Pascale Mosoni - Academia.edu (original) (raw)
Papers by Pascale Mosoni
Microbiology (Reading, England), 2002
This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previou... more 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 microscop...
Reproduction, nutrition, development, 1994
Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Ap... more Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Apart from ester bonds involving phenolic acids, which seem to play a major role in grasses, little is known about the other types of linkages that must exist but have proved difficult to demonstrate. The chemical nature of possible LCC linkages is presented and the various mechanisms through which LCCs in the cell-wall architecture may interfere with carbohydrate utilisation by rumen microorganisms are discussed.
Microbiology, 2005
Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesi... more 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.
Microbial Ecology, 2012
Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetoge... more 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 preinoculation 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 (1,000fold increase in methanogens) did not substantially affect acetogen diversity.
Journal of the Science of Food and Agriculture, 1994
ABSTRACT The lower halves of apical internodes of wheat harvested at the flowering stage were lab... more ABSTRACT The lower halves of apical internodes of wheat harvested at the flowering stage were labelled with [U-14C] phenylalanine (phe) or with [O14CH3] sinapic acid (sin). Cell wall residues (CWR) and saponified residues (SR) were incubated in a fermenter simulating the rumen for 7 days with rumen fluid or without microorganisms (controls). PheCWR was labelled in all lignin units (measured as aldehydes from nitrobenzene oxidation), in phenolic acids and slightly in proteins. Labelling of pheSR was more lignin-specific. SinCWR and sinSR were specifically labelled in syringyl units of lignin. The fermentation of CWR resulted in phenylpropane-derived unit losses in the following decreasing order: ferulic acid>p-coumaric acid>syringaldehyde>vanillin>p-hydroxybenzaldehyde. If allowance is made for slight losses in controls, 61, 52, 61 and 63% of the phenylpropanes of pheCWR, sinCWR, pheSR and sinSR, respectively, were transformed into an acid-precipitable fraction, an acid-soluble fraction and 14CO2. The comparison of pheCWR and sinCWR degradation showed that syringyl units were solubilised into acid-precipitable molecules to a greater extent than the other lignin units; demethylation of the syringyl units of lignins was also evident from the different productions of 14CO2. Alkali-resistant lignins of SR were mainly transformed into acid-precipitable molecules and were weakly degraded. Lignin solubilisation and degradation seem to be governed by different mechanisms which depend on both cell wall structure and rumen microflora.
Journal of the Science of Food and Agriculture, 1994
U-14C] phenylalanine (phe*) and [014CH3] sinapic acid (sin*) were infused into the cut ends of no... more U-14C] phenylalanine (phe*) and [014CH3] sinapic acid (sin*) were infused into the cut ends of normal and bm3 maizes (anthesis stage) under or above the last node or at mid-internode, with or without the leaf, in light or in darkness. Radioactivity was measured in the organs, and in phenolic constituents of the cell wall and saponified residues of the bases and tops of the apical internode. In both maize genotype labelled under the node the radioactivity was distributed more evenly in the organs with sin* than with phe*. Infusion above the node and at mid-internode greatly increased radioactivity in the bases and tops, respectively. Removal of the leaf only slightly increased the radioactivity, mainly in the bases, and no clear-cut effect of darkness was observed. Phe* labelled the phenolic acids and the three lignin units, but the syringyl units of bm3 maize were only slightly labelled. Sin* specifically labelled the syringyl units, which represented the least condensed fraction of lignins. Both the native and labelled lignins were highly alkali soluble. There were differences in lignin biogenesis between the bases and tops, and between normal and bm3 maizes. The newly formed lignins were slightly different from the native lignins but had similar types of heterogeneity, with variations in the internode and between genotypes similar to those in native lignins. Provided due allowance is made for the distinguishing characteristics of newly formed lignins, the ['4C-lignin] cell walls, which are strongly labelled on complementary structures, seem suitable model substrates for fermentation studies.
Journal of Microbiological Methods, 2009
An improved RNA isolation method based on the acid guanidinium-phenol-chloroform (AGPC) procedure... more 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.
Journal of Applied Microbiology, 2007
Aim: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacter... more Aim: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. Methods and Results: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease ()1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two-to fourfold) of the Ruminococci. Conclusion: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. Significance and Impact of the Study: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.
![Research paper thumbnail of Wheat lignin labeling using [U-14C]phenylalanine or [O-14CH3]sinapic acid for fermentation studies](https://attachments.academia-assets.com/42294713/thumbnails/1.jpg)
](Journal of Agricultural and Food Chemistry, 1993
ABSTRACT [(U-C)-C-14]Phenylalanine (Phe*) or [(O-CH3)-C-14]sinapic acid (Sin*) was infused in whe... more ABSTRACT [(U-C)-C-14]Phenylalanine (Phe*) or [(O-CH3)-C-14]sinapic acid (Sin*) was infused in wheat stems under the node of the apical fraction, with and without leaf, or at midinternode. (C-14-Lignin)Pronase-treated cell walls of the upper and lower halves of the internode were prepared and analyzed. With Phe*, the labeling had a greater variability than with Sin* and a different distribution in the organs; cell wall residues were labeled on lignin units with a high specific radioactivity, on phenolic acids, and on residual proteins. Labeling of saponified residues was more lignin-specific. With Sin*, syringyl units were specifically labeled. The infusion at midinternode considerably increased labeling yields, while the elimination of the leaf had very slight effect. Radiochemical and chemical results obtained on internode segments are discussed in terms of lignin labeling validity and lignin biogenesis. These substrates, strongly labeled on complementary structures, are potentially useful tools to study the fate of lignins during ruminal fermentation.
FEMS Microbiology Letters, 2009
Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of he... more Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of herbivores. It was described to synthesize two major glycosidehydrolases (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.
Archives of Microbiology, 2009
The objective of this study was to identify and characterize other proteins than Wmbrial proteins... more The objective of this study was to identify and characterize other proteins than Wmbrial 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 identiWed by a proteomic approach. In strain 20, Cel9B and Cel48A were identiWed as two major CBPs and as bacterial cell-associated proteins.
Applied Microbiology and Biotechnology, 2010
A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A a... more 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 Histagged 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 µmol min −1 mg −1 on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH6.0, 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.
Animal Feed Science and Technology, 2013
Supplementation of diets for ruminants with methionine analogues such as 2-hydroxy-4-(methylthio)... more Supplementation of diets for ruminants with methionine analogues such as 2-hydroxy-4-(methylthio) butanoic acid (HMB) and its isopropyl ester (HMBi) positively affect milk composition and yield, potentially partly through ruminal actions. Our objective was to investigate effects of HMB and HMBi on the rumen microbial ecosystem, with special emphasis on fibrolytic activities and fibrolytic microorganisms. Six ruminally cannulated Holstein dry cows were randomly assigned to three treatments in a 3 × 3 Latin square design with two cows per cell. Cows were fed twice a day at 7.5 kg/d dry matter (DM) intake with a control diet based on hay and wheat (50/50 on a DM basis) supplemented or not with HMB (Rhodimet ® AT88, Adisseo at 1.25 g/kg DM intake) or HMBi (MetaSmart TM , Adisseo, at 2.50 g/kg DM intake). Substrate degradability (i.e., corn grain, corn silage) was evaluated by in sacco incubation and effective degradability measurements of DM, crude protein, starch and neutral detergent fibre. Fermentation products (i.e., volatile fatty acids (VFAs), ammonia, lactate) and fibrolytic enzyme activities (i.e., carboxymethylcellulase (CMCase), xylanase) were analyzed in rumen content samples before (−1 h) and after (+3 h) feeding. Protozoal populations were counted by microscopy. Abundances in total and fibrolytic bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in total rumen contents or adhering to in sacco residues were estimated using quantitative polymerase-chain reaction (qPCR). HMB and HMBi supplementation increased VFA concentrations in the rumen as well as the ruminal abundance of F. succinogenes (P=0.03) and R. flavefaciens (P=0.05), although these increases had no effect on the CMCase and xylanase activities of the rumen contents, nor on in sacco bacterial colonization and degradation of the two corn substrates. Under our experimental conditions, Met analogues enhanced growth of two cellulolytic bacterial representatives, but did not improve rumen fibrolytic activity.
PLoS ONE, 2014
Background: A complex community of microorganisms is responsible for efficient plant cell wall di... more 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.
Microbiology (Reading, England), 2002
This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previou... more 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 microscop...
Reproduction, nutrition, development, 1994
Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Ap... more Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Apart from ester bonds involving phenolic acids, which seem to play a major role in grasses, little is known about the other types of linkages that must exist but have proved difficult to demonstrate. The chemical nature of possible LCC linkages is presented and the various mechanisms through which LCCs in the cell-wall architecture may interfere with carbohydrate utilisation by rumen microorganisms are discussed.
Microbiology, 2005
Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesi... more 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.
Microbial Ecology, 2012
Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetoge... more 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 preinoculation 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 (1,000fold increase in methanogens) did not substantially affect acetogen diversity.
Journal of the Science of Food and Agriculture, 1994
ABSTRACT The lower halves of apical internodes of wheat harvested at the flowering stage were lab... more ABSTRACT The lower halves of apical internodes of wheat harvested at the flowering stage were labelled with [U-14C] phenylalanine (phe) or with [O14CH3] sinapic acid (sin). Cell wall residues (CWR) and saponified residues (SR) were incubated in a fermenter simulating the rumen for 7 days with rumen fluid or without microorganisms (controls). PheCWR was labelled in all lignin units (measured as aldehydes from nitrobenzene oxidation), in phenolic acids and slightly in proteins. Labelling of pheSR was more lignin-specific. SinCWR and sinSR were specifically labelled in syringyl units of lignin. The fermentation of CWR resulted in phenylpropane-derived unit losses in the following decreasing order: ferulic acid>p-coumaric acid>syringaldehyde>vanillin>p-hydroxybenzaldehyde. If allowance is made for slight losses in controls, 61, 52, 61 and 63% of the phenylpropanes of pheCWR, sinCWR, pheSR and sinSR, respectively, were transformed into an acid-precipitable fraction, an acid-soluble fraction and 14CO2. The comparison of pheCWR and sinCWR degradation showed that syringyl units were solubilised into acid-precipitable molecules to a greater extent than the other lignin units; demethylation of the syringyl units of lignins was also evident from the different productions of 14CO2. Alkali-resistant lignins of SR were mainly transformed into acid-precipitable molecules and were weakly degraded. Lignin solubilisation and degradation seem to be governed by different mechanisms which depend on both cell wall structure and rumen microflora.
Journal of the Science of Food and Agriculture, 1994
U-14C] phenylalanine (phe*) and [014CH3] sinapic acid (sin*) were infused into the cut ends of no... more U-14C] phenylalanine (phe*) and [014CH3] sinapic acid (sin*) were infused into the cut ends of normal and bm3 maizes (anthesis stage) under or above the last node or at mid-internode, with or without the leaf, in light or in darkness. Radioactivity was measured in the organs, and in phenolic constituents of the cell wall and saponified residues of the bases and tops of the apical internode. In both maize genotype labelled under the node the radioactivity was distributed more evenly in the organs with sin* than with phe*. Infusion above the node and at mid-internode greatly increased radioactivity in the bases and tops, respectively. Removal of the leaf only slightly increased the radioactivity, mainly in the bases, and no clear-cut effect of darkness was observed. Phe* labelled the phenolic acids and the three lignin units, but the syringyl units of bm3 maize were only slightly labelled. Sin* specifically labelled the syringyl units, which represented the least condensed fraction of lignins. Both the native and labelled lignins were highly alkali soluble. There were differences in lignin biogenesis between the bases and tops, and between normal and bm3 maizes. The newly formed lignins were slightly different from the native lignins but had similar types of heterogeneity, with variations in the internode and between genotypes similar to those in native lignins. Provided due allowance is made for the distinguishing characteristics of newly formed lignins, the ['4C-lignin] cell walls, which are strongly labelled on complementary structures, seem suitable model substrates for fermentation studies.
Journal of Microbiological Methods, 2009
An improved RNA isolation method based on the acid guanidinium-phenol-chloroform (AGPC) procedure... more 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.
Journal of Applied Microbiology, 2007
Aim: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacter... more Aim: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. Methods and Results: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease ()1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two-to fourfold) of the Ruminococci. Conclusion: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. Significance and Impact of the Study: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.
Journal of Agricultural and Food Chemistry, 1993
ABSTRACT [(U-C)-C-14]Phenylalanine (Phe*) or [(O-CH3)-C-14]sinapic acid (Sin*) was infused in whe... more ABSTRACT [(U-C)-C-14]Phenylalanine (Phe*) or [(O-CH3)-C-14]sinapic acid (Sin*) was infused in wheat stems under the node of the apical fraction, with and without leaf, or at midinternode. (C-14-Lignin)Pronase-treated cell walls of the upper and lower halves of the internode were prepared and analyzed. With Phe*, the labeling had a greater variability than with Sin* and a different distribution in the organs; cell wall residues were labeled on lignin units with a high specific radioactivity, on phenolic acids, and on residual proteins. Labeling of saponified residues was more lignin-specific. With Sin*, syringyl units were specifically labeled. The infusion at midinternode considerably increased labeling yields, while the elimination of the leaf had very slight effect. Radiochemical and chemical results obtained on internode segments are discussed in terms of lignin labeling validity and lignin biogenesis. These substrates, strongly labeled on complementary structures, are potentially useful tools to study the fate of lignins during ruminal fermentation.
FEMS Microbiology Letters, 2009
Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of he... more Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of herbivores. It was described to synthesize two major glycosidehydrolases (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.
Archives of Microbiology, 2009
The objective of this study was to identify and characterize other proteins than Wmbrial proteins... more The objective of this study was to identify and characterize other proteins than Wmbrial 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 identiWed by a proteomic approach. In strain 20, Cel9B and Cel48A were identiWed as two major CBPs and as bacterial cell-associated proteins.
Applied Microbiology and Biotechnology, 2010
A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A a... more 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 Histagged 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 µmol min −1 mg −1 on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH6.0, 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.
Animal Feed Science and Technology, 2013
Supplementation of diets for ruminants with methionine analogues such as 2-hydroxy-4-(methylthio)... more Supplementation of diets for ruminants with methionine analogues such as 2-hydroxy-4-(methylthio) butanoic acid (HMB) and its isopropyl ester (HMBi) positively affect milk composition and yield, potentially partly through ruminal actions. Our objective was to investigate effects of HMB and HMBi on the rumen microbial ecosystem, with special emphasis on fibrolytic activities and fibrolytic microorganisms. Six ruminally cannulated Holstein dry cows were randomly assigned to three treatments in a 3 × 3 Latin square design with two cows per cell. Cows were fed twice a day at 7.5 kg/d dry matter (DM) intake with a control diet based on hay and wheat (50/50 on a DM basis) supplemented or not with HMB (Rhodimet ® AT88, Adisseo at 1.25 g/kg DM intake) or HMBi (MetaSmart TM , Adisseo, at 2.50 g/kg DM intake). Substrate degradability (i.e., corn grain, corn silage) was evaluated by in sacco incubation and effective degradability measurements of DM, crude protein, starch and neutral detergent fibre. Fermentation products (i.e., volatile fatty acids (VFAs), ammonia, lactate) and fibrolytic enzyme activities (i.e., carboxymethylcellulase (CMCase), xylanase) were analyzed in rumen content samples before (−1 h) and after (+3 h) feeding. Protozoal populations were counted by microscopy. Abundances in total and fibrolytic bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in total rumen contents or adhering to in sacco residues were estimated using quantitative polymerase-chain reaction (qPCR). HMB and HMBi supplementation increased VFA concentrations in the rumen as well as the ruminal abundance of F. succinogenes (P=0.03) and R. flavefaciens (P=0.05), although these increases had no effect on the CMCase and xylanase activities of the rumen contents, nor on in sacco bacterial colonization and degradation of the two corn substrates. Under our experimental conditions, Met analogues enhanced growth of two cellulolytic bacterial representatives, but did not improve rumen fibrolytic activity.
PLoS ONE, 2014
Background: A complex community of microorganisms is responsible for efficient plant cell wall di... more 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.