Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J

Abstract

Lactobacilus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in Agtll and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and H14 lysogens produced 0-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in XEMBL3 with the insert DNA from phage 1P4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A BglM fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into LactobaciUus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an activity spectrum that was the same as that of helveticin J.

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

“…There are several reports of plasmidassociated bacteriocin production from lactic acid bacteria [7,19,25], whereas in other cases, bacteriocin production has been linked to chromosomal DNA [14]. The results of this study suggest that the production of thermophilin ST-1 was not associated with a 2.7 kb plasmid, which is harbored in the S. thermophilus ACA-DC strain.…”

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).…”

Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides

“…Bacteriocin purification was in some cases achieved, but only a few primary structures were directly determined or deduced from their gene sequence, e.g. lactacin F (Muriana & Klaenhammer, 1991), helveticin J (Joerger & Klaenhammer, 1990), lactocin S (Mortvedt et al, 1991), lactococcin A and recently leucocin A-UAL 187 . Among those well-characterized bacteriocins, only leucocin A-UAL 187 appears inhibitory to Listeria monocytogenes.…”