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Background to this practical:
The practical outlined below includes the procedures for the production of an antimicrobial peptide (bacteriocin) by Staphylococcus warneri in the 2L or 10L fermenter. The 2L fermenters and the 10L fermenter are located in the National Pharmaceutical Education Centre.
The operation of both fermenters is very similar the primary difference being the fact that the laboratory 2L fermenter must be sterilised by placing it in the laboratory autoclave whereas the 10L fermenter has sterilisation in place (SIP) i.e. the medium is made up and autoclaved in the fermenter.

Introduction
Bacteriocins
Although commonly confused in the past with antibiotics the term bacteriocin refers to bacterial peptides that are ribosomally made or derived from gene-encoded precursor peptides and towards which the producing strain possesses a specific self-protection mechanism / immunity. Initially bacteriocins were produced by E.coli as early as 1925, these substances were named colicins. The colicins were used as the prototype bacteriocins from which all other bacteriocin analysis was compared Nisin produced by Lactococcus lactis was discovered soon after, in 1928, followed by subtilin an analogue of nisin, in 1948.. Since then the area of bacteriocin research has been studied intensely. However, detailed knowledge of bacteriocins is limited to a relatively small number of producing species of bacteria. These are species that are easy to grow and analyse, and organisms that are important in the medical and food industries, such as Enterobacteriaceae (producers of colicins), Pseudomonas (producers of pyocins), Staphylococcus and Streptococcus species (producers of lantibiotics) and the most widely studied, lactic acid bacteria, commonly referred to as LAB, that include members of the genera Lactobacillus, Lactococcus, Carnobacterium, Enterococcus and Pediococcus (producers of lactococcins and lantibiotics).Bacteriocins produced by Gram-negative bacteria have been proven to be different, in respect to size and specificity, to the bacteriocins produced by Gram-positive bacteria This is most likely due to their cell wall structural differences between Gram positive and Gram negative cells..

Bacteriocins produced by Staphylococcal species
This practical looks at the production of a bacteriocin from Staphylococcus warneri.
Although the majority of research has focused on bacteriocins produced by the lactic acid bacteria, due to their potential use in food preservation, there are several studies on bacteriocins isolated from Staphylococcus species. Below are some example of the bacteriocins from Sytaphylococci already studied.
Staphylococcus epidermis bacteriocins.

Epidermin: is produced by Staphylococcus epidermis and along with Pep5 and epilancin are the most thoroughly studied of the staphylococcal bacteriocins. The lantibiotic epidermin contains two lanthionine residues and one 3-methyllanthionine residue and has a molecular weight of 2164Da.
Epicidin 280 is a lantibiotic with activity dependant on two peptides with molecular weights of 3,133Da and 3,136Da. There is 75% similarity in amino acid composition between Epicidin 280 and Pep5, and cross immunity between the two S.epidermis bacteriocins has been reported.
Pep5: The bacteriocin Pep5 contains lanthionine and /or 3-metyllanthionine residues, which allowed for classification of Pep5 as a lantibiotic. Pep5 has a molecular weight of 3488Da. Production of the lantibiotic is plasmid linked to a 18.6kb plasmid. There are actually five separate plasmids identified for this strain. The gene responsible for Pep5 production and the immunity gene are located on the same gene. Other characteristics of the strain, for example penicillin resistance, are associated with the other plasmids

Epilacin K7: this bacteriocin was classified as a lantibiotic. Epilancin K7 contains one lanthionine and two 3-methyllanthionine residues and has a molecular weight of 3032Da
Staphylococcin 1580: With a molecular weight of 2165Da this bacteriocin was proven to be identical to the lantibiotic epidermin.

Staphylococcus aureus bacteriocins.
Most of the characterised antimicrobial compounds from Staphylococcus aureus are associated with the bacteriophage lytic group II, phage type 71 strains examples of which include the staphylococcins 412, staphylococcin 462, staphylococcin C55 and BacR1 as described below.
Staphylococcin C55: The activity of staphylococcin C55 is dependent on two lantibiotic staphylococcins C55? and C55? with molecular weights of 3,339Da and 2,993Da respectively (Dajani et al, 1970). These staphylococcins subunits are plasmid associated from a 32kb plasmid and are both required for antimicrobial activity (Navaratna et al, 1999). Navaratna also reported that the bacteriocins produced by Staphylococcus aureus C55 and the BacR1 producer Staphylococcus aureus UT0007 were identical and that this type of bacteriocin is widely distributed in Staphylococcus aureus.

BacR1: Bac R1 has a molecular weight of 3336Da. BacR1 was identical to one of the two-peptide bacteriocins of staphylococcin C55 (C55?).
Bac 1829: Unlike most other S.aureus bacteriocins, Bac 1829 is not produced by the bacteriophage lytic group II phage type 71 strains. Bac 1829 has a molecular weight of 6418Da and has inhibitory properties that are comparable with that of BacR1 although there is no other link between the two substances reported.
Bac 201: The bacteriocin is a large class III bacteriocin of 41,000Da and is active against Gram-positive and negative indicator organisms. Production of this bacteriocin is plasmid linked from a 4.8Mb plasmid.

Auracin A70: Auracin A70 demonstrates antimicrobial activity against Listeria monocytogenes, a characteristic that should classify it as Class IIA, but this bacteriocin is a multi-peptide non-lantibiotic bacteriocin. Auracin is composed of four peptides with molecular weights of 2,797Da, 2,924Da, 2954Da, and 3086Da, all four are required for activity and all four peptides are plasmid linked, from a single 8kb plasmid.

Aureocin A53: Aureocin A53 demonstrates antimicrobial active against Listeria monocytogenes. Activity is due to a single peptide of 6012.5Da that is plasmid linked from a 10.4kb plasmid. Class IIA bacteriocins are the most plentiful of bacteriocins isolated to date, however this bacteriocin is the only Staphylococcus bacteriocin that has been classified into this group.
Staphylococcin 462: This bacteriocin was isolated from bacteriophage lytic group II, phage type 71 Staphylococcus aureus.
Staphylococcin: 412: This bacteriocin is a multi-peptide complex, like aureocin A70, with an overall mass of 200,000Da.
Staphylococcus gallidermin Bacteriocin.

Gallidermin: Gallidermin is classified as a lantibiotic because of the presence of lanthionine and 3-methyllanthioine residues. The molecular weight of gallidermin is 2164Da. Gallidermin was discovered to be a natural variant of the lantibiotic epidermin although unlike the plasmid-linked epidermin, gallidermin production is linked to the chromosome of the producing strain Staphylococcus gallidermin .
Staphylococcus cohnii Bacteriocin.

Staphylococcin T: The lantibiotic staphylococcin T has a molecular weight of 2166Da. While staphylococcin T, gallidermin and epidermin have been isolated from three different strains of Staphylococcus, S.cohnii, S.gallidermin and S.epidermis respectively, the three bacteriocins seem to be natural variants, with similar structural homology

Staphylococcus warneri Bacteriocin.
Nukacin ISK-1: Nukacin is one of only a limited number of bacteriocins produced by S. warneri that has been reported to date. The bacteriocin was proven to contain three molecules of lanthionine and/or 3-methyllanthionine and is therefore classified as a lantibiotic. The molecular weight of Nukacin ISK-1 is 2900Da and amino acid sequence of nukacin shows strong similarities to the structure of lacticin-481

Production of nukacin by microbial fermentation:
There have been limited published studies on the production of antimicrobial peptides by Staphylococcus warneri. Nukacin produced by a Japanese culture collection of S warneri was produced using MRS broth. The inoculum stage involved the use of an overnight inoculum which was subsequently inoculated for six hours only into fresh MRS broth after six hours this inoculum was used to inoculate the fermenter. This practical studies the production of a bacteriocin by Staphylococci warneri. The medium used is TSB broth.
The nukacin is excreted by the cell into the extracellular medium. Its production begins in the exponential phase of growth and continues into the stationary phase.

 

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