Biomedical subjects

Paul J Weimer

Publications and source records attributed to Paul J Weimer.

8 recordsLinked to original sources

PubMed2006

Studies of the extracellular glycocalyx of the anaerobic cellulolytic bacterium Ruminococcus albus 7.

Anaerobic cellulolytic bacteria are thought to adhere to cellulose via several mechanisms, including production of a glycocalyx containing extracellular polymeric substances (EPS). As the compositions and structures of these glycocalyces have not been elucidated, variable-pressure scanning electron microscopy (VP-SEM) and chemical analysis were used to characterize the glycocalyx of the ruminal bacterium Ruminococcus albus strain 7. VP-SEM revealed that growth of this strain was accompanied by the formation of thin cellular extensions that allowed the bacterium to adhere to cellulose, followed by formation of a ramifying network that interconnected individual cells to one another and to the unraveling cellulose microfibrils. Extraction of 48-h-old whole-culture pellets (bacterial cells plus glycocalyx [G] plus residual cellulose [C]) with 0.1 N NaOH released carbohydrate and protein in a ratio of 1:5. Boiling of the cellulose fermentation residue in a neutral detergent solution removed almost all of the adherent cells and protein while retaining a residual network of adhering noncellular material. Trifluoroacetic acid hydrolysis of this residue (G plus C) released primarily glucose, along with substantial amounts of xylose and mannose, but only traces of galactose, the most abundant sugar in most characterized bacterial exopolysaccharides. Linkage analysis and characterization by nuclear magnetic resonance suggested that most of the glucosyl units were not present as partially degraded cellulose. Calculations suggested that the energy demand for synthesis of the nonprotein fraction of EPS by this organism represents only a small fraction (<4%) of the anabolic ATP expenditure of the bacterium.

Anaerobiosis↗

PubMed2006

Interactions between Euphorbia esula toxins and bovine ruminal microbes.

Cattle generally avoid grazing leafy spurge (LS; Euphorbia esula), whereas sheep and goats will often eat it. Understanding metabolism of toxic phytochemicals in LS by bovine rumen microflora may help explain why cattle often develop aversions to LS after initially eating it. Toxicity of LS compounds after in vitro fermentation with normal vs. antibiotic-modified bovine rumen digesta was evaluated at different lengths of fermentation. Levels of toxic and aversion-inducing ingenols were determined for fermented and nonfermented mixtures of LS and bovine rumen digesta, and the toxicity of an aversion-inducing extract of LS to rumen microbial species that are common in cattle, sheep, and goats was evaluated. Fermentation of LS with bovine digesta increased the toxicity of extracted compounds. Introduction of neomycin (an antibiotic that preferentially inhibits gram-negative bacteria) into the LS and bovine rumen digesta mixtures did not appear to affect toxicities regardless of fermentation length. Levels of ingenol were observed in LS and bovine digesta mixtures (both fermented and nonfermented) that were consistent with levels of ingenols reported for LS. Finally, a toxic extract of LS had little or no negative effect on the growth of several common species of rumen bacteria. The results indicate that LS is not generally toxic to the ruminal bacteria, but that microbial activity in the rumen may be responsible for enhancing LS toxicity to cattle.

Animals↗

PubMed2005

Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover.

Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.

DNA, Bacterial↗

PubMed2005

Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture.

Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in C. thermocellum ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a approximately 10-fold range of dilution rates (0.013 to 0.16 h(-1)). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h(-1): cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.

Bacterial Proteins↗

PubMed2005

Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots.

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.

Crenarchaeota↗

PubMed2004

Albusin B, a bacteriocin from the ruminal bacterium Ruminococcus albus 7 that inhibits growth of Ruminococcus flavefaciens.

An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.

Amino Acid Sequence↗

PubMed2002

Microbial cellulose utilization: fundamentals and biotechnology.

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

Bacteria↗

PubMedDate not supplied

Lactic acid bacteria used in inoculants for silage as probiotics for ruminants.

Many studies have shown the beneficial effects on ruminant performance of feeding them with silages inoculated with lactic acid bacteria (LAB). These benefits might derive from probiotic effects. The purpose of the current study was to determine whether LAB included in inoculants for silage can survive in rumen fluid (RF), as the first step in studying their probiotic effects. Experiments were conducted in the United States and Israel with clarified (CRF) and strained RF (SRF) that were inoculated at 10(6)-10(8) microorganisms/mL with and without glucose at 5 g/L. RF with no inoculants served as control. Ten commercial inoculants were used. The RF was incubated at 39 degrees C and sampled in duplicates at 6, 12, 24, 48, 72, and 96 h for pH and LAB counts. The results indicate that with glucose the pH of the RF decreased during the incubation period. In the SRF, the pH of the inoculated samples was higher than that of the controls in most cases. This might be a clue to the mechanism by which LAB elicit the enhancement in animal performance. LAB counts revealed that the inoculants survived in the RF during the incubation period. The addition of glucose resulted in higher LAB counts.

Animals↗