Mapping Pectic-Polysaccharide Epitopes in Cell Walls of Forage Chicory (Cichorium intybus) Leaves.

The cell walls of forage chicory (Cichorium intybus) leaves are known to contain high proportions of pectic polysaccharides. However, little is known about the distribution of pectic polysaacharides among walls of different cell types/tissues and within walls. In this study, immunolabelling with four monoclonal antibodies was used to map the distribution of pectic polysaccharides in the cell walls of the laminae and midribs of these leaves. The antibodies JIM5 and JIM7 are specific for partially methyl-esterified homogalacturonans; LM5 and LM6 are specific for (1→4)-ß-galactan and (1→5)-α-arabinan side chains, respectively, of rhamnogalacturonan I. All four antibodies labelled the walls of the epidermal cells with different intensities. JIM5 and JIM7, but not LM5 or LM6, labelled the middle lamella, tricellular junctions, and the corners of intercellular spaces of ground, xylem and phloem parenchyma. LM5, but not LM6, strongly labelled the walls of the few sclerenchyma fibres in the phloem of the midrib and lamina vascular bundles. The LM5 epitope was absent from some phloem parenchyma cells. LM6, but not LM5, strongly labelled the walls of the stomatal guard cells. The differential distribution of pectic epitopes among walls of different cell types and within walls may reflect the deposition and modification of these polysaccharides which are involved in cell wall properties and cell development.

Stratification of Archaea in the deep sediments of a freshwater meromictic lake: vertical shift from methanogenic to uncultured archaeal lineages.

As for lineages of known methanogens, several lineages of uncultured archaea were recurrently retrieved in freshwater sediments. However, knowledge is missing about how these lineages might be affected and structured according to depth. In the present study, the vertical changes of archaeal communities were characterized in the deep sediment of the freshwater meromictic Lake Pavin. For that purpose, an integrated molecular approach was performed to gain information on the structure, composition, abundance and vertical stratification of archaeal communities thriving in anoxic freshwater sediments along a gradient of sediments encompassing 130 years of sedimentation. Huge changes occurred in the structure and composition of archaeal assemblages along the sediment core. Methanogenic taxa (i.e. Methanosaeta and Methanomicrobiales) were progressively replaced by uncultured archaeal lineages (i.e. Marine Benthic Group-D (MBG-D) and Miscellaneous Crenarchaeal Group (MCG)) which are suspected to be involved in the methane cycle.

Methanobacterium lacus sp. nov., isolated from the profundal sediment of a freshwater meromictic lake.

An autotrophic, hydrogenotrophic methanogen, designated strain 17A1(T), was isolated from the profundal sediment of the meromictic Lake Pavin, France. The cells of the novel strain, which were non-motile, Gram-staining-negative rods that measured 2-15 µm in length and 0.2-0.4 µm in width, grew as filaments. Strain 17A1(T) grew in a mineral medium and its growth was stimulated by the addition of yeast extract, vitamins, acetate or rumen fluid. Penicillin, vancomycin and kanamycin reduced growth but did not completely inhibit it. Growth occurred at 14-41 °C (optimum 30 °C), at pH 5.0-8.5 (optimum pH 6.5) and with 0-0.4 M NaCl (optimum 0.1 M). The novel strain utilized H(2)/CO(2) and methanol/H(2) as substrates but not formate, acetate, methylamine/H(2), isobutanol or 2-propanol. Its genomic DNA G+C content was 37.0 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, strain 17A1(T) appeared to be a member of the genus Methanobacterium, with Methanobacterium beijingense 8-2(T) (96.3% sequence similarity) identified as the most closely related established species. Based on phenotypic and phylogenetic data, strain 17A1(T) represents a novel species of methanogen within the genus Methanobacterium, for which the name Methanobacterium lacus sp. nov. is proposed. The type strain is 17A1(T) (=DSM 24406(T)=JCM 17760(T)).

Establishment and development of ruminal hydrogenotrophs in methanogen-free lambs.

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10(7) to 10(8) cells g(-1)) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H(2) utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H(2) utilization was similar to that in conventional lambs. H(2) utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H(2) utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H(2) in the rumen.

Analysis of methanogen diversity in the rumen using temporal temperature gradient gel electrophoresis: identification of uncultured methanogens.

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 10(5)g(-1). Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (10(6)-10(-10) g(-1)). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.

Phylogenetic diversity of archaea and bacteria in the anoxic zone of a meromictic lake (Lake Pavin, France).

The compositions of archaeal and bacterial populations at different depths (60 m [mixolimnion-chemocline interface], 70 m [chemocline-subchemocline interface], 90 m, and 92 m [the water-sediment interface]) in the anoxic zone of the water column in Lake Pavin, a freshwater permanently stratified mountain lake in France, were determined. Phylogenetic trees were constructed from sequences to assess archaeal and bacterial diversity at the four sites.

16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs.

The population densities and identities of methanogens colonising new-born lambs in a grazing flock were determined from rumen samples collected at regular intervals after birth. Methanogen colonisation was found at the first sampling (1-3 days after birth) and population densities reached around 10(4) methanogens per gram at 1 week of age. Population densities increased in an exponential manner to a maximum of 10(8)-10(9) per gram at 3 weeks of age. To identify methanogens, PCR primers specific for each of the Archaea; a grouping of the orders Methanomicrobiales, Methanosarcinales and Methanococcales; the order Methanobacteriales; the order Methanococcales; the order Methanosarcinales; the genus Methanobacterium; and the genus Methanobrevibacter were designed. Primer-pair specificities were confirmed in tests with target and non-target micro-organisms. PCR analysis of DNA extracts revealed that all the detectable ruminal methanogens belonged to the order Methanobacteriales, with no methanogens belonging to the Methanomicrobiales, the Methanosarcinales, or the Methanococcales being detected. In 3 lambs, the initial colonising methanogens were Methanobrevibacter spp. and in 2 lambs were a mixture of Methanobrevibacter and Methanobacterium spp. In the latter case, the initial colonising Methanobacterium spp. subsequently disappeared and were not detectable 12-19 days after birth. Seven weeks after birth, lambs contained only Methanobrevibacter spp. This study, the first to provide information on the identities of methanogens colonising pre-ruminants, suggests that the predominant methanogens found in the mature rumen establish very soon after birth and well before a functioning rumen develops.

Degradation of fresh ryegrass by methanogenic co-cultures of ruminal fungi grown in the presence or absence of Fibrobacter succinogenes.

The ability of five ruminal fungi in syntrophic co-culture with the methanogen Methanobrevibacter smithii to degrade perennial ryegrass ( Lolium perenne) stem fragments and leaf blades was studied to determine the susceptibilities of non-autoclaved fresh tissues to fungal degradation. Autoclaving did not significantly increase fungal degradation of stem fragments but strongly increased degradation of leaf blades by a species of Caecomyces. In methanogenic co-cultures, non-autoclaved stem fragments were degraded more extensively by Neocallimastix frontalis and Piromyces isolates than by Caecomyces isolates. The N. frontalis and Piromyces isolates showed the greatest rates of stem degradation. When interactions between Fibrobacter succinogenes and methanogenic co-cultures of fungi growing on ryegrass stem were investigated, N. frontalis inhibited F. succinogenes. This has not been observed previously. In contrast, a Caecomyces species interacted positively with F. succinogenes to increase stem degradation, suggesting that F. succinogenes and Caecomyces spp. may have complementary fibrolytic activities. All five fungi tested failed to grow on fresh non-autoclaved leaf blades. In a repeat experiment with leaves from a separate harvest, leaf blades were degraded by N. frontalis but not by a Caecomyces species. We suggest that ryegrass leaf blades may contain natural anti-fungal compounds. Our results confirm the superiority of fungi in the degradation of intact stem and indicate that in vitro studies with non-autoclaved forage tissues may yield new information on forage factors affecting rumen microbes.