THE USE OF 16S RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES TO STUDYCOMPETITION BETWEEN RUMINAL FIBROLYTIC BACTERIA - DEVELOPMENT OF PROBES FOR RUMINOCOCCUS SPECIES AND EVIDENCE FOR BACTERIOCIN PRODUCTION

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococ cus albus and Ruminococcus flavefaciens. On the basis of specificity s tudies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB 1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed culture s for cellobiose as the carbon source. In dicultures containing R. alb us 8 and F. succinogenes S85, competition was not observed. However, R . flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when ce llobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 w ere grown together on cellobiose medium, R. albus 8 outcompeted R. fla vefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA onl y 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Fur ther, when R. albus 8, R. flavefaciens FD-I, and F. succinogenes S85 w ere grown together in a triculture, R. flavefaciens FD 1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. suc cinogenes S85 showed a similar competition pattern to that of the dicu ltures. The results show that the Ruminococcus probes were effective i n the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succi nogenes. Moreover, R. albus 8 was shown to produce a heat stable prote in factor which causes zones of inhibition in R. flavefaciens FD-1 bac terial lawns. This is the first demonstration of the production of a b acteriocin-like substance by a ruminal bacterium. It is postulated tha t bacteriocin production by ruminal fibrolytic bacteria is a mechanism used to compete for nutrients.