Related Experiment Videos
Propionicin SM1, a bacteriocin from Propionibacterium jensenii DF1: isolation and characterization of the protein and
S Miescher1, M P Stierli, M Teuber
1Laboratorium für Lebensmittelmikrobiologie, Institut für Lebensmittelwissenschaften, Eidgenössische Technische Hochschule, ETH-Zentrum, Zurich, Switzerland.
Abstract:
We purified a bacteriocin from the cell-free supernatant of Propionibacterium jensenii DF1 isolated from Swiss raw milk, and named it propionicin SM1. The heat-stable protein was strongly bactericidal against P. jensenii DSM20274. On the basis of the N-terminal amino acid sequence of the purified protein, a degenerate oligonucleotide probe was designed to locate and clone the corresponding gene of P. jensenii DF1. It hybridized exclusively with the DF1l-resident plasmid pLME106, but not with chromosomal DNA. Sequencing of the 6.9-kb plasmid revealed the targeted amino acid sequence within an open reading frame (ORF4) of 207 amino acids (molecular mass, 22,865 Da). The corresponding gene was named ppnA. It encodes the prepeptide PpnA that is processed to the mature protein (19,942 Da) propionicin SM1. No sequence homology is detectable with known proteins. However, the proposed leader peptide sequence containing 27 amino acids has typical signal peptide features and shows good homology to the leader peptide of Usp45, a protein excreted from Lactococcus lactis (VAN ASSELDONK et al., 1993). Plasmid pLME106 contains at least 9 ORFs, some exhibiting significant homologies to plasmid-encoded functions from other bacteria. The highest identity values were found for ORF1 with the theta replicase (acc. no. U39878) of Brevibacterium linens (58.8%) and ORF6 with the recombinase/invertase (acc. no. AF060871) found in Rhodococcus rhodochrous (46.4%).
Insights
Researchers purified propionicin SM1, a heat-stable bacteriocin from Propionibacterium jensenii, demonstrating its potent bactericidal activity. The gene encoding this novel antimicrobial peptide was identified on a plasmid, offering insights into bacterial defense mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacteriocins are ribosomally synthesized antimicrobial peptides with diverse industrial and therapeutic applications.
- Propionibacterium species, found in fermented foods like Swiss raw milk cheese, are known producers of bioactive compounds.
- Understanding bacteriocin production and genetics is crucial for developing novel food preservation and antimicrobial strategies.
Purpose of the Study:
- To purify and characterize a bacteriocin from Propionibacterium jensenii DF1.
- To identify and clone the gene responsible for bacteriocin production.
- To analyze the genetic context and potential functions of the encoding plasmid.
Main Methods:
- Bacteriocin purification from cell-free supernatant using standard protein purification techniques.
- N-terminal amino acid sequencing to design degenerate oligonucleotide probes.
- Gene cloning and sequencing of the encoding plasmid (pLME106).
- Bioinformatic analysis of open reading frames (ORFs) and predicted protein sequences.
Main Results:
- Purification of propionicin SM1, a heat-stable bacteriocin with strong bactericidal activity against P. jensenii DSM20274.
- Identification and cloning of the propionicin SM1 gene (ppnA) located on the plasmid pLME106.
- The ppnA gene encodes a prepeptide processed into a mature protein of 19,942 Da.
- Plasmid pLME106 contains multiple ORFs with homology to known bacterial plasmid functions, including theta-replicase and recombinase/invertase.
Conclusions:
- Propionicin SM1 is a novel bacteriocin produced by Propionibacterium jensenii DF1.
- The bacteriocin gene is plasmid-encoded, suggesting a role in plasmid maintenance or bacterial defense.
- The genetic analysis of pLME106 provides insights into the molecular mechanisms of propionibacteria.