Clones expressing activity against xylan or 0(1-3,1-4)glucan (lichenan) were isolated from a library of Ruminococcusflavefaciens 17 DNA made in bacteriophage A EMBL3. Hybridization analyses indicated the recovery of four separate genes encoding xylanases that showed no detectable associated carboxylmethylcellulase activity. One of these genes was associated with clones that also expressed P(1-3,1-4)glucanase and I-xylosidase activities. Ruminococcus flavefaciens is an important plant cell wall-degrading bacterium found in the rumen and hind gut of mammals (3). Many strains are known to be capable of causing extensive degradation of highly crystalline forms of cellulose, and this has led to interest in the enzymology (6, 17, 18) and genetic determination (1) of cellulases in this species. In addition, strains of R. flavefaciens are capable of extensive degradation of the noncellulosic polysaccharide components of plant cell walls (15) because they produce a variety of other polysaccharidases, including xylanase and pectinase (17, 21). These activities are likely to contribute substantially to the overall rate and extent of cell wall digestion even though the degradation products are often not utilized for growth. This report describes the analysis of genomic DNA clones from recently isolated R. flavefaciens 17 that express activities against two noncellulosic plant cell wall polysaccharides, xylan and 3(1-3,1-4)glucan. MATERIALS AND METHODS Strains. R. flavefaciens 17 is a recent isolate obtained nonselectively from the rumen of a fistulated cow that was receiving a diet of grass cubes, hay, and concentrates. Fermentation products, cell morphology and Gram stain, colony pigmentation, and the range of substrates fermented all agree with accepted characteristics for the species (3). Anaerobic methods. The M2 medium of Hobson (8) containing 0.2% cellobiose as the energy source was used to grow R. flavefaciens by the methods of Bryant (2). DL-Threonine (10 mM) was included in cultures used for DNA extraction. Library screening and molecular biology procedures. Chromosomal DNA was extracted from R. flavefaciens 17 by standard methods (19), but mutanolysin was used to achieve lysis. Size-fractionated Sau3A-cut DNA (average length,

Selective plating procedures were used to follow the fate of rifampicin‐resistant mutant strains of the obligately anaerobic species Bacteroides multiacidus and Selenomonas ruminantium after their introduction at numbers around 107/ml into the rumen of sheep. Bacteroides multiacidus strain F100 showed an initially rapid rate of loss (49%/h) but subsequently numbers declined more gradually approaching the limits of detection (103/ml) after 100 h. Viable cell numbers also decreased in vitro upon addition of F100 cells to whole rumen contents, but remained stable upon addition to cell‐free rumen fluid, suggesting protozoal predation. F100 cells were able to grow in vitro in whole rumen contents in the presence of an added utilizable substrate such as sorbitol, but addition of sorbitol to the rumen failed to enhance survival in vivo. In the case of S. ruminantium, introduced rifR strains persisted in the rumen at levels around 106 ml for at least 30 days.

Tetracycline resistant (TcR) strains of three of the major species of strictly anaerobic rumen bacteria Megasphaera elsdenii, Selenomonas ruminantium and Butyrivibrio fibrisolvens, were recovered with an isolation medium containing 20 μg/ml tetracycline. Only two of 14 strains of these species from other sources, isolated without antibiotic selection, showed tetracycline resistance. Evidence was found for the presence of plasmids in two tetracycline‐resistant strains of M. elsdenii, and in some strains of S. ruminantium.

Tetracycline resistance was transferred at frequencies between l0-7 and 10-6 per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23, belonging to B. ruminicola subsp. ruminicola.

Non‐denaturing polyacrylamide gel electrophoresis was used to detect genetic variation at loci coding for there intracellular enzymes in the obligately anaerobic rumen bacterium Selenomonas ruminantium. Four mobility variants were detected for lactate dehydrigenase, seven for glucokinase and at least five for NADP‐dependent glutamate dehydrogenase among 28 newly isolated, and eitght previously isolated strains from sheep and cattle. No evidence was found for an exclusive association of any particular electrophoretic mobility type with variable metabolic traits such as the ability to utilise lactate, to reduce nitrate or to ferment trehalose, sorbitol, rhamnose or glycerol. The most commonly occurring electromorph type was recovered from more than one animal, while most animals examined were show to harbour more than one electromorph type.