- Email: [email protected]
Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens
G Model
ARTICLE IN PRESS
BIOTEC 6788 1–2
Journal of Biotechnology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome announcement
1
Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens
2
3
4 5 6
Q1
Jeong-A Lim a , Dong Hwan Lee a , Byoung-Young Kim b , Sunggi Heu a,∗ a b
Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea Business Planning Dept, CHUNLAB INC, Gwanak-gu, Seoul 151-742, Republic of Korea
7
8 19
a r t i c l e
i n f o
a b s t r a c t
9 10 11 12 13
Article history: Received 17 July 2014 Accepted 24 July 2014 Available online xxx
14 15 16 17 18
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Keywords: Pantoea agglomerans Biocontrol agent Genome analysis
Pantoea agglomerans R190, isolated from an apple orchard, showed antibacterial activity against various spoilage bacteria, including Pectobacterium carotovorum subsp. carotovorum, and foodborne pathogens such as Escherichia coli O157:H7. Here, we report the genome sequence of P. agglomerans R190. This report will raise the value of P. agglomerans as an agent for biocontrol of disease. © 2014 Published by Elsevier B.V.
Pantoea agglomerans (formerly Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae) is a Gram-negative bacterium belonging to the Enterobacteriaceae (Rezzonico et al., 2009). The saprophyte P. agglomerans has long been known to be an opportunistic pathogen of plants and humans, however, P. agglomerans has also been focused on as a beneficial bacterium due to its metabolic capabilities including the production of antibiotics such as phenazine, pantocin, herbicolin, and dapdiamide antibiotics (Giddens et al., 2002; Jin et al., 2003; Kamber et al., 2012; Mavrodi et al., 2010; Smith et al., 2013). With these antibacterial activities, several strains of P. agglomerans are being sold commercially to control the pathogenic bacterium Erwinia amylovora, which causes fire blight on apple trees (Johnson and Stockwell, 1998). P. agglomerans R190 (KACC 91765P) was isolated from an apple orchard in Chuncheon, Korea. The strain showed strong antibacterial activity against various spoilage bacteria, including Clavibacter michiganensis, Burkholderia andropogonis, Chryseobacterium balustinum, Dickeya zeae, Pectobacterium carotovorum, and Pseudomonas putida, and against foodborne pathogens such as Salmonella enterica and Escherichia coli O157:H7. The human pathogens Klebsiella pneumoniae and Yersinia enterocolitica have also been controlled by treatment with P. agglomerans R190. Since it has been suggested that P. agglomerans R190 carries a wide range of antibacterial substances, full genome sequencing has been
∗ Corresponding author. Tel.: +82 31 290 0455; fax: +82 31 290 0407. E-mail addresses: [email protected], [email protected] (S. Heu).
conducted. Here, we report the genome sequence of P. agglomerans R190. The genomic library of P. agglomerans R190 was sequenced using GS-FLX pyrosequencing, Illumina HiSeq, and PacBio platforms. The generated paired-end sequencing reads (229,894 reads, 12,051,218 reads, and 48,640 reads, respectively) were assembled using the CLC genomics workbench 6.5.1 (CLC bio, Denmark). The contigs and PCR-based long reads were combined through manual curation using CodonCode Aligner 3.7.1 (CodonCode Corp., Dedham, MA, USA). To check errors and dubious regions, the final genome sequence was corrected by remapping with raw reads. The coding sequences (CDSs) were predicted by Glimmer 3.02 (Delcher et al., 2007). tRNAs and rRNAs were identified by tRNA-Scan-SE (Lowe and Eddy, 1997), and HMMER with EzTaxon-e rRNA profiles (Eddy, 1998; Kim et al., 2012). The predicted CDSs were compared to catalytic families (catFams) and National Center for Biotechnology Information (NCBI) clusters of orthologous groups (COGs) by rpsBLAST, and to NCBI reference sequences (RefSeq) and SEED databases by BLASTP for functional annotation (Overbeek et al., 2005; Pruitt et al., 2009; Tatusov et al., 2000; Yu et al., 2009). The draft genome sequence of P. agglomerans R190 comprised 5 contigs. The genome size was 5,002,566 bp at 309× coverage, with an N50 of 3,393,498 bp. The G + C content was 55.05%. There were 4,778 predicted open reading frames, 23 rRNA genes, and 77 tRNA genes. The total number of ORFs with predicted functions was 3,636. Among the five contigs, three (contigs 3, 4, and 5) were predicted plasmids. Contig 4 contained a gene cluster for a phenazine antibiotic. The gene cluster consisted of 16 total genes and the gene products play roles in phenazine core biosynthesis (ehpA,
http://dx.doi.org/10.1016/j.jbiotec.2014.07.440 0168-1656/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Lim, J.-A., et al., Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.440
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
G Model
ARTICLE IN PRESS
BIOTEC 6788 1–2
J.-A. Lim et al. / Journal of Biotechnology xxx (2014) xxx–xxx
2 Table 1 Genome features of P. agglomerans R190. Genome Information
Total
Contig 1
Genome size (bp) GC ratio (%) Protein coding genes (CDS) tRNA genes rRNA genes
5,002,566 55.05 4,778 77 23
3,393,498 55.49 3,213 60 13
ehpB, ehpC, ehpD, and ehpE), phenazine modification (ehpF, ehpG, ehpH, ehpI, ehpK, ehpL, ehpM, ehpN, and ehpO), transport (ehpJ), and producer resistance (ehpR) (Yu et al., 2011). Genes related to 76 other antibiotics known to be produced by P. agglomerans, such 77 as pantocin or herbicolin, were not predicted, thus it is possible 78 that all antibiotic activity of P. agglomerans strain R190 comes from 79 80Q2 phenazine antibiotics (Table 1). Nucleotide sequence accession number: This Whole Genome 81 Shotgun project has been deposited in GenBank under the acces82 sion number JNGC00000000. The version described in this paper is 83 version JNGC01000000. 84 74 75
85
Acknowledgments
88
This work was supported by the Rural Development Administration (RDA) fund (PJ008751) and the postdoctoral fellowships of J.L. and D.L.
89
References
86 87
90 91 92 93 94 95 96 97 98 99 100
Delcher, A.L., Bratke, K.A., Powers, E.C., Salzberg, S.L., 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23, 673–679. Eddy, S.R., 1998. Profile hidden Markov models. Bioinformatics 14, 755–763. Giddens, S.R., Feng, Y., Mahanty, H.K., 2002. Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087. Mol. Microbiol. 45, 769–783. Jin, M., Wright, S.A., Beer, S.V., Clardy, J., 2003. The biosynthetic gene cluster of Pantocin a provides insights into biosynthesis and a tool for screening. Angew. Chem. Int. Ed. Engl. 42, 2902–2905. Johnson, K.B., Stockwell, V.O., 1998. Management of fire blight: a case study in microbial ecology. Annu. Rev. Phytopathol. 36, 227–248. Kamber, T., Lansdell, T.A., Stockwell, V.O., Ishimaru, C.A., Smits, T.H., Duffy, B., 2012. Characterization of the biosynthetic operon for the antibacterial
Contig 2 681,307 55.83 623 17 10
Contig 3 538,297 53.42 556
Contig 4 212,535 51.97 197
Contig 5 176,929 52.46 189
peptide herbicolin in Pantoea vagans biocontrol strain C9-1 and incidence in Pantoea species. Appl. Environ. Microbiol. 78, 4412–4419. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J., 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62, 716–721. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Mavrodi, D.V., Peever, T.L., Mavrodi, O.V., Parejko, J.A., Raaijmakers, J.M., Lemanceau, P., Mazurier, S., Heide, L., Blankenfeldt, W., Weller, D.M., Thomashow, L.S., 2010. Diversity and evolution of the phenazine biosynthesis pathway. Appl. Environ. Microbiol. 76, 866–879. Overbeek, R., Begley, T., Butler, R.M., Choudhuri, J.V., Chuang, H.Y., Cohoon, M., de Crecy-Lagard, V., Diaz, N., Disz, T., Edwards, R., Fonstein, M., Frank, E.D., Gerdes, S., Glass, E.M., Goesmann, A., Hanson, A., Iwata-Reuyl, D., Jensen, R., Jamshidi, N., Krause, L., Kubal, M., Larsen, N., Linke, B., McHardy, A.C., Meyer, F., Neuweger, H., Olsen, G., Olson, R., Osterman, A., Portnoy, V., Pusch, G.D., Rodionov, D.A., Ruckert, C., Steiner, J., Stevens, R., Thiele, I., Vassieva, O., Ye, Y., Zagnitko, O., Vonstein, V., 2005. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 33, 5691–5702. Pruitt, K.D., Tatusova, T., Klimke, W., Maglott, D.R., 2009. NCBI Reference Sequences: current status, policy and new initiatives. Nucleic Acids Res. 37, D32–D36. Rezzonico, F., Smits, T.H., Montesinos, E., Frey, J.E., Duffy, B., 2009. Genotypic comparison of Pantoea agglomerans plant and clinical strains. BMC Microbiol. 9, 204. Smith, D.D., Kirzinger, M.W., Stavrinides, J., 2013. Draft Genome Sequence of the Antibiotic-Producing Cystic Fibrosis Isolate Pantoea agglomerans Tx10. Genome Announ. 1, e00904–e00913. Tatusov, R.L., Galperin, M.Y., Natale, D.A., Koonin, E.V., 2000. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33–36. Yu, C., Zavaljevski, N., Desai, V., Reifman, J., 2009. Genome-wide enzyme annotation with precision control: catalytic families (CatFam) databases. Proteins 74, 449–460. Yu, S., Vit, A., Devenish, S., Mahanty, H.K., Itzen, A., Goody, R.S., Blankenfeldt, W., 2011. Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin/mitomycin C resistance in other bacteria. BMC Struct. Biol. 11, 33.
Please cite this article in press as: Lim, J.-A., et al., Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.440
101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138
ARTICLE IN PRESS
BIOTEC 6788 1–2
Journal of Biotechnology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome announcement
1
Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens
2
3
4 5 6
Q1
Jeong-A Lim a , Dong Hwan Lee a , Byoung-Young Kim b , Sunggi Heu a,∗ a b
Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea Business Planning Dept, CHUNLAB INC, Gwanak-gu, Seoul 151-742, Republic of Korea
7
8 19
a r t i c l e
i n f o
a b s t r a c t
9 10 11 12 13
Article history: Received 17 July 2014 Accepted 24 July 2014 Available online xxx
14 15 16 17 18
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Keywords: Pantoea agglomerans Biocontrol agent Genome analysis
Pantoea agglomerans R190, isolated from an apple orchard, showed antibacterial activity against various spoilage bacteria, including Pectobacterium carotovorum subsp. carotovorum, and foodborne pathogens such as Escherichia coli O157:H7. Here, we report the genome sequence of P. agglomerans R190. This report will raise the value of P. agglomerans as an agent for biocontrol of disease. © 2014 Published by Elsevier B.V.
Pantoea agglomerans (formerly Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae) is a Gram-negative bacterium belonging to the Enterobacteriaceae (Rezzonico et al., 2009). The saprophyte P. agglomerans has long been known to be an opportunistic pathogen of plants and humans, however, P. agglomerans has also been focused on as a beneficial bacterium due to its metabolic capabilities including the production of antibiotics such as phenazine, pantocin, herbicolin, and dapdiamide antibiotics (Giddens et al., 2002; Jin et al., 2003; Kamber et al., 2012; Mavrodi et al., 2010; Smith et al., 2013). With these antibacterial activities, several strains of P. agglomerans are being sold commercially to control the pathogenic bacterium Erwinia amylovora, which causes fire blight on apple trees (Johnson and Stockwell, 1998). P. agglomerans R190 (KACC 91765P) was isolated from an apple orchard in Chuncheon, Korea. The strain showed strong antibacterial activity against various spoilage bacteria, including Clavibacter michiganensis, Burkholderia andropogonis, Chryseobacterium balustinum, Dickeya zeae, Pectobacterium carotovorum, and Pseudomonas putida, and against foodborne pathogens such as Salmonella enterica and Escherichia coli O157:H7. The human pathogens Klebsiella pneumoniae and Yersinia enterocolitica have also been controlled by treatment with P. agglomerans R190. Since it has been suggested that P. agglomerans R190 carries a wide range of antibacterial substances, full genome sequencing has been
∗ Corresponding author. Tel.: +82 31 290 0455; fax: +82 31 290 0407. E-mail addresses: [email protected], [email protected] (S. Heu).
conducted. Here, we report the genome sequence of P. agglomerans R190. The genomic library of P. agglomerans R190 was sequenced using GS-FLX pyrosequencing, Illumina HiSeq, and PacBio platforms. The generated paired-end sequencing reads (229,894 reads, 12,051,218 reads, and 48,640 reads, respectively) were assembled using the CLC genomics workbench 6.5.1 (CLC bio, Denmark). The contigs and PCR-based long reads were combined through manual curation using CodonCode Aligner 3.7.1 (CodonCode Corp., Dedham, MA, USA). To check errors and dubious regions, the final genome sequence was corrected by remapping with raw reads. The coding sequences (CDSs) were predicted by Glimmer 3.02 (Delcher et al., 2007). tRNAs and rRNAs were identified by tRNA-Scan-SE (Lowe and Eddy, 1997), and HMMER with EzTaxon-e rRNA profiles (Eddy, 1998; Kim et al., 2012). The predicted CDSs were compared to catalytic families (catFams) and National Center for Biotechnology Information (NCBI) clusters of orthologous groups (COGs) by rpsBLAST, and to NCBI reference sequences (RefSeq) and SEED databases by BLASTP for functional annotation (Overbeek et al., 2005; Pruitt et al., 2009; Tatusov et al., 2000; Yu et al., 2009). The draft genome sequence of P. agglomerans R190 comprised 5 contigs. The genome size was 5,002,566 bp at 309× coverage, with an N50 of 3,393,498 bp. The G + C content was 55.05%. There were 4,778 predicted open reading frames, 23 rRNA genes, and 77 tRNA genes. The total number of ORFs with predicted functions was 3,636. Among the five contigs, three (contigs 3, 4, and 5) were predicted plasmids. Contig 4 contained a gene cluster for a phenazine antibiotic. The gene cluster consisted of 16 total genes and the gene products play roles in phenazine core biosynthesis (ehpA,
http://dx.doi.org/10.1016/j.jbiotec.2014.07.440 0168-1656/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Lim, J.-A., et al., Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.440
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
G Model
ARTICLE IN PRESS
BIOTEC 6788 1–2
J.-A. Lim et al. / Journal of Biotechnology xxx (2014) xxx–xxx
2 Table 1 Genome features of P. agglomerans R190. Genome Information
Total
Contig 1
Genome size (bp) GC ratio (%) Protein coding genes (CDS) tRNA genes rRNA genes
5,002,566 55.05 4,778 77 23
3,393,498 55.49 3,213 60 13
ehpB, ehpC, ehpD, and ehpE), phenazine modification (ehpF, ehpG, ehpH, ehpI, ehpK, ehpL, ehpM, ehpN, and ehpO), transport (ehpJ), and producer resistance (ehpR) (Yu et al., 2011). Genes related to 76 other antibiotics known to be produced by P. agglomerans, such 77 as pantocin or herbicolin, were not predicted, thus it is possible 78 that all antibiotic activity of P. agglomerans strain R190 comes from 79 80Q2 phenazine antibiotics (Table 1). Nucleotide sequence accession number: This Whole Genome 81 Shotgun project has been deposited in GenBank under the acces82 sion number JNGC00000000. The version described in this paper is 83 version JNGC01000000. 84 74 75
85
Acknowledgments
88
This work was supported by the Rural Development Administration (RDA) fund (PJ008751) and the postdoctoral fellowships of J.L. and D.L.
89
References
86 87
90 91 92 93 94 95 96 97 98 99 100
Delcher, A.L., Bratke, K.A., Powers, E.C., Salzberg, S.L., 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23, 673–679. Eddy, S.R., 1998. Profile hidden Markov models. Bioinformatics 14, 755–763. Giddens, S.R., Feng, Y., Mahanty, H.K., 2002. Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087. Mol. Microbiol. 45, 769–783. Jin, M., Wright, S.A., Beer, S.V., Clardy, J., 2003. The biosynthetic gene cluster of Pantocin a provides insights into biosynthesis and a tool for screening. Angew. Chem. Int. Ed. Engl. 42, 2902–2905. Johnson, K.B., Stockwell, V.O., 1998. Management of fire blight: a case study in microbial ecology. Annu. Rev. Phytopathol. 36, 227–248. Kamber, T., Lansdell, T.A., Stockwell, V.O., Ishimaru, C.A., Smits, T.H., Duffy, B., 2012. Characterization of the biosynthetic operon for the antibacterial
Contig 2 681,307 55.83 623 17 10
Contig 3 538,297 53.42 556
Contig 4 212,535 51.97 197
Contig 5 176,929 52.46 189
peptide herbicolin in Pantoea vagans biocontrol strain C9-1 and incidence in Pantoea species. Appl. Environ. Microbiol. 78, 4412–4419. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J., 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62, 716–721. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Mavrodi, D.V., Peever, T.L., Mavrodi, O.V., Parejko, J.A., Raaijmakers, J.M., Lemanceau, P., Mazurier, S., Heide, L., Blankenfeldt, W., Weller, D.M., Thomashow, L.S., 2010. Diversity and evolution of the phenazine biosynthesis pathway. Appl. Environ. Microbiol. 76, 866–879. Overbeek, R., Begley, T., Butler, R.M., Choudhuri, J.V., Chuang, H.Y., Cohoon, M., de Crecy-Lagard, V., Diaz, N., Disz, T., Edwards, R., Fonstein, M., Frank, E.D., Gerdes, S., Glass, E.M., Goesmann, A., Hanson, A., Iwata-Reuyl, D., Jensen, R., Jamshidi, N., Krause, L., Kubal, M., Larsen, N., Linke, B., McHardy, A.C., Meyer, F., Neuweger, H., Olsen, G., Olson, R., Osterman, A., Portnoy, V., Pusch, G.D., Rodionov, D.A., Ruckert, C., Steiner, J., Stevens, R., Thiele, I., Vassieva, O., Ye, Y., Zagnitko, O., Vonstein, V., 2005. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 33, 5691–5702. Pruitt, K.D., Tatusova, T., Klimke, W., Maglott, D.R., 2009. NCBI Reference Sequences: current status, policy and new initiatives. Nucleic Acids Res. 37, D32–D36. Rezzonico, F., Smits, T.H., Montesinos, E., Frey, J.E., Duffy, B., 2009. Genotypic comparison of Pantoea agglomerans plant and clinical strains. BMC Microbiol. 9, 204. Smith, D.D., Kirzinger, M.W., Stavrinides, J., 2013. Draft Genome Sequence of the Antibiotic-Producing Cystic Fibrosis Isolate Pantoea agglomerans Tx10. Genome Announ. 1, e00904–e00913. Tatusov, R.L., Galperin, M.Y., Natale, D.A., Koonin, E.V., 2000. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33–36. Yu, C., Zavaljevski, N., Desai, V., Reifman, J., 2009. Genome-wide enzyme annotation with precision control: catalytic families (CatFam) databases. Proteins 74, 449–460. Yu, S., Vit, A., Devenish, S., Mahanty, H.K., Itzen, A., Goody, R.S., Blankenfeldt, W., 2011. Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin/mitomycin C resistance in other bacteria. BMC Struct. Biol. 11, 33.
Please cite this article in press as: Lim, J.-A., et al., Draft genome sequence of Pantoea agglomerans R190, a producer of antibiotics against phytopathogens and foodborne pathogens. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.440
101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138