Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/2/2004
Publication Date: 5/1/2004
Citation: Chen, J., Stevenson, D.M., Weimer, P.J. 2004. Albusin B, a bacteriocin from the ruminal bacterium Ruminococcus albus 7 that inhibits the growth of ruminococcus flavefaciens. Applied and Environmental Microbiology. 70(5):3167-3170. Interpretive Summary: Many rumen bacteria are known to produce bacteriocins, which are proteins that inhibit the growth of closely related species. Bacteriocins presumably allow the producing strain to compete effectively against sensitive species that are also present in the same habitat. We identified and characterized two bacteriocins that are produced by an abundant species of a fiber- digesting bacterium in the rumen that we had earlier shown to be positively correlated with milk production. The gene that allows the bacterium to produce one of the bacteriocins was characterized and shown to have some similarity to that from an unrelated non-ruminal bacterium from soil. These proteins appear to represent a new group of bacteriocins.These agents are the first bacteriocins to be purified from rumen cellulose-digesting bacteria. Because the population sizes of the producing species are known to be positively correlated with milk production, use of the bacteriocin may enhance the ability of this strain to compete against other, less desirable rumen bacteria to improve animal performance. Use of bacteriocins present producers with an alternative to antibiotics for beneficially altering ruminal micribial populations.
Technical Abstract: Ruminococcus albus and Ruminococcus flavefaciens are competitors for cellulose in the ruminal fermentation. R. albus typically dominates R. flavefaciens in binary culture and in the rumen, and while this outcome has been ascribed to production of bacteriocin-like substances by R. albus, these agents have not been purified. We obtained two proteins from culture supernatants of R. albus 7 that inhibited growth of R. flavefaciens FD-1. The first was purified over 131-fold to obtain a fraction that yielded two protein bands of MW ~36 kDa and ~45 kDa on SDS-PAGE gels, the former of which (albusin A) contained the inhibitory activity. The first 20 amino acids of the N-terminal sequence obtained from the ~36 kDa band were almost identical to the N-terminal sequence of a 50-kDa endoglucanase from R. albus F-40, although the albusin A fraction contained almost no endoglucanase activity. The second inhibitor (albusin B) of ~31 kDa was purified over 220-fold, and its N-terminal amino acid sequence was used to generate a probe for gene cloning, which was performed by a combination of traditional cloning and gene-walking PCR. The gene (albB) included an entire open reading frame encoding a protein with predicted molecular weight of 32168 Da and predicted pI of 7.7. BLAST analysis revealed moderate similarity to LlpA, a recently-described 30 kDa bacteriocin from Pseudomonas sp. BW11M1. Although weak sequence similarity was noted to lectins and mannose-binding proteins, activity of albusin B was not removed by treatment with mannose or glucomannan. BLAST search of the unfinished genome of R. albus (strain 8) identified two sequences with a similar-sized open reading frames having amino acid identity of 69% to albusin B, suggesting that R. albus 8 may produce one or more similar proteins.