Cyclic Bacteriocins next up previous contents
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Cyclic Bacteriocins

The bacteriocins are a heterogeneous group of proteins of microbial origin that are ribosomally produced and secreted extracellularly by many Gram-positive and Gram-negative bacteria. These proteins act mainly against bacteria of closely related species using a variety of inhibitory mechanisms such as inhibition of cell-wall synthesis, permeabilising cell membranes and inhibiting RNase or DNase activity [125]. In recent years a number of bacteriocins have been discovered that possess a head-to-tail cyclic backbone, including AS-48 from Enterococcus faecalis [127,126], gassericin A [129,128] from Lactobacillus gasseri LA39, and circularin A [131,130] from Clostridium beijerinckii. These proteins are between 58 and 70 amino acids in length and are among the largest ribosomally produced cyclic proteins yet discovered.

Figure 1.18: AS-48 and microcin bacteriocins Ribbon diagrams of A AS-48 showing the 5 helix structure and cyclisation point between Trp70 and Met1 and B microcin J25, which was formerly thought to be cyclic but was shown to possess a ``noose'' structure in which the C-terminus threads a ring formed by a N-terminus to Glu8 sidechain amide bond.

The best characterised of the cyclic bacteriocins, AS-48 is a highly basic 70 amino acid protein that contains a high proportion (49%) of hydrophobic amino acids and lacks cysteines or modified amino acids [132]. Its solution structure shows that it folds into five $ \alpha$-helices arranged according to the topology shown in Figure 1.18 [133]. Cyclisation occurs between residues Trp70 and Met1 in the middle of helix 5 and cyclisation appears to confer additional stability to the protein with its melting temperature being as high as 102$ ^\circ$C [134]. Along with gassericin A and circularin A, AS-48 is resistant to a number of proteases [131,136,135] and this could be explained by its compact globular structure. However, AS-48 did lose activity after incubation with trypsin, Glu-C and Lys-C [132,137] suggesting that, unlike the cyclotides, it is susceptible to endoproteinases in its native form. The combination of hydrophobic and cationic residues in AS-48 suggests an amphipatic structure and several crystallographic and biochemical studies have shown that it produces its effect via permeabilisation of the cell membrane (see [125] for a recent review).

Gassericin A, circularin A and AS-48 are expressed as larger precursors of 91, 72 and 105 amino acids respectively [131,138,139]. Interestingly, while AS-48 and grassericin A both possess precursors substantially larger than the mature product, the precursor of circularin A is only three residues longer than the mature form. In general bacteriocin production in bacteria is a complex process with additional genes required for the maturation and secretion of the mature peptide. AS-48 is encoded on the pMB2 plasmid [139] and transformation of a large portion of this plasmid into a non-AS-48 producing bacterium resulted in the production of AS-48 in the recipient [140]. Analysis of the region transformed into the recipient revealed nine genes, as-48BCC$ _1$DD$ _1$EFGH, that are involved in AS-48 production and immunity [141]. The results of these studies suggest that the full complement of enzymes needed for the production of AS-48 is encoded in the pMB2 plasmid.

To date little is known about the specifics of AS-48 production, although it is clear that it involves post-translational modification of a pre-propeptide into the mature cyclic protein. It has been proposed that an enzyme complex formed by As-48BC$ _1$D from the plasmid may cyclise AS-48 concomitant with the export of the molecule from the cell, although no biocatalytic domains involved in the cleavage and ligation have been identified [125]. Although expression of AS-48 can be induced by transformation of a plasmid containing the nine genes identified for AS-48 production it is interesting to note that in the sex pili system that produces VirB2, discussed further below, it has been shown that no plasmid borne enzyme is necessary for expression. Consequently, although the nine genes identified are necessary for expression of AS-48 the involvement of other host borne enzymes cannot be discounted.

Another class of bacteriocin contains peptides known as the microcins. These peptides are from the Enterobacteriaceae and are characterised by low molecular weights and resistance to high pH and high temperature. They are synthesised independent of the SOS system, which increases the capacity of the bacteria to produce error-free DNA repair on exposure to DNA damaging agents, and under nutrient deficient conditions. Approximately ten members have been identified and they display great diversity in structure and mechanisms of action (see [142,144,143]for recent reviews). One microcin, J25 from Escherichia coli, is a hydrophobic antibacterial peptide with an approximate mass of 2 kDa [145]. When originally characterised the lack of availability of either termini for sequencing and an 18 Da discrepancy in the expected mass led to the conclusion that it was a macrocyclic peptide [146], but, it was later shown that microcin J25 possessed a free C-terminal and that cyclisation was via an unusual N-terminus to Glu8 sidechain amide bond [149,147,148]. The structure was described simultaneously by three groups who showed that the C-terminal threaded a loop formed by the backbone and the Glu8-N-terminal amide bond, as can be seen in Figure 1.18. Hence although not a backbone cyclised peptide, as originally believed, microcin does possess a highly unusual structure and highlights the pitfalls in correctly identifying backbone cyclisation.

next up previous contents
Next: Sex pilus subunits Up: Other Cyclic Peptides Previous: Knottins and Cyclic trypsin
Jason Mulvenna