Functional analysis of circular and linear bacteriocins of Gram-positive bacteria

SUMMARY & GENERAL DISCUSSION Preservation methods such as drying and fermenting to store food products for longer periods of time have been used for centuries. Nowadays, people desire fresh and minimally processed food, to be prepared with minimal cooking. This sets new targets for the food industry, as such products can only contain a minimum of additives and at the same time need to have a shelf life that provides optimal consumer safety. One approach to accomplish this could be the addition of biological preservatives such as bacteriocins. Bacteriocins are antimicrobial peptides produced by bacteria that can kill or inhibit other bacteria. A few bacteriocins have already reached the stage of industrial application e.g. nisin and pediocin. New bacteriocins are identified on a regular basis and are studied to determine if they have novel properties that would make them desirable as preservative. Properties which are used to judge the effectiveness of bacteriocins as preservative are activity level, activity spectrum, biostability, and bioavailability. Chapter one presents an overview of the known anti-microbial peptides produced by bacteria. Genome organization, action mechanism, bacteriocin immunity by the producer and bacteriocin resistance are discussed. Chapter two describes the effects of amino acid changes on the activity of pediocin PA-1, produced by Pediococcus acidilactici. An inducible gene expression system for the production of pediocin PA-1 mutants with altered bactericidal activities was devolped in Lactococcus lactis. In a twin-plasmid system the expression of the pediocin structural gene was effectively uncoupled from the genes that encode the pediocin secretion machinery. The secretion machinery (encoded by the pedC and pedD genes) is expressed under the control of a NaCl-inducible promoter. The effects on bacteriocin activity of amino acid changes at certain positions in the pediocin PA-1 molecule are discussed. Replacing the alanine residue at position 34 in pediocin PA-1 by a positively or negatively charged residue strongly reduces bacteriocin activity. Changing the aspartic acid residue at position 17 alters target cell specificity. Chapter three describes the purification and (partial) amino acid sequencing of two novel antibacterial peptides which are active against the cheese spoilage bacterium Clostridium tyrobutyricum: closticin 574 and circularin A produced by C. tyrobutyricum ADRIAT 932 and C. beijerinckii ATCC 25752, respectively. Based on the obtained amino acid sequences the structural genes encoding closticin 574 and circularin A were identified. Closticin 574 is synthesized as a pre-proprotein of 309 amino acids that is possibly secreted via the general secretion pathway. After secretion it is likely to be hydrolyzed at an Asp-Pro site yielding a mature antimicrobial peptide of 82 amino acid residues. Circularin A is produced as a prepeptide of 72 amino acids. Cleavage between the third and fourth amino acid followed by a head-to-tail ligation between the resulting N- terminus and C-terminus creates a circular antimicrobial peptide (Fig. 1). The occurrence of circular proteins is rare but not unprecedented: Enterocin AS-48, a homologue of circularin A produced by Enterococcus faecalis S-48 is also cyclic. Other examples of circular proteins are cyclotides (plant defense peptides) (1), the pilus proteins TrbC (Escherichia coli) and VirB2 (Agrobacterium tumefaciens) (2). The identification of genes involved in the production of circularin A is described in Chapter four. Flanking the circularin A structural gene, a region of 12 kb containing 12 putative genes was identified and sequenced. Genes in this region are organized in overlapping open reading frames, which is indicative of translational coupling. Upon heterologous expression of circularin A in E. faecalis, five genes, i.e. cirABCDE, were identified to be minimally required for bacteriocin production and secretion. CirA is the structural gene and cirE confers immunity towards circularin A. CirBD is the putative transporter, which also confers partial resistance, and CirC putatively harbors the circularization activity, either alone or in concert with CirBD. Deletion of either of the three genes cirBCD prevents bacteriocin production. Upstream of cirA, four genes are present: cfgRK encode a two-component system, cfg01 encodes a protein with homology to AgrB, a membrane protein involved in post-translational modification of an auto-inducing quorum-sensing peptide (6) and cfg02 encodes a protein lacking any homology with proteins in the available databases. Downstream of the minimal region three genes (cirGHI) were identified, of which the function has not yet been clarified. Homologues (BacGHI) of the encoded proteins CirGHI are involved in increasing the expression of enterocin AS-48 (4), while other homologues (LolCDE) are involved in removing lipid-modified proteins from the membrane of E. coli (5). Heterologous expression of circularin A was initially attempted in L. lactis but this proved to be unsuccessful due to a deleterious effect of cirB. When cirB is omitted from the minimally required region the remainder can be introduced in L. lactis, but as expected, does not lead to circularin A production in this host. Other novel putative circular bacteriocins are described in Chapter 5. This was achieved by surveying genome databases for translation products with homology to CirC. CirC was used instead of CirA, as small peptides are less likely to yield homologies and have a tendency to be under-estimated in automatically annotated genome sequences, depending on the ORF-size cutoff used in the annotation procedure. Three novel bacteriocin coding regions were identified on the basis of CirC homologs and subsequent studies of the chromosomal region: one in each of four Staphylococcus aureus strains (Mu50, N315, MW2 and 467), one in the chromosome of Geobacillus stearothermophilus DSM13240 and one in the chromosome of Oenococcus oeni PSU-1. Functions of genes in these regions are assigned on the basis of homologies of the encoded proteins to those in the CirA encoding region. The probable head-to-tail ligation sites of the novel putative bacteriocins are predicted on the basis of homology to known circular bacteriocins. An interesting challenge for future work is to confirm or disprove aspects of the working model presented here and elucidate in particular the mode of action of CirA, the mechanism behind immunity, the regulation of CirA production and secretion and, most definitely, the bacteriocin circularization mechanism.