Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychrotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common. Lactic acid bacteria are commonly isolated from meats (7, 31, 32). These organisms can inhibit the natural microflora of meat, which include spoilage bacteria and, if present, pathogens such as Listeria monocytogenes and Staphylococcus aureus (3). Lactic acid bacteria can produce a variety of antimicrobial agents, including organic acids, diacetyl, and hydrogen peroxide (15). Minimally processed meat products which rely solely on refrigeration for preservation are particularly susceptible to growth by spoilage bacteria and pathogens that are psychrotrophic. Preservation of these products by production of organic acids or diacetyl would be organoleptically unacceptable. While it is not a highly conserved characteristic, some lactic acid bacteria produce bacteriocins (21). Bacteriocins produced by gram-positive bacteria are biologically active proteins demonstrating a bactericidal mode of action (34). Certain bacteriocins produced by lactic acid bacteria inhibit a variety of food-borne pathogens, including Bacillus cereus, Clostridium perfringens, Listeria species, and S. aureus (9, 17, 32, 33). This suggests that bacteriocin-producing lactic acid bacteria may be useful as natural preservatives. However, there have been no studies comparing the activity of these bacteriocins against the same panel of pathogens under well-controlled experimental conditions. L. monocytogenes and Aeromonas hydrophila grow at * Corresponding author. t This is manuscript D-10564-3-90 of the New Jersey State Agricultural Experiment Station.

The basal proton motive force (PMF) levels and the influence of the bacteriocin nisin on the PMF were determined in Listeria monocytogenes Scott A. In the absence of nisin, the interconversion of the pH gradient (ZApH) and the membrane potential (A4) led to the maintenance of a fairly constant PMF at-160 mV over the external pH range 5.5 to 7.0. The addition of nisin at concentrations of .5 tag/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 ig of nisin per ml, ApH was completely dissipated but A* decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only A*, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only ApH, under conditions in which nisin depleted both. Four other L. monocytogenes strains had basal PMF parameters similar to those of strain Scott A. Nisin (2.5 ,ug/ml) also completely dissipated PMF in these strains.

Bavaricin MN was purified from Lactobacillus sake culture supernatant 135-fold with a final yield of 11%. Sequence analysis revealed bavaricin MN to be a 42-amino-acid peptide having a molecular weight of 4,769 and a calculated pI of 10.0. Computer analysis indicated that the C-terminal region may form an ␣-helical structure with an amphipathic nature deemed important in the interaction of bacteriocins with biological membranes. Bavaricin MN rapidly depleted the membrane potential (⌬p) of energized Listeria monocytogenes cells in a concentration-dependent fashion. At a bavaricin MN concentration of 9.0 g/ml, the ⌬p decreased by 85%. Both the electrical potential (⌬⌿) and Z⌬pH components of the ⌬p were depleted, and this depletion was not dependent on a threshold level of proton motive force. In addition to studying the effect of bavaricin MN on the ⌬p of vegetative cells, bavaricin MN-induced carboxyfluorescein (CF) efflux from L. monocytogenes-derived lipid vesicles was also characterized. Bavaricin MN-induced CF leakage was also concentration dependent with an optimum of pH 6.0. The rate of CF efflux was 63% greater in lipid vesicles in which a ⌬⌿ was generated compared with that in lipid vesicles in the absence of a ⌬⌿.