The genome sequence of Streptomyces rochei 7434AN4, which carries a linear chromosome and three characteristic linear plasmids

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211 kb), -M (113 kb), and -S (18 kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G + C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406 bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas (23108515, 25108718 and 17H05446 to K.A.) from Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), Grants-in-Aid for Scientific Research (B) (16H04917 to K.A.) from the Japan Society for the Promotion of Science (JSPS), and the Sasakawa Scientific Research Grant from the Japan Science Society to Y.N. This work was partly supported by a JSPS A3 Foresight Program. A.A.F. and R.M. were supported by the Indonesia Endowment Fund for Education (LPDP). Sequencing analysis using an Illumina GAIIx sequencer was supported by the Grant in Aid for Scientific Research on Innovative Areas (22108010 to J.I.) from MEXT

放線菌二次代謝産物の高機能化を司るP450モノオキシゲナーゼの機能解明

広島大学(Hiroshima University)博士(工学)Doctor of Engineeringdoctora

Functional Analysis of P450 Monooxygenase SrrO in the Biosynthesis of Butenolide-Type Signaling Molecules in Streptomyces rochei

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics lankacidin and lankamycin, and their biosynthesis is tightly controlled by butenolide-type signaling molecules SRB1 and SRB2. SRBs are synthesized by SRB synthase SrrX, and induce lankacidin and lankamycin production at 40 nM concentration. We here investigated the role of a P450 monooxygenase gene srrO (orf84), which is located adjacent to srrX (orf85), in SRB biosynthesis. An srrO mutant KA54 accumulated lankacidin and lankamycin at a normal level when compared with the parent strain. To elucidate the chemical structures of the signaling molecules accumulated in KA54 (termed as KA54-SRBs), this mutant was cultured (30 L) and the active components were purified. Two active components (KA54-SRB1 and KA54-SRB2) were detected in ESI-MS and chiral HPLC analysis. The molecular formulae for KA54-SRB1 and KA54-SRB2 are C15H26O4 and C16H28O4, whose values are one oxygen smaller and two hydrogen larger when compared with those for SRB1 and SRB2, respectively. Based on extensive NMR analysis, the signaling molecules in KA54 were determined to be 6′-deoxo-SRB1 and 6′-deoxo-SRB2. Gel shift analysis indicated that a ligand affinity of 6′-deoxo-SRB1 to the specific receptor SrrA was 100-fold less than that of SRB1. We performed bioconversion of the synthetic 6′-deoxo-SRB1 in the Streptomyces lividans recombinant carrying SrrO-expression plasmid. Substrate 6′-deoxo-SRB1 was converted through 6′-deoxo-6′-hydroxy-SRB1 to SRB1 in a time-dependent manner. Thus, these results clearly indicated that SrrO catalyzes the C-6′ oxidation at a final step in SRB biosynthesis

Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function

The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane bound organelles but mere colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinih and elimination of the N terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease