Journal: Gut Microbes

Article Title: The mucin-degradation strategy of Ruminococcus gnavus : The importance of intramolecular trans -sialidases

doi: 10.1080/19490976.2016.1186334

Figure Lengend Snippet: Growth curves of R. gnavus ATCC 29149 and ATCC 35913 on pPGM. The growth curves represent the average growth, measured at OD600nm, of at least 3 biological replicates.

Article Snippet: Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans.

Techniques:

Journal: Gut Microbes

Article Title: The mucin-degradation strategy of Ruminococcus gnavus : The importance of intramolecular trans -sialidases

doi: 10.1080/19490976.2016.1186334

Figure Lengend Snippet: Comparison of the distribution of glycoside hydrolases (GHs) between R. gnavus strains. GHs are represented by light gray boxes for R. gnavus ATCC 35913, striped boxed for R. gnavus ATCC 29149 and dark gray boxes for R. gnavus E1.

Article Snippet: Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans.

Techniques:

Journal: Gut Microbes

Article Title: The mucin-degradation strategy of Ruminococcus gnavus : The importance of intramolecular trans -sialidases

doi: 10.1080/19490976.2016.1186334

Figure Lengend Snippet: Relative level of transcription of GH genes in R. gnavus ATCC 29149 (A) and ATCC 35913 (B). The transcriptomic analysis has been performed by RNASeq from R. gnavus grown in presence of pPGM and compared to Glc as sole carbon source. The relative level of transcription was expressed as the Log2 of the fold change in gene transcription and the figures showed averages of 4 biological replicates for the GH genes that exhibited increased transcription (Log2 fold change > 1). Data were analyzed by DESeq2. The significance of differential expression was determined by the Benjamini-Hochberg corrected p-values of the Wald test of the negative binomial test per each set of two conditions. The transcription level was considered significantly increased when p < 0.05 and a Log2 (fold change) >1 and significant results were labeled with *. Error bars were plotted as the standard error of the Log2 fold change.

Article Snippet: Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans.

Techniques: Expressing, Labeling

Journal: Gut Microbes

Article Title: The mucin-degradation strategy of Ruminococcus gnavus : The importance of intramolecular trans -sialidases

doi: 10.1080/19490976.2016.1186334

Figure Lengend Snippet: The Nan locus in R. gnavus ATCC 29149 and ATCC 35913. (A) Schematic representation of the nan genetic organization in ATCC 35913. RGNV35913_01299 encodes a putative GDSL-like protein. RGNV35913_01298encodes a putative sugar isomerase involved in sialic acid catabolism. RGNV35913_01297 encodes a protein with homology with transcriptional regulators of the AraC family. The following 3 genes code for a predicted solute-binding protein (RGNV35913_01296) and two putative permeases (RGNV35913_01295 and RGNV35913_01294), components of a sugar ABC transporter. The following gene has homology with oxidoreductases from the Gfo/Idh/MocA family. The sialidase gene nanH (RGNV35913_01292) predicted to encode the GH33 enzyme comes next. Then nanE (RGNV35913_01291), which encodes a predicted ManNAc-6-P epimerase is followed by nanA (RGNV35913_01290) encoding a putative Neu5Ac lyase. nanK (RGNV35913_01289) is the last gene of the cluster, coding for a predicted ManNAc kinase. The previously described R. gnavus ATCC 29149 nan cluster shares 99.9% identity with the one present in ATCC 35913. Level of transcription of nan genes in R. gnavus ATCC 29149 (B) or ATCC 35913 (C). R. gnavus was grown in basal YCFA medium supplemented with either glucose (Glc) or mucin (pPGM) as sole carbon source. Cells were collected during the exponential phase of growth; RNA was extracted from 4 biological replicates for each carbon sources. The level of transcription of each gene was determined by RNASeq. The transcription of each gene was compared when the bacterium grew with pPGM vs. Glc using the R package DESeqx; it was considered significantly increased when the transcript was present at least twice more frequently, with a padj value (p-value adjusted for multiple testing) <=0.05 (* padj<=0.05).

Article Snippet: Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans.

Techniques: Binding Assay

Journal: Gut Microbes

Article Title: The mucin-degradation strategy of Ruminococcus gnavus : The importance of intramolecular trans -sialidases

doi: 10.1080/19490976.2016.1186334

Figure Lengend Snippet: Proposed pathways for the catabolism of sialic acid in R. gnavus ATCC 29149 and ATCC 35913. Rg NanH releases 2,7-anhydro-Neu5Ac from α2–3 linked sialylated substrates. (A) It can be hypothesized that 2,7-anhydro-Neu5Ac is transported inside the bacterium via a 2,7-anhydro-Neu5Ac-specific ABC transporter composed of a solute-binding protein (RUMGNA_02698 in ATCC 29149;RGNV35913_01296 in ATCC 35913) and two putative permeases (RUMGNA_02697 and RUMGNA_02696 in ATCC 29149; RGNV35913_01295 and RGNV35913_01294 in ATCC 35913) and then hydrolyzed into Neu5Ac, possibly by the action of RUMGNA_02701 or RGNV35913_01299, before being catabolized into GlcNAc-6-P following the traditional pathway by the successive action of NanA (Neu5Ac lyase), NanK (ManNAc kinase) and NanE (ManNAc-6-P epimerase). (B) Alternatively, both 2,7-anhydro-Neu5Ac and Neu5Ac could enter the cells via the ABC transporter but NanA would either be inactive or specific for 2,7-anhydro-Neu5Ac, explaining the absence of growth of the bacteria on sialic acid.

Article Snippet: Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans.

Techniques: Binding Assay

Journal: Cell host & microbe

Article Title: A human gut bacterium antagonizes neighboring bacteria by altering their protein-folding ability.

doi: 10.1016/j.chom.2025.01.008

Figure Lengend Snippet: Figure 3. B. thetaiotaomicron PpiD (BT4371) directly binds to Bte1 and is required for Bte1 toxicity (A) Experimental scheme to isolate Bte1-binding proteins from B. thetaiotaomicron lysates. (B) Silver-stained SDS-PAGE analysis of proteins enriched by streptavidin pre- cipitation from MsyB-GFP or MsyB-Bte1 incubated with B. thetaiotaomicron lysates. Arrow indicates PpiD. (C) Bte1 binds to PpiD in vivo. Pull-downs were performed on B. thetaiotaomicron wild-type (WT) and ppiD-His strains expressing ss-bte1- HA and analyzed by western blot. (D) Outcome of competition assays between the indicated B. thetaiotaomicron recipient strains and Bf N Dvr2. Data represent means ± SD, from two bio- logical replicates on 3 separate days.

Article Snippet: Spectra were queried against the Bacteroides thetaiotaomicron database (Uniprot reference, downloaded: 01/2023, strain ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50), in addition to the protein sequence of Bte1 and Bti1 (Figure 5G), using the built-in SequestHT algorithm using a target-decoy strategy further paired with Percolator for peptide quality scoring metrics.

Techniques: Binding Assay, Staining, SDS Page, Incubation, In Vivo, Expressing, Western Blot

Journal: Cell host & microbe

Article Title: A human gut bacterium antagonizes neighboring bacteria by altering their protein-folding ability.

doi: 10.1016/j.chom.2025.01.008

Figure Lengend Snippet: Figure 4. PpiD and YfgM (BT4254) form a chaperone complex (A) Coomassie-stained SDS-PAGE analysis of proteins enriched by cobalt-affinity pull-downs from lysates of B. thetaiotaomicron WT or ppiD-His strains. Arrows indicate PpiD-His and YfgM. (B) Atomic structure of the PpiD-YfgM complex. YfgM depicted as a green ribbon; PpiD domains (NC, P1–P4) are labeled and colored as in (D). (C) Surface representation of the PpiD-YfgM complex. PpiD is shown in purple and YfgM is shown in green. (D) Domain architecture of PpiD. TM, transmembrane domain; NTD, N-terminal domain (yellow); parvulin domains P1 (lilac), P2 (orange), P3 (pink), and P4 (light blue); CTD, C-terminal domain (yellow). (E) Lysozyme aggregation in the presence or absence of YfgM or the PpiD-YfgM complex. (F) Isomerization-dependent refolding of RCM-T1 by YfgM or the PpiD-YfgM complex. (G) PpiD interacts with the Sec-translocon component SecG. Pull-downs from cells incubated with the crosslinker DSP are designated as ‘‘+ DSP.’’ Data in (E) and (F) represent means ± SD, from two biological replicates on 3 separate days.

Article Snippet: Spectra were queried against the Bacteroides thetaiotaomicron database (Uniprot reference, downloaded: 01/2023, strain ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50), in addition to the protein sequence of Bte1 and Bti1 (Figure 5G), using the built-in SequestHT algorithm using a target-decoy strategy further paired with Percolator for peptide quality scoring metrics.

Techniques: Staining, SDS Page, Labeling, Incubation

Journal: Cell host & microbe

Article Title: A human gut bacterium antagonizes neighboring bacteria by altering their protein-folding ability.

doi: 10.1016/j.chom.2025.01.008

Figure Lengend Snippet: Figure 5. Bte1 alters the chaperone and isomerase activity of the PpiD-YfgM complex (A) Bte1 interacts with the PpiD-YfgM complex in a PpiD-dependent manner. Pull-downs were performed on B. thetaiotaomicron WT, ppiD-His, yfgM-His, or DppiD-yfgM-His strains expressing ss-bte1-HA and analyzed by western blot 1 h after ss-bte1-HA induction. (B) Cryo-EM 3D reconstruction of the PpiD-YfgM-Bte1 complex (top). Below, the same 3D reconstruction is shown with each protein distinguished by color. (C) Domain architecture of PpiD in the cryo-EM 3D reconstructed PpiD-YfgM-Bte1 complex. PpiD domains are colored as in Figure 4D; YfgM and Bte1 are shown in green and blue, respectively. (D) Lysozyme aggregation in the presence of the PpiD-YfgM complex pre-incubated with purified Bte1 and two stable Bte1 mutants, which exhibit abrogated toxicity in cellular assays (Bte1indel; Bte1G217D). Data in gray (buffer) and green (PpiD-YfgM complex) are the same as in Figure 4E. (E) Isomerization-dependent refolding of RCM-T1 by the PpiD-YfgM complex in the presence or absence of purified Bte1. Data in gray (buffer) and green (PpiD- YfgM complex) are the same as in Figure 4F. Data in (D) and (E) represent means ± SD, from two biological replicates on 3 separate days. (F and G) Comparative proteomic analyses of (F) B. thetaiotaomicron WT and DppiD strains, and (G) B. thetaiotaomicron ss-bte1+ and ss-bte1+bti1+ strains 40 min after induction of ss-bte1 expression. Proteins with a fold-change > 2 and a FDR less than 0.01 (based on 3 biological replicates) are shown as larger data points. Among these proteins, those predicted to be secreted through the Sec translocon are shown in red; others are shown in black. See also Tables S2 and S3.

Article Snippet: Spectra were queried against the Bacteroides thetaiotaomicron database (Uniprot reference, downloaded: 01/2023, strain ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50), in addition to the protein sequence of Bte1 and Bti1 (Figure 5G), using the built-in SequestHT algorithm using a target-decoy strategy further paired with Percolator for peptide quality scoring metrics.

Techniques: Activity Assay, Expressing, Western Blot, Cryo-EM Sample Prep, Incubation

Journal:

Article Title: 1 H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5- f ]Quinoline by Human Intestinal Microbiota

doi: 10.1128/AEM.71.9.5116-5123.2005

Figure Lengend Snippet: Abilities of individual bacterial strains originating from the human digestive tract to convert IQ to 7-OH-IQ a

Article Snippet: Bacterial species and strain Origin Source or reference % of initial IQ degraded b Bacteroides thetaiotaomicron ATCC 29148 Human feces 29 74 9-3JE HMA rat cecum c This study d 52 Bacteroides vulgatus ATCC 8482 Human feces 29 0 Bifidobacterium bifidum B536 Infant feces 14 0 Bifidobacterium longum ATCC 15707 Adult intestine 29 0 Clostridium clostridiiforme 5-1JD1 HMA rat cecum This study 76 9-2JB HMA rat cecum This study 60 10-2JD2 HMA rat cecum This study 50 Clostridium nexile ATCC 27757 Human feces 29 0 Clostridium perfringens G22 Human feces F. Marcille, unpublished data 100 Eggertella lenta γ12 HMA rat cecum 2 0 Escherichia coli 2-1JC HMA rat cecum This study 61 8-1JA HMA rat cecum This study 80 8-1JC HMA rat cecum This study 61 10-2JB HMA rat cecum This study 47 Eubacterium rectale ATCC 33656 Human feces 29 0 Ruminococcus gnavus FRE1 Human feces 12 0 Open in a separate window a For incubation, resting-cell suspensions in PYE buffer were supplemented with 100 μM IQ (anoxic conditions, 37°C, 150 rpm). b At the end of incubation (72 h), the IQ concentration was determined by HPLC analysis. c HMA, human microbiota associated. d Among the 135 strains isolated from the mixed cecal contents of HMA rats and assayed for IQ degradation, only the 8 biodegradative strains are indicated in this table.

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