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Internal compartmentalization of metals is an important metal tolerance mechanism in many organisms. In plants and fungi, sequestration into the vacuole is a major detoxification mechanism for metals. Cation transport into the vacuole can be mediated by CAX (cation exchanger) transporters. The Arabidopsis thaliana AtCAX2 transporter was shown previously to transport Ca(2+), Cd(2+) and Mn(2+). To assess the conservation of the functional and regulatory characteristics of CAX2-like transporters in higher plants, we have characterized AtCAX2 orthologues from Arabidopsis (AtCAX5), tomato (LeCAX2) and barley (HvCAX2). Substrate specificity and regulatory activity were assessed using a yeast heterologous-expression assay. Each CAX could transport Ca(2+) and Mn(2+) into the yeast vacuole, but they each had different cation transport kinetics. Most notably, there was variation in the regulation of the transporters. As found with AtCAX2 previously, only expression of an N-terminally truncated form of AtCAX5 in yeast was able to mediate Ca(2+) and Mn(2+) transport, indicating that activity may be controlled by an autoregulatory region at the N-terminus. In contrast, either full-length or truncated LeCAX2 could efficiently transport Ca(2+), although Mn(2+) transport was controlled by the N-terminus. HvCAX2 did not appear to possess an N-terminal regulatory domain. Expression of AtCAX2 was not significantly modulated by metal stress; however, AtCAX5 and HvCAX2 were transcriptionally up-regulated by high Mn(2+) treatment, and by Ca(2+) and Na(+) stress respectively. It is therefore apparent that, despite the high sequence identity between plant CAX2 orthologues, there is significant diversity in their functional characteristics, particularly with regard to regulatory mechanisms.

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Seven citrus essential oils (EOs) were screened by disc diffusion assay for their antibacterial act against 11 serotypes/strains of Salmonella. The 3 most active oils were selected to determine the minimal inhibitory concentration (MIC) against the same Salmonella. Orange terpenes, single-folded d-limonene, and orange essence terpenes all exhibited inhibitory activity against the Salmonella spp. on the disc diffusion assay. EOs were stabilized in broth by the addition of 0.15% (w/v) agar for performance of the MIC tests. Orange terpenes and d-limonene both had MICs of 1%. The most active compound, terpenes from orange essence, produced an MIC that ranged from 0.125% to 0.5% against the 11 Salmonella tested. Gas chromatography-mass spectrometry (GC/MS) analysis revealed that this orange essence oil was composed principally of d-limonene, 94%, and myrcene at about 3%. EOs from citrus offer the potential for all natural antimicrobials for use in improving the safety of organic or all natural foods.

The objectives of this study were to screen activity of citrus essential oil fractions (EOs) alone and in combination with organic acids against 2 species of Listeria. Five citrus EOs were initially screened by disc diffusion assay for antibacterial activity. Cold pressed terpeneless Valencia orange oil (CP terpeneless oil) had the strongest bacteriostatic (MIC) and bactericidal (MBC) properties at 0.55% and 1.67%, respectively. Four organic acids were tested for effectiveness against Listeria. Citric and malic acids proved to be the most effective with MBC of 1.1% alone. Assays were conducted to determine synergistic effects of EOs and citric or malic acids. There was a significant decrease in MIC and MBC to 0.04% EO plus 0.12% malic or citric acid. EOs from citrus paired with organic acids offer the potential as an all-natural antimicrobial for improving the safety of all-natural foods.

Due to increasing concerns about the development of antimicrobial resistance amongst pathogenic bacteria, alternative strategies have been sought that do not use antibiotics to reduce pathogenic bacteria from foods and patients. A natural compound that has potent antimicrobial properties is citrus peel, which contains a variety of essential oils that inhibit the growth of or kill pathogenic bacteria. In the present study, seven citrus-based natural antimicrobials were evaluated for their ability to inhibit the growth of the pathogen Escherichia coli O157:H7. Zones of inhibition of E. coli O157:H7 by the citrus-derived fraction (10 microL/6 mm disk) were determined by a disk-diffusion assay on Sorbitol-MacConkey agar. Inhibition zones were observed after 48 h lawn growth of E. coli O157:H7 cells at 37 degrees C. Two citrus-based fractions, orange CP VAL terpeneless FAB 968611 and Limonene 1x Dist FAB 955430, inhibited E. coli O157:H7 with inhibition zones of approx. 11-24 mm dia. The remaining other five citrus-derived extracts (orange oil FL VAL 1121 ARR 974760, Orange 5x Conc VAL 4121 ARR 968374, orange terpenes ESS 1120 ARR 986259, orange terpenes CP 1100 ARR 986255, and orange terpenes OEO HP 1100 ARR 986257) were noninhibitory to E. coli O157:H7, yielding no clear inhibition zones. These studies show that citrus-derived natural compounds differ in their inhibitory activity against E. coli O157:H7 and some have potential applications as inhibitory agents against E. coli O157:H7 in various pathogen reduction strategies.

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