Immunochemical characterization of temperature-regulated production of enterocin L50 (EntL50A and EntL50B), enterocin P, and enterocin Q by Enterococcus faecium L50

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47 degrees C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32 degrees C, but production becomes negligible when the growth temperature is above 37 degrees C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47 degrees C. Maximum EntL50A and EntL50B production was detected at 25 degrees C, while EntP and EntQ are maximally produced at 37 and 47 degrees C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.     

Complete Sequence of the Enterocin Q-Encoding Plasmid pCIZ2 from the Multiple Bacteriocin Producer Enterococcus faecium L50 and Genetic Characterization of Enterocin Q Production and Immunity

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.     

Complete sequence of the enterocin Q-encoding plasmid pCIZ2 from the multiple bacteriocin producer Enterococcus faecium L50 and genetic characterization of enterocin Q production and immunity

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.     

Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q

Enterococcus faecium L50 grown at 16 to 32 degrees C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47 degrees C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Havarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.     

Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1_s) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter P_GAL1. The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFα1_s-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system.Lactic acid bacteria (LAB) are widely known for their ability to produce a variety of ribosomally synthesized proteins or peptides, referred to as bacteriocins, displaying antimicrobial activity against a broad range of gram-positive bacteria and, to a lesser extent, gram-negative bacteria, including spoilage and food-borne pathogenic microorganisms (11, 19, 33, 34, 36, 37). These antimicrobials may be classified into three main classes: (i) the lantibiotics, or posttranslationally modified peptides; (ii) the nonmodified, small, heat-stable peptides; and (iii) the large, heat-labile protein bacteriocins. Class II bacteriocins are further grouped into five subclasses: the subclass IIa (pediocin-like bacteriocins containing the N-terminal conserved motif YGNGVxC), the subclass IIb (two-peptide bacteriocins), the subclass IIc (leaderless bacteriocins), the subclass IId (circular bacteriocins), and the subclass IIe (other peptide bacteriocins) (17, 19, 21, 37). All lantibiotics and most class II bacteriocins are synthesized as biologically inactive precursors containing an N-terminal extension (the so-called double-glycine-type leader sequence or the Sec-dependent signal peptide), which is cleaved off concomitantly with externalization of biologically active bacteriocins by a dedicated ATP-binding cassette transporter and its accessory protein or by the Sec system and the signal peptidases, respectively (11, 17). Interestingly, only a few bacteriocins described to date are synthesized without an N-terminal extension, including enterocin L50 (L50A and L50B) (8), enterocin Q (EntQ) (10), enterocin EJ97 (41), and the bacteriocin LsbB (20).In recent years, there has been an increasing interest in the application of bacteriocinogenic microorganisms and/or their bacteriocins as biopreservatives to guarantee the safety and quality of foods and beverages, such as fermented vegetables and meats, dairy and fish products, and wine and beer (12, 15, 16, 39, 47). Three main strategies for the use of bacteriocins as food biopreservatives have been proposed: (i) addition of a purified/semipurified bacteriocin preparation as a food additive; (ii) use of a substrate previously fermented by a bacteriocin-producing strain as a food ingredient; and/or (iii) inoculation of a culture to produce the bacteriocin in situ in fermented foods (13, 15). The lantibiotic nisin A is the most widely characterized bacteriocin and the only one that has been legally approved in more than 48 countries as a food additive for use in certain types of cheeses (13, 16). Likewise, nisin A has been approved as a beer additive in Australia and New Zealand (16). However, the difficulties encountered in addressing the regulatory approval of new bacteriocins as food additives have spurred the development of the other bacteriocin-based food biopreservation strategies (13, 17).Beer is a beverage with a remarkable microbiological stability and is considered as a food substrate difficult to spoil. However, some LAB, such as Lactobacillus brevis, Lactobacillus lindneri, and Pediococcus damnosus, are able to spoil beer and are recognized as the most hazardous bacteria for breweries, being responsible for approximately 70% of microbial beer spoilage incidents (40, 47). The ever-growing consumer demand for less-processed and less chemically preserved foods and beverages is promoting the development of alternative biocontrol strategies, such as those based on the use of bacteriocins as biopreservatives (12, 15, 39, 47). However, beyond the strict requirements to fulfill legal regulations, the commercial application of bacteriocins as beer additives is hindered mainly by low bacteriocin production yields and increases in production costs (44). Considering that Saccharomyces cerevisiae is commonly used as starter culture for brewing (24, 28, 35), a novel beer biopreservation strategy based on the development of bactericidal S. cerevisiae brewing strains has been proposed to overcome the aforementioned challenges (44, 46, 47). In this respect, the heterologous production of LAB bacteriocins, namely, pediocin PA-1 (PedPA-1) from Pediococcus acidilactici PAC1.0 and plantaricin 423 from Lactobacillus plantarum 423, by laboratory strains of S. cerevisiae has been reported (44, 46).Enterocin L50 (EntL50) is a commonly found bacteriocin composed of two highly related leaderless antimicrobial peptides, enterocin L50A (EntL50A) and enterocin L50B (EntL50B), which possesses a broad antimicrobial spectrum against LAB, food-borne pathogenic bacteria, and human and animal clinical pathogens (8, 9, 10, 11). Previous work by our group showed that EntL50 (EntL50A and EntL50B) may be used as a beer biopreservative to inhibit the growth of beer spoilage bacteria (1). Therefore, genetically engineered strains of S. cerevisiae heterologously expressing and secreting EntL50A and EntL50B have been developed in this work. For this purpose, we constructed the segregationally stable expression and secretion vector pYABD01, which allowed the secretion of biologically active EntL50A and EntL50B directed by MFα1_s through the S. cerevisiae Sec system.     

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.     

Sec-Mediated Secretion of Bacteriocin Enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Improved Antimicrobial Activities of Synthetic-Hybrid Bacteriocins Designed from Enterocin E50-52 and Pediocin PA-1

Two hybrid bacteriocins, enterocin E50-52/pediocin PA-1 (EP) and pediocin PA-1/enterocin E50-52 (PE), were designed by combining the N terminus of enterocin E50-52 and the C terminus of pediocin PA-1 and by combining the C terminus of pediocin PA-1 and the N terminus of enterocin E50-52, respectively. Both hybrid bacteriocins showed reduced MICs compared to those of their natural counterparts. The MICs of hybrid PE and EP were 64- and 32-fold lower, respectively, than the MIC of pediocin PA-1 and 8- and 4-fold lower, respectively, than the MIC of enterocin E50-52. In this study, the effect of hybrid as well as wild-type (WT) bacteriocins on the transmembrane electrical potential (ΔΨ) and their ability to induce the efflux of intracellular ATP were investigated. Enterocin E50-52, pediocin PA-1, and hybrid bacteriocin PE were able to dissipate ΔΨ, but EP was unable to deplete this component. Both hybrid bacteriocins caused a loss of the intracellular concentration of ATP. EP, however, caused a faster efflux than PE and enterocin E50-52. Enterocin E50-52 and hybrids PE and EP were active against the Gram-positive and Gram-negative bacteria tested, such as Micrococcus luteus, Salmonella enterica serovar Enteritidis 20E1090, and Escherichia coli O157:H7. The hybrid bacteriocins designed and described herein are antimicrobial peptides with MICs lower those of their natural counterparts. Both hybrid peptides induce the loss of intracellular ATP and are capable of inhibiting Gram-negative bacteria, and PE dissipates the electrical potential. In this study, the MIC of hybrid bacteriocin PE decreased 64-fold compared to the MIC of its natural peptide counterpart, pediocin PA-1. Inhibition of Gram-negative pathogens confers an additional advantage for the application of these peptides in therapeutics.     

Phenotypic and genotypic characterization of bacteriocins in clinical <Emphasis Type="Italic">enterococcal</Emphasis> isolates of Tunisia

The presence of bacteriocin structural genes (entA, entB, entP, entQ, entAS-48, entL50A/B, bac31, and cylL) encoding different bacteriocins (enterocin A, enterocin B, enterocin P, enterocin Q, enterocin AS-48, enterocin L50A/B, bacteriocin 31 and cytolysin L, respectively), and the production of bacteriocin activity were analysed in 139 E. faecalis and 41 E. faecium clinical isolates of Tunisia. Forty-eight of 139 E. faecalis isolates (34%) and 7 of 41 of E. faecium isolates (17%) were bacteriocin producers. Sixty-two per cent of the bacteriocin-producing enterococci showed inhibitory activity against L. monocytogenes. Different combinations of entA, entB, entP, and entL50A/B genes were detected among the seven bacteriocin-producer E. faecium isolates, and more that one gene were identified in all the isolates. The entA gene was associated in most of the cases with entB gene in E. faecium isolates. Cyl _LS were the unique genes detected among E. faecalis (in 24 of 48 bacteriocin-producer isolates, 50%). A β-hemolytic activity was demonstrated in 19 of the 24 cyl _LS -positive E. faecalis isolates (79%), this activity being negative in the remaining five isolates. The presence of different bacteriocin structural genes and the production of antimicrobial activities seems to be a common trait of clinical enterococci.     

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Effect of Immersion Solutions Containing Enterocin AS-48 on Listeria monocytogenes in Vegetable Foods

The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of Listeria monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 μg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 μg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15°C, as well as green asparagus at 15°C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22°C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P₃₂, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.     

Seedling growth, mitochondrial characteristics, and alternative respiratory capacity of corn genotypes differing in cold tolerance

Four maize (Zea mays L.) inbreds representing genetic differences in seedling cold tolerance were used to determine the effect of growth temperatures on dry weight accumulation and mitochondrial properties, especially the alternative oxidase capacity. Seedlings were grown in darkness at 30°C (constant), 14°C (constant), and 15°C for 16 hours and 8°C for 8 hours. Inbreds B73 and B49 were characterized as cold tolerant while G50 and G84 were cold sensitive. Shoot growth rate of cold-sensitive inbreds in the lower temperatures was slower relative to the tolerant inbreds. Mesocotyl tissue was particularly sensitive to low temperatures during growth after germination. There were no significant differences in relative rates of mitochondrial respiration in the cold-tolerant compared to cold-sensitive inbreds measured at 25°C. Mitochondria from all seedlings grown at all temperatures had the ability to phosphorylate as indicated by the observation of respiratory control. This result indicated that differences in low temperature growth were probably not related to mitochondrial function at low temperatures. Alternative oxidase capacity was higher in mitochondria from seedlings of all inbreds grown at 14°C compared to 30°C. Capacities in seedlings of 14°C-grown B73 and G50 were higher than in B49 and G84. Capacities in seedlings grown for 16 hours at 15°C and 8 hours at 8°C were similar to those from 14°C-grown except in G50 which was lower and similar to those grown at 30°C. Mesocotyl tissue was the most responsive tissue to low growth temperature. Coleoptile plus leaf tissue responded similarly but contained lower capacities. Antibody probing of western blots of mitochondrial proteins confirmed that differences in alternative oxidase capacities were due to differences in levels of the alternative oxidase protein. Male sterile lines of B73 were also grown under the three different temperature regimes. These lines grew equally as well as the normal B73 at all temperatures and the response of alternative oxidase capacity and protein to low growth temperature was similar to normal B73.     

Chilling-Induced Ethylene Production in Cucumbers (Cucumis sativus L.)

1-Aminocyclopropane-1-carboxylic acid (ACC) level, ACC synthase activity, and ethylene production in cucumbers (Cucumis sativus L.) remain low while the fruit are held at a temperature which causes chilling injury (2.5°C) and increase rapidly only upon transfer to warmer temperatures. The increase in ACC synthase activity during the warming period is inhibited by cycloheximide but not cordycepin or α-amanitin. Our data indicate that the synthesis of ACC synthase, which results in increased ACC levels and accelerated ethylene production, occurs only upon warming, possibly from a message produced or unmasked during the chilling period. Ethylene production by chilled (2.5°C) cucumbers increased very little upon transfer to 25°C if the fruit were chilled for more than 4 days. The fruit held for 4 days or longer showed a large increase in ACC levels but little ethylene production even in the presence of exogenous ACC. This suggests that the system which converts ACC to ethylene is damaged by prolonged exposure to the chilling temperature. Cucumbers stored at a low but nonchilling temperature (13°C) showed very little change in ACC level, ethylene production, or ACC synthase activity even after transfer to 25°C.     

Bacterial Resistance to Ultrasonic Waves under Pressure at Nonlethal (Manosonication) and Lethal (Manothermosonication) Temperatures

The decimal reduction times of Streptococcus faecium, Listeria monocytogenes, Salmonella enteritidis, and Aeromonas hydrophila corresponding to heat treatment at 62°C were 7.1, 0.34, 0.024, and 0.0096 min, and those corresponding to manosonication treatment (40°C, 200 kPa, 117 μm) were 4.0, 1.5, 0.86, and 0.90 min, respectively. The manosonication decimal reduction times of the four species investigated decreased sixfold when the amplitude was increased from 62 to 150 μm and fivefold when the relative pressure was raised from 0 to 400 kPa. In L. monocytogenes, S. enteritidis, and A. hydrophila, the lethal effect of manothermosonication was the result of the addition of the lethal effects of heat and manosonication, whereas in S. faecium it was a synergistic effect.     

NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V_1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V_1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.     

Temperature response of photosynthesis in different drug and fiber varieties of Cannabis sativa L.

The temperature response on gas and water vapour exchange characteristics of three medicinal drug type (HP Mexican, MX and W1) and four industrial fiber type (Felinq 34, Kompolty, Zolo 11 and Zolo 15) varieties of Cannabis sativa, originally from different agro-climatic zones worldwide, were studied. Among the drug type varieties, optimum temperature for photosynthesis (T_opt) was observed in the range of 30–35 °C in high potency Mexican HPM whereas, it was in the range of 25–30 °C in W1. A comparatively lower value (25 °C) for T_opt was observed in MX. Among fiber type varieties, T_opt was around 30 °C in Zolo 11 and Zolo 15 whereas, it was near 25 °C in Felinq 34 and Kompolty. Varieties having higher maximum photosynthesis (P_{N max}) had higher chlorophyll content as compared to those having lower P_{N max}. Differences in water use efficiency (WUE) were also observed within and among the drug and fiber type plants. However, differences became less pronounced at higher temperatures. Both stomatal and mesophyll components seem to be responsible for the temperature dependence of photosynthesis (P_N) in this species, however, their magnitude varied with the variety. In general, a two fold increase in dark respiration with increase in temperature (from 20 °C to 40 °C) was observed in all the varieties. However, a greater increase was associated with the variety having higher rate of photosynthesis, indicating a strong association between photosynthetic and respiratory rates. The results provide a valuable indication regarding variations in temperature dependence of P_N in different varieties of Cannabis sativa L.