Bacteriocin Markers for Propionibacteria Gene Transfer Systems

The antibotulinal baceriocins, propionicin PLG-1 and jenseniin G., were the first to be identified, purified and characterized for the dairy propionibaceria and are produced by Propionibacterium thoenii P127 and P. thoenii/jensenii P126, respectively. Objectives of this project were to (a) produce polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1; (b) identify, clone and characterize the propionicin PLG-1 (plg-1) and jenseniin G (jnG) genes; and (3) develop gene transfer systems for dairy propionibacteria using them as models. Polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1 were produced in rabbits. Anti-PLG-1 antiserum had high titers (256,000 to 512,000), neutralized PLG-1 activity, and detected purified PLG-1 at 0.10 mg/ml (indirect ELISA) and 0.033 mg/ml (competitive indirect ELISA). Thirty-nine of 158 strains (most P. thoenii or P. jensenii) yielded cross-reacting material; four strains of P. thoenii, including two previously unidentified bacteriocin producers, showed biological activity. Eight propionicin-negative P127 mutants produced neither ELISA response nor biological activity. Western blot analyses of supernates detected a PLG-1 band at 9.1 kDa and two additional protein bands with apparent molecular weights of 16.2 and 27.5 kDa. PLG-1 polyclonal antibodies were used for detection of jenseniin G. PLG-1 antibodies neutralized jenseniin G activity and detected a jenseniin G-sized, 3.5 kDa peptide. Preliminary immunoprecipitation of crude preparations with PLG-1 antibodies yielded three proteins including an active 3-4 kDa band. Propionicin PLG-1 antibodies were used to screen a P. jensenii/thoenii P126 genomic expression library. Complete sequencing of a cloned insert identified by PLG-1 antibodies revealed a putative response regulator, transport protein, transmembrane protein and an open reading frame (ORF) potentially encoding jenseniin G. PCR cloning of the putative plg-1 gene yielded a 1,100 bp fragment with a 355 bp ORF encoding 118 amino acids; the deduced N-terminus was similar to the known PLG-1 N-terminus. The 118 amino acid sequence deduced from the putative plg-1 gene was larger than PLG-1 possibly due to post-translational processing. The product of the putative plg-1 gene had a calculated molecular weight of 12.8 kDa, a pI of 11.7, 14 negatively charged residues (Asp+Glu) and 24 positively charged residues (Arg+Lys). The putative plg-1 gene was expressed as an inducible fusion protein with a six-histidine residue tag. Metal affinity chromatography of the fused protein yielded a homogeneous product. The fused purified protein sequence matched the deduced putative plg-1 gene sequence. The data preliminarily suggest that both the plg-1 and jnG genes have been identified and cloned. Demonstrating that antibodies can be produced for propionicin PLG-1 and that those antibodies can be used to detect, monitor and compare activity throughout growth and purification was an important step towards monitoring PLG-1 concentrations in food systems. The unexpected but fortunate cross-reactivity of PLG-1 antibodies with jenseniin G led to selective recovery of jenseniin G by immunoprecipitation. Further refinement of this separation technique could lead to powerful affinity methods for rapid, specific separation of the two bacteriocins and thus facilitate their availability for industrial or pharmaceutical uses. Preliminary identification of genes encoding the two dairy propionibacteria bacteriocins must be confirmed; further analysis will provide means for understanding how they work, for increasing their production and for manipulating the peptides to increase their target species. Further development of these systems would contribute to basic knowledge about dairy propionibacteria and has potential for improving other industrially significant characteristics.