Abstract
Mycoplasma hyorhinis most commonly causes polyserositis and arthritis in swine and is a common contaminant during the cell culture in the laboratory. In our continuing research for diverse bioactive compounds from Bacillus subtilis 109GGC020, we discovered uncommon cyclic lipotetrapeptides showing inhibitory activities against M. hyorhinis with similar structures to previously reported bacilotetrins A and B. Bacilotetrins C–E (1–3), new cyclic lipodepsipeptides, were isolated from the EtOAc extract obtained from the fermentation of marine-derived Bacillus subtilis isolated from a marine sponge sample collected from the Gageo reef, Republic of Korea. The structures of 1–3, consisting of three leucine residues, one glutamic acid, and a β-hydroxy fatty acid, were elucidated by detailed analysis of 1D, 2D NMR, and HR-ESIMS data. The absolute configurations of the amino acids and β-hydroxy fatty acid were established by advanced Marfey’s method and Mosher’s method, respectively. The localization of L- and D-amino acids within the compounds was determined by retention time comparison of each purchased dipeptide standard to the partial hydrolysate products using LC-MS. Compounds 1–3 exhibited anti-mycoplasma activity, with an MIC value of 31 μg/mL, twofold stronger than that of the positive control, BioMycoX®. Detailed analysis and comparison of the spectroscopic data between bacilotetrins A (4) and B (5) and 1–3 led us to revise the structures of 4 and 5.
Highlights
1–3 exhibited anti-mycoplasma activity, with an Compounds 1–3 exhibited anti-mycoplasma activity, with an minimum inhibitory concentration (MIC) value of 31μg/mL. These results revealed that the type of branch of β-OH fatty acids does not affect their. These results revealed that the type of branch of β-OH fatty acids does not affect their inhibitory activity against M. hyorhinis, and the cyclic lipodepsipeptide core plays a more inhibitory activity against M. hyorhinis, and the cyclic lipodepsipeptide core plays a more important role
The aqueous layer was concentrated in vacuo and purified by LR-LCMS (YMC ODS-A, 250 × 4.6 mm, 5 μm, 0.5 mL/min, UV: 224 nm) using a gradient MeCN–H2 O (+0.02% TFA) solvent system (20% MeCN for 10 min, 20–100% MeCN over 40 min, and 100% MeCN for 10 min) to obtain three fragments (P1: Glu–Leu, tR 7.6 min, m/z 261 [M + H]+ ; P2: Leu–Leu, tR 23.0 min, m/z 245 [M + H]+ ; P3: β-OH-acid–Leu, tR 28.6 min, m/z 358 [M + H]+ ) (Figure S26)
Each mixture was dissolved in methylene chloride (MC) and washed with 1N HCl solution, saturated NaHCO3 solution and brine
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Marine-derived Bacillus subtilis 109GGC020 has been reported to produce interesting secondary metabolites, including macrolactins (gageomacrolactins [4]), linear lipopeptides (gageotetrins A–C [5], gageopeptides A–D [6], and gageostatins A–C [7]), and cyclic lipopeptides (gageopeptins A and B [8], and bacilotetrins A and B [9]) with antibacterial and antifungal activities. Mycoplasma species infect animals, plants, insects, and humans and are often found in research laboratories. Mycoplasma species infect animals, plants, insects, and humans and are often found in research laboratories as contaminants in cell culture mycoplasmas, M. hyorhinis is a comas contaminants in cell culture [11,13].[11,13].
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