Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12 after treatments with metal ions

Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e., Mn2+, Na+, Mg2+, Zn2+, Cu2+, Fe2+, Fe3+ and Al3+) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated. Results: Three most effective metal ions (Zn2+, Cu2+ and Mg2+) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2+, Cu2+ and Mg2+ were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn2+, Cu2+ and Mg2+ on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium. Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy

Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12 after treatments with metal ions

Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e., Mn2+, Na+, Mg2+, Zn2+, Cu2+, Fe2+, Fe3+ and Al3+) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated. Results: Three most effective metal ions (Zn2+, Cu2+ and Mg2+) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2+, Cu2+ and Mg2+ were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn2+, Cu2+ and Mg2+ on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium. Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy

Development of Colloidal Gold‐Based Lateral Flow Immunoassay for Rapid Qualitative and SemiQuantitative Analysis of Ustiloxins A and B in Rice Samples

Rice false smut is a worldwide devastating rice disease infected by the fungal pathogen Villosiclava virens. Ustiloxin A (UA) and ustiloxin B (UB), cyclopeptide mycotoxins, were the major ustiloxins isolated from the rice false smut balls (FSBs) that formed in the pathogen‐infected rice spikelets. Based on the specific monoclonal antibodies (mAbs) 2D3G5 and 1B5A10, respectively, against UA and UB, the lateral flow immunoassays (LFIAs) were developed, and the indicator ranges for UA and UB both were 50-100 ng/mL. The cross‐reactivities of UB for UA LFIA, and UA for UB LFIA were 5% and 20%, respectively, which were consistent with the icELISA results reported previously. Even at 50,000 ng/mL, none of other commonly existent metabolites in rice samples caused noticeable inhibition. The LFIAs were used for determination of UA and UB contents in rice FSBs and rice grains, and the results were agreeable with those by HPLC and icELISA. There was no change in the sensitivity of either dipstick stored at 4 °C) after at least three months. The developed LFIA has specificity and sensitivity for detecting UA and UB as well as simplicity to use. It will be a potential point‐of‐care device for rapid evaluation of the rice samples contaminated by UA and UB

Antibodies with Higher Bactericidal Activity Induced by a <i>Neisseria gonorrhoeae</i> Rmp Deletion Mutant Strain

<div><p><i>Neisseria gonorrhoeae</i> (<i>N. gonorrhoeae</i>) outer membrane protein reduction modifiable protein (Rmp) has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of <i>N. gonorrhoeae</i> by gene homologous recombination. The 261–460 nucleotide residues of Rmp gene amplified from <i>N. gonorrhoeae</i> WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into <i>N. gonorrhoeae</i> WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.</p></div

Primers used to construct pMD19▵<i>rmp</i>::<i>Kan.</i>

<p>Primers used to construct pMD19▵<i>rmp</i>::<i>Kan.</i></p

Detection of the genetic stability of <i>N. gonorrhoeae</i> Rmp deletion mutant strain.

<p>Same amounts of every 5 generations of culture products subcultured in ordinary culture medium were inoculated in FB liquid culture media with and without kanamycin for counting (n = 5).</p

Western-blotting analysis of the expression of Rmp protein in wild-type and Rmp deletion mutant strains of <i>N. gonorrhoeae</i>.

<p>Lysates of wild-type (WT) and mutant (MT) strains were subjected to SDS-PAGE and were transferred to PVDF membranes to react with anti-rrRmp (+) and normal mouse serum (–), respectively.</p

Growth curve of <i>N. gonorrhoeae</i> Rmp deletion mutant and wild-type strains.

<p>Same amounts of Rmp deletion mutant and wild-type strains were inoculated to FB liquid culture medium, from which 10 µl was sampled to measure the OD₆₀₀ value every one hour.</p