C. Class IIc bacteriocins
1.4 Bacteriocins Produced by Leuconostoc Species
Some of the Leuconostoc strains may sometimes even produce more than one type of bacteriocin. Leuconostoc mesenteroides subsp. mesenteroides FR52 is a good example of this scenario as this strain produces mesentericin 52A (which is identical to mesentericin Y105) mesentericin 52B, and leucocin C (Corbier et al., 2001; Fimland et al., 2002b). Furthermore, mesentericin 52B has been detected in culture extracts of L. mesenteroides Y105.
Papthanasopoulos et al. (1998) reported that L. mesenteroides TA33a produces leucocin A-;
B- and C-TA33a. This study found that the partial sequence of leucocin C-TA33a shares 80 to 78% homology with leucocin A UAL187-22, leucocin B-TA11a, and mesentericin Y105 (table 1.2). In addition, it was also found that leucocin C-TA33a appears to be similar to other Leuconostoc bacteriocins at the N-terminus, but more similar to other known class II bacteriocins at the C-terminus (Papthanasopoulos et al., 1998). This high homology indicates the close relation within the different strains of the same species.
Table 1.2. Multiple alignment of the amino acid sequence of leucocin C-TA33a and other class IIa bacteriocins. Dashes represent gaps introduced to optimize the alignment, and consensus amino acids are highlighted (Papthanasopoulos et al., 1998).
Bacteriocin Sequence
Leucocin C-TA33a --KNYGNG-VHCTKKGCSVDWGYAAT---NIANNSVMNGLTG---
Leucocin B-TA11a --KYYGNG-VHCTKSGCSVNWGEAFS---AGVHRLANGGNGFW--
Leucocin A-UAL187-22 --KYYGNG-VHCTKSGCSVNWGEAFS---AGVHRLANGGNGFW--
Mesentericin Y105 --KYYGNGVVHCTKSGCSVNWGEAAS---AGIHRLANGGNGFW--
1.4.1 Leuconostoc gelidum
Leucocin A is a class IIa bacteriocin produced by Leuconostoc gelidum UAL187-22, which is isolated from vacuum packaged meat (Hastings et al., 1991). This bacteriocin inhibits a wide spectrum of LAB, meat spoilage bacteria, and Listeria monocytogenes, a food-borne pathogen widely distributed in the environment (van Belkum & Stiles, 1995; Gaeng et al., 2000).
Molecular characterization of the genes involved in bacteriocin production revealed that the genetic determinants are located on two of the three plasmids namely pLG 7.6 and pLG 9.2 (van Belkum & Stiles, 1995). Studies involving molecular cloning revealed that the genetic determinants for leucocin A production are arranged in an operon like structure arranged as two open reading frames (ORFs) (figure 1.5a) (Stiles, 1993). The red arrow on figure 1.5a represents the ORF encoding 61 amino acids and was identified as the leucocin structural gene. Van Belkum and Stiles (1995) determined that the second ORF (lcaB) encodes the immunity protein, which is responsible for conferring immunity to the producer organism.
Molecular cloning by van Belkum and Stiles (1995) further confirmed the presence of additional open reading frames termed lcaC, lcaD, and lcaE. Homology comparisons of lcaC with other proteins showed distinctive similarities with secretory proteins that differ from those involved in the general signal sequence-dependent export pathway. LcaC contains a highly conserved ATP-binding domain in the C-terminus and several hydrophobic domains towards the N-terminus. Furthermore, a high degree of homology of lcaC with other ABC transporters including mesD and pedD was also observed. This data indicated that lcaC belongs to the family of ABC-transporters and is responsible for the extracellular translocation and activation of leucocin A (Fregeau-Gallagher et al., 1997). LcaD was shown to be associated with bacteriocin production while the role of lcaE remains unclear (van Belkum & Stiles, 1995).
1.4.2 Leuconostoc mesenteroides
Leuconostoc mesenteroides subsp. mesenteroides is used industrially to produce dextrans and is the most common species isolated from fermented feed and food products. In addition, its recognized antagonistic or synergistic properties in mixed microflora populations have led to studies on its importance in some starter cultures (Moschetti et al., 2000).
Mesentericin Y105 is a 37 amino acid class IIa bacteriocin produced by Leuconostoc mesenteroides Y105 (Morisset et al., 2004). The structural gene encoding mesentericin Y105 designated mesY is located on a 35-kb plasmid (pHY30). All genes responsible for mesentericin Y105 production are organized in an operon like fashion constituting five ORFs (figure 1.5b). Each cluster, however, formed a separate operon that was surprisingly transcribed in a different and opposite direction from the other (Fremaux et al., 1995). The immunity gene (mesI) is situated directly downstream from mesY. A unique feature of these gene clusters was the presence of a stem loop structure, downstream from mesI. This structure is believed to be the terminator flanking mesI gene (Morisset et al., 2004). Three additional ORFs have also been located and have been termed mesC, mesD and mesE. MesD resembled atypical ATP-dependent transporter, while mesE encodes for the accessory protein that acts as a membrane anchor and assists the transporter protein. The function of mesC however, remains unknown (Fremaux et al., 1995).
A large amount of studies have been conducted on the genes encoding bacteriocins, mesentericin Y105 and leucocin A (Morisset et al., 2004). These revealed the presence of sequence homology between mesI and lcaB. Additional homology was found between mesD and the gene encoding an ATP-dependent transporter for leucocin A (lcaC) (van Belkum &
Stiles, 1995). This raised questions about the similarities existing between mesentericin Y105 and leucocin-A UAL187 both on their antimicrobial activities and structure.
Figure 1.5. Organization of the gene clusters involved in the production and immunity of (a) leucocin A and (b) mesentericin Y105. Open reading frames are represented by arrows with the corresponding colors: bacteriocin pre-peptide (red), immunity genes (black), ABC- transporter (dark blue), product with unidentified function (white). The number of amino acid residues encoded by each ORF is indicated below each arrow. Promoters are represented by green boxes and terminators by red lollipops. With respect to carnobacteriocin B2 and BM1, the same ORFs exist (Ennahar et al., 2000).