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Genome sequence of the nicotine-degrading Agrobacterium tumefaciens S33
Journal of Biotechnology 228 (2016) 1–2
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome announcement
Genome sequence of the nicotine-degrading Agrobacterium tumefaciens S33 Wenjun Yu a , Huili Li a , Kebo Xie a , Haiyan Huang a,b , Huijun Xie c , Shuning Wang a,∗ a b c
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People’s Republic of China Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan, People’s Republic of China Environment Research Institute, Shandong University, Jinan 250100, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 12 April 2016 Accepted 13 April 2016 Available online 20 April 2016 Keywords: Genome sequence Nicotine Degradation Agrobacterium tumefaciens Functionalized pyridine Tobacco wastes
a b s t r a c t Agrobacterium tumefaciens S33 is capable of growing with nicotine as the sole source of carbon and nitrogen, and has the potential to dispose of tobacco wastes and transform nicotine into functionalized pyridines intermediates, which are important precursors for some valuable drugs and insecticides. Here we report the complete genome sequence of strain S33 and predict the gene cluster involved in nicotine catabolism according to the annotation. © 2016 Elsevier B.V. All rights reserved.
Microbial degradation of the natural alkaloid nicotine has recently received attention because it represents a promising strategy to detoxify tobacco industry waste and to remove nicotine from tobacco products (Brandsch, 2006; Wang et al., 2012). Interestingly, it also attracts attention from chemists since nicotine can be used as a starting material to produce various chemicals of pharmaceutical importance, especially, nicotine can be transformed into renewable functionalized pyridines by biocatalytic processes that are difficult to synthesized by chemical methods (Roduit et al., 1997; Wang et al., 2005). Various bacteria degrading nicotine have been isolated and characterized such as Arthrobacter sp. and Pseudomonas sp., which decompose nicotine mainly through the pyridine pathway and pyrrolidine pathway, respectively, whose molecular mechanisms have been well characterized (Brandsch, 2006; Tang et al., 2013). Previously, we isolated Agrobacterium tumefaciens S33 from the rhizospheric soil of a tobacco plant, which was capable of utilizing nicotine as sole source of carbon and nitrogen (Wang et al., 2009). A novel fused nicotine degradation pathway of pyridine pathway and pyrrolidine pathway was discovered in strain S33 based on the intermediates identification and enzyme assays (Wang
∗ Corresponding author at: State Key Laboratory of Microbial Technology, Shandong University, 27 Shan Da South Road, Jinan 250100, People’s Republic of China. E-mail address: [email protected] (S. Wang). http://dx.doi.org/10.1016/j.jbiotec.2016.04.029 0168-1656/© 2016 Elsevier B.V. All rights reserved.
et al., 2012). Further, we purified and characterized 6-hydroxy-3succinoylpyridine hydroxylase (Hsh) and the complex of nicotine dehydrogenase (NdhAB) and 6-hydroxypseudooxynicotine oxidase (Pno) involved in the pathway (Li et al., 2014; Li et al., 2016). However, the molecular mechanism responsible for the novel fused pathway in A. tumefaciens S33 is still unclear. Here, we present the complete genome sequence of strain S33, obtained using the Pacific Biosciences (PacBio) sequencing technology. Genomic DNA from strain S33 was extracted using the Wizard Genomic DNA Purification Kit (Promega) and evaluated for its quality and quantity on the Bioanalyzer 2100 (Agilent). A 10kb insert SMRT-bell library was constructed and then sequenced by PacBio RS II sequencer (Pacific Biosciences) (Eid et al., 2009). After quality control, a total of 90,720 polymerase reads with a mean read length of 11,519 bases were generated, which led to a total of 1,045,009,373 bases with a 190-fold average coverage. Then the reads were de novo assembled using SMRT analysis software version 2.3.0 (Pacific Biosciences) (Chin et al., 2013). The assembled genome sequence was annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) version 3.1 (http://www. ncbi.nlm.nih.gov/genome/annotation prok/) and the Rapid Annotations using Subsystems Technology (RAST) server (Aziz et al., 2008). The final genome sequence of the strain S33 comprises 5,481,595 bases, which were assembled into 2 chromosomes, one circular chromosome (2.5 Mb) and one linear chromosome
2
W. Yu et al. / Journal of Biotechnology 228 (2016) 1–2
Table 1 Genome features of A. tumefaciens S33.
We also thank Professor Ping Xu from Shanghai Jiao Tong University for his valuable suggestion and supports.
Features
Chromosome 1
Chromosome 2
Molecular shape Size (bp) GC content (%) Total number of the genes Protein coding genes (CDSs) rRNAs (5S, 16S, 23S) tRNAs Pseudogenes GenBank accession number
circular 2,497,934 59.0 2244 2173 6 14 50 CP014259.1
linear 2,983,661 59.3 2897 2818 6 41 29 CP014260.1
(2.98 Mb) with GC content of 59.0% and 59.3%, respectively (Table 1). In total, 5141 genes were predicted using the PGAP, including 4991 protein-coding sequences (CDSs), 79 pseudo genes, 55 tRNAs, and 12 rRNA genes. According to the annotation and our previous study, some genes, which locate on the circular chromosome, were identified or predicted to be involved in nicotine degradation. Among them, the ORFs with locus tag as AWN88 01360, 01355, 01220, and 01205 have been experimentally verified to encode NdhB, NdhA, Pno, and Hsh, respectively (Li et al., 2014; Li et al., 2016). The ORFs with locus tag as AWN88 01345 and 01320 encode two proteins almost identical to 6-hydroxynicotine oxidase and 2,5-dihydroxypyridine dioxygenase from nicotine-degrading Ochrobactrum sp. SJY1, respectively (Yu et al., 2015). The arrangement of these ORFs in strain S33 suggests that the genes for nicotine degradation form a big gene cluster, which are not found in other strains of A. tumefaciens by comparison using RAST server. The genome sequence presented here will be helpful for elucidating the molecular mechanism on nicotine catabolism in A. tumefaciens S33 and developing new biological methods for transforming nicotine into valuable compounds. Strain and nucleotide sequence accession numbers The strain S33 has been deposited at China Center for Type Culture Collection under the accession number CCTCC AB 2016054 (originally CCTCC M 206131). This genome sequence has been deposited at GenBank under the accession numbers CP014259.1 and CP014260.1. Acknowledgements This work was supported by the grants from National Natural Science Foundation of China (Grant No. 30970027), the Excellent Middle-Aged and Youth Scientist Award Foundation of Shandong Province (No. BS2009SW006), and the Fundamental Research Funds of Shandong University (Grant No. 2014JC023).
References Aziz, R., Bartels, D., Best, A., DeJongh, M., Disz, T., Edwards, R., Formsma, K., Gerdes, S., Glass, E., Kubal, M., Meyer, F., Olsen, G., Olson, R., Osterman, A., Overbeek, R., McNeil, L., Paarmann, D., Paczian, T., Parrello, B., Pusch, G., Reich, C., Stevens, R., Vassieva, O., Vonstein, V., Wilke, A., Zagnitko, O., 2008. The RAST server: rapid annotations using subsystems technology. BMC Genom. 9, 75. Brandsch, R., 2006. Microbiology and biochemistry of nicotine degradation. Appl. Microbiol. Biotechnol. 69, 493–498. Chin, C.S., Alexander, D.H., Marks, P., Klammer, A.A., Drake, J., Heiner, C., Clum, A., Copeland, A., Huddleston, J., Eichler, E.E., Turner, S.W., Korlach, J., 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569. Eid, J., Fehr, A., Gray, J., Luong, K., Lyle, J., Otto, G., Peluso, P., Rank, D., Baybayan, P., Bettman, B., Bibillo, A., Bjornson, K., Chaudhuri, B., Christians, F., Cicero, R., Clark, S., Dalal, R., Dewinter, A., Dixon, J., Foquet, M., Gaertner, A., Hardenbol, P., Heiner, C., Hester, K., Holden, D., Kearns, G., Kong, X., Kuse, R., Lacroix, Y., Lin, S., Lundquist, P., Ma, C., Marks, P., Maxham, M., Murphy, D., Park, I., Pham, T., Phillips, M., Roy, J., Sebra, R., Shen, G., Sorenson, J., Tomaney, A., Travers, K., Trulson, M., Vieceli, J., Wegener, J., Wu, D., Yang, A., Zaccarin, D., Zhao, P., Zhong, F., Korlach, J., Turner, S., 2009. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138. Li, H., Xie, K., Huang, H., Wang, S., 2014. 6-Hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33. PLoS One 9, e103324. Li, H., Xie, K., Yu, W., Hu, L., Huang, H., Xie, H., Wang, S., 2016. Nicotine dehydrogenase complexed with 6-hydroxypseudooxynicotine oxidase involved in the hybrid nicotine-degrading pathway in Agrobacterium tumefaciens S33. Appl. Environ. Microbiol., http://dx.doi.org/10.1128/aem. 03909-15. Roduit, J.P., Wellig, A., Kiener, A., 1997. Renewable functionalized pyridines derived from microbial metabolites of the alkaloid (S)-nicotine. Heterocycles 45, 1687–1702. Tang, H., Wang, L., Wang, W., Yu, H., Zhang, K., Yao, Y., Xu, P., 2013. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genet. 9, e1003923. Wang, S.N., Xu, P., Tang, H.Z., Meng, J., Liu, X.L., Qing, C., 2005. Green route to 6-hydroxy-3-succinoyl-pyridine from (S)-nicotine of tobacco waste by whole cells of a Pseudomonas sp. Environ. Sci. Technol. 39, 6877–6880. Wang, S.N., Liu, Z., Xu, P., 2009. Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33. J. Appl. Microbiol. 107, 838–847. Wang, S., Huang, H., Xie, K., Xu, P., 2012. Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway. Appl. Microbiol. Biotechnol. 95, 1567–1578. Yu, H., Tang, H., Zhu, X., Li, Y., Xu, P., 2015. Molecular mechanism of nicotine degradation by a newly isolated strain: Ochrobactrum sp. strain SJY1. Appl. Environ. Microbiol. 81, 272–281.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome announcement
Genome sequence of the nicotine-degrading Agrobacterium tumefaciens S33 Wenjun Yu a , Huili Li a , Kebo Xie a , Haiyan Huang a,b , Huijun Xie c , Shuning Wang a,∗ a b c
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People’s Republic of China Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan, People’s Republic of China Environment Research Institute, Shandong University, Jinan 250100, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 12 April 2016 Accepted 13 April 2016 Available online 20 April 2016 Keywords: Genome sequence Nicotine Degradation Agrobacterium tumefaciens Functionalized pyridine Tobacco wastes
a b s t r a c t Agrobacterium tumefaciens S33 is capable of growing with nicotine as the sole source of carbon and nitrogen, and has the potential to dispose of tobacco wastes and transform nicotine into functionalized pyridines intermediates, which are important precursors for some valuable drugs and insecticides. Here we report the complete genome sequence of strain S33 and predict the gene cluster involved in nicotine catabolism according to the annotation. © 2016 Elsevier B.V. All rights reserved.
Microbial degradation of the natural alkaloid nicotine has recently received attention because it represents a promising strategy to detoxify tobacco industry waste and to remove nicotine from tobacco products (Brandsch, 2006; Wang et al., 2012). Interestingly, it also attracts attention from chemists since nicotine can be used as a starting material to produce various chemicals of pharmaceutical importance, especially, nicotine can be transformed into renewable functionalized pyridines by biocatalytic processes that are difficult to synthesized by chemical methods (Roduit et al., 1997; Wang et al., 2005). Various bacteria degrading nicotine have been isolated and characterized such as Arthrobacter sp. and Pseudomonas sp., which decompose nicotine mainly through the pyridine pathway and pyrrolidine pathway, respectively, whose molecular mechanisms have been well characterized (Brandsch, 2006; Tang et al., 2013). Previously, we isolated Agrobacterium tumefaciens S33 from the rhizospheric soil of a tobacco plant, which was capable of utilizing nicotine as sole source of carbon and nitrogen (Wang et al., 2009). A novel fused nicotine degradation pathway of pyridine pathway and pyrrolidine pathway was discovered in strain S33 based on the intermediates identification and enzyme assays (Wang
∗ Corresponding author at: State Key Laboratory of Microbial Technology, Shandong University, 27 Shan Da South Road, Jinan 250100, People’s Republic of China. E-mail address: [email protected] (S. Wang). http://dx.doi.org/10.1016/j.jbiotec.2016.04.029 0168-1656/© 2016 Elsevier B.V. All rights reserved.
et al., 2012). Further, we purified and characterized 6-hydroxy-3succinoylpyridine hydroxylase (Hsh) and the complex of nicotine dehydrogenase (NdhAB) and 6-hydroxypseudooxynicotine oxidase (Pno) involved in the pathway (Li et al., 2014; Li et al., 2016). However, the molecular mechanism responsible for the novel fused pathway in A. tumefaciens S33 is still unclear. Here, we present the complete genome sequence of strain S33, obtained using the Pacific Biosciences (PacBio) sequencing technology. Genomic DNA from strain S33 was extracted using the Wizard Genomic DNA Purification Kit (Promega) and evaluated for its quality and quantity on the Bioanalyzer 2100 (Agilent). A 10kb insert SMRT-bell library was constructed and then sequenced by PacBio RS II sequencer (Pacific Biosciences) (Eid et al., 2009). After quality control, a total of 90,720 polymerase reads with a mean read length of 11,519 bases were generated, which led to a total of 1,045,009,373 bases with a 190-fold average coverage. Then the reads were de novo assembled using SMRT analysis software version 2.3.0 (Pacific Biosciences) (Chin et al., 2013). The assembled genome sequence was annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) version 3.1 (http://www. ncbi.nlm.nih.gov/genome/annotation prok/) and the Rapid Annotations using Subsystems Technology (RAST) server (Aziz et al., 2008). The final genome sequence of the strain S33 comprises 5,481,595 bases, which were assembled into 2 chromosomes, one circular chromosome (2.5 Mb) and one linear chromosome
2
W. Yu et al. / Journal of Biotechnology 228 (2016) 1–2
Table 1 Genome features of A. tumefaciens S33.
We also thank Professor Ping Xu from Shanghai Jiao Tong University for his valuable suggestion and supports.
Features
Chromosome 1
Chromosome 2
Molecular shape Size (bp) GC content (%) Total number of the genes Protein coding genes (CDSs) rRNAs (5S, 16S, 23S) tRNAs Pseudogenes GenBank accession number
circular 2,497,934 59.0 2244 2173 6 14 50 CP014259.1
linear 2,983,661 59.3 2897 2818 6 41 29 CP014260.1
(2.98 Mb) with GC content of 59.0% and 59.3%, respectively (Table 1). In total, 5141 genes were predicted using the PGAP, including 4991 protein-coding sequences (CDSs), 79 pseudo genes, 55 tRNAs, and 12 rRNA genes. According to the annotation and our previous study, some genes, which locate on the circular chromosome, were identified or predicted to be involved in nicotine degradation. Among them, the ORFs with locus tag as AWN88 01360, 01355, 01220, and 01205 have been experimentally verified to encode NdhB, NdhA, Pno, and Hsh, respectively (Li et al., 2014; Li et al., 2016). The ORFs with locus tag as AWN88 01345 and 01320 encode two proteins almost identical to 6-hydroxynicotine oxidase and 2,5-dihydroxypyridine dioxygenase from nicotine-degrading Ochrobactrum sp. SJY1, respectively (Yu et al., 2015). The arrangement of these ORFs in strain S33 suggests that the genes for nicotine degradation form a big gene cluster, which are not found in other strains of A. tumefaciens by comparison using RAST server. The genome sequence presented here will be helpful for elucidating the molecular mechanism on nicotine catabolism in A. tumefaciens S33 and developing new biological methods for transforming nicotine into valuable compounds. Strain and nucleotide sequence accession numbers The strain S33 has been deposited at China Center for Type Culture Collection under the accession number CCTCC AB 2016054 (originally CCTCC M 206131). This genome sequence has been deposited at GenBank under the accession numbers CP014259.1 and CP014260.1. Acknowledgements This work was supported by the grants from National Natural Science Foundation of China (Grant No. 30970027), the Excellent Middle-Aged and Youth Scientist Award Foundation of Shandong Province (No. BS2009SW006), and the Fundamental Research Funds of Shandong University (Grant No. 2014JC023).
References Aziz, R., Bartels, D., Best, A., DeJongh, M., Disz, T., Edwards, R., Formsma, K., Gerdes, S., Glass, E., Kubal, M., Meyer, F., Olsen, G., Olson, R., Osterman, A., Overbeek, R., McNeil, L., Paarmann, D., Paczian, T., Parrello, B., Pusch, G., Reich, C., Stevens, R., Vassieva, O., Vonstein, V., Wilke, A., Zagnitko, O., 2008. The RAST server: rapid annotations using subsystems technology. BMC Genom. 9, 75. Brandsch, R., 2006. Microbiology and biochemistry of nicotine degradation. Appl. Microbiol. Biotechnol. 69, 493–498. Chin, C.S., Alexander, D.H., Marks, P., Klammer, A.A., Drake, J., Heiner, C., Clum, A., Copeland, A., Huddleston, J., Eichler, E.E., Turner, S.W., Korlach, J., 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569. Eid, J., Fehr, A., Gray, J., Luong, K., Lyle, J., Otto, G., Peluso, P., Rank, D., Baybayan, P., Bettman, B., Bibillo, A., Bjornson, K., Chaudhuri, B., Christians, F., Cicero, R., Clark, S., Dalal, R., Dewinter, A., Dixon, J., Foquet, M., Gaertner, A., Hardenbol, P., Heiner, C., Hester, K., Holden, D., Kearns, G., Kong, X., Kuse, R., Lacroix, Y., Lin, S., Lundquist, P., Ma, C., Marks, P., Maxham, M., Murphy, D., Park, I., Pham, T., Phillips, M., Roy, J., Sebra, R., Shen, G., Sorenson, J., Tomaney, A., Travers, K., Trulson, M., Vieceli, J., Wegener, J., Wu, D., Yang, A., Zaccarin, D., Zhao, P., Zhong, F., Korlach, J., Turner, S., 2009. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138. Li, H., Xie, K., Huang, H., Wang, S., 2014. 6-Hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33. PLoS One 9, e103324. Li, H., Xie, K., Yu, W., Hu, L., Huang, H., Xie, H., Wang, S., 2016. Nicotine dehydrogenase complexed with 6-hydroxypseudooxynicotine oxidase involved in the hybrid nicotine-degrading pathway in Agrobacterium tumefaciens S33. Appl. Environ. Microbiol., http://dx.doi.org/10.1128/aem. 03909-15. Roduit, J.P., Wellig, A., Kiener, A., 1997. Renewable functionalized pyridines derived from microbial metabolites of the alkaloid (S)-nicotine. Heterocycles 45, 1687–1702. Tang, H., Wang, L., Wang, W., Yu, H., Zhang, K., Yao, Y., Xu, P., 2013. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genet. 9, e1003923. Wang, S.N., Xu, P., Tang, H.Z., Meng, J., Liu, X.L., Qing, C., 2005. Green route to 6-hydroxy-3-succinoyl-pyridine from (S)-nicotine of tobacco waste by whole cells of a Pseudomonas sp. Environ. Sci. Technol. 39, 6877–6880. Wang, S.N., Liu, Z., Xu, P., 2009. Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33. J. Appl. Microbiol. 107, 838–847. Wang, S., Huang, H., Xie, K., Xu, P., 2012. Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway. Appl. Microbiol. Biotechnol. 95, 1567–1578. Yu, H., Tang, H., Zhu, X., Li, Y., Xu, P., 2015. Molecular mechanism of nicotine degradation by a newly isolated strain: Ochrobactrum sp. strain SJY1. Appl. Environ. Microbiol. 81, 272–281.