Actinobacteria (Actinomycetes) are a significant and interesting group of gram-positive bacteria. They are regular, though infrequent, members of the microbial life in the rumen and represent up to 3 % of total rumen bacteria; there is considerable lack of information about ecology and biology of rumen actinobacteria. During the characterization of variability of rumen treponemas using non-cultivation approach, we also noted the variability of rumen actinobacteria. By using Treponema-specific primers a specific 16S rRNA gene library was prepared from cow and sheep rumen total DNA. About 10 % of recombinant clones contained actinobacteria-like sequences. Phylogenetic analyses of 11 clones obtained showed the high variability of actinobacteria in the ruminant digestive system. While some sequences are nearly identical to known sequences of actinobacteria, we detected completely new clusters of actinobacteria-like sequences, representing probably new, as yet undiscovered, group of rumen Actinobacteria. Further research will be necessary for understanding their nature and functions in the rumen.

Complete 16S rRNA sequences were determined of recently proposed new species of treponemes designated strain S and T. Sequence comparison indicated that both species belong to the Treponema saccharophilum cluster, having thus at least 5 cultivable representatives. Phylogenetic analysis of available GenBank 16S rRNA sequences revealed two phylogenetically distant treponema clusters (T. saccharophilum cluster and T. bryantii cluster). Surprisingly, while among cultivated treponemes dominate T. saccharophilum cluster members, detailed analysis showed that all treponema-like sequences obtained by culture independent 16S rRNA methods belong to the T. bryantii cluster, from which only two cultivable representatives have so far been known. Meta-analysis of available data revealed that treponemes are an infrequent and minor group of bacteria, representing less than 2.4% of total rumen bacteria.

Genome analysis of Treponema zioleckii proved that, in this bacterium, besides chromosomal DNA, a relatively small extrachromosomal DNA element is present. This element was shown to be a double-stranded circular plasmid DNA of approximately 7 kbp; it was designated as pKT. The plasmid was characterized by molecular and bioinformatic analysis. No pKT homologous DNA sequences were detected in other rumen Treponema strains. The overall G+C content of the pKT plasmid is approximately 56 %, which is higher than in other Treponema plasmids or genomes. The Rep module of the pKT plasmid consisting of the rep gene and the region of repeats was identified within a 1.6-kbp fragment. The putative rep gene encodes the replication protein belonging to the pfam04796 RepA_C family of proteins with the highest similarity (25 % within 249 amino acids) to the RepA protein from the green sulfur bacterium Prosthecochloris aestuarii.