TOR coordinates nucleotide availability with ribosome biogenesis in plants.

TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic Ser/Thr protein kinase that coordinates growth and metabolism with nutrient availability. We conducted a medium-throughput functional genetic screen to discover essential genes that promote TOR activity in plants, and identified a critical regulatory enzyme, cytosolic phosphoribosyl pyrophosphate (PRPP) synthetase (PRS4). PRS4 synthesizes cytosolic PRPP, a key upstream metabolite in nucleotide synthesis and salvage pathways. We found that prs4 knockouts are embryo-lethal in Arabidopsis thaliana, and that silencing PRS4 expression in Nicotiana benthamiana causes pleiotropic developmental phenotypes, including dwarfism, aberrant leaf shape, and delayed flowering. Transcriptomic analysis revealed that ribosome biogenesis is among the most strongly repressed processes in prs4 knockdowns. Building on these results, we discovered that TOR activity is inhibited by chemical or genetic disruption of nucleotide biosynthesis, but that this effect can be reversed by supplying plants with nucleobases. Finally, we show that TOR transcriptionally promotes nucleotide biosynthesis to support the demands of ribosomal RNA synthesis. We propose that TOR coordinates ribosome biogenesis with nucleotide availability in plants to maintain metabolic homeostasis and support growth.

Use of Pantoea agglomerans ASB05 as a biocontrol agent to inhibit the growth of Salmonella enterica on intact cantaloupe melons.

AIMS: To identify biocontrol agents to prevent the growth of Salmonella serotype Enterica on cantaloupe melons during the pre- and postharvest periods. METHODS AND RESULTS: We created a produce-associated bacterial library containing 8736 isolates and screened it using an in-vitro fluorescence inhibition assay to identify bacteria that inhibit the growth of S. Enterica. One isolate, Pantoea agglomerans ASB05, was able to grow, persist, and inhibit the growth of S. Enterica on intact cantaloupe melons under simulated pre- and postharvest conditions. We also demonstrated that the growth inhibition of S. Enterica by P. agglomerans ASB05 was due to the production of a phenazine type antibiotic. CONCLUSIONS: Pantoea agglomerans ASB05 is an effective biocontrol agent for the prevention of S. Enterica growth on intact cantaloupe melons in both the pre- and postharvest environments.

Bacillus amyloliquefaciens ALB65 Inhibits the Growth of Listeria monocytogenes on Cantaloupe Melons.

Listeria monocytogenes is a foodborne pathogen that causes high rates of hospitalization and mortality in people infected. Contamination of fresh, ready to eat produce by this pathogen is especially troubling because of the ability of this bacterium to grow on produce under refrigeration temperatures. In this study, we created a library of over 8,000 plant phyllosphere-associated bacteria and screened them for the ability to inhibit the growth of L. monocytogenes in an in vitro fluorescence-based assay. One isolate, later identified as Bacillus amyloliquefaciens ALB65, was able to inhibit the fluorescence of L. monocytogenes by >30-fold in vitro. B. amyloliquefaciens ALB65 was also able to grow, persist, and reduce the growth of L. monocytogenes by >1.5 log CFU on cantaloupe melon rinds inoculated with 5 × 103 CFU at 30°C and was able to completely inhibit its growth at temperatures below 8°C. DNA sequence analysis of the B. amyloliquefaciens ALB65 genome revealed six gene clusters that are predicted to encode genes for antibiotic production; however, no plant or human virulence factors were identified. These data suggest that B. amyloliquefaciens ALB65 is an effective and safe biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons and possibly other types of produce.IMPORTANCEListeria monocytogenes is estimated by the Centers for Disease Control and Prevention and the U.S. Food and Drug Administration to cause disease in approximately 1,600 to 2,500 people in the United States every year. The largest known outbreak of listeriosis in the United States was associated with intact cantaloupe melons in 2011, resulting in 147 hospitalizations and 33 deaths. In this study, we demonstrated that Bacillus amyloliquefaciens ALB65 is an effective biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons under both pre- and postharvest conditions. Furthermore, we demonstrated that B. amyloliquefaciens ALB65 can completely inhibit the growth of L. monocytogenes during cold storage (<8°C).

Genomic Insight into Natural Inactivation of Shiga Toxin 2 Production in an Environmental Escherichia coli Strain Producing Shiga Toxin 1.

Shiga toxin-producing Escherichia coli (STEC) consists of a group of diverse strains differing greatly in genetic make-up and pathogenicity potential. Here, we investigated production of Shiga toxins (Stxs) in a bovine isolate carrying multiple Shiga toxin genes (stxs) after exposure to several antibiotics commonly used in food animals. Strain RM10809-C3 was co-isolated with a STEC O145:H28 strain from cattle feces near a leafy greens-growing region in California. The genome of RM10809-C3 is composed of a 5,128,479-bp chromosome and a 122,641-bp plasmid, encoding 5108 coding sequences. Strain RM10809-C3 belongs to serotype O22:H8 and is clustered together with two STEC O168:H8 food isolates using either multilocus sequence type or core genome-based phylogenetic analysis. Six intact prophages were identified in the genome of RM10809-C3, among which prophage 4 contained two sets of stx2d; whereas prophage 9 carried one set of stx1a. Increased production of Stx1 was detected in RM10809-C3 after exposure to mitomycin C and enrofloxacin, but not in cells exposed to tetracycline. In contrast, Stx2 remained undetectable in cells treated with any of the antibiotics examined. Comparison of Stx-converting prophages in strain RM10809-C3 with those in strain EDL933 revealed altered stx2 promoters in RM10809-C3, including deletion of the late promoter PR' and the mutations in qut, the binding site of antitermination protein Q. In contrast, both PR' and qut within the promoter of stx1 in RM10809-C3 were identical to the corresponding one in EDL933. Further, the protein Q encoded by Stx1-prophage in RM10809-C3 exhibited >94% identity with either of the two EDL933 protein Q; whereas both protein Q encoded by Stx2-prophage in RM10809-C3 were distantly related to any of the EDL933 protein Q. Natural silence of Stx2 production in strain RM10809-C3 emphasizes that not only the stx coding regions but also their regulatory factors are important in STEC risk assessment.

Biocontrol of Escherichia coli O157:H7 by Enterobacter asburiae AEB30 on intact cantaloupe melons.

Escherichia coli O157:H7 causes >73,000 foodborne illnesses in the United States annually, many of which have been associated with fresh ready-to-eat produce including cantaloupe melons. In this study, we created a produce-associated bacterial (PAB) library containing >7500 isolates and screened them for the ability to inhibit the growth of E. coli O157:H7 using an in vitro fluorescence-based growth assay. One isolate, identified by 16S and whole-genome sequence analysis as Enterobacter asburiae, was able to inhibit the growth of E. coli by ~30-fold in vitro and produced zones of inhibition between 13 and 21 mm against 12 E. coli outbreak strains in an agar spot assay. We demonstrated that E. asburiae AEB30 was able to grow, persist and inhibit the growth of E. coli on cantaloupe melons under simulated pre- and post-harvest conditions. Analysis of the E. asburiae AEB30 genome revealed an operon encoding a contact-dependent growth inhibition (CDI) system that when mutated resulted in the loss of E. coli growth inhibition. These data suggest that E. asburiae AEB30 is a potential biocontrol agent to prevent E. coli contamination of cantaloupe melons in both pre- and post-harvest environments and that its mode of action is via a CDI system.

Complete genome sequence of Citrobacter braakii ASE1 generated by PacBio sequencing.

We report the complete genome sequence of Citrobacter braakii ASE1 generated by the PacBio Sequel II platform. This bacterium was isolated from the soil of a lettuce farm in Salinas, CA, USA, in 2020. The genome consists of a single circular chromosome of 5,021,820 bp with a 52.2% GC content.

Conformation-dependent high-affinity monoclonal antibodies to prion proteins.

Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP(Sc), an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP(Sc) commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP(Sc) from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K(D) values for mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.

Sensitive and selective detection of peanut allergen Ara h 1 by ELISA and lateral flow immunoassay.

The Ara h1 protein is a peanut allergen and it provides a useful biomarker for the detection of peanut protein. In this manuscript, we describe the generation of monoclonal antibodies (MAbs) against the Ara h1 protein and their development into sensitive and selective immunoassays for peanut detection. Our enzyme-linked immunosorbent assay (sELISA) detects a peanut meal standard with a sensitivity of 10 ng/mL and 500 ng/mL by lateral flow immunoassay (LFIA). MAb Ara h1 binding epitopes were identified, and immunoassay detection was limited to peanut meal varieties irrespective of thermal treatment. No binding was observed from tree nut meals (100-0.4 µg/mL). Peanut allergen detection during food manufacturing can limit the incidence of product recall resulting from cross-contact contamination or improper labeling of finished food products. Detection of Ara h1 by immunoassay can provide a cost-effective method for rapid surveillance of peanut during food production and prior to consumption.

A rapid method to improve protein detection by indirect ELISA.

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.

Complete Genome Sequence of Enterobacter asburiae Strain AEB30, Determined Using Illumina and PacBio Sequencing.

The complete genome sequence of Enterobacter asburiae strain AEB30 is presented. The strain was isolated from store-bought ginger in Albany, CA, in 2016.

A rapid and sensitive lateral flow immunoassay (LFIA) for the detection of gluten in foods.

The gluten protein found in a variety of cereal grains is a food allergen that can elicit a spectrum of immuno-inflammatory responses in people. Consumer awareness has prompted changes in food labeling requirements, expanded gluten-free food product availability and increased demand for effective gluten testing methodologies. To meet the challenges associated with gluten testing from diverse and complex foods we developed a lateral flow immunoassay (LFIA) using a pair of novel gliadin monoclonal antibodies (MAbs). Using a visual gold reporter, we show sensitive gluten detection (150 ng/mL) from complex food substrates using a fast (<5 min) and easy testing methodology. In this report we characterize the binding properties of a cohort of newly generated gliadin monoclonal antibodies suitable for gluten detection using multiple assay formats and introduce a novel plug-n-play test strip platform with integrated test components in a single-use format.

Complete Genome Sequence of Pantoea agglomerans ASB05 Using Illumina and PacBio Sequencing.

We present the complete genome sequence of Pantoea agglomerans ASB05 and three associated plasmids, generated using a combination of the Illumina and PacBio platforms. P. agglomerans ASB05 was isolated from fresh cherries purchased in Albany, CA, in 2016.

Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms.

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

X-Ray-Based Irradiation of Larvae and Pupae of the Navel Orangeworm (Lepidoptera: Pyralidae).

The suitability of adult male the navel orangeworm, Amyelois transitella (Walker) for Sterile Insect Technique (SIT) has been reported for both high energy gamma (>1 MeV) and low energy x-ray (90 keV) sterilization. However, research regarding sterilization of NOW larvae and pupae by gamma irradiation indicated nonsuitability due to high mortality. Here, NOW larvae and pupae were irradiated to doses up to 50 Gy with 90 keV x-rays, then paired with nonirradiated colony mates. Sterility of surviving insects was determined by the presence or absence of hatched neonates. While presence of offspring does not guarantee viability, the absence does guarantee sterility (as is appropriate for SIT) and was thus the measure used here. Early stage larvae experienced 77% mortality at a dose of 30 Gy, versus 20% for nonirradiated control. At 40 Gy, mortality reached 98%. Of surviving early stage larvae at 30 Gy, 29% of moth pairs produced offspring. For late stage larvae, no offspring were produced at 40 Gy, but mortality was 73%. For pupae, mortality reached 53% at 30 Gy with 13% still producing neonates, while mortality reached 98% at 40 Gy. These results are consistent with reported results for gamma irradiation of NOW larvae where sterility was observed somewhere between the 30 Gy and 60 Gy data points, but mortality was high. This further confirms the lack of suitability of NOW irradiated in the larval stage, whether by gamma or x-ray, and supports the hypothesis that x-ray and gamma treatments are biologically equivalent at equal doses.

Complete Genomic Sequences of Three Salmonella enterica subsp. enterica Serovar Muenchen Strains from an Orchard in San Joaquin County, California.

We present here the complete genome sequences of three Salmonella enterica subsp. enterica serovar Muenchen strains, LG24, LG25, and LG26. All three strains were isolated from almond drupes grown in an orchard in San Joaquin County, California, in 2016. These genomic sequences are nonidentical and will contribute to our understanding of S. enterica genomics.

Rapid Detection of Staphylococcal Enterotoxin-B by Lateral Flow Assay.

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose-response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.

Use of Phyllosphere-Associated Lactic Acid Bacteria as Biocontrol Agents To Reduce Salmonella enterica Serovar Poona Growth on Cantaloupe Melons.

Foodborne illness associated with fresh, ready-to-eat produce continues to be a significant challenge to public health. In this study, we created a phyllosphere-associated lactic acid bacteria (PLAB) library and screened it via a high-throughput in vitro fluorescent assay to identify bacteria capable of inhibiting the growth of the pathogenic bacterium Salmonella enterica. One isolate, 14B4, inhibited the growth of S. enterica by >45-fold in vitro; it was able to grow and persist on the surfaces of cantaloupe melons at both ambient (25°C) and refrigerator (5°C) temperatures. Isolate 14B4 inhibited the growth of S. enterica on the surfaces of cantaloupes by >3 log when incubated at 25°C for 24 h and by >4 log when the cantaloupes were stored at 5°C for 3 days and the temperature was shifted to 25°C for 2 days. Genomic DNA sequence analysis of isolate 14B4 revealed that it was Lactococcus lactis and that it did not contain any known antibiotic biosynthesis gene clusters, antibiotic resistance genes, or genes encoding any known virulence factors. Organic acid analysis revealed that L. lactis produces substantial amounts of lactic acid, which is likely the inhibitory substance that reduced the growth of Salmonella on the cantaloupes.

Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E.

Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A-G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of BoNT/E3. Monoclonal antibodies (mAbs) were generated against BoNT/E3 by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E3. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay was constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals.

Dosage-dependent effects of monensin on the rumen microbiota of lactating dairy cattle.

We examined the dose-dependent effects of feeding lactating dairy cows a standard diet supplemented with monensin at 175, 368, or 518 mg cow-1 day-1 on the rumen microbiota. For each dosage, 3 animals were randomly assigned into groups and fed the same basal total mixed ration diet supplemented with monensin, at the respective dose. After 20 days, rumen samples were taken and the effect on the microbiota was examined by 16S rRNA gene sequence analysis and qPCR. At the lowest dose no significant change in 16S rRNA gene sequences associated with any bacterial phyla was observed; however, at the medium and high dosages, we observed significant reductions in sequences associated with gram-positive bacteria and significant increases in those associated with gram-negative bacteria that were dosage dependent. All dosages reduced the levels of sequences associated with methanogenic archaea in the rumen, with the medium dosage showing the largest decline. No significant difference was observed for the 18S rRNA gene sequences associated with protozoa in any of the libraries. Our results indicate that with this diet the medium dosage of monensin was most efficacious for the reduction in methanogenic archaea in the rumen of lactating dairy.

A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins.

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL-1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for ß-amanitin (ß-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.