Pediocin AcH Production Research Articles

Gene organization and sequences of pediocin AcH/PA-1 production operons in Pediococcus and Lactobacillus plasmids

To determine the locations and sequences of pediocin AcH production genes in Pediococcus parvulus ATO77 from vegetables, Lactobacillus plantarum WHE92 from Muenster cheese, and a lactose-fermenting isolate Pediococcus pentosaceus S34 from buffalo milk. Plasmid curing, Southern blot hybridization, and DNA sequence analysis indicate that pediocin AcH production genes are encoded by highly similar operons in unique plasmids designated pATO77 from P. parvulus ATO77, pS34 from P. pentosaceus S34, and pWHE92 from Lact. plantarum WHE92. Structure, immunity and secretion system genes are linked together in the operons, and the promoter sequences are the same. The amino acid sequences of the encoded proteins are highly conserved between plasmids. Pediocin AcH production genes are located within a plasmid-borne operon cassette in all lactic acid bacterial strains examined to date. All four genes needed for production are present within a single plasmid in each strain. This is the first demonstration that the expression of a class IIa bacteriocin is directed by a common gene cassette that has been disseminated to unique plasmids in different genera of lactic acid bacteria. These plasmids should be useful for expressing pediocin AcH in Pediococcus and Lactobacillus strains used in food production.

Modelling and simulation of diffusional mass transfer of glucose during fermentative production of pediocin AcH from Pediococcus acidilactici H

Fermentative production of bacteriocin, pediocin AcH was studied in a batch fermenter using Pediococcus acidilactici H. An initial pH of 6.5 and a fermentation temperature of 37 °C were found to be favourable for optimum production up to 20 h of fermentation time. Rate of cell growth utilizing glucose as substrate during the exponential growth phase and stationary phase was studied experimentally and the data obtained were simulated into the proposed model. The mathematical model also considered the diffusional mass transfer of glucose into the active mass of the cell. Results showed that the model could adequately account for the transfer mechanism of glucose and the phenomenon of cell growth in the fermenter in exponential growth and stationary phases.

The pediocin AcH precursor is biologically active.

The properties of the pediocin AcH precursor, prepediocin AcH, have been studied to gain insight into how producer cells may protect themselves from the activity of intracellular prebacteriocins. The native 62-amino-acid precursor and the 44-amino-acid mature species were expressed in Escherichia coli host strains that lack the leader peptide processing enzyme, PapD. Both forms inhibited the growth of the test bacterium Listeria innocua Lin11, indicating that the native precursor is biologically active. The two species also were synthesized in the context of maltose-binding protein chimeric proteins to facilitate the measurement of their relative specific activities. The chimeric form of the precursor was approximately 80% as active as the chimeric mature species. Of relevance to cell protection and pediocin AcH production, it was determined that the precursor is strongly susceptible to inactivation by reducing agents and to degradation by chymotrypsin and endogenous E. coli proteases. Taken together, the results indicate that the activity of prepediocin AcH may have to be controlled prior to secretion to prevent toxicity to the host. Perhaps producer cells avoid membrane damage by maintaining the precursor in a reduced inactive state or by degrading molecules whose secretion is delayed.

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.

Analysis of the pediocin AcH gene cluster from plasmid pSMB74 and its expression in a pediocin-negative Pediococcus acidilactici strain.

The 3,500-bp pap operon in the 8,877-bp plasmid pSMB74 contains a cluster of four genes, papABCD, of which papA encodes prepediocin (A. M. Motlagh, M. Bukhtiyarova, and B. Ray, Lett. Appl. Microbiol. 18:305-312, 1994). The cluster without the promoter was cloned in the shuttle vector pHPS9. An Escherichia coli strain and a pediocin-sensitive Pediococcus acidilactici strain transformed with the recombinant plasmid, pMBR1.0, produced pediocin AcH. Deletion analysis by introducing mutations in the four genes in pMBR1.0 revealed that only papA and papD were required for pediocin AcH production and that the gene product of papD has both translocation and processing functions. In the transformed minicells of E. coli chi 925 the proteins of the pap cluster were synthesized, indicating no polar effect due to deletion.

Complete nucleotide sequence of pSMB 74, a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici.

Several Pediococcus acidilactici strains produce a plasmid-encoded bacteriocin, pediocin AcH. Previous studies have shown that this plasmid, designated as pSMB 74, encodes genes associated with the production of prepediocin, its post-translation processing to pediocin AcH, transmembrane translocation of these molecules, and immunity of producer cells against pediocin AcH. We report here the complete nucleotide sequence of pSMB 74. The plasmid has a total of 8877 bp. Four genes have been located on pSMB 74. The genes are arranged in a gene cluster of 3500 bp and share a common promoter and rho-independent stem-loop terminator. The four genes, each with independent ribosome binding sites (rbs), initiation and termination codons and spacer sequences in between, were designated as pap A, pap B, pap C and pap D and encode respectively for proteins of 62, 112, 174 and 724 amino acids. The results of this study can be useful either to introduce a suitable marker at a unique restriction site in pSMB 74 and use it as a vector or to clone the pap gene cluster in a suitable plasmid and transform desirable strains for pediocin AcH production. The gene sequence has been submitted to Gene Bank (Acc. No. U02482).

Determination of bacteriocin-encoding plasmids of Pediococcus acidilactici strains by Southern hybridization

A 3.5 kb (2.3 MDa) plasmid probe (pRBI) containing the pap gene (pediocin AcH production) was used in Southern hybridization tests to determine the presence or absence of a plasmid-encoded pediocin gene in various isolates of Pediococcus. This probe hybridized with a 9.4 kb (6.2 MDa) plasmid found present in all pediocin-producing Pediococcus acidilactici strains tested. Conversely, this probe showed no reactions with Ped. acidilactici RS2-1, AB6-1 and PC, strains which did not produce pediocin and lacked the 9.4 kb (6.2 MDa) plasmid. This probe also did not hybridize with the plasmids from a bacteriocin producing Ped. pentosaceus strain.

Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H.

N-terminal analysis of purified pediocin AcH produced a partial sequence of 23 amino acids. This sequence matched perfectly with a segment of 23 amino acids in a 62 amino acid molecule generated from the 186 nucleotide sequence open reading frame in a Hind III fragment in pSMB74 encoding pap-gene (pediocin AcH production). It is suggested that the molecule is translated as inactive prepediocin AcH of 62 amino acids. Then through enzymatic modifications the leader segment of 18 amino acids is removed from the NH2-terminal. The remaining segment of 44 amino acids is active pediocin AcH of 4628 M(r).

Influence of Growth Conditions on the Production of a Bacteriocin, Pediocin AcH, by Pediococcus acidilactici H

The influence of growth parameters on the production of pediocin AcH by Pediococcus acidilactici H was studied. This strain produced large quantities of pediocin AcH in TGE broth (Trypticase [1%], glucose [1%], yeast extract [1%], Tween 80 [0.2%], Mn [0.033 mM], Mg [0.02 mM] [pH 6.5]) within 16 to 18 h at 30 to 37 degrees C (final pH, 3.6 to 3.7). Pediocin AcH production was negligible when the pH of the medium was maintained at 5.0 or above, even in the presence of high cell mass.