Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481

Abstract

Lactobacillus helveticus 481 produced an antimicrobial agent active against five closely related species. The sensitive indicators included L. helveticus 1846 and 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. The antimicrobial compound was active at neutral pH under aerobic or anaerobic conditions, was sensitive to proteolytic enzymes and heat (30 min at 100°C), and demonstrated a bactericidal mode of action against sensitive indicators. These data confirmed that antimicrobial activity of L. helveticus 481 was mediated by a bacteriocin, designated helveticin J. Production of helveticin J was maximized in an anaerobic fermentor held lit a constant pH'of 5.5. Ultrafiltration experiments on cWuture supernatants containing the bacteriocin revealed that helveticin J was present as an aggregate with a molecular weight in excess of 300,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of helveticin J purified through Sephadex chromatography resolved a 37,000-dalton p,rotein band with bacteriocin activity. L. helveticus 481 was shown to harbor a single 8-megadalton plasmid (pMJ1008). Isolates cured of pMJ1008 were phenotypically identical to plasmid-bearing cells in fermentation patterns, helveticin J activity, and unmunity spectra. The data provided evidence for a chromosomal location of helveticin J and host immunity determinants.Lactobacillus helveticus and other lactobacilli have been shown to inhibit only closely related species (3,5,10,25). A narrow inhibitory spectrum, proteinaceous nature, and bactericidal mode of action are typical characteristics of antimicrobial agents classified as *bacteriocins (23). Very few lactobacifli have been confirmed to produce true bacteriocins within these criteria (3,4,6,25,26). Only a single bacteriocin has been identified and characterized within the L. helveticus species. Upreti and Hinsdill (25,26) confirmed the production of a bacteriogcin, designated lactocin LP27, by L. helveticus LP27. Lactocin LP27 exhibited a narrow activity spectrum by inhibiting strains of L. acidophilus and L. helveticus. A proteinaceous nature was demonstrated by inactivation of the bacteriocin with trypsin and pronase. In contrast to a bactericidal mode of action typical of bacteriocins, lactocin LP27 had a bacteriostatic effect on the indicator, L. helveticus LS18. Protein synthesis was terminated by lactocin LP27, but DNA and RNA synthesis and ATP levels were not affected.Bacteriocin production and resistance phenotypes offer excellent markers for study of genetic linkages and transfer systems. However, there is little information available on bacteriocins, genetic linkages, and gene transfer systems in Lactobacillus species. Gram-negative and gram-positive bacteria commonly harbor plasmid-borne genetic determinants of bacteriocin production and of host cell bacteriocin immunity (17,19,20,23,27). However, all attempts to locate plasmid determinants responsible for bacteriocin production and host immunity in lactobacilli have been unsuccessful. Difficulties in curing lactobaci...

Purification and characterization of thermophilin ST-1, a novel bacteriocin produced by Streptococcus thermophilus ACA-DC 0001

“…Cell death was not associated with lysis or leakage of the cell membrane, since no changes in optical density were observed. This finding is consistent with other reports, suggesting that thermophilin T, thermophilin A and several bacteriocins from different lactic acid bacteria can exert a similar effect [1,13,28,38]. However, several bacteriocins causing lysis of sensitive cells have also been reported for several species of lactic acid bacteria [2,23].…”

“…delbrueckii ssp. bulgaricus [13,15,34,35]. On the contrary, other bacteriocins from different S. thermophilus strains such as thermophilin A [38], thermophilin 347 [36], thermophilin ST-13 [18] and thermophilin T [1] seem to be included in the non-lantibiotic heat-stable group of bacteriocins with small molecular mass.…”

ef1097 and ypkK encode enterococcin V583 and corynicin JK, members of a new family of antimicrobial proteins (bacteriocins) with modular structure from Gram-positive bacteria

“…The former was suggested to have a monomeric molecular mass of 37 kDa and to form aggregates of .300 kDa, whereas the latter was reported to be a bacteriocin with multimeric molecular mass of 30-50 kDa and potentially composed of 17 kDa monomers. Helveticin J is active against other lactic acid bacteria such as lactobacilli and lactococci, is chromosomally encoded and was shown to be inactivated by proteolytic digestion and heat (Joerger & Klaenhammer, 1986, 1990, features consistent with its size and proteinaceous nature.…”

“…However, more recent reisolation of the inhibitory agent revealed the antimicrobial activity to be epidermin, a peptide bacteriocin (2164.6 Da) belonging to the lantibiotic subgroup, which co-purified with the higher molecular mass species (Sahl, 1994). Two additional high molecular mass bacteriocins from Grampositive bacteria have been reported: helveticin J from Lactobacillus helveticus 481 (Joerger & Klaenhammer, 1986, 1990) and a potentially related, unnamed compound from Lb. helveticus CNRZ450 (Thompson et al, 1996).…”

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…The class has been suggested to have another name: bacteriolysin, since these proteins were considered to degrade the targeted cell wall leading to cytolysis. However, helveticin J does not kill bacteria by lytic effect (Joerger and Klaenhammer 1986). (Simmonds et al 1997), enterolysin A from Enterococcus faecalis (Hickey et al 2003), millericin B from Streptococcus milleri (Beukes et al 2000) and linocin M18 produced by…”