from the initial inoculum and the collected feces samples, and the ON/OFF ratio of IVp-I/IVp-II in each sample was determined by qPCR. The data are expressed as means ± standard deviation. The bars labeled with different letters indicate significant differences at p < 0.05. (C) Competitive growth assay of IVp-I/Vp-II-locked B . fragilis mutant and its parental YCH46 stain. Equivalent cell mixture of wild and the OFF/ON mutant cells was inoculated into BALB/c germ-free mice. Colony PCR was performed on at least 96 colonies per sample with primer pair encompassing the deletion site in BF2766 to compare population levels of mutants (closed triangles) with wild type (closed circles) cells in the mouse intestine. In this experiment, mice were kept in a vinyl isolator to maintain their gnotobiotic conditions.

Article Snippet: The B . fragilis YCH46 DNA microarray from NimbleGen Systems, which includes 4,527 target genes with at least 8 unique 60-mer synthetic oligonucleotide probes for each gene, was used for the comparative transcriptomic analysis of the mutant strains.

Techniques: Growth Assay, Mutagenesis, Staining, In Vitro, In Vivo, Suspension, Standard Deviation, Labeling