G Model
ARTICLE IN PRESS
BIOTEC 7070 1–2
Journal of Biotechnology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
1
Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951)
2
3
4
Q1
5 6
Daniel Wibberg a , Rabeaa S. Alkhateeb a,b , Anika Winkler a , Andreas Albersmeier a , Sarah Schatschneider a,b,1 , Stefan Albaum a , Karsten Niehaus a,b , Gerd Hublik c , Alfred Pühler a , Frank-Jörg Vorhölter a,∗ a
Center for Biotechnology CeBiTec, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany Abteilung für Proteom-und Metabolomforschung, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany c Jungbunzlauer Austria AG, 2064 Wulzeshofen, Austria
7
b
8 9 10
a r t i c l e
11 24
i n f o
a b s t r a c t
12
Article history: Received 21 March 2015 Accepted 25 March 2015 Available online xxx
13 14 15 16 17
Keywords: Exopolysaccharide EPS Thickening agent Plant pathogen Systems biotechnology
18 19 20 21 22 23
Xanthomonas campestris NRRL B-1459 was used in pioneering studies related to the biotechnological production of xanthan, the commercially most important polysaccharide of bacterial origin. The analysis of its genome revealed a 5.1 Mb chromosome plus the first complete plasmid of an X. campestris strain applied in biotechnology. © 2015 Published by Elsevier B.V.
Xanthomonads are Gram-negative ␥-proteobacteria. As plant pathogens they affect important crops world-wide (Ryan et al., 2011). A characteristic feature of Xanthomonas bacteria is their 27 exopolysaccharide xanthan. Xanthan is an efficient thickening 28 agent (Jeanes et al., 1961). It is produced commercially by fermen29 tation of X. campestris pv. campestris (Hublik, 2012). A systematic 30 understanding of xanthan biosynthetic genes was obtained by ana31 lyzing the genome of X. campestris pv. campestris B100 (Vorhölter 32 33Q4 et al., 2008; Vorhölter et al., 2003) while complete genomes of the X. campestris pv. campestris strains 8004 and ATCC 33913 had 34 been analyzed with regard to plant pathology (Ryan et al., 2011). 35 Recently, draft genome data was made available for additional X. 36 campestris pv. campestris strains (Bolot et al., 2013a, 2013b). 37 The X. campestris isolate B-1459, initially characterized at the 38 Northern Regional Research Laboratory (NRRL) in Peoria, Illinois, 39 USA, was used in pioneering studies of xanthan biotechnology 40 (Jeanes et al., 1961) and when metabolomics and flux balance anal41 ysis (Létisse et al., 2002) were introduced to xanthomonads. Later, 42 omics analyses and systems biotechnology (Schatschneider et al., 43 25Q3 26
Q2
∗ Corresponding author. Tel.: +49 521 1068701. E-mail address: [email protected] (F.-J. Vorhölter). 1 Present address: Centre for Analytical Bioscience, Boots Science Building, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
2014) were continued with X. campestris pv. campestris strain B100 where complete genome data was available (Vorhölter et al., 2008; 2003). To analyze the genetic basis of xanthan biosynthesis in the early lab strain, the genome sequence of X. campestris B-1459 was determined. We extracted genomic DNA of X. campestris strain B-1459 and generated a whole genome shotgun fragment library plus two mate-pair fragment libraries (Maus et al., 2014) using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) and an Illumina Nextera Mate Pair Sample Preparation Kit, respectively. The libraries were sequenced on an Illumina MiSeq system following standard protocols as per the manufacturer’s instructions. The average fragment size of the WGS library turned out to be 666 ± 227 bp, while the mate-pair libraries had average fragment sizes of 6.246 ± 1.597 and 7.838 ± 2.183 bp. A 207-fold coverage was achieved for the genome of strain B-1459, which had a G+C content of 65.03%. Assembly with the GS de novo software (Newbler) covered 1,079,270,030 bases from 4,779,001 aligned individual reads; among them 2,087,030 paired-end reads, and resulted in 74 contigs, organized in two scaffolds. The two scaffolds represented a chromosome of 5,077,495 bp and a plasmid. The plasmid of 45,729 bp was completed in an in silico approach (Wibberg et al., 2011). Based on the assembled genome data an automated annotation was carried out with the GenDB software (Meyer et al., 2003) using
http://dx.doi.org/10.1016/j.jbiotec.2015.03.026 0168-1656/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Wibberg, D., et al., Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951). J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.026
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
G Model BIOTEC 7070 1–2
ARTICLE IN PRESS D. Wibberg et al. / Journal of Biotechnology xxx (2015) xxx–xxx
2
Table 1 Genome features of Xanthomonas campestris pv. campestris B-1459.
69 70 71 72 73 74 75 76
77 78
References
Feature
Chromosome
Plasmid
Number of contigs Number of scaffolds Size Protein coding genes (CDSs) CDSs with predicted functions tRNA genes
70 1 5,077,495 bp 4260 3179 54
1 1 45,729 bp 62 20 0
Prodigal to predict protein-coding genes as established for other xanthomonads (Wichmann et al., 2013). Resulting genome features are summarized in Table 1. Now the genetic blueprint of xanthan biosynthesis is available for five X. campestris pv. campestris strains, among them two that have been frequently applied in biotechnological research. This data will facilitate more detailed and comparative analyses to correlate xanthan production characteristics and genomic sequences. 1. Nucleotide sequence accession number and availability of Xanthomonas campestris NRRL B 1459
86
The genome sequence of X. campestris B-1459 was deposited in the EMBL database under the accession numbers CDNB00000000 (chromosome) and LN811400 (plasmid). The strain is available from the American Type Culture Collection, from the BCCM/LMG Bacteria Collection (Ghent, Belgium), and from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) under the accession numbers ATCC 13951, LMG 573, and DSM-19000, respectively.
87
Conflict of interest
79 80 81 82 83 84 85
88
89
No conflict of interests declared. Acknowledgement
The project benefited from financial support by Jungbunzlauer 91Q6 Austria AG. 90Q5
Bolot, S., Guy, E., Carrere, S., Barbe, V., Arlat, M., Noël, L.D., 2013a. Genome sequence of Xanthomonas campestris pv. campestris strain Xca5. Genome Announc. 1, pii: e00032-12. Bolot, S., Roux, B., Carrere, S., Jiang, B.L., Tang, J.L., Arlat, M., Noël, L.D., 2013b. Genome sequences of three atypical Xanthomonas campestris pv. campestris strains, CN14, CN15, and CN16. Genome Announc. 1, pii: e00465-13. Hublik, G., 2012. 10.11–Xanthan. In: Polymer Science: A Comprehensive Reference. Elsevier, Amsterdam, pp. 221–229. Jeanes, A., Pittsley, J.E., Senti, F.R., 1961. Polysaccharide B-1459: a new hydrocolloid polyelectrolyte produced from glucose by bacterial fermentation. J. Appl. Polym. Sci. 5, 519–526. Létisse, F., Chevallereau, P., Simon, J.L., Lindley, N., 2002. The influence of metabolic network structures and energy requirements on xanthan gum yields. J. Biotechnol. 99, 307–317. Maus, I., Stantscheff, R., Wibberg, D., Stolze, Y., Winkler, A., Pühler, A., König, H., Schlüter, A., 2014. Complete genome sequence of the methanogenic neotype strain Methanobacterium formicicum MFT . J. Biotechnol., http://dx.doi.org/ 10.1016/j.jbiotec.2014.09.018. Meyer, F., Goesmann, A., McHardy, A.C., Bartels, D., Bekel, T., Clausen, J., Kalinowski, J., Linke, B., Rupp, O., Giegerich, R., Pühler, A., 2003. GenDB – an open source genome annotation system for prokaryote genomes. Nucl. Acids Res. 31, 2187–2195. Ryan, R.P., Vorhölter, F.J., Potnis, N., Jones, J.B., Van Sluys, M.A., Bogdanove, A.J., Dow, J.M., 2011. Pathogenomics of Xanthomonas: understanding bacteriumplant interactions. Nat. Rev. Microbiol. 9, 344–355. Schatschneider, S., Huber, C., Neuweger, H., Watt, T.F., Pühler, A., Eisenreich, W., Wittmann, C., Niehaus, K., Vorhölter, F.J., 2014. Metabolic flux pattern of glucose utilization by Xanthomonas campestris pv. campestris: prevalent role of the Entner-Doudoroff pathway and minor fluxes through the pentose phosphate pathway and glycolysis. Mol. Biosyst. 10, 2663– 2676. Vorhölter, F.J., Schneiker, S., Goesmann, A., Krause, L., Bekel, T., Kaiser, O., Linke, B., Patschkowski, T., Rückert, C., Schmid, J., Sidhu, V.K., Sieber, V., Tauch, A., Watt, S.A., Weisshaar, B., Becker, A., Niehaus, K., Pühler, A., 2008. The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis. J. Biotechnol. 134, 33–45. Wibberg, D., Blom, J., Jaenicke, S., Kollin, F., Rupp, O., Scherf, B., Schneiker-Bekel, S., Sczcepanowski, R., Goesman, A., Setubal, J.C., Schmitt, R., Pühler, A., Schlüter, A., 2011. Complete sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plamid. J. Biotechnol. 155, 50–62. Wichmann, F., Vorhölter, F.J., Hersemann, L., Widmer, F., Blom, J., Niehaus, K., Reinhard, S., Conradin, C., Kölliker, R., 2013. The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection. Mol. Plant Pathol. 14, 576–588.
Please cite this article in press as: Wibberg, D., et al., Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951). J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.026
92
93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
ARTICLE IN PRESS
BIOTEC 7070 1–2
Journal of Biotechnology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
1
Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951)
2
3
4
Q1
5 6
Daniel Wibberg a , Rabeaa S. Alkhateeb a,b , Anika Winkler a , Andreas Albersmeier a , Sarah Schatschneider a,b,1 , Stefan Albaum a , Karsten Niehaus a,b , Gerd Hublik c , Alfred Pühler a , Frank-Jörg Vorhölter a,∗ a
Center for Biotechnology CeBiTec, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany Abteilung für Proteom-und Metabolomforschung, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany c Jungbunzlauer Austria AG, 2064 Wulzeshofen, Austria
7
b
8 9 10
a r t i c l e
11 24
i n f o
a b s t r a c t
12
Article history: Received 21 March 2015 Accepted 25 March 2015 Available online xxx
13 14 15 16 17
Keywords: Exopolysaccharide EPS Thickening agent Plant pathogen Systems biotechnology
18 19 20 21 22 23
Xanthomonas campestris NRRL B-1459 was used in pioneering studies related to the biotechnological production of xanthan, the commercially most important polysaccharide of bacterial origin. The analysis of its genome revealed a 5.1 Mb chromosome plus the first complete plasmid of an X. campestris strain applied in biotechnology. © 2015 Published by Elsevier B.V.
Xanthomonads are Gram-negative ␥-proteobacteria. As plant pathogens they affect important crops world-wide (Ryan et al., 2011). A characteristic feature of Xanthomonas bacteria is their 27 exopolysaccharide xanthan. Xanthan is an efficient thickening 28 agent (Jeanes et al., 1961). It is produced commercially by fermen29 tation of X. campestris pv. campestris (Hublik, 2012). A systematic 30 understanding of xanthan biosynthetic genes was obtained by ana31 lyzing the genome of X. campestris pv. campestris B100 (Vorhölter 32 33Q4 et al., 2008; Vorhölter et al., 2003) while complete genomes of the X. campestris pv. campestris strains 8004 and ATCC 33913 had 34 been analyzed with regard to plant pathology (Ryan et al., 2011). 35 Recently, draft genome data was made available for additional X. 36 campestris pv. campestris strains (Bolot et al., 2013a, 2013b). 37 The X. campestris isolate B-1459, initially characterized at the 38 Northern Regional Research Laboratory (NRRL) in Peoria, Illinois, 39 USA, was used in pioneering studies of xanthan biotechnology 40 (Jeanes et al., 1961) and when metabolomics and flux balance anal41 ysis (Létisse et al., 2002) were introduced to xanthomonads. Later, 42 omics analyses and systems biotechnology (Schatschneider et al., 43 25Q3 26
Q2
∗ Corresponding author. Tel.: +49 521 1068701. E-mail address: [email protected] (F.-J. Vorhölter). 1 Present address: Centre for Analytical Bioscience, Boots Science Building, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
2014) were continued with X. campestris pv. campestris strain B100 where complete genome data was available (Vorhölter et al., 2008; 2003). To analyze the genetic basis of xanthan biosynthesis in the early lab strain, the genome sequence of X. campestris B-1459 was determined. We extracted genomic DNA of X. campestris strain B-1459 and generated a whole genome shotgun fragment library plus two mate-pair fragment libraries (Maus et al., 2014) using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) and an Illumina Nextera Mate Pair Sample Preparation Kit, respectively. The libraries were sequenced on an Illumina MiSeq system following standard protocols as per the manufacturer’s instructions. The average fragment size of the WGS library turned out to be 666 ± 227 bp, while the mate-pair libraries had average fragment sizes of 6.246 ± 1.597 and 7.838 ± 2.183 bp. A 207-fold coverage was achieved for the genome of strain B-1459, which had a G+C content of 65.03%. Assembly with the GS de novo software (Newbler) covered 1,079,270,030 bases from 4,779,001 aligned individual reads; among them 2,087,030 paired-end reads, and resulted in 74 contigs, organized in two scaffolds. The two scaffolds represented a chromosome of 5,077,495 bp and a plasmid. The plasmid of 45,729 bp was completed in an in silico approach (Wibberg et al., 2011). Based on the assembled genome data an automated annotation was carried out with the GenDB software (Meyer et al., 2003) using
http://dx.doi.org/10.1016/j.jbiotec.2015.03.026 0168-1656/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Wibberg, D., et al., Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951). J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.026
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
G Model BIOTEC 7070 1–2
ARTICLE IN PRESS D. Wibberg et al. / Journal of Biotechnology xxx (2015) xxx–xxx
2
Table 1 Genome features of Xanthomonas campestris pv. campestris B-1459.
69 70 71 72 73 74 75 76
77 78
References
Feature
Chromosome
Plasmid
Number of contigs Number of scaffolds Size Protein coding genes (CDSs) CDSs with predicted functions tRNA genes
70 1 5,077,495 bp 4260 3179 54
1 1 45,729 bp 62 20 0
Prodigal to predict protein-coding genes as established for other xanthomonads (Wichmann et al., 2013). Resulting genome features are summarized in Table 1. Now the genetic blueprint of xanthan biosynthesis is available for five X. campestris pv. campestris strains, among them two that have been frequently applied in biotechnological research. This data will facilitate more detailed and comparative analyses to correlate xanthan production characteristics and genomic sequences. 1. Nucleotide sequence accession number and availability of Xanthomonas campestris NRRL B 1459
86
The genome sequence of X. campestris B-1459 was deposited in the EMBL database under the accession numbers CDNB00000000 (chromosome) and LN811400 (plasmid). The strain is available from the American Type Culture Collection, from the BCCM/LMG Bacteria Collection (Ghent, Belgium), and from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) under the accession numbers ATCC 13951, LMG 573, and DSM-19000, respectively.
87
Conflict of interest
79 80 81 82 83 84 85
88
89
No conflict of interests declared. Acknowledgement
The project benefited from financial support by Jungbunzlauer 91Q6 Austria AG. 90Q5
Bolot, S., Guy, E., Carrere, S., Barbe, V., Arlat, M., Noël, L.D., 2013a. Genome sequence of Xanthomonas campestris pv. campestris strain Xca5. Genome Announc. 1, pii: e00032-12. Bolot, S., Roux, B., Carrere, S., Jiang, B.L., Tang, J.L., Arlat, M., Noël, L.D., 2013b. Genome sequences of three atypical Xanthomonas campestris pv. campestris strains, CN14, CN15, and CN16. Genome Announc. 1, pii: e00465-13. Hublik, G., 2012. 10.11–Xanthan. In: Polymer Science: A Comprehensive Reference. Elsevier, Amsterdam, pp. 221–229. Jeanes, A., Pittsley, J.E., Senti, F.R., 1961. Polysaccharide B-1459: a new hydrocolloid polyelectrolyte produced from glucose by bacterial fermentation. J. Appl. Polym. Sci. 5, 519–526. Létisse, F., Chevallereau, P., Simon, J.L., Lindley, N., 2002. The influence of metabolic network structures and energy requirements on xanthan gum yields. J. Biotechnol. 99, 307–317. Maus, I., Stantscheff, R., Wibberg, D., Stolze, Y., Winkler, A., Pühler, A., König, H., Schlüter, A., 2014. Complete genome sequence of the methanogenic neotype strain Methanobacterium formicicum MFT . J. Biotechnol., http://dx.doi.org/ 10.1016/j.jbiotec.2014.09.018. Meyer, F., Goesmann, A., McHardy, A.C., Bartels, D., Bekel, T., Clausen, J., Kalinowski, J., Linke, B., Rupp, O., Giegerich, R., Pühler, A., 2003. GenDB – an open source genome annotation system for prokaryote genomes. Nucl. Acids Res. 31, 2187–2195. Ryan, R.P., Vorhölter, F.J., Potnis, N., Jones, J.B., Van Sluys, M.A., Bogdanove, A.J., Dow, J.M., 2011. Pathogenomics of Xanthomonas: understanding bacteriumplant interactions. Nat. Rev. Microbiol. 9, 344–355. Schatschneider, S., Huber, C., Neuweger, H., Watt, T.F., Pühler, A., Eisenreich, W., Wittmann, C., Niehaus, K., Vorhölter, F.J., 2014. Metabolic flux pattern of glucose utilization by Xanthomonas campestris pv. campestris: prevalent role of the Entner-Doudoroff pathway and minor fluxes through the pentose phosphate pathway and glycolysis. Mol. Biosyst. 10, 2663– 2676. Vorhölter, F.J., Schneiker, S., Goesmann, A., Krause, L., Bekel, T., Kaiser, O., Linke, B., Patschkowski, T., Rückert, C., Schmid, J., Sidhu, V.K., Sieber, V., Tauch, A., Watt, S.A., Weisshaar, B., Becker, A., Niehaus, K., Pühler, A., 2008. The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis. J. Biotechnol. 134, 33–45. Wibberg, D., Blom, J., Jaenicke, S., Kollin, F., Rupp, O., Scherf, B., Schneiker-Bekel, S., Sczcepanowski, R., Goesman, A., Setubal, J.C., Schmitt, R., Pühler, A., Schlüter, A., 2011. Complete sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plamid. J. Biotechnol. 155, 50–62. Wichmann, F., Vorhölter, F.J., Hersemann, L., Widmer, F., Blom, J., Niehaus, K., Reinhard, S., Conradin, C., Kölliker, R., 2013. The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection. Mol. Plant Pathol. 14, 576–588.
Please cite this article in press as: Wibberg, D., et al., Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951). J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.026
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