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Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach.

AIMS: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18:2) via conjugated 18:2 metabolites (mainly cis-9,trans-11-18:2, conjugated linoleic acid) to vaccenic acid (trans-11-18:1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18:0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. METHODS AND RESULTS: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. CONCLUSIONS: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. SIGNIFICANCE AND IMPACT OF THE STUDY: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

Xyn11A, a multidomain multicatalytic enzyme from Pseudobutyrivibrio xylanivorans Mz5T.

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.

EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain.

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945-1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

[Clinical-endoscopic-histologic correlations in gastric pathology. II]. / Correlaciones clínico-endoscópicas-histológicas en patología gástrica II.

Some authors do not accept as valid the clinical diagnosis of gastritis, due to the latter being mainly an anatomical-pathological term. Others, however, argue that such diagnosis may be establish through a correct anamnesis. A cross-sectional study of a hundred and fifty patients undergoing fibrosgastroscopy with antral biopsy and anamnesis, as well as clinical exploration in accordance with specific protocols, has been conducted. We have concluded that there does not exist as symptomatology associated to a concrete pathology, despite the presence of certain symptoms as, for example, the pain, that may be related with certain pathologies (14/20 of those developing H.H.E.D., 22/34 of those developing UB and 7/10 of those developing neoplasia). We also inferred that many of the patients (46/50) with endoscopic and histological normal findings (20/23), present as many as or more symptoms than those with pathological findings. This symptomatology may be due to a somatic disorder that could be hiding the process.

[Clinical-endoscopic-histological correlations in gastric pathology. I]. / Correlaciones clínico-endoscópicas-histológicas en patología gástrica. I.

We have conducted a blind cross-selectional study with 150 consecutive patients undergoing fibrogastroscopy and biopsy of the antral mucosa. The endoscopic sensitivity for the diagnosis of the several types of gastritis, is low (63%), although this technique is highly sensitive for other types of pathology. Consequently, we conclude that, in all endoscopic explorations, at least one biopsy of the antral mucosa must be done, given the anatomopathological definition of gastritis. In addition, the clinical use of this term should be restricted to the histologically demonstrated cases.

[Clinical characteristics of gastric infection caused by Helicobacter pylori]. / Características clínicas de la afección gástrica por Helicobacter pylori.

In a series of 150 patients submitted for diagnostic gastroscopy, a prospective study of nine anamnesis variables and four exploratory ones was done. Endoscopic and histopathologic diagnoses, as well as the clinical data, were obtained by blinded observers. Helicobacter pylory (Hp) presence in antral mucosa was determined by culture. A clear relationship between Hp presence and peptic ulcer disease, bulboduodenitis and histologic gastritis was found, as expected, but no clinical variable correlated positively with it. We conclude, therefore, that Hp presence in antral mucosa cannot be predicted clinically, a fact probably related to the unspecificity of symptoms in gastroduodenal disease and not to a lack of pathogenicity of this organism.

[Histopathologic changes associated with the presence of Helicobacter pylori in antral mucosa]. / Alteraciones histopatológicas asociadas a la presencia de Helicobacter pylori en mucosa antral.

Helicobacter pylori is a gram negative bacteria which has recently been associated to tissular changes of the upper digestive tract, however, the causal role has not yet been determined. Of 150 patients studied, 63 had tissular changes associated to Helicobacter pylori (Hp), 8 had Hp without tissular related changes (of whom 3 suffered bulbar ulcus and 1 gastric ulcus); the rest of the patients had hiatus hernia associated to distal esophagitis or pyloric stenosis; and only one patient was found to have normal tissue. A clear associated to distal esophagitis or pyloric stenosis; and only one patient was found to have normal tissue. A clear association between Hp and chronic or atrophic gastritis was determined, but no association was found between Hp and gastric cancer.