Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.Methanogens are classified under the kingdom Archaea and are divided into five major orders, Methanobacteriales, Methanosarcinales, Methanococcales, Methanomicrobiales, and Methanopyrales. Among these, Methanobacteriales dominates the rumen environment (36,41). Methanogens live under strictly anaerobic conditions and are the only organisms that derive all their metabolic energy from the reduction of CO 2 by hydrogen to produce methane. Based on phylogenomic analysis, 31 proteins are uniquely present in all methanogens, strongly indicating that all methanogenic archaea form a monophyletic group exclusive of other archaea and that this lineage likely evolved from Archaeoglobus (15). Methanogenesis serves as the terminal electron sink process during organic matter decomposition in the rumen (16, 17) and has long been considered a metabolic waste process accounting for 5 to 15% of metabolizable energy loss in ruminants (17). Anthropogenic methane production is of environmental concern, and efforts to reduce it have focused on the reduction of methane eructation from ruminants, with little succ...

This study evaluated the effects of selected essential oils on archaeal communities using the ovine rumen model. Forty weaned Canadian Arcott ewes, fed with barley-based diet, were allotted to one of three essential oil supplementation treatments or a control (10 ewes per treatment) for 13 weeks. The treatments were cinnamaldehyde, garlic oil, juniper berry oil, and a control with no additive. Rumen content was sampled after slaughter and grouped by treatment by combining subsamples from each animal. DNA was extracted from the pooled samples and analyzed for methanogenic archaea using quantitative polymerase chain reaction, denaturing gradient gel electrophoresis, cloning, and sequencing. Our results suggest that the total copy number of archaeal 16S rRNA was not significantly affected by the treatments. The phylogenetic analysis indicated a trend toward an increased diversity of methanogenic archaea related to Methanosphaera stadtmanae, Methanobrevibacter smithii, and some uncultured groups with cinnamaldehyde, garlic, and juniper berry oil supplementation. The trends in the diversity of methanogenic archaea observed with the essential oil supplementation may have resulted from changes in associated protozoal species. Supplementation of ruminant diets with essential oils may alter the diversity of rumen methanogens without affecting the methanogenic capacity of the rumen.

Enzymes that hydrolyze complex polysaccharides into simple sugars are modular in architecture and consist of single or multiple catalytic domains fused to targeting modules called carbohydrate-binding modules (CBMs). CBMs bind to their ligands with high affinity and increase the efficiency of the catalytic components by targeting the enzymes to its substrate. Here we utilized a multidisciplinary approach to characterize each of the two family 16 carbohydrate-binding domain components of the highly active mannanase from the thermophile Thermoanaerobacterium polysaccharolyticum. These represent the first crystal structures of family 16 CBMs. Calorimetric analysis showed that although these CBMs demonstrate high specificity toward ␤-1,4-linked sugars, they can engage both cello-and mannopolysaccharides. To elucidate the molecular basis for this specificity and selectivity, we have determined high resolution crystal structures of each of the two CBMs, as well as of binary complexes of CBM16-1 bound to either mannopentaose or cellopentaose. These results provide detailed molecular insights into ligand recognition and yield a framework for rational engineering experiments designed to expand the natural repertoire of these targeting modules.The turnover of photosynthetically fixed carbon through the action of microbial glycoside hydrolases has been estimated to be of the order of 10 11 tons annually (1). Consequently, glycoside hydrolases play a key role in the global carbon cycle, and their properties, if well harnessed, will enhance the release of substrates (monomeric sugars) critical to the use of cellulosic materials in the biofuel industry. For most glycoside hydrolases, such as bacterial cellulases and xylanases, the polypeptides are organized in a modular arrangement that usually consists of a catalytic domain and an associated carbohydrate-binding module (CBM).2 These modules may be joined through linkers that are rich in proline, serine, and threonine (2). The evolutionary rationale that led to these complex molecular architectures is currently unclear (3).It has been suggested that CBMs attain multivalency through multiplicity (4). Therefore, it could be hypothesized that multiple CBMs act synergistically to bind to their target ligand, leading to an increased accessibility of the catalytic domain to the target polysaccharide. This view is supported by studies on a recombinant double CBM, constructed by fusing family 1 CBMs of two Trichoderma reesei cellobiohydrolases via a linker peptide (5). A 5-10-fold increase in the affinity for crystalline cellulose was observed for the double CBM compared with the individual modules, and similar results were observed for two CBMs of Cellulomonas fimi xylanase 11A (3). Thus, in some arrangements, the affinity for substrates increases with multiplicity of CBMs, and this appears to occur frequently in hyperthermophiles and thermophiles (6). Contrary to these finding, a report on the product of a manA gene of Caldicellulosiruptor strain RT8B.4 with two N-terminal CBMs sho...

The hydrolysis of polysaccharides containing mannan requires endo-1,4-␤-mannanase and 1,4-␤-mannosidase activities. In the current report, the biochemical properties of two endo-␤-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-␤-mannanase and endo-1,4-␤-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-␤-mannanase activity and little endo-1,4-␤-glucanase activity; however, this enzyme also exhibited 1,4-␤-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of ␤-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.Bioenergy feedstocks consist primarily of the plant cell wall components cellulose and hemicellulose, and hydrolysis of these polysaccharides to their monomeric sugars involves a set of enzymes acting synergistically to cleave the different chemical linkages (9). Although cellulose consists of glucose units linked together in ␤-1,4-glycosidic linkages, the hemicellulosic component of feedstock may vary in chemical composition. Some feedstock hemicellulose molecules are mainly composed of ␤-1,4-linked xylose backbones with arabinose side chains, while others are comprised of a larger variety of sugars, including galactose and mannose existing as various forms of mannan (26). Mannans constitute a less significant portion of hemicellulose in bioenergy feedstocks, such as switchgrass. However, the presence of mannan may result in linkages that restrict release of fermentable sugars.A variety of mannans are found in nature. These include linear mannan, glucomannan, galactomannan, and glucogalactomannan. In each case, the polysaccharide contains a ␤-1,4-linked backbone of mannose residues that may be substituted up to 33% (or up to 50% in hardwoods) with glucose residues (32). In galact...

Sediments contain an abundance of microorganisms. However, the diversity and distribution of microorganisms associated with sediments are poorly understood, particularly in lacustrine environments. We used banding patterns from denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequences to assess the structure of bacterial communities in the Holocene sediments of a meromictic lake in Minnesota. Cluster analysis of the DGGE banding patterns indicates that the early- and middle-Holocene samples group separately from the late-Holocene samples. About 79% of the recovered bacterial sequences cluster with the alpha-, beta-, delta-, epsilon-, and gamma- Proteobacteriaceae and Firmicutes. The remaining approximately 21% lack cultured representatives. The taxonomic lineages of bacteria differ statistically among the early-, middle-, and late-Holocene samples, although the difference is smallest between early- and middle-Holocene samples. Early- and middle-Holocene samples are dominated by epsilon-Proteobacteriaceae, and late-Holocene samples are dominated by sequences from uncultured subphyla. We only recovered delta-Proteobacteriaceae in late-Holocene sediments and alpha- and gamma- Proteobacteriaceae in late- and middle-Holocene sediments. Diversity estimates derived from early-, middle-, and late-Holocene clone libraries indicate that the youngest (late-Holocene) samples had significantly greater bacterial diversity than the oldest (early-Holocene) samples, and the middle-Holocene samples contained intermediate levels of diversity. The observed patterns of diversity may be caused by increased bacterial niche-partitioning in younger sediments that contain a greater abundance of labile organic matter than older sediments.