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Draft Genome Sequence of Streptomyces fradiae olg1-1, a Strain Resistant to Nitrone-Oligomycin Aleksey A. Vatlin,a
Olga B. Bekker,a Ludmila N. Lysenkova,b Valery N. Danilenkoa
Vavilov Institute of General Genetics RAS, Moscow, Russiaa; Gause Institute of New Antibiotics, Moscow, Russiab
We report a draft genome sequence of Streptomyces fradiae olg1-1, a mutant strain derived from the model object S. fradiae ATCC 19609, which is resistant to nitrone-oligomycin and has a mutation in the DNA-binding domain of a transcriptional regulator PadR. Received 15 September 2015 Accepted 15 September 2015 Published 22 October 2015 Citation Vatlin AA, Bekker OB, Lysenkova LN, Danilenko VN. 2015. Draft genome sequence of Streptomyces fradiae olg1-1, a strain resistant to nitrone-oligomycin. Genome Announc 3(5):e01252-15. doi:10.1128/genomeA.01252-15. Copyright © 2015 Vatlin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Aleksey A. Vatlin, [email protected], or Valery N. Danilenko, [email protected].
M
acrolide antibiotic oligomycin A produces an antibacterial effect on pathogenic actinobacteria, causing actinomycosis, as well as on nonpathogenic strains of S. fradiae (1). A series of derivatives of oligomycin A was obtained (1, 2), including nitrone-oligomycin (3), for further research into new anticancer drugs (4) and drugs for the treatment of actinomycosis. There is evidence that the mechanism of action for oligomycin A is based on a chemical interaction with the C-subunit of F0F1-ATP synthase (5). However, research involving oligomycin A and its derivatives, carried out on the model object S. fradiae ATCC 19609, showed that it has additional biotargets (6, 7). The impossibility of obtaining mutants resistant to oligomycin A with a frequency of up to 10⫺11 confirmed the presence of two or more biological targets. The aim of this work was to obtain a mutant strain of S. fradiae that is resistant to nitrone-oligomycin and harbors only one resistance mutation. We carried out whole-genome sequencing of S. fradiae olg1-1, a mutant strain resistant to nitrone-oligomycin. The frequency of obtaining mutants was 1 to 2 ⫻ 109 CFU. The mutant was selected on solid medium containing 150 M of nitrone-oligomycin (MIC ⫽10 M) and phenotypically had Bld-type bad sporulation (8). Comparative genomic analysis of the mutant strain and the wild-type S. fradiae strain ATCC 19609 (9) revealed a mutation in padR H(24)R. PadR is a multifunctional transcriptional regulator, which is involved in the control of expression of genes associated with detoxification, virulence, and multidrug resistance in bacteria (10–12). This single nucleotide polymorphism was further confirmed by additional Sanger sequencing. No aerial mycelium developed in the phenotype mutant strain, which suggests that PadR is a transcriptional regulator possibly involved in the process of differentiation, direct resistance to nitrone-oligomycin or negative regulation of genes, and encoding the antibiotic’s biotargets. The inactivation of the padR gene may lead to overexpression of these genes, reducing the antibiotic susceptibility. The genome sequencing of S. fradiae strain olg1-1 was carried out using a whole-genome shotgun sequencing approach performed on a Roche 454-GS Junior instrument (Roche, Switzer-
September/October 2015 Volume 3 Issue 5 e01252-15
land). A total of 344,794 reads were generated. All reads were assembled to an initial draft genome of 7,667,096 nucleotides at 21-fold coverage using the GS de novo assembler version 3.0 (Roche). The resulting draft genome sequence consists of 208 contigs (193 contigs ⬎500 bp; largest contig, 316,524 bp; overall GC content, 72.8%). The automatic functional annotation results were obtained using the NCBI Prokaryotic Genome Annotation Pipeline (PGAAP, http://www.ncbi.nlm.nih.gov/genome /annotation_prok). The S. fradiae olg1-1 genome contains 6,474 predicted genes, 4 rRNA operons, and 65 tRNAs. A total of 5,781 coding sequences (CDSs), 617 pseudogenes, 7 noncoding RNAs (ncRNAs), 4 clustered regularly interspaced short palindromic repeats (CRISPRs), and 77 frameshifted genes were predicted using PGAAP. In addition, 10 insertion sequence (IS) elements were found. The only region of phage activity was identified in the genome sequence (PHAST). This mutation will give a deeper insight to the mechanisms responsible for the resistance of bacteria to nitrone-oligomycin and oligomycin A and can be applied to the development of new antibiotics with a novel mechanism of action. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number LGSP00000000. The version described in this paper is the first version, LGSP01000001. ACKNOWLEDGMENTS We thank D. M. Maslov, K. V. Shur, and N. V. Zakharevich for their assistance. This work was supported by the Russian Science Foundation under State Contract 15-15-00141.
REFERENCES 1. Lysenkova LN, Turchin KF, Korolev AM, Dezhenkova LG, Bekker OB, Shtil AA, Danilenko VN, Preobrazhenskaya MN. 2013. Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain. Bioorg Med Chem 21:2918 –2924. http://dx.doi.org/10.1016/j.bmc.2013.03.081. 2. Lysenkova LN, Turchin KF, Korolev AM, Danilenko VN, Bekker OB, Dezhenkova LG, Shtil AA, Preobrazhenskaya MN. 2014. Study on retroaldol degradation products of antibiotic oligomycin A. J Antibiot 67: 153–158. http://dx.doi.org/10.1038/ja.2013.92. 3. Lysenkova LN, Turchin KF, Danilenko VN, Korolev AM, Preobrazhen-
Genome Announcements
genomea.asm.org 1
Vatlin et al.
4.
5. 6.
7.
skaya MN. 2010. The first examples of chemical modification of oligomycin A. J Antibiot 63:17–22. http://dx.doi.org/10.1038/ja.2009.112. Zu Y, Zhao Q, Zhao X, Zu S, Meng L. 2011. Process optimization for the preparation ofoligomycin-loaded folate-conjugated chitosan nanoparticles as a tumor-targeted drug delivery system using a two-level factorial design method. Int J Nanomed 6:3429 –3441. http://dx.doi.org/10.2147/ IJN.S27157. Symersky J, Osowski D, Walters DE, Mueller DM. 2012. Oligomycin frames a common drug-binding site in the ATP synthase. Proc Natl Acad Sci U S A 109:13961–13965. http://dx.doi.org/10.1073/pnas.1207912109. Alekseeva MG, Elizarov SM, Bekker OB, Lubimova IK, Danilenko VN. 2009. F0F1 ATP synthase of streptomycetes: modulation of activity and oligomycin resistance by protein Ser/Thr kinases. Biochem (Mosc) Suppl Ser A Membr Cell Biol 3:16 –23. http://dx.doi.org/10.1134/ S1990747809010036. Alekseeva MG, Mironcheva TA, Mavletova DA, Elizarov SM, Zakharevich NV, Danilenko VN. 2015. F0F1-ATP synthase of Streptomyces fradiae ATCC 19609: structural, biochemical, and functional characterization. Biochem (Mosc) 80:296 –309. http://dx.doi.org/10.1134/ S0006297915030050.
2 genomea.asm.org
8. Elliot MA, Bibb MJ, Buttner MJ, Leskiw BK. 2001. BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2). Mol Microbiol 40:257–269. http://dx.doi.org/10.1046/j.1365 -2958.2001.02387.x. 9. Bekker OB, Klimina KM, Vatlin AA, Zakharevich NV, Kasianov AS, Danilenko VN. 2014. Draft genome sequence of Streptomyces fradiae ATCC 19609, a strain highly sensitive to antibiotics. Genome Announc 2(6):e01247-14. http://dx.doi.org/10.1128/genomeA.01247-14. 10. Fibriansah G, Kovács ÁT, Pool TJ, Boonstra M, Kuipers OP, Thunnissen AWH. 2012. Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily. PLoS One 7:e48015. http://dx.doi.org/10.1371/journal.pone.0048015. 11. Flórez AB, Álvarez S, Zabala D, Braña AF, Salas JA, Méndez C. 2015. Transcriptional regulation of mithramycin biosynthesis in Streptomyces argillaceus: dual role as activator and repressor of the PadR-like regulator MtrY. Microbiol 161:272–284. http://dx.doi.org/10.1099/mic.0.080895-0. 12. Morabbi Heravi K, Lange J, Watzlawick H, Kalinowski J, Altenbuchner J. 2015. Transcriptional regulation of the vanillate utilization genes (vanABK operon) of Corynebacterium glutamicum by VanR, a PadR-like repressor. J Bacteriol 197:959 –972. http://dx.doi.org/10.1128/JB.02431-14.
Genome Announcements
September/October 2015 Volume 3 Issue 5 e01252-15
Draft Genome Sequence of Streptomyces fradiae olg1-1, a Strain Resistant to Nitrone-Oligomycin Aleksey A. Vatlin,a
Olga B. Bekker,a Ludmila N. Lysenkova,b Valery N. Danilenkoa
Vavilov Institute of General Genetics RAS, Moscow, Russiaa; Gause Institute of New Antibiotics, Moscow, Russiab
We report a draft genome sequence of Streptomyces fradiae olg1-1, a mutant strain derived from the model object S. fradiae ATCC 19609, which is resistant to nitrone-oligomycin and has a mutation in the DNA-binding domain of a transcriptional regulator PadR. Received 15 September 2015 Accepted 15 September 2015 Published 22 October 2015 Citation Vatlin AA, Bekker OB, Lysenkova LN, Danilenko VN. 2015. Draft genome sequence of Streptomyces fradiae olg1-1, a strain resistant to nitrone-oligomycin. Genome Announc 3(5):e01252-15. doi:10.1128/genomeA.01252-15. Copyright © 2015 Vatlin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Aleksey A. Vatlin, [email protected], or Valery N. Danilenko, [email protected].
M
acrolide antibiotic oligomycin A produces an antibacterial effect on pathogenic actinobacteria, causing actinomycosis, as well as on nonpathogenic strains of S. fradiae (1). A series of derivatives of oligomycin A was obtained (1, 2), including nitrone-oligomycin (3), for further research into new anticancer drugs (4) and drugs for the treatment of actinomycosis. There is evidence that the mechanism of action for oligomycin A is based on a chemical interaction with the C-subunit of F0F1-ATP synthase (5). However, research involving oligomycin A and its derivatives, carried out on the model object S. fradiae ATCC 19609, showed that it has additional biotargets (6, 7). The impossibility of obtaining mutants resistant to oligomycin A with a frequency of up to 10⫺11 confirmed the presence of two or more biological targets. The aim of this work was to obtain a mutant strain of S. fradiae that is resistant to nitrone-oligomycin and harbors only one resistance mutation. We carried out whole-genome sequencing of S. fradiae olg1-1, a mutant strain resistant to nitrone-oligomycin. The frequency of obtaining mutants was 1 to 2 ⫻ 109 CFU. The mutant was selected on solid medium containing 150 M of nitrone-oligomycin (MIC ⫽10 M) and phenotypically had Bld-type bad sporulation (8). Comparative genomic analysis of the mutant strain and the wild-type S. fradiae strain ATCC 19609 (9) revealed a mutation in padR H(24)R. PadR is a multifunctional transcriptional regulator, which is involved in the control of expression of genes associated with detoxification, virulence, and multidrug resistance in bacteria (10–12). This single nucleotide polymorphism was further confirmed by additional Sanger sequencing. No aerial mycelium developed in the phenotype mutant strain, which suggests that PadR is a transcriptional regulator possibly involved in the process of differentiation, direct resistance to nitrone-oligomycin or negative regulation of genes, and encoding the antibiotic’s biotargets. The inactivation of the padR gene may lead to overexpression of these genes, reducing the antibiotic susceptibility. The genome sequencing of S. fradiae strain olg1-1 was carried out using a whole-genome shotgun sequencing approach performed on a Roche 454-GS Junior instrument (Roche, Switzer-
September/October 2015 Volume 3 Issue 5 e01252-15
land). A total of 344,794 reads were generated. All reads were assembled to an initial draft genome of 7,667,096 nucleotides at 21-fold coverage using the GS de novo assembler version 3.0 (Roche). The resulting draft genome sequence consists of 208 contigs (193 contigs ⬎500 bp; largest contig, 316,524 bp; overall GC content, 72.8%). The automatic functional annotation results were obtained using the NCBI Prokaryotic Genome Annotation Pipeline (PGAAP, http://www.ncbi.nlm.nih.gov/genome /annotation_prok). The S. fradiae olg1-1 genome contains 6,474 predicted genes, 4 rRNA operons, and 65 tRNAs. A total of 5,781 coding sequences (CDSs), 617 pseudogenes, 7 noncoding RNAs (ncRNAs), 4 clustered regularly interspaced short palindromic repeats (CRISPRs), and 77 frameshifted genes were predicted using PGAAP. In addition, 10 insertion sequence (IS) elements were found. The only region of phage activity was identified in the genome sequence (PHAST). This mutation will give a deeper insight to the mechanisms responsible for the resistance of bacteria to nitrone-oligomycin and oligomycin A and can be applied to the development of new antibiotics with a novel mechanism of action. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number LGSP00000000. The version described in this paper is the first version, LGSP01000001. ACKNOWLEDGMENTS We thank D. M. Maslov, K. V. Shur, and N. V. Zakharevich for their assistance. This work was supported by the Russian Science Foundation under State Contract 15-15-00141.
REFERENCES 1. Lysenkova LN, Turchin KF, Korolev AM, Dezhenkova LG, Bekker OB, Shtil AA, Danilenko VN, Preobrazhenskaya MN. 2013. Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain. Bioorg Med Chem 21:2918 –2924. http://dx.doi.org/10.1016/j.bmc.2013.03.081. 2. Lysenkova LN, Turchin KF, Korolev AM, Danilenko VN, Bekker OB, Dezhenkova LG, Shtil AA, Preobrazhenskaya MN. 2014. Study on retroaldol degradation products of antibiotic oligomycin A. J Antibiot 67: 153–158. http://dx.doi.org/10.1038/ja.2013.92. 3. Lysenkova LN, Turchin KF, Danilenko VN, Korolev AM, Preobrazhen-
Genome Announcements
genomea.asm.org 1
Vatlin et al.
4.
5. 6.
7.
skaya MN. 2010. The first examples of chemical modification of oligomycin A. J Antibiot 63:17–22. http://dx.doi.org/10.1038/ja.2009.112. Zu Y, Zhao Q, Zhao X, Zu S, Meng L. 2011. Process optimization for the preparation ofoligomycin-loaded folate-conjugated chitosan nanoparticles as a tumor-targeted drug delivery system using a two-level factorial design method. Int J Nanomed 6:3429 –3441. http://dx.doi.org/10.2147/ IJN.S27157. Symersky J, Osowski D, Walters DE, Mueller DM. 2012. Oligomycin frames a common drug-binding site in the ATP synthase. Proc Natl Acad Sci U S A 109:13961–13965. http://dx.doi.org/10.1073/pnas.1207912109. Alekseeva MG, Elizarov SM, Bekker OB, Lubimova IK, Danilenko VN. 2009. F0F1 ATP synthase of streptomycetes: modulation of activity and oligomycin resistance by protein Ser/Thr kinases. Biochem (Mosc) Suppl Ser A Membr Cell Biol 3:16 –23. http://dx.doi.org/10.1134/ S1990747809010036. Alekseeva MG, Mironcheva TA, Mavletova DA, Elizarov SM, Zakharevich NV, Danilenko VN. 2015. F0F1-ATP synthase of Streptomyces fradiae ATCC 19609: structural, biochemical, and functional characterization. Biochem (Mosc) 80:296 –309. http://dx.doi.org/10.1134/ S0006297915030050.
2 genomea.asm.org
8. Elliot MA, Bibb MJ, Buttner MJ, Leskiw BK. 2001. BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2). Mol Microbiol 40:257–269. http://dx.doi.org/10.1046/j.1365 -2958.2001.02387.x. 9. Bekker OB, Klimina KM, Vatlin AA, Zakharevich NV, Kasianov AS, Danilenko VN. 2014. Draft genome sequence of Streptomyces fradiae ATCC 19609, a strain highly sensitive to antibiotics. Genome Announc 2(6):e01247-14. http://dx.doi.org/10.1128/genomeA.01247-14. 10. Fibriansah G, Kovács ÁT, Pool TJ, Boonstra M, Kuipers OP, Thunnissen AWH. 2012. Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily. PLoS One 7:e48015. http://dx.doi.org/10.1371/journal.pone.0048015. 11. Flórez AB, Álvarez S, Zabala D, Braña AF, Salas JA, Méndez C. 2015. Transcriptional regulation of mithramycin biosynthesis in Streptomyces argillaceus: dual role as activator and repressor of the PadR-like regulator MtrY. Microbiol 161:272–284. http://dx.doi.org/10.1099/mic.0.080895-0. 12. Morabbi Heravi K, Lange J, Watzlawick H, Kalinowski J, Altenbuchner J. 2015. Transcriptional regulation of the vanillate utilization genes (vanABK operon) of Corynebacterium glutamicum by VanR, a PadR-like repressor. J Bacteriol 197:959 –972. http://dx.doi.org/10.1128/JB.02431-14.
Genome Announcements
September/October 2015 Volume 3 Issue 5 e01252-15
