- Short genome report
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Complete genome sequence of Novosphingobium pentaromativorans US6-1T
Standards in Genomic Sciences volume 10, Article number: 107 (2015)
Novosphingobium pentaromativorans US6-1T is a species in the family Sphingomonadaceae. According to the phylogenetic analysis based on 16S rRNA gene sequence of the N. pentaromativorans US6-1T and nine genome-sequenced strains in the genus Novosphingobium, the similarity ranged from 93.9 to 99.9 % and the highest similarity was found with Novosphingobium sp. PP1Y (99.9 %), whereas the ANI value based on genomes ranged from 70.9 to 93 % and the highest value was 93 %. This microorganism was isolated from muddy coastal bay sediments where the environment is heavily polluted by polycyclic aromatic hydrocarbons (PAHs). It was previously shown to be capable of degrading multiple PAHs, including benzo[a]pyrene. To further understand the PAH biodegradation pathways the previous draft genome of this microorganism was revised to obtain a complete genome using Illumina MiSeq and PacBio platform. The genome of strain US6-1T consists of 5,457,578 bp, which includes the 3,979,506 bp chromosome and five megaplasmids. It comprises 5110 protein-coding genes and 82 RNA genes. Here, we provide an analysis of the complete genome sequence which enables the identification of new characteristics of this strain.
The polycyclic aromatic hydrocarbons are widely distributed in the environment as one of the persistent organic pollutants and are generated by natural combustion processes as well as human activities . Benzo(a)pyrene is of environmental concern due to its high carcinogenic  and bioaccumulation potential . Biodegradation in contaminated environments is one of the important processes of remediation. Therefore, isolation of potent biodegradation strains and elucidation of the biodegradation pathways have drawn attention for a long time [4–6]. Novosphingobium pentaromativorans US6-1T, a Gram negative halophilic marine bacterium, is one of the potent strains capable of utilizing a series of high molecular weight PAHs as sole carbon and energy sources. Strain US6-1T showed an especially high degradation ability for benzo(a)pyrene . To understand the PAH biodegradation pathways, genomic and proteomic approaches were conducted on this strain [8, 9]. In the genomic study it was reported that strain US6-1T contained at least two large plasmids and most of the coding genes associated with PAH degradation were located in the larger plasmid pLA1 . However, the draft genome sequence was inadequate to understanding the degradation processes for high-molecular-weight compounds of PAH and their regulation mechanism. Therefore, completion of the strain US6-1T genome was carried-out and the genomic repertoire is reported in here.
Classification and features
At the time of writing, the genus Novosphingobium contains 30 species including N. pentaromativorans US6-1T. Phylogenetic analysis based on the 16S rRNA gene sequences using the neighbor-joining, maximum-likelihood and maximum-parsimony methods showed that N. pentaromativorans US6-1T formed a clade with other members within the genus Novosphingobium (Fig. 1). N. pentaromativorans US6-1T shared the 16S rRNA gene identity with the type strains, N. aquaticum FNE08-86T and N. mathurense SM117T, in the range of 93.9 and 98.7 %, respectively. The strain PP1Y , one of the whole-genome sequenced strains in genus Novosphingobium , was most closely related to N. pentaromativorans US6-1T with 99.9 % similarity.
Strain US6-1T cells are Gram-negative, non-motile rods (Table 1). Cells are 0.36–0.45 μm in width and 0.97–1.95 μm in length. Colonies on ZoBell 2216 agar and trypticase soy agar medium are yellowish and circular. Optimal growth occurred at 30 °C and was retarded below 20 °C. The organism tolerates pH values from 6 to 9 and optimal growth occurs at pH 6.5. Strain US6-1T grows in the range of 1–6 % NaCl with optimal growth at 2.5 % NaCl. The isolate can grow under anaerobic conditions but growth is retarded .
N. pentaromativorans US6-1T utilizes cyclodextrin, dextrin, Tween 40, Tween 80, α-D-glucose, maltose, D-trehalose, sucrose, psicose, methyl pyruvate, β-hydroxybutyric acid, α-ketobutyric acid, propionic acid, acetic acid, quinic acid, L-alanine, L-alanyl glycine, L-aspartic acid, L-glutamic acid, L-proline, L-threonine and L-phenylalanine . These phenotypes were confirmed by genomic methods.
Genome sequencing information
Genome project history
The genome of N. pentaromativorans US6-1T was sequenced in 2009 using a 454 GS FLX Titanium sequencing platform. The assembly and annotation of draft genome sequences were completed on August 11, 2011 and the GenBank data was released on September 5, 2011. The genome project has been deposited at DDBJ/EMBL/GenBank under the accession number AGFM00000000 . On January 1, 2014, N. pentaromativorans US6-1T was selected for complete genome sequencing using Illumina MiSeq and PacBio RS II sequencing technology. The complete genome was annotated on May 26, 2014 by ChunLab Inc., South Korea and the sequence was deposited in GenBank on October 10, 2014 (CP009291, CP009292, CP009293, CP009294, CP009295, CP009296). Table 2 represents the project information and its association with MIGS version 2.0 compliance .
Growth conditions and genomic DNA preparation
US6-1T (=KCTC 10454T ) was cultivated for 1 day at 30 °C in 100 ml ZoBell medium (5 g peptone, 1 g yeast extract, 0.01 g FePO4 per liter of 20 % distilled water and 80 % filtered aged seawater) by shaking incubation (150 rpm). Cell was harvested by centrifugation at 6000 × g for 15 min at 4 °C and then washed twice with sterilized seawater. The genomic DNA isolation prepared by using a Wizard® genomic DNA purification kit (Promega, USA) according to the manufacturer’s instructions. Genomic DNA quantified using the PicoGreen® fluometric quantification kit (Molecular Probes) and preserved at −20 °C for sequencing.
Genome sequencing and assembly
The genomic DNA was fragmented using dsDNA fragmentase to generate DNA pieces suitable for library construction. The DNA fragments were processed with a TruSeq DNA sample preparation kit v2 (Illumina Inc., USA) following the manufacturer’s instructions. The final library was quantified by a Bioanalyzer 2100 (Agilent, USA) and the average library size was 300 bp. The genomic library was sequenced by Illumina MiSeq (Illumina Inc., USA) and a PacBio RS II sequencer (Pacific Biosciences, USA). Generated Illumina sequencing reads (8,767,104 reads, total read length 2,156,191,562 bp) and PacBio reads (1,362,072 reads, total read length 703,045,197 bp) were assembled using the CLC genomics workbench 7.0.4 (CLC bio, Denmark) and the PacBio SMRT Analysis Pipeline 2.2.0. Finally, we obtained 6 contigs. The contigs and PCR-based long reads were combined through manual curation using CodonCode Aligner 3.7.1 (CodonCode Corp., USA). The final plasmid sequences were corrected by remapping with raw reads to check errors and dubious regions.
The genes in the assembled genome were predicted using Prodigal  as part of the DOE-JGI genome annotation pipeline [13, 14], followed by a round of manual curation using the JGI GenePRIMP pipeline . tRNAs were identified by tRNA-Scan-SE , and the search for rRNAs used HMMER with EzTaxon-e rRNA profiles [17, 18]. The predicted CDSs were compared to catalytic families, NCBI COG by rpsBLAST, NCBI reference sequences and SEED databases by BLASTP, for functional annotation [19–22]. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes-Expert Review (IMG-ER) platform .
The total length of the complete genome sequence is 5,457,578 bp, which includes a 3,979,506 bp chromosome and five plasmids pLA 1 (0.18 Mb), pLA 2 (0.06 Mb), pLA 3 (0.75 Mb), pLA 4 (0.33 Mb), and pLA 5 (0.13 Mb) (Table 3). The DNA G + C content was determined to be 63.02 %. There are 82 RNA genes which includes 9 rRNAs, 54 tRNAs and 19 miscRNAs (Table 4). All of the amino acid coding genes are located on the chromosome. From the gene prediction results, 5110 CDSs were identified. The statistics of the genome based on the IMG (ID: 59347) are summarized in Table 4 and the distribution of genes into COG functional categories is presented in Fig. 2 and Table 5.
Insights from the genome sequence
In this study, the relationship between 16S rRNA gene sequence similarity and ANI value of the N. pentaromativorans US6-1T was examined for nine genome-sequenced strains in the genus Novosphingobium . The 16S rRNA gene sequence similarity ranged from 93.9 to 99.9 % whereas the ANI values ranged from 70.9 to 93 % (Fig. 3). All interspecies relations (plot number 1–8 in Fig. 3) coincided with the species delineation, while the relation (plot number 9 in Fig. 3) between N. pentaromativorans US6-1T and Novosphingobium sp. PP1Y showed the discrepancy of the species delineation in terms of 16S rRNA gene sequence similarities and ANI values. This evidence suggests that the strains US6-1T and PP1Y are likely different species, because ANI (93 %) is lower than 95 % in spite of the 99.9 % 16S rRNA gene sequence similarity . However, Gan et al.  demonstrated that these two strains may belong to the same species on the basis of average amino acid identity, dinucleotide relative abundance values and genome signature dissimilarity. Kim et al.  reported several exceptional cases of the proposed standard for species delineation. Among them a high number of cases (39 %) with >98.65 % 16S rRNA gene sequence similarity, and <95 % ANI, were found for strains that are known to have high intraspecific or intragenomic variations between multiple 16S rRNA genes in the genome. The same case was found between N. pentaromativorans US6-1T and Novosphingobium sp. PP1Y in the current study even though the intraspecific or intragenomic variations between multiple 16S rRNA genes in those genomes were low. At present, it is not clear how 16S rRNA gene sequence similarity between these two strains has been conserved despite having relatively divergent genomes.
Strain US6-1T has two different extradiol pathways . A previous analysis found that genes involved in the catechol 2,3-dioxygenase pathway are encoded in plasmid pLA1, whereas those of the protocatechuate 4,5-dioxygenase pathway are located in the chromosomal genome. Based on the completed genome data, however, it was discovered that most of the protocatechuate 4,5-dioxygenase genes are encoded in pLA3 (three alpha-subunits and two beta-subunits are in pLA3, with one beta-subunit in the chromosome) and that both extradiol biodegradation pathways are encoded separately in two plasmids. Additional gene such as a copy of naphthalene 1,2-dioxygenase involved in aromatic hydrocarbon degradation is encoded in the chromosomal genome.
N. pentaromativorans US6-1T was isolated from marine sediments and it showed halophilic characteristics. This strain is capable of degrading multi-ring aromatic compounds including benzo[a]pyrene. By completing the genome sequencing, the genomic composition of N. pentaromativorans US6-1T was revised from one chromosome and two plasmids to one chromosome and five plasmids, and the total size was changed from approximately 5.1 to 5.5 Mb. The relationship between 16S rRNA gene sequence similarities and ANI values of the N. pentaromativorans US6-1T and nine genome-sequenced strains in the genus Novosphingobium indicated that all interspecies relations coincided with the species delineation, while the relation between N. pentaromativorans US6-1T and Novosphingobium sp. PP1Y did not. The two extradiol pathways are distributed on two of the plasmids and some dioxygenase genes such as a copy of protocatechuate 4,5-dioxygenase beta-subunit and naphthalene 1,2-dioxygenase genes involved in aromatic hydrocarbon degradation are encoded in chromosomal DNA. The current findings using this complete genome sequence of N. pentaromativorans US6-1T show that the PAHs biodegradation pathway genes are distributed on two plasmids. This result differs from the findings of the draft genome sequence we previously reported . Further research is required to reveal the full pathway of high-molecular-mass aromatic hydrocarbon degradation and its regulation mechanism.
Average nucleotide identity
Polycyclic aromatic hydrocarbons
Mohn WW, Westerberg K, Cullen WR, Reimer KJ. Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms. Appl Environ Microbiol. 1997;63:3378–84.
National Toxicological Program (NTP). Tenth report on carcinogens, Report of the NTP on carcinogens. Washington: National Academy Press; 2002.
McElroy AE, Farrington JW, Teal JM. Bioavailability of polycyclic aromatic hydrocarbons in the aquatic environment. In: Varanasi U, editor. Metabolism of polycyclic aromatic hydrocarbons in the aquatic environment. Boca Raton: CRC Press Inc; 1989. p. 1–39.
Kweon O, Kim SJ, Holland RD, Chen H, Kim DW, Gao Y, et al. Polycyclic aromatic hydrocarbon metabolic network in Mycobacterium vanbaalenii PYR-1. J Bacteriol. 2001;193:4326–37.
Rodríguez-Blanco A, Vetion G, Escande M-L, Delille D, Ghiglione J-F. Gallaecimonas pentaromativorans gen. nov., sp. nov., a bacterium carrying 16S rRNA gene heterogeneity and able to degrade high-molecular-mass polycyclic aromatic hydrocarbons. Int J Syst Evol Microbiol. 2010;60:504–9.
Kim S-J, Kwon KK, Hyun J-H, Svetashev VI. Bioremediation of PAHs in marine sediment. J Ocean Sci Tech. 2004;1:7–13.
Sohn JH, Kwon KK, Kang J-H, Jung H-B, Kim S-J. Novosphingobium pentaromativorans sp. nov., a high-molecular-mass polycyclic aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment. Int J Syst Evol Microbiol. 2004;54:1483–7.
Luo YR, Kang SG, Kim S-J, Kim M-R, Li N, Lee J-H, et al. Genome sequence of Benzo(a)pyrene-degrading bacterium Novosphingobium pentaromativorans US6-1. J Bacteriol. 2011;194:907.
Yun SH, Choi C-W, Lee S-Y, Lee YG, Kwon J, Leem SH, et al. Proteomic characterization of plasmid pLA1 for biodegradation of polycyclic aromatic hydrocarbons in the marine bacterium, Novosphingobium pentaromativorans US6-1. PLoS One. 2014;9:e90812.
D’Argenio V, Notomista E, Petrillo M, Cantiello P, Cafaro P, Izzo V, et al. Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern. BMC Genomics. 2013;15:384.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiati on site identification. BMC Bioinformatics. 2010;11:119.
Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009;1:63–7.
Chen IM, Markowitz VM, Chu K, Anderson I, Mavromatis K, Kyrpides NC, et al. Improving microbial genome annotations in an integrated database context. PLoS One. 2013;8:e54859.
Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, et al. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010;7:455–7.
Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25:955–64.
Eddy SR. Profile hidden Markov models. Bioinformatics. 1998;14:755–63.
Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012;62:716–21.
Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, et al. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 2005;33:5691–702.
Pruitt KD, Tatusova T, Klimke W, Maglott DR. NCBI Reference Sequences: current status, policy and new initiatives. Nucleic Acids Res. 2009;37:32–6.
Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28:33–6.
Yu C, Zavaljevski N, Desai V, Reifman J. Genome-wide enzyme annotation with precision control: catalytic families (CatFam) databases. Proteins. 2009;74:449–60.
Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009;25:2271–8.
Kim M, Oh H-S, Park S-C, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol. 2014;64:346–51.
Gan HM, Hudson AO, Rahman AYA, Chan KG, Savka MA. Comparative genomic analysis of six bacteria belonging to the genus Novosphingobium: insights into marine adaptation, cell-cell signaling and bioremediation. BMC Genomics. 2014;14:431.
Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–25.
Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol. 1981;17:368–76.
Kluge AG, Farris JS. Quantitative phyletics and the evolution of anurans. Syst Zool. 1969;18:1–32.
Felsenstein J. Confidence limits on phylogenies: an approach using bootstrap. Evolution. 1985;39:783–91.
Jukes T, Cantor CR. Evolution of protein molecules. Mamm Protein Metab. 1969;3:21–132.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.
Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A. 2009;106:19126–31.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.
Garrity GM, Bell JA, Lilbum T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 1.
Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 1.
Validation List No. 107: List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006; 56:1-6.
Yabuuchi E, Kosako Y. Order IV. Sphingomonadales ord. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 230–3.
Kosako Y, Yabuuchi E, Naka T, Fujiwara N, Kobayashi K. Proposal of Sphingomonadaceae fam. nov., consisting of Sphingomonas Yabuuchi et al. 1990, Erythrobacter Shiba and Shimidu 1982, Erythromicrobium Yurkov et al. 1994, Porphyrobacter Fuerst et al. 1993, Zymomonas Kluyver and van Niel 1936, and Sandaracinobacter Yurkov et al. 1997, with the type genus Sphingomonas Yabuuchi et al. 1990. Microbiol Immunol. 2000;44:563–75.
Validation List no. 77: Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int J Syst Evol Microbiol. 2000; 50:1953.
Takeuchi M, Hamana K, Hiraishi A. 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. 2001;51:1405–17.
Gomila M, Gascó J, Busquets A, Gil J, Bernabeu R, Buades JM, et al. Identification of culturable bacteria present in haemodialysis water and fluid. FEMS Microbiol Ecol. 2005;52:101–14.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Gene Ontol Consortium Nat Genet. 2000;25:25–9.
This work was supported by the KIOST in-house program (PE99314) and the Marine Genomics 100+ Korea Program. The authors thank Dr. J.P. van der Meer for English correction and A. Patra for genome analysis.
The authors declare that they have no competing interests.
DHC performed the genomic analysis and drafted the manuscript. YMK performed the phylogenetic analysis with additional genomic analysis and finalized the manuscript. KKK participated in the design and discussion of this study. SJK oversaw the project and was responsible for finalizing the manuscript. All authors read and approved the manuscript.
Dong Hee Choi and Yong Min Kwon contributed equally to this work.
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Choi, D.H., Kwon, Y.M., Kwon, K.K. et al. Complete genome sequence of Novosphingobium pentaromativorans US6-1T . Stand in Genomic Sci 10, 107 (2015). https://doi.org/10.1186/s40793-015-0102-1
- Polycyclic aromatic hydrocarbon
- Extradiol dioxygenase