The plant material rich in complex polysaccharides is an important component of the diet of herbivorous and omnivorous mammals. Mammals do not possess their own digestive enzymes for the break down of plant cell wall polysaccharides. Polymer hydrolysis is provided by microbial communities in digestive tract. The conditions in gastrointestinal tract favor the development of dense microbial communities with high abundance completely dominated by strict and obligatory anaerobic microorganisms. The main products of anaerobic fermentation are short chain fatty acids, which contribute up to 70% of the energy required for growth and development in the case of large ruminants. Microbial degradation of the gastrointestinal tract of herbivorous mammals, particularly in the rumen, is much faster than the rest, indicating that the extremely high, rapid and efficient degradation capacity of lignocellulosic feedstock of the gut microbiota. As a result, there is outstanding interest in the industrial exploitation of enzymes involved in the conversion of plant polysaccharides.

Wild alpine ruminants forage on extremely complex plant polymers most of the year: poor quality vascular plants, mosses, lichens and fungi. This suggests that they are tolerant to fiber and tannin-rich plants and capable of digesting the recalcitrant plant polymers from their winter forage. Alpine wild ruminants therefore represent a very special microbial ecosystem with maximum degradation efficiency of recalcitrant plant material, but their rumen bacteria and dietary preferences have not been comprehensively studied yet.

Within the framework of the international project "Global Census of Yellow Microbial Diversity Project" AgResearch, New Zealand, samples of the contents of ruminants from different locations around the world, of distinct species, breeding, sex and feed where collected, and samples of wild ruminants, camels and macropods represented a special case of microbial ecosystem with maximum degradation efficiency of plant material. Bioinformatic analyses of deep sequencing data from phylogenetic markers targeting bacteria, archaea, protozoa, fungi, and of selected functional genes, were used to determine phylogenetic composition of microbial communities, define the core communities, identify diversity of microbial groups and their dependence on the type of occurrence, nutrition, breeding, feed conversion efficiency and other measured parameters.

We intend to perform deep sequencing of prokaryotic transcriptome to determine the extent and type of microbial transcripts with denovo assembly. This will serve as a basis for the identification of transcripts of functional genes in the mammalian gastrointestinal tract involved in the degradation of the plant cell wall polysaccharides, their nature and dynamics of the expression of key genes for the degradation of complex polysaccharides. Microbial community samples collected from the digestive tracts of wild ruminants with high efficiency degradation of plant polymers will serve as the baseline. The analysis of datasets produced in this study, their time­line and co­occurrence assembly and correlation to metabolites is going to provide an approach to identification of yet undescribed or putative enzyme coding sequences amenable for subcloning. Direct cell­independent expression system is going to enable us the production of sufficient amounts of targeted enzymes that are going to be available for tests of specific activities on plant substrates and for expression in live host organisms (E.coli, Prevotella ruminicola, Bacteroides sp).

Scientific background 

Degradation of plant food in the gastrointestinal tract takes place gradually from the hydrolysis of complex polysaccharides up to the final products of the fermentation. Different metabolic groups of microorganisms are involved in each step. Polisacharidases (hydrolases, cellulases, ...) are enzymes that break the coarse and partially decomposed plant residues. Mammals do not possess these enzymes and therefore their recovery of energy from plant foodstuff depends solely on the performance of their gut microbiota. It has been shown that increasing the amount of dietary fiber increases the activity of the gut microbiota and that the composition of the feed is an important factor shaping the structure of microbial communities. Previous studies have been conducted only at the level of individual cell cultures of isolated cellulolytic bacteria from herbivores. The surprisingly large number of genes encoding polisaharidases (including over 100 species in the genera Fibrobacter and Prevotella ) and the few detailed functional and molecular studies of degradation and exploitation of polysaccharides conducted for a few types of anaerobic bacteria support the notion of a unprecedent metabolic potential of microbial communities residing in intestinal tract of mammals. Historical development of molecular techniques has facilitated the identification and tracking of microorganisms including the unculturable majority in all complex ecosystems, as well as gastrointestinal tract. Recent developments in the preparation of complex microbial samples for total RNA preservation, shot gun sequencing and direct denovo assembly (databases independent reconstruction) of the entire transcriptome of microbial communities of complex systems have shown that complete operons are being transcribed enabling direct integration of the different identities of functional genes, their cooccurrence and relative representation of expression and to a great extent also the relative by t identification of organisms from which they arise . 

Problem identification: 

Prokaryotic and eukaryotic organisms in gastrointestinal tracts harbor a diverse plethora of polysaccharidase enzymes, however, the information about their expression, frequency, relative distribution, dynamics and harmonization of the whole range of these enzymes, it is lacking. Five species of wild ruminants (roebuck, deer, mouflon, capricorn, chamois) represent a natural gradient in capacity for degradation of recalcitrant plant polymers and their mixtures in the fuminant feed. Recalcitrance is related to altitude of plant growth and seasonality, in which the autumn ­winter substrates represent the least degradable. A collection of plant substrates in natural gradient and autumn season thus represent mapping of increasing complexity of plant composition with increasing altitude. Based on previous research from other microbial systems it is anticipated different conclusions from those currently accepted are going to be established regarding the transcription of transporters of organic acids, degradation products, nitrogen, different oxidoreductase and hydrolase complexes which are involved in the degradation processes of organic polymers in the mammalian gastrointestinal tracts. The results of this study will represent a key step in detecting new coding regions, establishing enzyme identity, dynamics, coexpression, depending on the environmental parameters, biochemical properties of substrates and their cumulative degradation efficiency of various plant substrates, all aiming at their industrial use for improvement of lignocellulose depolymeriazion proces either as stand­alone or in combination with existing instrial enzyme preparations. 

Objectives: 

The objective of the study is to identify and record the metatranscriptome of the realistic microbial communities in the gastrointestinal tract of wild ruminants that are characterized by high efficiency in using a variety of plant polymers, to assess their intertemporal connectivity and dependence on the type of plant polymers and their degradation products (metabonome). For this purpose analyses of the initial and all subsequent states of samples taken from five species of wild ruminants in the Slovenian mountains are going to be conducted in a controlled laboratory experiment under anaerobic conditions.