GENOME ANNOUNCEMENT
Draft genome sequence of Sphingobium sp. strain ba1, resistant to kanamycin and nickel ions Caterina Manzari1, Matteo Chiara2, Alessandra Costanza1, Claudia Leoni1, Mariateresa Volpicella1, Ernesto Picardi1,3,4, Anna Maria D’Erchia1, Antonio Placido3, Massimo Trotta5, David S. Horner2, Graziano Pesole1,3,4,6 & Luigi R. Ceci3 1
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy; 2Department of Biosciences, University of Milan, Milan, Italy; 3Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy; 4National Institute of Biostructures and Biosystems (INBB), Rome, Italy; 5Institute for Chemical-Physical Processes, National Research Council, Bari, Italy; and 6Center of Excellence in Comparative Genomics, University of Bari, Bari, Italy
Correspondence: Luigi R. Ceci, Institute of Biomembranes and Bioenergetics, National Research Council, Via Amendola 165/A, 70126 Bari, Italy. Tel.: +39 0805443311; fax: +39 0805443403; e-mail: [email protected] Received 29 August 2014; revised 29 September 2014; accepted 1 October 2014. Final version published online 31 October 2014.
Abstract The genome sequence of a Sphingobium strain capable of tolerating high concentrations of Ni ions, and exhibiting natural kanamycin resistance, is presented. The presence of a transposon derived kanamycin resistance gene and several genes for efflux-mediated metal resistance may explain the observed characteristics of the new Sphingobium isolate.
DOI: 10.1111/1574-6968.12618
MICROBIOLOGY LETTERS
Editor: David Studholme Keywords antibiotic resistance; metal resistance; transposon.
The genus Sphingobium consists of Gram-negative, strictly aerobic, chemo-heterotrophic, rod-shaped, yellowpigmented bacteria, with membranes rich of glycosphingolipids (Yabuuchi et al., 1990). It is one of four closely related genera (Sphingomonas sensu stricto, Sphingobium, Novosphingobium, and Sphingopyxis) into which the previous more comprehensive genus Sphingomonas was subdivided on the basis of 16S rRNA gene sequences and biochemical features, such as fatty acid and polyamine composition, nitrate reduction, etc. (Takeuchi et al., 2001). In our laboratory, a spontaneous cell growth in the form of yellow round colonies was observed on Sistromagar plate containing 50 lg mL1 kanamycin and 7 mM NiCl2 incubated at 30 °C for 5–6 days. Analysis of 16S rRNA gene sequences from single colonies allowed assignment of the isolate to the genus Sphingobium with the highest sequence identity (99%) observed with the 16S DNA gene of S. xenophagum.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
The capacity of the novel Sphingobium isolate to tolerate high concentrations of Ni ions was further confirmed by growth in either Sistrom or LB liquid cultures supplemented with kanamycin and increasing concentrations of NiCl2, at 30 °C for 48 h. The strain (termed ba1) is able to grow up 20 mM salt concentration (higher concentrations were not tested due to precipitation of insoluble greenish nickel compounds). While several species of Sphingobium are known for their capacity to degrade xenobiotic compounds, particularly poly-aromatic hydrocarbons (Li et al., 2013), information about their growth in the presence of high concentrations of metal ions is scarce. Only S. cupriresistens, a species isolated from copper mine soil, has been reported to tolerate high concentrations of a metal ion (0.9 mM Cu2+) (Li et al., 2013). A member of the closely related genus Novosphingobium was also reported to tolerate 2.5 mM NiCl2 (D’Argenio et al., 2014).
FEMS Microbiol Lett 361 (2014) 8–9
Draft genome sequence of Sphingobium sp. strain ba1
DNA extracted from a single colony of Sphingobium ba1 was subjected to whole genome shotgun sequencing using the Nextera XT library preparation workflow (Illumina, San Diego, CA) and 2 9 250-nucleotide paired-end reads were generated on Illumina MiSeq instrument. The reads were quality trimmed using the sliding window mode of the TRIMMOMATIC (Bolger et al., 2014) program, with a cutoff value of 20. De novo genome assembly was performed using VELVET 1.2.10 (Zerbino & Birney, 2008) with a comprehensive grid search used for parameter optimization. The draft genome sequence is 4.46 Mb in length (c. 509 coverage, 62.9% G + C) and consists of 493 contigs. Scaffolding produced 87 supercontigs, of which the largest three (3.21 Mb, 600 Kb and 317 Kb) represented 93% of the total assembly length. Genome annotation was performed by merging predictions from XBASE (Chaudhuri & Pallen, 2006) using S. japonicum (Nagata et al., 2010) as reference species and by the Rapid Annotation Subsystem Technology (RAST) server (Aziz et al., 2008). 4657 protein coding genes, 48 transfer RNA genes, and 6 or 7 copies of the ribosomal RNA operon were identified. Kanamycin resistance is not typical of the genus Sphingobium (Vaz-Moreira et al., 2011). A predicted aph (30 )I homolog encoding an aminoglycoside 30 -phosphotransferase (GenBank accession number CAA23656) and an associated downstream region of about 1100 bp (GenBank accession number JPPQ01000086.1, locus tag IL54_4593) are identical to a two-thousand bp sequence of the Tn903 transposon (Oka et al., 1981). This locus may explain the presence and origin of kanamycin resistance in Sphingobium sp. strain ba1. Several homologs of CzcCBA resistance-nodulation division (RND) transporters, implicated in the efflux of metal ions (Nies, 2003), were detected. However, these genes are quite common in bacteria, and their effective role in conferring Ni-resistance has to be confirmed. Nucleotide sequence accession number. This Whole Genome Shotgun project has been deposited at DDBJ/ EMBL/GenBank under the accession JPPQ00000000. The version described in this study is version JPPQ01000000. Raw data are available from the Sequence Read Archive (SRA) under the accession number SRX708527.
Acknowledgements
9
Authors’ contribution C.M. and M.C. contributed equally to this work.
References Aziz RK, Bartels D, Best AA et al. (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9: 75. Bolger AM, Lohse M & Usadel B (2014) TRIMMOMATIC: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114–2120. Chaudhuri RR & Pallen MJ (2006) XBASE, a collection of online databases for bacterial comparative genomics. Nucleic Acids Res 34: D335–D337. D’Argenio V, Notomista E, Petrillo M et al. (2014) Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern. BMC Genomics 15: 384. Li L, Liu H, Shi Z & Wang G (2013) Sphingobium cupriresistens sp. nov., a copper-resistant bacterium isolated from copper mine soil, and emended description of the genus Sphingobium. Int J Syst Evol Microbiol 63: 604–609. Nagata Y, Ohtsubo Y, Endo R, Ichikawa N, Ankai A, Oguchi A, Fukui S, Fujita N & Tsuda M (2010) Complete genome sequence of the representative gamma-hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26. J Bacteriol 192: 5852–5853. Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27: 313–339. Oka A, Sugisaki H & Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147: 217–226. Takeuchi M, Hamana K & Hiraishi A (2001) Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51: 1405–1417. Vaz-Moreira I, Nunes OC & Manaia CM (2011) Diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water. Appl Environ Microbiol 77: 5697–5706. Yabuuchi E, Yano I, Oyaizu H, Hashimoto Y, Ezaki T & Yamamoto H (1990) Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol 34: 99–119. Zerbino DR & Birney E (2008) VELVET: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821–829.
This work was supported by Ministero dell’Istruzione, Universita e Ricerca (Micromap [PON01_02589]) and by the Molecular Biodiversity Lab of Lifewatch-Italia.
FEMS Microbiol Lett 361 (2014) 8–9
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Draft genome sequence of Sphingobium sp. strain ba1, resistant to kanamycin and nickel ions Caterina Manzari1, Matteo Chiara2, Alessandra Costanza1, Claudia Leoni1, Mariateresa Volpicella1, Ernesto Picardi1,3,4, Anna Maria D’Erchia1, Antonio Placido3, Massimo Trotta5, David S. Horner2, Graziano Pesole1,3,4,6 & Luigi R. Ceci3 1
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy; 2Department of Biosciences, University of Milan, Milan, Italy; 3Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy; 4National Institute of Biostructures and Biosystems (INBB), Rome, Italy; 5Institute for Chemical-Physical Processes, National Research Council, Bari, Italy; and 6Center of Excellence in Comparative Genomics, University of Bari, Bari, Italy
Correspondence: Luigi R. Ceci, Institute of Biomembranes and Bioenergetics, National Research Council, Via Amendola 165/A, 70126 Bari, Italy. Tel.: +39 0805443311; fax: +39 0805443403; e-mail: [email protected] Received 29 August 2014; revised 29 September 2014; accepted 1 October 2014. Final version published online 31 October 2014.
Abstract The genome sequence of a Sphingobium strain capable of tolerating high concentrations of Ni ions, and exhibiting natural kanamycin resistance, is presented. The presence of a transposon derived kanamycin resistance gene and several genes for efflux-mediated metal resistance may explain the observed characteristics of the new Sphingobium isolate.
DOI: 10.1111/1574-6968.12618
MICROBIOLOGY LETTERS
Editor: David Studholme Keywords antibiotic resistance; metal resistance; transposon.
The genus Sphingobium consists of Gram-negative, strictly aerobic, chemo-heterotrophic, rod-shaped, yellowpigmented bacteria, with membranes rich of glycosphingolipids (Yabuuchi et al., 1990). It is one of four closely related genera (Sphingomonas sensu stricto, Sphingobium, Novosphingobium, and Sphingopyxis) into which the previous more comprehensive genus Sphingomonas was subdivided on the basis of 16S rRNA gene sequences and biochemical features, such as fatty acid and polyamine composition, nitrate reduction, etc. (Takeuchi et al., 2001). In our laboratory, a spontaneous cell growth in the form of yellow round colonies was observed on Sistromagar plate containing 50 lg mL1 kanamycin and 7 mM NiCl2 incubated at 30 °C for 5–6 days. Analysis of 16S rRNA gene sequences from single colonies allowed assignment of the isolate to the genus Sphingobium with the highest sequence identity (99%) observed with the 16S DNA gene of S. xenophagum.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
The capacity of the novel Sphingobium isolate to tolerate high concentrations of Ni ions was further confirmed by growth in either Sistrom or LB liquid cultures supplemented with kanamycin and increasing concentrations of NiCl2, at 30 °C for 48 h. The strain (termed ba1) is able to grow up 20 mM salt concentration (higher concentrations were not tested due to precipitation of insoluble greenish nickel compounds). While several species of Sphingobium are known for their capacity to degrade xenobiotic compounds, particularly poly-aromatic hydrocarbons (Li et al., 2013), information about their growth in the presence of high concentrations of metal ions is scarce. Only S. cupriresistens, a species isolated from copper mine soil, has been reported to tolerate high concentrations of a metal ion (0.9 mM Cu2+) (Li et al., 2013). A member of the closely related genus Novosphingobium was also reported to tolerate 2.5 mM NiCl2 (D’Argenio et al., 2014).
FEMS Microbiol Lett 361 (2014) 8–9
Draft genome sequence of Sphingobium sp. strain ba1
DNA extracted from a single colony of Sphingobium ba1 was subjected to whole genome shotgun sequencing using the Nextera XT library preparation workflow (Illumina, San Diego, CA) and 2 9 250-nucleotide paired-end reads were generated on Illumina MiSeq instrument. The reads were quality trimmed using the sliding window mode of the TRIMMOMATIC (Bolger et al., 2014) program, with a cutoff value of 20. De novo genome assembly was performed using VELVET 1.2.10 (Zerbino & Birney, 2008) with a comprehensive grid search used for parameter optimization. The draft genome sequence is 4.46 Mb in length (c. 509 coverage, 62.9% G + C) and consists of 493 contigs. Scaffolding produced 87 supercontigs, of which the largest three (3.21 Mb, 600 Kb and 317 Kb) represented 93% of the total assembly length. Genome annotation was performed by merging predictions from XBASE (Chaudhuri & Pallen, 2006) using S. japonicum (Nagata et al., 2010) as reference species and by the Rapid Annotation Subsystem Technology (RAST) server (Aziz et al., 2008). 4657 protein coding genes, 48 transfer RNA genes, and 6 or 7 copies of the ribosomal RNA operon were identified. Kanamycin resistance is not typical of the genus Sphingobium (Vaz-Moreira et al., 2011). A predicted aph (30 )I homolog encoding an aminoglycoside 30 -phosphotransferase (GenBank accession number CAA23656) and an associated downstream region of about 1100 bp (GenBank accession number JPPQ01000086.1, locus tag IL54_4593) are identical to a two-thousand bp sequence of the Tn903 transposon (Oka et al., 1981). This locus may explain the presence and origin of kanamycin resistance in Sphingobium sp. strain ba1. Several homologs of CzcCBA resistance-nodulation division (RND) transporters, implicated in the efflux of metal ions (Nies, 2003), were detected. However, these genes are quite common in bacteria, and their effective role in conferring Ni-resistance has to be confirmed. Nucleotide sequence accession number. This Whole Genome Shotgun project has been deposited at DDBJ/ EMBL/GenBank under the accession JPPQ00000000. The version described in this study is version JPPQ01000000. Raw data are available from the Sequence Read Archive (SRA) under the accession number SRX708527.
Acknowledgements
9
Authors’ contribution C.M. and M.C. contributed equally to this work.
References Aziz RK, Bartels D, Best AA et al. (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9: 75. Bolger AM, Lohse M & Usadel B (2014) TRIMMOMATIC: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114–2120. Chaudhuri RR & Pallen MJ (2006) XBASE, a collection of online databases for bacterial comparative genomics. Nucleic Acids Res 34: D335–D337. D’Argenio V, Notomista E, Petrillo M et al. (2014) Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern. BMC Genomics 15: 384. Li L, Liu H, Shi Z & Wang G (2013) Sphingobium cupriresistens sp. nov., a copper-resistant bacterium isolated from copper mine soil, and emended description of the genus Sphingobium. Int J Syst Evol Microbiol 63: 604–609. Nagata Y, Ohtsubo Y, Endo R, Ichikawa N, Ankai A, Oguchi A, Fukui S, Fujita N & Tsuda M (2010) Complete genome sequence of the representative gamma-hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26. J Bacteriol 192: 5852–5853. Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27: 313–339. Oka A, Sugisaki H & Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147: 217–226. Takeuchi M, Hamana K & Hiraishi A (2001) Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51: 1405–1417. Vaz-Moreira I, Nunes OC & Manaia CM (2011) Diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water. Appl Environ Microbiol 77: 5697–5706. Yabuuchi E, Yano I, Oyaizu H, Hashimoto Y, Ezaki T & Yamamoto H (1990) Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol 34: 99–119. Zerbino DR & Birney E (2008) VELVET: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821–829.
This work was supported by Ministero dell’Istruzione, Universita e Ricerca (Micromap [PON01_02589]) and by the Molecular Biodiversity Lab of Lifewatch-Italia.
FEMS Microbiol Lett 361 (2014) 8–9
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
