Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing

Abstract

It is well recognized that a dynamic biofilm develops upon plant biomass in the herbivore gastrointestinal tract, but this component of the microbiome has not previously been specifically sampled, or directly compared with the biodiversity present in the planktonic fraction of digesta. In this study, the digesta collected from four sheep fed two different diets was separated into three fractions: the planktonic phase, and the microbial populations either weakly or tightly adherent to plant biomass. The community DNA prepared from each fraction was then subjected to both ribosomal intergenic spacer analysis (RISA) and denaturing gradient gel electrophoresis (DGGE). Both types of analysis showed that dietary factors influence community structure, and that the adherent fractions produced more complex profiles. The RIS-clone libraries prepared from the planktonic and adherent populations were then subjected to restriction fragment length polymorphism (RFLP) and DNA sequence analyses, which resulted in a far greater degree of discrimination among the fractions. Although many of the sequenced clones from the adherent populations were assigned to various clusters within the low G+C Gram-positive bacteria, the clone libraries from animals consuming an all-grass diet were largely comprised of novel lineages of Clostridium, while in animals consuming the starch-containing diet, Selenomonas and Ruminococcus spp. were the dominant low G+C Gram-positive bacteria. Additionally, the libraries from hay-fed animals also contained clones most similar to asaccharolytic Clostridia, and other Gram-positive bacteria that specialize in the transformation of plant phenolic compounds and the formation of cinnamic, phenylacetic and phenylpropionic acids. These results reveal, for the first time, the phylogeny of adherent subpopulations that specialize in the transformation of plant lignins and other secondary compounds, which potentiate polysaccharide hydrolysis by other members of the biofilm.

Microbial ecosystem and methanogenesis in ruminants

“…The majority of these microbes have not yet been cultivated; however, using molecular biology methods it has been estimated that, for the bacteria alone there are at least between 300 and 400 phylotypes (Edwards et al, 2004;Larue et al, 2005;Yu et al, 2006). In addition, protozoa, fungi, methanogenic archaea and bacteriophages also contribute to the diversity and functioning of the rumen microbial ecosystem.…”

Use of ‘natural’ products as alternatives to antibiotic feed additives in ruminant production

“…The rumen ecosystem normally harbours about 10 10 microorganisms, and is composed of several hundred species (Edwards et al, 2004;Larue et al, 2005). These species possess a vast array of enzymes acting concertedly to degrade the plant substrates present in feeds.…”

Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases

“…Ϸ3 l of whole rumen digesta (fiber-adherent (FA) and liquid associated microbes) was collected from the dorsal third rumen 6 weeks after the beginning of the study, 1 hour after the morning feeding. Samples were then partitioned into FA fractions and liquid fractions before DNA extraction (14,51). The pooled sample (PL) (derived from a mixture of liquid fractions from each of the 3 steers) was thoroughly mixed before the DNA extraction (14,52).…”

“…Samples were then partitioned into FA fractions and liquid fractions before DNA extraction (14,51). The pooled sample (PL) (derived from a mixture of liquid fractions from each of the 3 steers) was thoroughly mixed before the DNA extraction (14,52). Samples were stored at Ϫ80°C until DNA extraction.…”

“…Bacteria predominate the rumen, with a variety of anaerobic protozoa and fungi (1), and the associated occurrence of bacteriophage is well documented (2). The use of small subunit (SSU) rRNA sequence analysis has allowed for a more complete description of the rumen microbiome and these inventories have demonstrated that a large microbial component remains uncultured (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and that a high proportion of the fibrolytic population has not been thoroughly described (7,8,13,14). The rumen habitat contains a consortium of microbes that harbor the complex lignocellulosic degradation system for the microbial attachment and digestion of plant biomass.…”