ACC Deaminase from <i>Lysobacter gummosus</i> OH17 Can Promote Root Growth in <i>Oryza sativa</i> Nipponbare Plants

Although <i>Lysobacter</i> species are a remarkable source of natural compounds with antibacterial and antifungal activities, the ability of these bacteria to produce plant growth promoters remains practically unknown. In this work, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) has been isolated from the secretions of <i>Lysobacter gummosus</i> OH17, indicating the presence of an ACC deaminase, which was shown to be encoded in the gene <i>peg_1256</i>. The recombinant enzyme could not only deaminate ACC to provide 2-oxobutanoic acid but also catalyzed the amination of the 2-oxobutanoic acid, demonstrating, for the first time, that ACC deaminases can produce ACC. After the treatment of rice <i>Oryza sativa</i> Nipponbare plants with OH17 ACC deaminase, the ethylene production levels were 44% higher in comparison with the control experiments, allowing significant improvements in root, 10%, and stem, 14%, growth

Production of Antifungal <i>p</i>‑Aminobenzoic Acid in <i>Lysobacter antibioticus</i> OH13

Among <i>Lysobacter</i> species, <i>Lysobacter antibioticus</i> has been demonstrated to be an interesting source of antimicrobial metabolites for the biocontrol of plant diseases. Although the antibacterial activity was attributed to <i>N</i>-oxide phenazines, the active compounds involved in the antifungal function remained unknown. In this work, an antifungal compound was isolated and identified as <i>p</i>-aminobenzoic acid (pABA). Antifungal activity screening revealed that pABA shows activity against a number of plant pathogens. The genes involved in the synthetic route of this compound in OH13 were identified. Further, the production of pABA was optimized by modification of the carbon source using engineered <i>L. antibioticus</i> OH13 strains

Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in <i>Lysobacter enzymogenes</i> OH11

The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by <i>Lysobacter</i> and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production. We first used the CRISPR/dCas9 system to increase the expression of five cotranscribed genes (<i>orf1–5</i>) in the WAP gene cluster, by fusing the omega subunit of RNA polymerase with dCas9 that targets the operon’s promoter region. This led to the transcription of the genes increased by 5–48 folds in strain dCas9-ω3. We then refactored four putative self-protection genes (<i>orf6</i>, <i>orf7</i>, <i>orf9</i> and <i>orf10</i>) by reorganizing them into an operon under the control of a strong <i>Lysobacter</i> promoter, P_HSAF. The refactored operon was introduced into strain dCas9-ω3, and the transcription of the self-protection genes increased by 20–60 folds in the resultant engineered strains. The yield of the three main WAP-8294A compounds, WAP-8294A1, WAP-8294A2, and WAP-8294A4, increased by 6, 4, and 9 folds, respectively, in the engineered strains. The data also showed that the yield increase of WAP-8294A compounds was mainly due to the increase of the extracellular distribution. WAP-8294A2 exhibited potent (MIC 0.2–0.8 μg/mL) and specific activity against <i>S. aureus</i> among a battery of clinically relevant Gram-positive pathogens (54 isolates)

Development of an Enzyme Linked Immunosorbent Assay and an Immunochromatographic Assay for Detection of Organophosphorus Pesticides in Different Agricultural Products

<div><h3>Objective</h3><p>Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment.</p> <h3>Methods</h3><p>A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively.</p> <h3>Results</h3><p>Under the optimized conditions, the IC₅₀ values of the ELISA ranged from 3.7 to 162.2 ng mL^–1 for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC₅₀ values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL⁻¹. The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides.</p> <h3>Conclusions</h3><p>The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.</p> </div

Molecular structures of the haptens.

<p>Hapten 1 was used as immunizing hapten and homologous coating hapten, hapten 2 to 12 were used as heterogenous coating hapten candidate. ni: no atom.</p

Schematic diagram of ICA test strip and result judgment.

<p>The ICA test strip was composed of the NC membrane, MAb-gold-conjugated pad, sample pad and absorbent pad, which were pasted onto an adhesive plastic backing. Result judgment was based on the color changed on the T and C line zone.</p

Matrix interference of greengrocery in ICA test strip.

<p>ICA test strip detection of parathion-methyl with 10-, 20-, 40-fold dilutions of greengrocery matrix interference and buffer. Standard solutions of parathion-methyl at each final concentration of 128, 64, 32, 16, 8, 4, 2 and 0 ng mL-1 (numbers across the top of strips from left to right) were tested.</p

Reading time curves.

<p>Each point on the y axis represents the average value for the G/Peak - ROD, and the x axis values are the time after the addition of the working buffers (n = 3).</p

Recovery studies of samples spiked with parathion-methyl, parathion, and fenitrothion by multi-analyte ELISA (<i>n</i> = 3).

<p>Recovery studies of samples spiked with parathion-methyl, parathion, and fenitrothion by multi-analyte ELISA (<i>n</i> = 3).</p

Matrix interference study and correlation of recoveries between the ICA and ELISA.

<p>A: Standard inhibition curves for parathion-methyl in the buffer and different matrices using the multi-analyte ICA, Chinese cabbage and Apple samples were diluted 5-fold, and greengrocery samples were diluted 20-fold; B: Standard inhibition curves for parathion-methyl in the buffer and different matrices using the multi-analyte ELISA, Chinese cabbage, Apple and greengrocery samples were diluted 40-fold; C: The degree of correlation between the multi-analyte ICA and ELISA for analyses of samples spiked with parathion-methyl, parathion, and fenitrothion.</p