Sensitization of Gram-Negative and Gram-Positive Bacteria to Jenseniin G by Sublethal Injury

Jenseniin G, a bacteriocin produced by Propionibacterium thoenii P126, is active against related propionibacteria and some lactic acid bacteria and is sporostatic to botulinal spores. The objective of this study was to evaluate the effects of sublethal stress on jenseniin G activity. Bacillus cereus, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, Shigella flexneri, Staphylococcus aureus, and Yersinia enterocolitica were subjected to temperature, acid, and osmotic stresses in the presence of jenseniin G. The bacteriocin reduced the viability of sublethally injured cultures, although the extent of reduction varied with strain and treatment. E. faecalis was the most sensitive to temperature stress; no reduction of viable counts occurred in the absence of jenseniin G, and a 1.5-log reduction occurred in the presence of jenseniin G. B. cereus, L. monocytogenes, and S. aureus were more sensitive to jenseniin G when exposed to heat stress than when exposed to cold stress, whereas E. coli, Salmonella Typhimurium, and S. flexneri were more sensitive to jenseniin G when exposed to cold stress than when exposed to heat stress. When comparing an acid stress test alone to a combination of acid stress and jenseniin G, E. faecalis and L. monocytogenes showed the greatest sensitivities with 4.87- and 2.82-log reductions, respectively, after 7 days. All cultures except for S. aureus were adversely affected by the combination of salt stress and jenseniin G. Salmonella Typhimurium showed the greatest sensitivity to salt stress with jenseniin G (a 1.54-log reduction at day 7) when compared to salt stress alone (a 0.55-log reduction at day 7). Jenseniin G, like bacteriocins produced by other gram-positive species, has broader activity against stressed organisms.

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.

Characterization of Bacteriocins Produced by Food Bioprocessing Propionobacteria

Objectives were to further characterize activity spectra of dairy propionibacteria bacteriocins, jenseniin G and propionicin PLG-1, purify them, examine the role of cell walls in resistance, examine their interactions with cytoplasmic membrane, explain producer immunity, and clone the responsible genes. Inhibitory spectra of both bacteriocins were further characterized. Propionicin was most effective in controlling Gram-positive, rather than Gram-negative organisms; it controlled growth of sensitive cells both in a culture medium and a model food system. Jenseniin inhibited yogurt cultures and may help prevent yogurt over-acidification. Both were active against botulinal spores; jenseniin was sporostatic; propionicin was sporicidal. Jenseniin was produced in broth culture, was stable to pH and temperature extremes, and was purified. Its molecular mass (3649 Da) and partial amino acid composition (74%) were determined. A blocked jenseniin N-terminus prevented sequencing. Methods to produce propionicin in liquid culture were improved, and large scale culture protocols to yield high titers were developed. Methods to detect and quantify propionicin activity were optimized and standardized. Stability of partially purified propionicin was demonstrated and an improved purification scheme was developed. Purified propionicin had a 9328-Da molecular mass, contained 99 amino acids, and was significantly hydrophobic; ten N-terminal amino acids were identified. Propionicin and Jenseniin interacted with cytoplasmic membranes; resistance of insensitive species was cell wall-related. Propionicin and jenseniin acted similarly; their mode of action appeared to differ from nisin. Spontaneous jenseniin-resistant mutants were resistant to propionicin but nisin-sensitive. The basis for producer immunity was not resolved. Although bacteriocin genes were not cloned, a jenseniin producer DNA clone bank and three possible vectors for cloning genes in propionibacteria were constructed. In addition, transposon Tn916 was conjugatively transferred to the propionicin producer from chromosomal and plasmid locations at transfer frequencies high enough to permit use of Tn916 for insertional mutagenesis or targeting genes in propionibacteria. The results provide information about the bacteriocins that further supports their usefulness as adjuncts to increase food safety and/or quality.