Phage resistance and altered growth habit in a strain of Streptococcus bovis.

Bacteriophage (phi Sb01) of Streptococcus bovis, isolated from pooled rumen fluid of cattle, was a small siphovirus of morphotype B1. It contained double-stranded DNA of length 30.9 kb, which was digested by the restriction endonucleases, EcoRI, HindIII, and PvuII. Bacteria which survived phi Sb01 infection (strain 2BAr) grew in long chains (100-200 cells), ultimately forming large clumps of cells. This growth habit was in distinct contrast to that of the parent host strain which grew predominantly in the form of single cells or diplococci. Strain 2BAr was genetically stable, resistant to phi Sb01 attack, and the observed differences in the growth characteristics of the parent strain and 2BAr indicated that cells of 2BAr were more adherent. In the rumen ecosystem, the selection of phage-resistant bacteria with altered growth characteristics may be a factor in modifying bacterial phenotypes, and thus increasing variability among bacteria which are closely related genetically.

Morphology of three polycentric rumen fungi and description of a procedure for the induction of zoosporogenesis and release of zoospores in cultures.

Three polycentric rumen fungi, LL, LC2 and Ruminomyces elegans (C2), isolated from the rumen of cattle were grown in six culture media. LL and LC2 were morphologically similar. Their characteristics resembled those of Orpinomyces and Neocallimastix joyonii, and they grew well and produced sporangia after 3-4 d growth in all the media. R. elegans differed morphologically from LL and LC2, but although it also grew well in all media, abundant sporangia occurred only after 2-3 d growth in media containing cellulose. Undifferentiated sporangia were produced by all three isolates; differentiation of the sporangia did not occur in the spent growth media. However, if thalli possessing recently-formed sporangia were transferred to, or flooded with, fresh liquid medium or rumen fluid, zoosporogenesis and liberation of zoospores occurred within 17-20 min for isolates LL and LC2 and 30 min for R. elegans. Procedures for inducing zoosporogenesis by polycentric anaerobic fungi are described.

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.

The isolation and comparison of cellulase genes from two strains of Ruminococcus albus.

Endo-1,4-beta-glucanase genes have been cloned from two strains of Ruminococcus albus recently isolated in this laboratory. Although the strains were phenotypically similar, cross-hybridization studies between them showed significant genetic differences, with only 20% of the genome forming DNA heteroduplexes. Heteroduplexes displayed an average dissociation temperature 9 degrees C lower than that of the homoduplex. Consistent with this, restriction maps of the two endoglucanase genes showed no similarity, and hybridization work using the endoglucanase genes as probes revealed that neither gene was present in the genome of the other isolate of R. albus. Comparative enzyme characterization showed differences between the enzymes in their response to temperature, pH and substrate preference.

Biology of gut anaerobic fungi.

The obligately anaerobic nature of the gut indigenous fungi distinguishes them from other fungi. They are distributed widely in large herbivores, both in the foregut of ruminant-like animals and in the hindgut of hindgut fermenters. Comparative studies indicate that a capacious organ of fermentative digestion is required for their development. These fungi have been assigned to the Neocallimasticaceae, within the chytridiomycete order Spizellomycetales. The anaerobic fungi of domestic ruminants have been studied most extensively. Plant material entering the rumen is rapidly colonized by zoospores that attach and develop into thalli. The anaerobic rumen fungi have been shown to produce active cellulases and xylanases and specifically colonise and grow on plant vascular tissues. Large populations of anaerobic fungi colonise plant fragment in the rumens of cattle and sheep on high-fibre diets. The fungi actively ferment cellulose which results in formation of a mixture of products including acetate, lactate, ethanol, formate, succinate, CO2 and H2. The properties of the anaerobic fungi together with the extent of their populations on plant fragments in animals on high-fibre diets indicates a significant role for the fungi in fibre digestion.

Morphological diversity of ruminal bacteriophages from sheep and cattle.

Large numbers of bacteriophages (2 x 10(7) to 1 x 10(8)/ml) were present in ruminal fluid from sheep and cattle. Twenty-six distinct types were identified and placed in three morphological groups; several phages possessed unusual structural features. The large numbers and diversity of phages observed indicates a possible role in bacterial lysis and hence in the population dynamics of the ruminal bacteria.

Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae.

Vegetative and reproductive stages of Caecomyces equi gen. nov., sp. nov. isolated from the horse caecum were examined by light and electron microscopy. This organism, which is similar to isolates known as Sphaeromonas communis, produces uniflagellate, uninucleate zoospores whose perikinetosomal structures, i.e. circumflagellar ring, spur, struts and scoop, are similar in many respects to those described in species of Neocallimastix. Microtubular roots extend basally from the spur and associate with hydrogenosomes and the nucleus. Another group of microtubules radiates laterally in a fan-shaped array close to the plasmalemma. Zoospores encyst, shedding their flagella with basal bodies, and germinate to diglobular thalli. Either coralloid or bulbous rhizoids form in plant material, but only the latter in axenic culture. Incipient zoospores are produced from a multinucleate eucarpic thallus and devlop within cleavage vacuoles containing flagella. An isolate from the cow rumen was found to be similar to C. equi in morphology and zoospore ultrastructure. On the basis of zoospore ultrastructure, we assign the new genus to the Neocallimasticaceae of the order Spizellomycetales. Organisms previously described as Sphaeromonas communis and Piromonas communis are renamed Caecomyces communis and Piromyces communis and assigned to the same family.

Basal body loss during fungal zoospore encystment: evidence against centriole autonomy.

The controversial question of the possible autonomy of centrioles, as shown by the persistence of all or part of them in the generative cell line throughout the life cycle of organisms, remains unresolved. All previous reports on shedding or withdrawal of cilia and flagella showed that their basal bodies (= centrioles) were retained in the cells where they may, or may not, subsequently disassemble. We show that in the fungus Neocallimastix sp. the basal bodies are discarded with the flagella when zoospores encyst. This shedding of basal bodies argues against centriolar persistence in any form and thus against their autonomy and endosymbiotic origin.

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates.

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.

Fermentation of Cellulose to Methane and Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri.

The fermentation of cellulose by a rumen anaerobic fungus in the presence of Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri strain 227 resulted in the formation of 2 mol each of methane and carbon dioxide per mol of hexose fermented. Coculture of the fungus with either Methanobrevibacter sp. or M. barkeri produced 0.6 and 1.3 mol of methane per mol of hexose, respectively. Acetate, formate, ethanol, hydrogen, and lactate, which are major end products of cellulose fermentation by the fungus alone, were either absent or present in very low quantities at the end of the triculture fermentation (

Cellulose fermentation by a rumen anaerobic fungus in both the absence and the presence of rumen methanogens.

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.

Rumen anaerobic fungi of cattle and sheep.

Plant fragments obtained from natural rumen digesta of fistulated cattle and sheep were examined by scanning electron microscopy. Various plant materials suspended in the rumen for different times were examined likewise. By 2 h large numbers of phycomycetous fungal zoospores were found attached to fibrous plant fragments, particularly vascular tissues. The subsequent development of these fungi resulted in production of thalli with extensive rhizoids and with sporangia up to 175 mum long. Scanning electron microscope examination of plant fragments randomly selected from natural rumen contents of both cattle and sheep demonstrated widespread colonization by large populations of these anaerobic fungi. Furthermore, all plant fragments suspended in nylon bags in the rumen were also extensively colonized. These findings demonstrate that plant fragments in the rumen are the sites of colonization and development by the anaerobic phycomycetous fungi. In addition, the results suggest that these fungi may form a significant part of the rumen microbiota in cattle and sheep fed on fibrous diets and suggest that they may be important in fiber digestion.

The rumen ciliate epidinium in primary degradation of plant tissues.

Pieces of lucerne stem suspended in a sheep rumen in nylon bags were removed after different time intervals and examined by scanning electron microscopy. By 15 min large numbers of the ciliate protozoan Epidinium Crawley were attached to damaged areas of the stem, although a complex protozoal fauna was present in the rumen contents. Highest concentrations were on cortex and phloem tissues, with densely packed protozoa forming a complete ring around the transversely cut end of the stem between the epidermis and the vascular cylinder. Within 2 h there was extensive degradation of thin-walled tissues, as indicated by the amount of exposed vascular cylinder. Epidermis was not degraded but slid down the side of the stem as the underlying tissues were removed. This rapid degradation of plant tissue is explained by direct ingestion of tissues by the protozoa on the plant fragments. Many of the epidinia attached to the stem pieces were ingesting phloem elements and chlorophyllous tissue. The rumen protozoa have not previously been shown to participate on this scale in the physical degradation of plant material.

Attachment of the ciliate Epidinium Crawley to plant fragments in the sheep rumen.

High concentrations of the ciliate Epidinium Crawley are associated with damaged regions of fresh plant material undergoing digestion in the sheep rumen. This finding supports that postulate that sequestration in the rumen explains the low rate of passage of protozoa despite the high flow rate of liquid from the rumen. The maintenance of Epidinium in the rumen, despite their slow growth rate, is also explained.

Scanning electron microscope study of bacteria associated with the rumen epithelium of sheep.

Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed.

Formate as an intermediate in the bovine rumen fermentation.

An average of 11 (range, 2 to 47) mumoles of formate per g per hr was produced and used in whole bovine rumen contents incubated in vitro, as calculated from the product of the specific turnover rate constant, k, times the concentration of intercellular formate. The latter varied between 5 and 26 (average, 12) nmoles/g. The concentration of formate in the total rumen contents was as much as 1,000 times greater, presumably owing to formate within the microbial cells. The concentration of formate in rumen contents minus most of the plant solids was varied, and from the rates of methanogenesis the Michaelis constant, K(m), for formate conversion to CH(4) was estimated at 30 nmoles/g. Also, the dissolved H(2) was measured in relation to methane production, and a K(m) of 1 nmole/g was obtained. A pure culture of Methanobacterium ruminantium showed a K(m) of 1 nmole of H(2)/g, but the K(m) for formate was much higher than the 30 nmoles for the rumen contents. It is concluded that nonmethanogenic microbes metabolize intercellular formate in the rumen. CO(2) and H(2) are the principal substrates for rumen methanogenesis. Eighteen per cent of the rumen methane is derived from formate, as calculated from the intercellular concentration of hydrogen and formate in the rumen, the Michaelis constants for conversion of these substrates by rumen liquid, and the relative capacities of whole rumen contents to ferment these substrates.