Peter Pristaš

Assessment of ciliates in the sheep rumen by DGGE

Aims:  This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep.

Production of enterolysin A by rumen Enterococcus faecalis strain and occurrence of enlA homologues among ruminal Gram‐positive cocci

Aims:  Purification and partial characterization of an extracellular bacteriocin produced by the ruminal isolate Enterococcus faecalis II/1 and determine the frequency of occurrence of enterolysin A structural gene within the ruminal cocci.

A bacteriocin-mediated antagonism by Enterococcus faecium BC25 against ruminal Streptococcus bovis

A bacteriocin-like activity produced by Enterococcus faecium BC25, isolated from the the rumen of a cow, was partially purified and characterized. The active substance was prepared by ammonium sulfate and chloroform/methanol precipitation of culture supernatant. The bacteriocin was a protein of molecular mass 17.5 kDa. Activity was inactivated by trypsin and proteinase K. The bacteriocin BC25 inhibited growth of amylolytic ruminal strains of Streptococcus bovis, including S. bovis AO 24/85. Results showed that bacteriocine substance BC25 has a bacteriostatic effect when present in concentrations exceeding 125 AU/ml. Agar overlays and batch culture growth experiments proved that E. faecium BC25 was producing bacteriocin that inhibited the growth of S. bovis.

Effects of high- and low-fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees.

We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n=2) eating low‐fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high‐fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (P<0.001). On the other hand, HFD inoculum increased the production of total short‐chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet‐to‐be‐sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production. Am. J. Primatol. 71:548–557, 2009.

The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

BackgroundThe hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I.ResultsThe [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome.ConclusionThe hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.

Bacteriocins of ruminal bacteria.

Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strainE. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strainS. bovis II/I) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci.

Isolation and characterization of enterocin BC25 and occurrence of the ent A gene among ruminal Gram-positive cocci

Summary Enterocin BC25, a bacteriocin produced by Enterococcus faecium BC25 isolated from the rumen of cow was purified to homogeneity and sequenced. Twenty amino acids were identified in the peptide chain (TTHSGKYYGNGVYCT-KNKCT), identical to the N-terminal sequence of enterocin A. The DNA sequence of the enterocin BC25 structural gene and putative immunity protein exhibited high similarity to the ent A gene. The occurrence of a 726 bp amplicon containing the enterocin A structural gene was studied among gram-positive ruminal cocci by PCR. Our results showed wide occurrence of the ent A structural gene among ruminal enterococcal and streptococcal bacterial strains tested, and indicate variable ability to express bacteriocin production and resistance.

The symbiotic intestinal ciliates and the evolution of their hosts

The evolution of sophisticated differentiations of the gastro-intestinal tract enabled herbivorous mammals to digest dietary cellulose and hemicellulose with the aid of a complex anaerobic microbiota. Distinctive symbiotic ciliates, which are unique to this habitat, are the largest representatives of this microbial community. Analyses of a total of 484 different 18S rRNA genes show that extremely complex, but related ciliate communities can occur in the rumen of cattle, sheep, goats and red deer (301 sequences). The communities in the hindgut of equids (Equus caballus, Equus quagga), and elephants (Elephas maximus, Loxodonta africanus; 162 sequences), which are clearly distinct from the ruminant ciliate biota, exhibit a much higher diversity than anticipated on the basis of their morphology. All these ciliates from the gastro-intestinal tract constitute a monophyletic group, which consists of two major taxa, i.e. Vestibuliferida and Entodiniomorphida. The ciliates from the evolutionarily older hindgut fermenters exhibit a clustering that is specific for higher taxa of their hosts, as extant species of horse and zebra on the one hand, and Africa and Indian elephant on the other hand, share related ciliates. The evolutionary younger ruminants altogether share the various entodiniomorphs and the vestibuliferids from ruminants.

Treponema zioleckii sp. nov., a novel fructan-utilizing species of rumen treponemes

During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.

Morphological and phylogenetical studies on a new soil hypotrich ciliate: Kahliella matisi spec. nov. (Hypotrichia, Kahliellidae).

The morphology, ontogenesis, encystment, and 18S rRNA gene sequence of a new soil hypotrich ciliate, Kahliella matisi, were studied. Main characteristics of K. matisi are: (1) two short and six longitudinal cirral rows right of the adoral zone of membranelles and four longitudinal rows left of it; (2) three dorsal kineties, of which kinety 1 extends along the left cell margin, kinety 2 runs in a slightly sigmoidal line, and kinety 3 is distinctly shortened posteriorly. Ontogenesis is similar to that in congeners, especially in the development of the marginal rows and long dorsal kineties, the preservation of some old cirral rows after division, and the direction of the neokinetal wave. However, there are some peculiarities: (1) reorganization of the proximal parental adoral membranelles; (2) splitting of opisthes anlage II into the cirral streak II and III; and (3) formation of the parental cirral row R3 from anlagen IV and V. During encystment, the body diminishes and becomes globular, the nuclear apparatus is reorganized, and the ciliature is resorbed. In our molecular phylogenies, the family Kahliellidae is polyphyletic and the position of K. matisi is rather poorly resolved, indicating a relationship with oxytrichids.