BOVICIN HC5, A BACTERIOCIN FROM STREPTOCOCCUS BOVIS HC5

Authors: MANTOVANI, H - CORNELL UNIVERSITY; HU, H - CORNELL UNIVERSITY; WOROBO, R - CORNELL UNIVERSITY; Russell, James

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2002
Publication Date: 10/13/2002
Citation: MANTOVANI, H.C., HU, H., WOROBO, R.W., RUSSELL, J.B. 2002. BOVICIN HC5, A BACTERIOCIN FROM STREPTOCOCCUS BOVIS HC5. MICROBIOLOGY. 148:3347-3352.

Interpretive Summary: Cattle in the U.S. are often fed antibiotics, but the widespread use of antibiotics in animal feed has been criticized. Antibiotics are primarily targeted against gram-positive gut bacteria. Gram-positive ruminal bacteria produce large amounts of hydrogen a precursor of methane, ammonia, a wasteful end-product of amino acid degradation, and lactic acid, an acid that causes ruminal acidosis, ruminal ulcers, founder and even death of the animal. Some gram-positive bacteria produce peptides (bacteriocins) that can inhibit other gram-positive bacteria, and bacteriocins have been proposed an alternative to antibiotics. We previously isolated a bacterium with a very active and heat stable bacteriocin. We have now purified it and determined its N-terminal amino acid sequence. Research on bacteriocins has the potential to decrease the need for antibiotic in animal feed and lessen the risk of antibiotic resistance in human medicine.

Technical Abstract: Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin resistant S. bovis JB1 cells could be strongly inhibited. Agar overlays showed that S. bovis HC5 inhibited a variety of gram-positive bacteria, and the spectrum of activity was similar to monensin, a commonly used feed additive. The activity could be released from the cell surface by Tween, precipitated by ammonium sulfate and dialyzed. The partially purified extract was inactivated by Pronase E and trypsin, but it was resistant to heat, proteinase K and a-chymotrypsin. The partially purified extract caused potassium efflux from glycolyzing S. bovis JB1 cells, and Tricine-SDS-PAGE gels indicated that the active peptide had a molecular weight of less than 3000 Da. When Tween was omitted from the basal medium, the activity remained cell associated, and simple wash steps eliminated most contaminating peptides. Because this latter activity could be liberated from washed cells by acidic NaCl (100 mM, pH 2.0) without significant cell lysis, the purification was greatly improved. The bacteriocin (bovicin HC5) was then purified by high pressure liquid chromatography. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis indicated that purified bovicin HC5 had a molecular weight of approximately 2440 Da, and the terminal amino acid sequence was VG-RYAS-PG-SWKYV-F. Amino acid residues that did not correspond to amino acids commonly found in proteins had approximately the same position as dehydroalanines found in some lantibiotics. The N-terminal amino acid sequence of bovicin HC5 showed similarity to a lantibiotic precursor of S. pyogenes SF370, but the identity was only 55%. Further work will be needed to locate the gene, but bovicin HC5 appears to be a novel bacteriocin.