Journal: Neurogastroenterology and Motility

Article Title: GABA ‐producing Bifidobacterium dentium modulates visceral sensitivity in the intestine

doi: 10.1111/nmo.12904

Figure Lengend Snippet: Microbial glutamate decarboxylase gene ( gadB ) in the human microbiome. (A) The relative abundance of the gadB gene among different body sites in 96 healthy adult individuals is depicted as a bar graph. The vertical bars indicate the mean relative abundances (and body site) of gadB (± SEM ). The chemical structure of glutamate and GABA and conversion of glutamate to GABA by GadB are shown within the graph. The colored horizontal bars indicate body sites. (B) Bacterial genera/species of the human gut microbiome harboring putative glutamate decarboxylases are depicted as a pie chart. The prevalence of glutamate decarboxylases among the members of the healthy human gut microbiome was estimated from data deposited at the Integrated Microbial Genomes/Human Microbiome Project ( IMG / HMP ) database ( http://img.jgi.doe.gov ). Percentages displayed represent genus‐level distribution among these genomes, with species‐level distribution shown for the Bifidobacterium species

Article Snippet: Bifidobacterium strains ( B. dentium ATCC 27678, B. breve NCIMB 8807, and B. breve NCIMB8807 pESHgadB) were grown in culture conditions described above.

Techniques:

Journal: Neurogastroenterology and Motility

Article Title: GABA ‐producing Bifidobacterium dentium modulates visceral sensitivity in the intestine

doi: 10.1111/nmo.12904

Figure Lengend Snippet: GABA production by commensal intestinal strains and Bifidobacterium dentium in the human gut microbiome. (A) Screening of 16 different intestinal commensal and/or probiotic isolates identified B. dentium ATCC 27678 as a major GABA producer. GABA concentrations were measured using LC ‐ MS after 48 hours of anaerobic growth at 37°C in either regular MRS (dark green) or MRS medium supplemented with 1% w/v glutamate (light green). Error bars represent standard error of 3 independently performed experiments. B. dentium was the only species that produced significantly more GABA ( P <.0001; two‐way ANOVA with Bonferroni correction for multiple comparisons). (B) B. dentium in the healthy human gut microbiome, as detected by Metaphlan profiling of shotgun metagenomic sequence libraries. Different colors represent subject cohorts, while bars show proportion of sequences with hits to B. dentium found in each individual. The horizontal bars represent the average relative abundance (as determined by the proportion of sequences with hits to B. dentium ) in each cohort

Article Snippet: Bifidobacterium strains ( B. dentium ATCC 27678, B. breve NCIMB 8807, and B. breve NCIMB8807 pESHgadB) were grown in culture conditions described above.

Techniques: Liquid Chromatography with Mass Spectroscopy, Produced, Sequencing

Journal: Neurogastroenterology and Motility

Article Title: GABA ‐producing Bifidobacterium dentium modulates visceral sensitivity in the intestine

doi: 10.1111/nmo.12904

Figure Lengend Snippet: In vitro and in vivo activity of glutamate decarboxylase (GadB) from Bifidobacterium dentium . (A) GadB 3D structure with its proposed active site highlighted. Dark blue color depicts catalytic lysine at position 289 (K289), while threonine (T225) and aspartate (D256) are colored in light blue and red, respectively. Position of co‐factor pyridoxal phosphate, PLP , is shown in green. (B) Site directed mutagenesis effect on recombinant GadB activity. pQE 60—negative control of crude extract from Escherichia coli strain harboring empty pQE 60 vector. GadB‐ WT —crude extract with recombinant wild‐type GadB overexpressed in E. coli . GadB‐T, GadB‐D, and GadB‐K are crude extracts of recombinant GadB with mutated amino acids from T225, D256, and K289 to alanine, respectively. Bars demonstrate GABA (dark green) or l ‐glutamate (light green) concentration. Error bars represent standard error of 3 independently performed experiments (* P <.01, ** P <.05; one‐way ANOVA of log transformed data, Bonferroni correction for multiple comparisons). (C) Expression of GadB from B. dentium in B. breve by complementation. gadB from B. dentium ATCC 27678 ( BD ) was cloned into the pESH 46 ( pESH gadB) expression vector and transformed into B. breve NCIMB 8807 ( BB ) allowing for constitutive expression ( BB gadB). GABA was measured via LC ‐ MS method (Data S1). Error bars represent standard error of 3 independently performed experiments (**** P <.0001, ns=not significant; one‐way ANOVA of log transformed data, Bonferroni correction for multiple comparisons). (D) Six‐week‐old male Swiss Webster mice (n=6–8 per group) were orally administered 1% glutamate plus B. dentium ATCC 27678 ( BD ), B. breve NCIMB 8807 ( BB ), B. breve NCIMB 8807 pESH gadB ( BB gadB), or saline ( PBS ) for 5 days. Mice administered B. breve pESH gadB had significantly more GABA in their cecal content as measured by ELISA (** P <.01; one‐way ANOVA with Bonferroni correction)

Article Snippet: Bifidobacterium strains ( B. dentium ATCC 27678, B. breve NCIMB 8807, and B. breve NCIMB8807 pESHgadB) were grown in culture conditions described above.

Techniques: In Vitro, In Vivo, Activity Assay, Mutagenesis, Recombinant, Negative Control, Plasmid Preparation, Concentration Assay, Transformation Assay, Expressing, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Saline, Enzyme-linked Immunosorbent Assay

Journal: Neurogastroenterology and Motility

Article Title: GABA ‐producing Bifidobacterium dentium modulates visceral sensitivity in the intestine

doi: 10.1111/nmo.12904

Figure Lengend Snippet: Neuromodulatory effects of GABA ‐producing Bifidobacterium dentium ATCC 27678 administration on colonic sensory neuron activity. Control (Sham) and fecal retention ( FR ) rats were gavaged daily with GABA ‐producing, gadB ‐positive B. dentium or gadB ‐negative B. breve strains (n=4 rats and 20–24 neurons per treatment group). Colon‐specific DRG neurons were isolated and used for the measurements of cell excitability by patch clamp recordings. The following parameters are displayed: (A) Resting membrane potential ( RMP ), (B) rheobase, (C) action potential spikes at 2× rheobase, (D) action potential spikes at 3× rheobase, (E) cell diameter, (F) membrane capacitance, (G) input resistance, (H) action potential threshold, (I) action potential amplitude, (J) action potential overshoot, (K) action potential duration, (L) action potential latency. Bars represent mean values with standard error (* P <.05, ** P <.01, *** P <.001; Kruskal‐Wallis with Dunn correction for multiple comparison)

Article Snippet: Bifidobacterium strains ( B. dentium ATCC 27678, B. breve NCIMB 8807, and B. breve NCIMB8807 pESHgadB) were grown in culture conditions described above.

Techniques: Activity Assay, Control, Isolation, Patch Clamp, Membrane, Comparison

Journal: Neurogastroenterology and Motility

Article Title: GABA ‐producing Bifidobacterium dentium modulates visceral sensitivity in the intestine

doi: 10.1111/nmo.12904

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Bifidobacterium strains ( B. dentium ATCC 27678, B. breve NCIMB 8807, and B. breve NCIMB8807 pESHgadB) were grown in culture conditions described above.

Techniques: Mutagenesis, Recombinant, Expressing, Plasmid Preparation, Cloning