Transcriptome dynamics of alternative splicing events revealed early phase of apoptosis induced by methylparaben in H1299 human lung carcinoma cells.

Methylparaben is most frequently used as an antimicrobial preservative in pharmaceuticals and foods. Methylparaben has been subjected to toxicological studies owing to the increasing concern regarding its possible impact on the environment and human health. However, the cytotoxicity and underlying mechanisms of methylparaben exposure in human lung cells have not been explored. Here, we investigated the effect of methylparaben on cell cycle, apoptotic pathways, and changes in the transcriptome profiles in human lung cells. Our results demonstrate that treatment with methylparaben causes inhibition of cell growth. In addition, methylparaben induced S- and G2/M-phase arrest as a result of enhanced apoptosis. Transcriptome analysis using RNA-seq revealed that mRNA expression of ER stress- and protein misfolding-related gene sets was upregulated in methylparaben-treated group. RNA splicing- and maturation-related gene sets were significantly down-regulated by methylparaben treatment. Interestingly, RNA-seq analysis at the transcript level revealed that alternative splicing events, especially retained intron, were markedly changed by a low dose of methylparaben treatment. Altogether, these data show that methylparaben induces an early phase of apoptosis through cell cycle arrest and downregulation of mRNA maturation.

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in Kitasatospora cheerisanensis KCTC2395.

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B1 contains 2-amino- 3-hydroxycyclopent-2-enone (C5N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C5N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.

Enzymatic synthesis of avermectin B1a glycosides for the effective prevention of the pine wood nematode Bursaphelenchus xylophilus.

Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP)-glycosyltransferase from Bacillus licheniformis with various UDP sugars. As a result, the following four avermectin B1a glycosides were produced: avermectin B1a 4″-ß-D-glucoside, avermectin B1a 4″-ß-D-galactoside, avermectin B1a 4″-ß-L-fucoside, and avermectin B1a 4″-ß-2-deoxy-D-glucoside. The avermectin B1a glycosides were structurally analyzed based on HR-ESI MS and 1D and 2D nuclear magnetic resonance spectra, and the anti-nematodal effect of avermectin B1a 4″-ß-D-glucoside was found to exhibit the highest activity (IC50 = 0.23 µM), which was approximately 32 times greater than that of avermectin B1a (IC50 = 7.30 µM), followed by avermectin B1a 4″-ß-2-deoxy-D-glucoside (IC50 = 0.69 µM), avermectin B1a 4″-ß-L-fucoside (IC50 = 0.89 µM), and avermectin B1a 4″-ß-D-galactoside (IC50 = 1.07 µM). These results show that glycosylation of avermectin B1a effectively enhances its in vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.

Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics.

Kitasatospora cheerisanensis KCTC 2395, producing bafilomycin antibiotics belonging to plecomacrolide group, was isolated from a soil sample at Mt. Jiri, Korea. The draft genome sequence contains 8.04 Mb with 73.6% G+C content and 7,810 open reading frames. All the genes for aerial mycelium and spore formations were confirmed in this draft genome. In phylogenetic analysis of MurE proteins (UDP-N-acetylmuramyl-(L)-alanyl-(D)-glutamate:DAP ligase) in a conserved dcw (division of cell wall) locus, MurE proteins of Kitasatospora species were placed in a separate clade between MurEs of Streptomyces species incorporating (LL)-diaminopimelic acid (DAP) and MurEs of Saccharopolyspora erythraea as well as Mycobacterium tuberculosis ligating meso-DAP. From this finding, it was assumed that Kitasatospora MurEs exhibit the substrate specificity for both (LL)-DAP and meso-DAP. The bafilomycin biosynthetic gene cluster was located in the left subtelomeric region. In 71.3 kb-long gene cluster, 17 genes probably involved in the biosynthesis of bafilomycin derivatives were deduced, including 5 polyketide synthase (PKS) genes comprised of 12 PKS modules.

Draft Genome Sequence of Kitasatospora cheerisanensis KCTC 2395, Which Produces Plecomacrolide against Phytopathogenic Fungi.

Kitasatospora cheerisanensis KCTC 2395, which produces antifungal metabolites with bafilomycin derivatives, including bafilomycin C1-amide, was isolated from a soil sample at Mt. Jiri, South Korea. Here, we report its draft genome sequence, which contains 8.04 Mb with 73.6% G+C content and 7,810 protein-coding genes.

Organization and characterization of a biosynthetic gene cluster for bafilomycin from Streptomyces griseus DSM 2608.

Streptomyces griseus DSM 2608 produces bafilomycin, an antifungal plecomacrolide antibiotic. We cloned and sequenced an 87.4-kb region, including a polyketide synthase (PKS) region, methoxymalonate genes, flavensomycinate genes, and other putative regulatory genes. The 58.5kb of PKS region consisting 12 PKS modules arranged in five different PKS genes, was assumed to be responsible for the biosynthesis of plecomacrolide backbone including 16-membered macrocyclic lactone. All the modules showed high similarities with typical type I PKS genes. However, the starting module of PKS gene was confirmed to be specific for isobutyrate by sequence comparison of an acyltransferase domain. In downstream of PKS region, the genes for methoxymalonate biosynthesis were located, among which a gene for FkbH-like protein was assumed to play an important role in the production of methoxymalonyl-CoA from glyceryl-CoA. Further the genes encoding flavensomycinyl-ACP biosynthesis for the post-PKS tailoring were also found in the upstream of PKS region. By gene disruption experiments of a dehydratase domain of module 12 and an FkbH-like protein, this gene cluster was confirmed to be involved in the biosynthesis of bafilomycin.