Shujin Wang

Characterization of two anti-fungal lipopeptides produced by Bacillus amyloliquefaciens SH-B10

Bacillus amyloliquefaciens SH-B10 isolated from deep-sea sediment produces two anti-fungal lipopeptides that were purified by bioactivity-guided fractionation based on acid precipitation, vacuum flash chromatography and semi-preparative HPLC. The two compounds were identified by tandem Q-TOF mass spectroscopy as C16 fengycin A (1) and a new fengycin with an aminobutyric acid at position 6 of the peptide backbone (2). Both compounds showed significant inhibitory activities against five plant fungal pathogens in paper-agar disk diffusion assay. This is the first report on the anti-fungal activities of the rare 6-Abu fengycin lipopeptides, and at the same time provided an insight into the potential of marine microbial resource in biological control and sustainable agriculture.

Optimization for the Production of Surfactin with a New Synergistic Antifungal Activity

Background Two of our long term efforts are to discover compounds with synergistic antifungal activity from metabolites of marine derived microbes and to optimize the production of the interesting compounds produced by microorganisms. In this respect, new applications or mechanisms of already known compounds with a high production yield could be continually identified. Surfactin is a well-known lipopeptide biosurfactant with a broad spectrum of antimicrobial and antiviral activity; however, there is less knowledge on surfactin’s antifungal activity. In this study, we investigated the synergistic antifungal activity of C15-surfactin and the optimization of its production by the response surface method. Methodology/Principal Findings Using a synergistic antifungal screening model, we found that the combination of C15-surfactin and ketoconazole (KTC) showed synergistic antifungal effect on Candida albicans SC5314 when the concentrations of C15-surfactin and KTC were 6.25 µg/mL and 0.004 µg/mL, respectively. These concentrations were lower than their own efficient antifungal concentrations, which are >100 µg/mL and 0.016 µg/mL, respectively. The production of C15-surfactin from Bacillus amyloliquefaciens was optimized by the response surface methodology in shaker flask cultivation. The Plackett-Burman design found sucrose, ammonium nitrate and NaH2PO4.2H2O to have significant effects on C15-surfactin production. The optimum values of the tested variables were 21.17 g/L sucrose, 2.50 g/L ammonium nitrate and 11.56 g/L NaH2PO4·2H2O. A production of 134.2 mg/L, which were in agreement with the prediction, was observed in a verification experiment. In comparison to the production of original level (88.6 mg/L), a 1.52-fold increase had been obtained. Conclusion/Significance This work first found that C15-surfactin was an efficient synergistic antifungal agent, and demonstrated that response surface methodology was an effective method to improve the production of C15-surfactin.

Isolation and characterization of a new iturinic lipopeptide, mojavensin A produced by a marine-derived bacterium Bacillus mojavensis B0621A

Three lipopeptides were isolated by bioactivity-guided fractionation from the fermentation broth of Bacillus mojavensis B0621A. A new iturinic lipopeptide, named mojavensin A (1), was tentatively characterized by 1D, 2D NMR and MS spectroscopy, Marfey’s method containing a novel peptide backbone of L-Asn1, D-Tyr2, D-Asn3, L-Gln4, L-Pro5, D-Asn6, L-Asn7 and an anteiso-type of the saturated β-fatty acid side chain. Compound 2 and 3 were tentatively identified as iso-C16 fengycin B and anteiso-C17 fengycin B, respectively. These lipopeptides displayed dose-dependent antifungal activity against a broad spectra of phytopathogens and were weakly antagonistic to Staphylococcus aureus. Moreover, they all revealed cytotoxic activities against the human leukemia (HL-60) cell line. Mojavensin A, iso-C16 fengycin B, and anteiso-C17 fengycin B inhibited the growth of HL-60 with IC50 of 100, 100 and 1.6 μM, respectively.

New Spirotetronate Antibiotics, Lobophorins H and I, from a South China Sea-Derived Streptomyces sp. 12A35

Strain 12A35 was isolated from a deep-sea sediment collected from the South China Sea and showed promising antibacterial activities. It was identified as Streptomyces sp. by the 16S rDNA sequence analysis. Bioassay-guided fractionation using HP20 adsorption, flash chromatography over silica gel and octadecylsilyl (ODS) and semi-preparative HPLC, led to the isolation and purification of five metabolites from the fermentation culture of 12A35. Two new spirotetronate antibiotics, lobophorins H (1) and I (2), along with three known analogues, O-β-kijanosyl-(1→17)-kijanolide (3), lobophorins B (4) and F (5) were characterized by 1D, 2D-NMR and MS data. These compounds exhibited significant inhibitory activities against Bacillus subtilis. Compounds 1 and 5 exhibited moderate activities against Staphylococcus aureus. In particular, the new compound lobophorin H (1) showed similar antibacterial activities against B. subtilis CMCC63501 to ampicillin.

Effect of monospecific and mixed Cunninghamia lanceolata plantations on microbial community and two functional genes involved in nitrogen cycling

Conversion of native broadleaf forest (NF) and introduction of broadleaf species into monospecific Cunninghamia lanceolata plantations are silvicultural practices driven by the increasing demand for timber production. This study was conducted to evaluate the impacts of successive planting of C. lanceolata and mixed plantations (C. lanceolata-Michelia macclurei, CFM; C. lanceolata-Alnus cremastogyne, CFA; C. lanceolata-Kalopanax septemlobus, CFK) on microbial community diversity. Microbial biomass (MBC) was assessed using chloroform fumigation-extraction. Using denaturing gradient gel electrophoresis (DGGE), we examined the biodiversity within eubacterial (16S rRNA gene) and fungal (28S rRNA gene) species and two genes involved in N cycling: nifH and amoA. Microbial community diversities and microbial biomass decreased as NF was substituted by successive plantings of C. lanceolata plantations, whereas the trend reversed after introducing the broadleaf, M. macclurei, into pure C. lanceolata plantations. A strong positive correlation between MBC changes and total organic C (TOC), total organic N (TON), available N and extractable C (Cext) were seen, which suggests that MBC was tightly coupled with the content of soil organic matter. The Shannon index showed that bacterial diversity and two functional genes (nifH and amoA) diversities associated with monospecific C. lanceolata plantations were lower than that of NF or mixed C. lanceolata plantations, such as CFM and CFA, whereas the opposite was seen for fungal diversity. Bacterial diversity was positively correlated with pH, TOC, TON, Cext and NH4+-N; while fungal diversity was positively correlated with C/N ratio and negatively correlated with pH. Both nitrogen fixing and ammonia oxidizing bacterial diversities were positively correlated with pH. Thus, soil pH was not only significantly positively correlated with bacterial diversity (r = 0.502, P < 0.05), nifH gene diversity (r = 0.564, P < 0.01) and amoA gene diversity (r = 0.659, P < 0.001), but also negatively correlated with fungal diversity (r = − 0.505, P < 0.05), which seemed to be responsible for the discrimination of the soil microbial communities among these plantations. These findings suggest that different silvicultural practices have significant impacts on the soil microbial community through influences on soil chemical properties.

Cytotoxic Polyphenols from a Sponge‐Associated Fungus Aspergillus versicolor Hmp‐48

A new dibenzo[1,4]dioxin 1, and two new prenylated diphenyl ethers, 2 and 3, together with six known compounds, 4–9, were isolated from a sponge‐associated fungus Aspergillus versicolor Hmp‐F48 by bioactivity‐guided fractionation. Their structures were elucidated by 1D‐ and 2D‐NMR, and MS analyses. The compounds showed potent cell growth inhibitory activities against HL‐60 cell line.

NMR spectroscopic and MS/MS spectrometric characterization of a new lipopeptide antibiotic bacillopeptin B1 produced by a marine sediment-derived Bacillus amyloliquefaciens SH-B74.

Bacillus species have the ability to secrete lipopeptide antibiotics under both in vivo and in vitro conditions.1, 2, 3, 4 Bacillus lipopeptides are versatile antibiotics composed mainly of three families—iturins, plipastatins (or fengcyins) and surfactins. Iturins and plipastatins are fungicides and have potential utilization in the biological control of fungal plant pathogens.5 Marine-derived microorganisms have arisen as a focus of research during recent decades, and numerous novel metabolites with excellent biological activity have been reported.6, 7 In the present work, compound 1 was isolated and purified from the fermentation broth of a marine sediment-derived B. amyloliquefaciens SH-B74. The structure of compound 1 was characterized by one-dimensional and two-dimensional NMR spectroscopy as well as tandem mass spectrometry (MS/MS) analysis. Finally, the antagonistic activities of compound 1 to several plant pathogens were also evaluated in vitro by the paper disc-agar diffusion assay.

Molecular characterization of the duck enteritis virus UL4 gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.