Publications by authors named "Pascale Mosoni"

18 Publications

  • Page 1 of 1

Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses.

mBio 2021 03 3;12(2). Epub 2021 Mar 3.

Université Clermont Auvergne, INRAE, UMR 454 MEDIS, Clermont-Ferrand, France

, , and are the three predominant cellulolytic bacterial species found in the rumen. studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with S85 and sp. strain 87.7, and 5 months later, the lambs were inoculated with 8 and FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while and preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response. Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, , , and , has been extensively studied to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of competitions between and the two species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.
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http://dx.doi.org/10.1128/mBio.03533-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092306PMC
March 2021

Tripartite relationship between gut microbiota, intestinal mucus and dietary fibers: towards preventive strategies against enteric infections.

FEMS Microbiol Rev 2021 03;45(2)

Université Clermont Auvergne, UMR 454 INRAe, Microbiology, Digestive Environment and Health (MEDIS), Clermont-Ferrand, France.

The human gut is inhabited by a large variety of microorganims involved in many physiological processes and collectively referred as to gut microbiota. Disrupted microbiome has been associated with negative health outcomes and especially could promote the onset of enteric infections. To sustain their growth and persistence within the human digestive tract, gut microbes and enteric pathogens rely on two main polysaccharide compartments, namely dietary fibers and mucus carbohydrates. Several evidences suggest that the three-way relationship between gut microbiota, dietary fibers and mucus layer could unravel the capacity of enteric pathogens to colonise the human digestive tract and ultimately lead to infection. The review starts by shedding light on similarities and differences between dietary fibers and mucus carbohydrates structures and functions. Next, we provide an overview of the interactions of these two components with the third partner, namely, the gut microbiota, under health and disease situations. The review will then provide insights into the relevance of using dietary fibers interventions to prevent enteric infections with a focus on gut microbial imbalance and impaired-mucus integrity. Facing the numerous challenges in studying microbiota-pathogen-dietary fiber-mucus interactions, we lastly describe the characteristics and potentialities of currently available in vitro models of the human gut.
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http://dx.doi.org/10.1093/femsre/fuaa052DOI Listing
March 2021

Procyanidin-Cell Wall Interactions within Apple Matrices Decrease the Metabolization of Procyanidins by the Human Gut Microbiota and the Anti-Inflammatory Effect of the Resulting Microbial Metabolome In Vitro.

Nutrients 2019 Mar 19;11(3). Epub 2019 Mar 19.

Université Clermont Auvergne, INRA, UMR 0454 MEDIS, F-63000 Clermont-Ferrand, France.

B-type oligomeric procyanidins in apples constitute an important source of polyphenols in the human diet. Their role in health is not known, although it is suggested that they generate beneficial bioactive compounds upon metabolization by the gut microbiota. During apple processing, procyanidins interact with cell-wall polysaccharides and form stable complexes. These interactions need to be taken into consideration in order to better assess the biological effects of fruit constituents. Our objectives were to evaluate the impact of these interactions on the microbial metabolization of cell walls and procyanidins, and to investigate the potential anti-inflammatory activity of the resulting metabolome, in addition to analyzing the taxonomical changes which the microbiota undergo. In vitro fermentation of three model apple matrices with microbiota from 4 healthy donors showed that the binding of procyanidins to cell-wall polysaccharides, whether covalently or non-covalently, substantially reduced procyanidin degradation. Although cell wall-unbound procyanidins negatively affected carbohydrate fermentation, they generated more hydroxyphenylvaleric acid than bound procyanidins, and increased the abundance of and genera. The best results in terms of production of anti-inflammatory bioactive metabolites were observed from the apple matrix with no bonds between procyanidins and cell wall polysaccharides, although the matrix with non-covalent bonds was not far behind.
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http://dx.doi.org/10.3390/nu11030664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471247PMC
March 2019

Food Chemicals Disrupt Human Gut Microbiota Activity And Impact Intestinal Homeostasis As Revealed By In Vitro Systems.

Sci Rep 2018 Jul 20;8(1):11006. Epub 2018 Jul 20.

MEDIS, Université Clermont Auvergne, INRA, Clermont-Ferrand, France.

Growing evidence indicates that the human gut microbiota interacts with xenobiotics, including persistent organic pollutants and foodborne chemicals. The toxicological relevance of the gut microbiota-pollutant interplay is of great concern since chemicals may disrupt gut microbiota functions, with a potential impairment of host homeostasis. Herein we report within batch fermentation systems the impact of food contaminants (polycyclic aromatic hydrocarbons, polychlorobiphenyls, brominated flame retardants, dioxins, pesticides and heterocyclic amines) on the human gut microbiota by metatranscriptome and volatolome i.e. "volatile organic compounds" analyses. Inflammatory host cell response caused by microbial metabolites following the pollutants-gut microbiota interaction, was evaluated on intestinal epithelial TC7 cells. Changes in the volatolome pattern analyzed via solid-phase microextraction coupled to gas chromatography-mass spectrometry mainly resulted in an imbalance in sulfur, phenolic and ester compounds. An increase in microbial gene expression related to lipid metabolism processes as well as the plasma membrane, periplasmic space, protein kinase activity and receptor activity was observed following dioxin, brominated flame retardant and heterocyclic amine exposure. Conversely, all food contaminants tested induced a decreased in microbial transcript levels related to ribosome, translation and nucleic acid binding. Finally, we demonstrated that gut microbiota metabolites resulting from pollutant disturbances may promote the establishment of a pro-inflammatory state in the gut, as stated with the release of cytokine IL-8 by intestinal epithelial cells.
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http://dx.doi.org/10.1038/s41598-018-29376-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054606PMC
July 2018

FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota.

Front Microbiol 2018 13;9:215. Epub 2018 Feb 13.

UMR 454 MEDIS, INRA, Université Clermont Auvergne, Clermont-Ferrand, France.

Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme) families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6) were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi) represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.
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http://dx.doi.org/10.3389/fmicb.2018.00215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816793PMC
February 2018

Metatranscriptomics Reveals the Active Bacterial and Eukaryotic Fibrolytic Communities in the Rumen of Dairy Cow Fed a Mixed Diet.

Front Microbiol 2017 31;8:67. Epub 2017 Jan 31.

UR454 Unité de Microbiologie, INRA Saint-Genès-Champanelle, France.

Ruminants have a unique ability to derive energy from the degradation of plant polysaccharides through the activity of the rumen microbiota. Although this process is well studied , knowledge gaps remain regarding the relative contribution of the microbiota members and enzymes . The present study used RNA-sequencing to reveal both the expression of genes encoding carbohydrate-active enzymes (CAZymes) by the rumen microbiota of a lactating dairy cow and the microorganisms forming the fiber-degrading community. Functional analysis identified 12,237 CAZymes, accounting for 1% of the transcripts. The CAZyme profile was dominated by families GH94 (cellobiose-phosphorylase), GH13 (amylase), GH43 and GH10 (hemicellulases), GH9 and GH48 (cellulases), PL11 (pectinase) as well as GH2 and GH3 (oligosaccharidases). Our data support the pivotal role of the most characterized fibrolytic bacteria ( and ), and highlight a substantial, although most probably underestimated, contribution of fungi and ciliate protozoa to polysaccharide degradation. Particularly these results may motivate further exploration of the role and the functions of protozoa in the rumen. Moreover, an important part of the fibrolytic bacterial community remains to be characterized since one third of the CAZyme transcripts originated from distantly related strains. These findings are used to highlight limitations of current metatranscriptomics approaches to understand the functional rumen microbial community and opportunities to circumvent them.
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http://dx.doi.org/10.3389/fmicb.2017.00067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5281551PMC
January 2017

Mannoside recognition and degradation by bacteria.

Biol Rev Camb Philos Soc 2017 Nov 20;92(4):1969-1990. Epub 2016 Dec 20.

LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077, Toulouse, France.

Mannosides constitute a vast group of glycans widely distributed in nature. Produced by almost all organisms, these carbohydrates are involved in numerous cellular processes, such as cell structuration, protein maturation and signalling, mediation of protein-protein interactions and cell recognition. The ubiquitous presence of mannosides in the environment means they are a reliable source of carbon and energy for bacteria, which have developed complex strategies to harvest them. This review focuses on the various mannosides that can be found in nature and details their structure. It underlines their involvement in cellular interactions and finally describes the latest discoveries regarding the catalytic machinery and metabolic pathways that bacteria have developed to metabolize them.
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http://dx.doi.org/10.1111/brv.12316DOI Listing
November 2017

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

BMC Genomics 2016 05 4;17:326. Epub 2016 May 4.

Institut National de la recherche Agronomique (INRA), UR454 Microbiologie, Centre de Clermont-Ferrand-Theix, 63122, Saint-Genès-Champanelle, France.

Background: Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1A(T).

Results: Transcriptomic analyses of B. xylanisolvens XB1A(T) grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70.

Conclusion: This study shows that xylan degradation by B. xylanisolvens XB1A(T) is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1A(T) has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health.
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http://dx.doi.org/10.1186/s12864-016-2680-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855328PMC
May 2016

Unraveling the pectinolytic function of Bacteroides xylanisolvens using a RNA-seq approach and mutagenesis.

BMC Genomics 2016 Feb 27;17:147. Epub 2016 Feb 27.

Institut National de la Recherche Agronomique (INRA), UR454 Microbiologie, Centre de Clermont-Ferrand/Theix, 63122, Saint-Genès Champanelle, France.

Background: Diet and particularly dietary fibres have an impact on the gut microbiome and play an important role in human health and disease. Pectin is a highly consumed dietary fibre found in fruits and vegetables and is also a widely used additive in the food industry. Yet there is no information on the effect of pectin on the human gut microbiome. Likewise, little is known on gut pectinolytic bacteria and their enzyme systems. This study was undertaken to investigate the mechanisms of pectin degradation by the prominent human gut symbiont Bacteroides xylanisolvens.

Results: Transcriptomic analyses of B. xylanisolvens XB1A grown on citrus and apple pectins at mid- and late-log phases highlighted six polysaccharide utilization loci (PUL) that were overexpressed on pectin relative to glucose. The PUL numbers used in this report are those given by Terrapon et al. (Bioinformatics 31(5):647-55, 2015) and found in the PUL database: http://www.cazy.org/PULDB/. Based on their CAZyme composition, we propose that PUL 49 and 50, the most overexpressed PULs on both pectins and at both growth phases, are involved in homogalacturonan (HG) and type I rhamnogalacturonan (RGI) degradation, respectively. PUL 13 and PUL 2 could be involved in the degradation of arabinose-containing side chains and of type II rhamnogalacturonan (RGII), respectively. Considering that HG is the most abundant moiety (>70%) within pectin, the importance of PUL 49 was further investigated by insertion mutagenesis into the susC-like gene. The insertion blocked transcription of the susC-like and the two downstream genes (susD-like/FnIII). The mutant showed strong growth reduction, thus confirming that PUL 49 plays a major role in pectin degradation.

Conclusion: This study shows the existence of six PULs devoted to pectin degradation by B. xylanisolvens, one of them being particularly important in this function. Hence, this species deploys a very complex enzymatic machinery that probably reflects the structural complexity of pectin. Our findings also highlight the metabolic plasticity of B. xylanisolvens towards dietary fibres that contributes to its competitive fitness within the human gut ecosystem. Wider functional and ecological studies are needed to understand how dietary fibers and especially plant cell wall polysaccharides drive the composition and metabolism of the fibrolytic and non-fibrolytic community within the gut microbial ecosystem.
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http://dx.doi.org/10.1186/s12864-016-2472-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769552PMC
February 2016

Rumen cellulosomics: divergent fiber-degrading strategies revealed by comparative genome-wide analysis of six ruminococcal strains.

PLoS One 2014 3;9(7):e99221. Epub 2014 Jul 3.

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.

Background: A complex community of microorganisms is responsible for efficient plant cell wall digestion by many herbivores, notably the ruminants. Understanding the different fibrolytic mechanisms utilized by these bacteria has been of great interest in agricultural and technological fields, reinforced more recently by current efforts to convert cellulosic biomass to biofuels.

Methodology/principal Findings: Here, we have used a bioinformatics-based approach to explore the cellulosome-related components of six genomes from two of the primary fiber-degrading bacteria in the rumen: Ruminococcus flavefaciens (strains FD-1, 007c and 17) and Ruminococcus albus (strains 7, 8 and SY3). The genomes of two of these strains are reported for the first time herein. The data reveal that the three R. flavefaciens strains encode for an elaborate reservoir of cohesin- and dockerin-containing proteins, whereas the three R. albus strains are cohesin-deficient and encode mainly dockerins and a unique family of cell-anchoring carbohydrate-binding modules (family 37).

Conclusions/significance: Our comparative genome-wide analysis pinpoints rare and novel strain-specific protein architectures and provides an exhaustive profile of their numerous lignocellulose-degrading enzymes. This work provides blueprints of the divergent cellulolytic systems in these two prominent fibrolytic rumen bacterial species, each of which reflects a distinct mechanistic model for efficient degradation of cellulosic biomass.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099221PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081043PMC
November 2015

Methanogen colonisation does not significantly alter acetogen diversity in lambs isolated 17 h after birth and raised aseptically.

Microb Ecol 2012 Oct 2;64(3):628-40. Epub 2012 Mar 2.

CSIRO Livestock Industries, St. Lucia, QLD 4067, Australia.

Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17 h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen acetogen population since they lacked cultivable representatives of methanogens. Putative acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential acetogen diversity in isolated lambs and conventional sheep was different. Potential acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect acetogen diversity.
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http://dx.doi.org/10.1007/s00248-012-0024-zDOI Listing
October 2012

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A.

Appl Microbiol Biotechnol 2010 Aug 8;87(6):2097-105. Epub 2010 Jun 8.

INRA, UR Unité de Microbiologie, Centre de Recherches de Clermont-Ferrand/Theix, Saint-Genès-Champanelle, France.

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K (m) and V (max) of 1.6 mg ml(-1) and 118 micromol min(-1) mg(-1) on oat spelt xylan, and its optimal temperature and pH for activity were 37 degrees C and pH 6.0, respectively. Its catalytic properties (k (cat)/K (m) = 3,300 ml mg(-1) min(-1)) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.
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http://dx.doi.org/10.1007/s00253-010-2694-0DOI Listing
August 2010

Differential translocation of green fluorescent protein fused to signal sequences of Ruminococcus albus cellulases by the Tat and Sec pathways of Escherichia coli.

FEMS Microbiol Lett 2009 May 1;294(2):239-44. Epub 2009 Apr 1.

Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of herbivores. It was described to synthesize two major glycoside-hydrolases (Cel9B and Cel48A), which are exported and anchored at the cell surface. In bacteria, proteins destined to cross the cytoplasmic membrane are synthesized as precursors and possess a signal sequence (SS) directing them to the 'Sec' (general secretory) or 'Tat' (twin arginine translocation) pathway. SS composition of Cel9B and Cel48A suggests that these two enzymes translocate using different secretory pathways. In order to confirm this hypothesis, the SSs of Cel9B and Cel48A were fused to the green fluorescent protein (GFP) and expressed in wild-type Escherichia coli and in its Tat and Sec isogenic mutants. The SS cleavage and the formation of the mature protein were then followed by Western blot and fluorescence microscopy. This study shows that the SS of Cel9B directs the preprotein to the 'Tat' translocation pathway while the GFP fused to the SS of Cel48A is exported through the 'Sec' machinery. These observations suggest that R. albus possess a Tat pathway, in addition to the general secretory pathway.
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http://dx.doi.org/10.1111/j.1574-6968.2009.01576.xDOI Listing
May 2009

Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep.

J Microbiol Methods 2009 Apr 31;77(1):8-16. Epub 2009 Jan 31.

INRA, UR454 Unité de Microbiologie, F-63122 Saint-Genès-Champanelle, France.

An improved RNA isolation method based on the acid guanidinium-phenol-chloroform (AGPC) procedure using saline precipitation but no column purification was evaluated for quantifying microbial gene expression using reverse transcription quantitative PCR (RT-qPCR) in rumen contents. The method provided good RNA integrity and quantity extracts. The transcript levels of eight glycoside hydrolase (GH) genes of the major rumen fibrolytic bacterium Fibrobacter succinogenes were quantified in the complex microbiota of a conventional sheep and in a gnotobiotic lamb harboring a microflora containing F. succinogenes S85 as the sole cellulolytic microorganism. This study validated the improved RNA isolation method, RT-qPCR conditions to quantify GH transcripts using either the F. succinogenes S85 tuf gene or the 16S rRNA-encoding gene (rrs) as the reference gene, and demonstrated the need to work with good quality RNAs. Transcripts from all the selected genes cel3, endA(FS), celF and endB endoglucanase genes, cedA cellodextrinase gene, mlg lichenase gene, and xynC and xynD xylanase genes of F. succinogenes S85 were detected and quantified at varying levels in the rumen content of the two animal models. This study opens new perspectives in studying microbial gene expression in the rumen of both conventional and gnotobiotic sheep.
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http://dx.doi.org/10.1016/j.mimet.2008.11.009DOI Listing
April 2009

Proteomic identification of CBM37-containing cellulases produced by the rumen cellulolytic bacterium Ruminococcus albus 20 and their putative involvement in bacterial adhesion to cellulose.

Arch Microbiol 2009 Apr 21;191(4):379-88. Epub 2009 Feb 21.

INRA, UR454 Unité de Microbiologie, 63122 Saint-Genès-Champanelle, France.

The objective of this study was to identify and characterize other proteins than fimbrial proteins potentially involved in R. albus 20 adhesion to cellulose using an adhesion-related antiserum preparation (i.e. anti-Adh serum). From protein fractions of R. albus 20 grown on cellulose, the serum recognized at least 10 cellulose-binding proteins (CBPs), among which homologs of glycoside hydrolases (family 5, 9 and 48) of R. albus 8 (i.e. Cel5G, Cel9B and Cel48A) were identified by a proteomic approach. In strain 20, Cel9B and Cel48A were identified as two major CBPs and as bacterial cell-associated proteins. The anti-Adh serum was also shown to target the C-terminal family 37 carbohydrate-binding module (CBM37) of Cel9B and Cel48A, indicating that this module, unique to R. albus, may play a significant role in bacterial adhesion to cellulose as suggested previously for R. albus 8. Overall, our results support the hypothesis of an adhesion mechanism involving the CBM37 of Cel9B and Cel48A. This adhesion mechanism may not be restricted to these two enzymes but may also involve other CBM37-containing proteins such as Cel5G and the other uncharacterised proteins recognized by the anti-Adh serum.
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http://dx.doi.org/10.1007/s00203-009-0463-1DOI Listing
April 2009

The Ruminococcus albus pilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose.

Microbiology (Reading) 2005 Apr;151(Pt 4):1291-9

Unité de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France.

Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesion to cellulose. The subunit protein has been identified in strain 8 (CbpC) and strain 20 (GP25) and both are type IV fimbrial (Pil) proteins. The presence of a pil locus that is organized similarly in both strains is reported here together with the results of an initial examination of a second Pil protein. Downstream of the cbpC/gp25 gene (hereafter referred to as pilA1) is a second pilin gene (pilA2). Northern blot analysis of pilA1 and pilA2 transcripts showed that the pilA1 transcript is much more abundant in R. albus 8, and real-time PCR was used to measure pilA1 and pilA2 transcript abundance in R. albus 20 and its adhesion-defective mutant D5. Similar to the findings with R. albus 8, the relative expression of pilA1 in the wild-type strain was 73-fold higher than that of pilA2 following growth with cellobiose, and there were only slight differences between the wild-type and mutant strain in pilA1 and pilA2 transcript abundances, indicating that neither pilA1 nor pilA2 transcription is adversely affected in the mutant strain. Western immunoblots showed that the PilA2 protein is localized primarily to the membrane fraction, and the anti-PilA2 antiserum does not inhibit bacterial adhesion to cellulose. These results suggest that the PilA2 protein plays a role in the synthesis and assembly of type IV fimbriae-like structures by R. albus, but its role is restricted to cell-associated functions, rather than as part of the externalized fimbrial structure.
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http://dx.doi.org/10.1099/mic.0.27735-0DOI Listing
April 2005

Adhesion to cellulose of the Gram-positive bacterium Ruminococcus albus involves type IV pili.

Microbiology (Reading) 2002 Jun;148(Pt 6):1871-1880

Unité de Microbiologie1 and Unité de Recherches sur la Viande, Equipe Microbiologie2, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France.

This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previously shown to be underproduced by a spontaneous adhesion-defective mutant D5 of Ruminococcus albus 20. An antiserum against wild-type strain 20 was adsorbed with the mutant D5 to enrich it in antibodies 'specific' to adhesion structures of R. albus 20. The resulting antiserum, called anti-Adh serum, blocked adhesion of R. albus 20 and reacted mainly with GP25 in bacterial and extracellular protein fractions of R. albus 20. The N-terminal sequence of purified GP25 was identical to that of CbpC, a 21 kDa cellulose-binding protein (CBP) of R. albus 8. The nucleotide sequence of the gp25 gene was determined by PCR and genomic walking procedures. The gp25 gene encoded a protein of 165 aa with a calculated molecular mass of 16940 Da that showed 72.9% identity with CbpC and presented homologies with type IV pilins of Gram-negative pathogenic bacteria. Negative-staining electron microscopy revealed fine and flexible pili surrounding R. albus 20 cells while mutant cells were not piliated. In addition, immunoelectron microscopy showed that the anti-Adh serum probing mainly GP25, completely decorated the pili surrounding R. albus 20, thereby showing that GP25 was a major pilus subunit. This study shows for the first time the presence of pili at the surface of R. albus and identifies GP25 as their major protein subunit. Though GP25 was not identified as a CBP, isolated pili were shown to bind cellulose. In conclusion, these pili, which belong to the family of type IV pili, mediate adhesion of R. albus 20 to cellulose.
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http://dx.doi.org/10.1099/00221287-148-6-1871DOI Listing
June 2002