Bacteriocin

Classification of bacteriocins

Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins, the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins (formerly called 'colicines,' meaning 'coli killers'). They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. In fact, one of the oldest known so-called colicins was called colicin V and is now known as microcin V. It is much smaller and produced and secreted in a different manner than the classic colicins.

This naming system is problematic for a number of reasons. First, naming bacteriocins by what they putatively kill would be more accurate if their killing spectrum were contiguous with genus or species designations. The bacteriocins frequently possess spectra that exceed the bounds of their named taxa and almost never kill the majority of the taxa for which they are named. Further, the original naming is generally derived not from the sensitive strain the bacteriocin kills, but instead the organism that produces the bacteriocin. This makes the use of this naming system a problematic basis for theory; thus the alternative classification systems.

Bacteriocins that contain the modified amino acid lanthionine as part of their structure are called lantibiotics.

Methods of classification

Alternative methods of classification include: method of killing (pore-forming, nuclease activity, peptidoglycan production inhibition, etc.), genetics (large plasmids, small plasmids, chromosomal), molecular weight and chemistry (large protein, peptide, with/without sugar moiety, containing atypical amino acids such as lanthionine), and method of production (ribosomal, post-ribosomal modifications, non-ribosomal).

One method of classification fits the bacteriocins into Class I, Class IIa/b/c, and Class III.

Class I bacteriocins

The class I bacteriocins are small peptide inhibitors and include nisin and other lantibiotics.

Class II bacteriocins

The class II bacteriocins are small (<10 kDa) heat-stable proteins. This class is subdivided into five subclasses. The class IIa bacteriocins (pediocin-like bacteriocins) are the largest subgroup and contain an N-terminal consensus sequence -Tyr-Gly-Asn-Gly-Val-Xaa-Cys across this group. The C-terminal is responsible for species-specific activity, causing cell-leakage by permeabilizing the target cell wall.

Class IIa bacteriocins have a large potential for use in food preservation as well medical applications due to their strong anti-Listeria activity and broad range of activity. One example of Class IIa bacteriocin is pediocin PA-1. The class IIb bacteriocins (two-peptide bacteriocins) require two different peptides for activity. One such an example is lactococcin G, which permeabilizes cell membranes for monovalent sodium and potassium cations, but not for divalent cations. Almost all of these bacteriocins have a GxxxG motifs. This motif is also found in transmembrane proteins, where they are involved in helix-helix interactions. Accordingly, the bacteriocin GxxxG motifs can interact with the motifs in the membranes of the bacterial cells, killing the cells. Class IIc encompasses cyclic peptides, in which the N-terminal and C-terminal regions are covalentely linked. Enterocin AS-48 is the prototype of this group. Class IId cover single-peptide bacteriocins, which are not post-translationally modified and do not show the pediocin-like signature. The best example of this group is the highly stable aureocin A53. This bacteriocin is stable under highly acidic conditions, high temperatures, and is not affected by proteases.

The most recently proposed subclass is the Class IIe, which encompasses those bacteriocins composed by three or four non-pediocin like peptides. The best example is aureocin A70, a four-peptide bacteriocin, highly active against Listeria monocytogenes, with potential biotechnological applications.

Class III bacteriocins

Class III bacteriocins are large, heat-labile (>10 kDa) protein bacteriocins. This class is subdivided in two subclasses: subclass IIIa or bacteriolysins and subclass IIIb. Subclass IIIa comprises those peptides that kill bacterial cells by cell wall degradation, thus causing cell lysis. The best studied bacteriolysin is lysostaphin, a 27 kDa peptide that hydrolises the cell walls of several Staphylococcus species, principally S. aureus. Subclass IIIb, in contrast, comprises those peptides that do not cause cell lysis, killing the target cells by disrupting the membrane potential, which causes ATP efflux .

Class IV bacteriocins

Class IV bacteriocins are defined as complex bacteriocins containing lipid or carbohydrate moieties. Confirmation by experimental data was established with the characterisation of sublancin and glycocin F (GccF) by two independent groups.

Databases

Two databases of bacteriocins are available: BAGEL and BACTIBASE.

Medical significance

Bacteriocins are of interest in medicine because they are made by non-pathogenic bacteria that normally colonize the human body. An example of this would be genus Lactobacilli. These bacteria inhabit the normal, lower reproductive tract of women. Loss of these harmless bacteria following antibiotic use may allow opportunistic pathogenic bacteria to invade the human body.

Bacteriocins have also been suggested as a cancer treatment. They have shown distinct promise as a diagnostic agent for some cancers, but their status as a form of therapy remains experimental and outside the mainsteam of cancer research. This is partly due to questions about their mechanism of action and the presumption that anti-bacterial agents have no obvious connection to killing mammalian tumor cells. Some of these questions have been addressed, at least in part.

Bacteriocins were tested as AIDS drugs around 1990, but did not progress beyond in-vitro tests on cell lines.

Bacteriocins have been proposed as a replacement for antibiotics to which pathogenic bacteria have become resistant. Potentially, the bacteriocins could be produced by bacteria intentionally introduced into the patient to combat infection.

In spite of these promising advantages, nisin is the only bacteriocin generally recognized as safe by the Food and Drug Administration and is currently used as a food preservative in several countries.This limitation in bacteriocins availability in the market as preservatives and antimicrobials can be attributed to multiple factors, including: (i) the high cost of their commercial production; (ii) the loss of their activity by proteolytic enzymes; (iii) their unfavorable interactions with other food constituents, which decreases the availability and necessitates a huge amount of the peptide to be added; (iv) the alterations of the chemical and physical properties of these compounds during the various food-processing stages; (v) the low yield of these compounds due to ineffective recovery by traditional purification methods; and (vi) the narrow spectrum of activity observed for most of the tested bacteriocins against pathogenic bacteria. In the last years, several studies on bacteriocins have demonstrated that the optimization of their production conditions, their purification methods, their combinations with other antimicrobial agents, the hurdle technology approach, and nanotechnology formulations, could all represent solutions to some of the previously mentioned problems.

Production

There are many ways to demonstrate bacteriocin production, depending on the sensitivity and labor intensiveness desired. To demonstrate their production, technicians stab inoculate multiple strains on separate multiple nutrient agar Petri dishes, incubate at 30 °C for 24 h., overlay each plate with one of the strains (in soft agar), incubate again at 30 °C for 24 h. After this process, the presence of bacteriocins can be inferred if there are zones of growth inhibition around stabs. This is the simplest and least sensitive way. It will often mistake phage for bacteriocins. Some methods prompt production with UV radiation, Mitomycin C, or heat shock. UV radiation and Mitomycin C are used because the DNA damage they produce stimulates the SOS response. Cross streaking may be substituted for lawns. Similarly, production in broth may be followed by dripping the broth on a nascent bacterial lawn, or even filtering it. Precipitation (ammonium sulfate) and some purification (e.g. column or HPLC) may help exclude lysogenic and lytic phage from the assay.