Listeriocin 743A

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.

Staphylococcus aureus A70 produces a heat-stable bacteriocin designated aureocin A70. Aureocin A70 is encoded within a mobilisable 8 kb plasmid, pRJ6, and is active against Listeria monocytogenes. Experiments of transposition mutagenesis and gene cloning had shown that aureocin A70 production and immunity were associated with the HindIII-A and B fragments of pRJ6. Therefore, a 6332 bp region of the plasmid, encompassing both these fragments, was sequenced using a concatenation DNA sequencing procedure. DNA sequence and genetic analyses revealed the presence of three transcriptional units that appear to be involved in bacteriocin activity. The first transcriptional unit contains a single gene, aurT, which encodes a protein that resembles an ATP-dependent transporter, similar to those involved in lantibiotic export. AurT is required for aureocin A70 production and it appears to be essential for mobilisation of pRJ6. The second putative operon contains two open reading frames (ORFs); the first gene, orfA, is predicted to encode a protein similar to small repressor proteins found in some Archaea, whose function remains to be elucidated. The second gene, orfB, codes for an 138 amino acid residue protein which shares a number of characteristics (high pI and hydrophobicity profile) with proteins associated with immunity, needed for self-protection against bacteriocin. Four other genes are present in the third operon, aurABCD. aurABCD encode four related peptides that are small (30-31 amino acid residues), strongly cationic (pI of 9.85 to 10.04) and highly hydrophobic. Theses peptides also have a high content of small amino acid residues like glycine and alanine, and no cysteine residue. Tn917-lac insertional mutations, which affected aureocin A70 activity, reside within operon aurABCD. Analysis of purified bacteriocin preparations by mass spectrometry demonstrated that all four peptides encoded by aurABCD operon are produced, expressed and excreted without post-translational modifications. Thus, aureocin A70 is a multi-peptide non-lantibiotic bacteriocin, which is transported without processing.

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.