- Open Access
High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway
Standards in Genomic Sciencesvolume 9, pages676–686 (2014)
Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.
The filamentous and Gram-positive genus Streptomyces, belonging to the phylum Actinobacteria , are attractive organisms for bioprospecting being the largest antibiotic-producing genus discovered in the microbial world so far . These species have also been exploited for heterologous expression of a variety of secondary metabolites . Additionally, these species harbor genes coding for enzymes that can be applicable in industry and biotechnology [4,5].
Since the first, complete Streptomyces genome was published , a number of strains isolated from terrestrial environments have been reported [7–11]. Genomic investigations on Streptomyces from marine sources have, however, just recently begun [12–16].
Here, we present the draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a marine source, together with the description of genome properties and annotation. Results from functional enzyme screening of the bacterium are also reported.
Classification and features
The Streptomyces sp. strain AW19M42 was identified in a biota sample collected from the internal organs of a sea squirt (class Ascidiacea, subphylum Tunicate, phylum Chordata). The tunicate was isolated using an Agassiz trawl at a depth of 77m in Hellmofjorden, in the sub-Arctic region of Norway (Table 1). The trawling was done during a research cruise with R/V Jan Mayen in April 2010.
The bacterium was isolated during four weeks of incubation at 4–15°C on humic acid containing agar media that is selective for growth of actinomycetes [29,30]. For isolation and nucleic acid extraction the bacterium was cultivated in autoclaved media containing 0.1% (w/v) malt extract, 0.1% (v/v) glycerol, 0.1% (w/v) peptone, 0.1% (w/v) yeast extract, 2% (w/v) agar in 50% (v/v) natural sea water and 50% (v/v) distilled water, pH 8.2 . The gene encoding16S rRNA was amplified by using two universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG) and 1492R (5′-GGTTACCTTGTTACGACTT) , in a standard Taq polymerase driven PCR (VWR) on crude genomic DNA prepared by using InstaGene Matrix (BioRad). Following PCR purification by PureLink PCR Purification (Invitrogen), sequencing was carried out with the BigDye terminator kit version 3.1 (Applied Biosystems) and a universal 515F primer (5′-GTGCCAGCMGCCGCGGTAA) . Using the 16S rRNA sequence data in a homology search by BLAST  indicated that the isolate belonged to the Streptomyces genus, among the Streptomycetaceae family of Actinobacteria. A phylogenetic tree was reconstructed from the 16S rRNA gene sequence together with other Streptomyces homologues (Figure 1) using the MEGA 5.10 software suit . The evolutionary history was inferred using the UPGMA method  and the evolutionary distances were computed using the Maximum Composite Likelihood method . The phylogenetic analysis confirmed that the isolate AW19M42 belongs to the genus Streptomyces. The closest neighbor with a reported, complete genome sequence is Streptomyces griseus subsp. griseus , however, the phylogenetic tree indicates that the Streptomyces sp. strain AW19M42 isolate belongs to a closely related but separate clade. Draft genomes have not been reported for this clade previously.
Genome sequencing and annotation
The organism was selected for genome sequencing on the basis of its phylogenetic position. The genome project is part of a Norwegian bioprospecting project called Molecules for the Future (MARZymes) which aims to search Arctic and sub-Arctic regions for marine bacterial isolates that might serve as producers of novel secondary metabolites and enzymes. High quality genomic DNA for sequencing was isolated with the GenElute Bacterial Genomic DNA Kit (Sigma) according to the protocol for extraction of nucleic acids from gram positive bacteria. A 700 bp paired-end library was prepared and sequenced using the HiSeq 2000 (Illumina) paired-end technology (Table 2). This generated 13.94 million paired-end reads that were assembled into 670 contigs larger than 500 bp using the CLC Genomics Workbench 5.0 software package . Gene prediction was performed using Glimmer 3  and gene functions were annotated using an in-house genome annotation pipeline.
The total size of the genome is 8,008,851 bp and has a GC content of 70.57% (Table 3), similar to that of other sequenced Streptomyces isolates. A total of 7,727 coding DNA sequences (CDSs) were predicted (Table 3). Of these, 6,400 could be assigned to a COG number (Table 4). In addition, 62 tRNAs and 8 copies of the rRNA operons were identified.
All putative protein coding sequences were assigned KEGG orthology , and mapped onto pathways using the KEGG Automatic Annotation Server (KAAS) server . The analysis revealed that Streptomyces sp. strain AW19M42 harbors several genes related to biosynthesis of secondary metabolites. We have identified genes that map to the streptomycin biosynthesis pathway (glucose-1-phosphate thymidylyltransferase (EC 18.104.22.168), dTDP-glucose 4,6-dehydratase (EC 22.214.171.124) and dTDP-4-dehydrorhamnose reductase (EC 126.96.36.199)). Also, several genes map to the pathways for biosynthesis of siderophore group nonribosomal peptides, biosynthesis of type II polyketide product pathway and polyketide sugar unit biosynthesis. Interestingly, two clusters, comprising five genes, both mapped to the biosynthesis of type II polyketide backbone pathway. These genes clusters comprise genes STREP_3146-3150 and STREP_4370-4374. This suite of genes may contribute to a distinct profile of secondary metabolites production.
Insights from the Genome Sequence
The isolate was successfully screened for lipase, caseinase, gelatinase, chitinase, amylase and DNase activities (Figure 2), by using marine broth (Difco) agar plates incubated at 20°C [41–46]. The plates were supplemented with 1% (v/v) tributyrin, 1% (w/v) skim milk, 0.4% (w/v) gelatin, 0.5% (w/v) chitin or 2% (w/v) starch, respectively (all substrates from Sigma), whereas DNase test agar (Merck) was supplemented with 0.3M NaCl, representing sea water salt concentration, before screening for DNase activity. Putative genes coding for these activities were identified in the genome based on annotation or by homology search (Table 5).
The 8 Mb draft genome belonging to Streptomyces sp. strain AW19M42, originally isolated from a marine sea squirt in the sub-Arctic region of Norway has been deposited at ENA/DDBJ/GenBank under accession number CBRG000000000. The isolate was successfully screened for several enzymatic activities that are applicable in biotechnology and candidate genes coding for the enzyme activities were identified in the genome. Streptomyces sp. strain AW19M42 will serve as a source of functional enzymes and other bioactive chemicals in future bioprospecting projects.
Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119–169.
de Lima Procópio RE, Silva IR, Martins MK, Azevedo JL, Araujo JM. Antibiotics produced by Streptomyces. Braz J Infect Dis 2012; 16:466–471. PubMed http://dx.doi.org/10.1016/j.bjid.2012.08.014
Baltz RH. Streptomyces and Saccharopolysporahosts for heterologous expression of secondary metabolite gene clusters. J Ind Microbiol Biotechnol 2010; 37:759–772. PubMed http://dx.doi.org/10.1007/s10295-010-0730-9
Joshi AP, Deshmukh SS. Streptomycesnucleases. Crit Rev Microbiol 2011; 37:227–236. PubMed http://dx.doi.org/10.3109/1040841X.2011.562173
Sinha S, Tripathi P, Chand S. A new bifunctional chitosanase enzyme from Streptomyces sp. and its application in production of antioxidant chitooligosaccharides. Appl Biochem Biotechnol 2012; 167:1029–1039. PubMed http://dx.doi.org/10.1007/s12010-012-9546-6
Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, et al. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 2002; 417:141–147. PubMed http://dx.doi.org/10.1038/417141a
Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda H, Yamashita A, Hattori M, Horinouchi S. Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 2008; 190:4050–4060. PubMed http://dx.doi.org/10.1128/JB.00204-08
Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 2003; 21:526–531. PubMed http://dx.doi.org/10.1038/nbt820
Pethick FE, Macfadyen AC, Tang Z, Sangal V, Liu TT, Chu J, Kosec G, Petkovic H, Guo M, Kirby R, et al. Draft genome sequence of the oxytetracycline-producing bacterium Streptomyces rimosus ATCC 10970. Genome Announc 2013; 1:e0006313. PubMed http://dx.doi.org/10.1128/genomeA.00063-13
Erxleben A, Wunsch-Palasis J, Gruning BA, Luzhetska M, Bechthold A, Gunther S. Genome sequence of Streptomyces sp. strain Tu6071. J Bacteriol 2011; 193:4278–4279. PubMed http://dx.doi.org/10.1128/JB.00377-11
Wang XJ, Yan YJ, Zhang B, An J, Wang JJ, Tian J, Jiang L, Chen YH, Huang SX, Yin M, et al. Genome sequence of the milbemycin-producing bacterium Streptomycesbingchenggensis. J Bacteriol 2010; 192:4526–4527. PubMed http://dx.doi.org/10.1128/JB.00596-10
Li F, Jiang P, Zheng H, Wang S, Zhao G, Qin S, Liu Z. Draft genome sequence of the marine bacterium Streptomyces griseoaurantiacus M045, which produces novel manumycin-type antibiotics with a pABA core component. J Bacteriol 2011; 193:3417–3418. PubMed http://dx.doi.org/10.1128/JB.05053-11
Zhao X, Yang T. Draft genome sequence of the marine sediment-derived actinomycete Streptomyces xinghaiensis NRRL B24674T. J Bacteriol 2011; 193:5543. PubMed http://dx.doi.org/10.1128/JB.05689-11
Fan L, Liu Y, Li Z, Baumann HI, Kleinschmidt K, Ye W, Imhoff JF, Kleine M, Cai D. Draft genome sequence of the marine Streptomyces sp. strain PP-C42, isolated from the Baltic Sea. J Bacteriol 2011; 193:3691–3692. PubMed http://dx.doi.org/10.1128/JB.05097-11
Xiong ZQ, Wang Y. Draft genome sequence of the marine Streptomyces sp. strain AA1529, isolated from the Yellow Sea. J Bacteriol 2012; 194:5474–5475. PubMed http://dx.doi.org/10.1128/JB.01247-12
Qin S, Zhang H, Li F, Zhu B, Zheng H. Draft genome sequence of marine Streptomyces sp. strain W007, which produces angucyclinone antibiotics with a benz[a]anthracene skeleton. J Bacteriol 2012; 194:1628–1629. PubMed http://dx.doi.org/10.1128/JB.06701-11
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576
Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a New Hierarchic Classification System, Actinobacteriaclassis nov. Int J Syst Bacteriol 1997; 47:479–491. http://dx.doi.org/10.1099/00207713-47-2-479
Zhi XY, Li WJ, Stackebrandt E. An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol 2009; 59:589–608. PubMed http://dx.doi.org/10.1099/ijs.0.65780-0
Buchanan RE. Studies in the Nomenclature and Classification of the Bacteria II. The Primary Subdivisions of the Schizomycetes. J Bacteriol 1917; 2:155–164. PubMed
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. http://dx.doi.org/10.1099/00207713-30-1-225
Kim SB, Lonsdale J, Seong CN, Goodfellow M. Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici (1943)AL) emend. Rainey et al. 1997. Antonie van Leeuwenhoek 2003; 83:107–116. PubMed http://dx.doi.org/10.1023/A:1023397724023
Waksman SA, Henrici AT. The Nomenclature and Classification of the Actinomycetes. J Bacteriol 1943; 46:337–341. PubMed
Pridham TG, Tressner HD. Genus I. Streptomyces Waksman and Henrici 1943, 339. In: Buchanan RE, Gibbons NE (eds), Bergey’s Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 748–829.
Witt D, Stackebrandt E. Unification of the genera Streptoverticillium and Streptomyces, and amendation of Streptomyces Waksman and Henrici 1943, 339 AL. Syst Appl Microbiol 1990; 13:361–371. http://dx.doi.org/10.1016/S0723-2020(11)80234-1
Wellington EMH, Stackebrandt E, Sanders D, Wolstrup J, Jorgensen NOG. Taxonomic status of Kitasatosporia, and proposed unification with Streptomyces on the basis of phenotypic and 16S rRNA analysis and emendation of Streptomyces Waksman and Henrici 1943, 339AL. Int J Syst Bacteriol 1992; 42:156–160. PubMed http://dx.doi.org/10.1099/00207713-42-1-156
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556
Bredholt H, Fjaervik E, Johnsen G, Zotchev SB. Actinomycetesfrom sediments in the Trondheim fjord, Norway: diversity and biological activity. Mar Drugs 2008; 6:12–24. PubMed http://dx.doi.org/10.3390/md6010012
Hayakawa M, Nonomura H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol 1987; 65:501–509. http://dx.doi.org/10.1016/0385-6380(87)90108-7
Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E and Goodfellow M (eds.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, United Kingdom, 1991, p. 115–175
Turner S, Pryer KM, Miao VPW, Palmer JD. Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis. J Eukaryot Microbiol 1999; 46:327–338. PubMed http://dx.doi.org/10.111550-7408.1999.tb04612.x
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410. PubMed
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; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121
Sneath PH, Sokal RR. Numerical taxonomy. Nature 1962; 193:855–860. PubMed http://dx.doi.org/10.1038/193855a0
Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 2004; 101:11030–11035. PubMed http://dx.doi.org/10.1073/pnas.0404206101
Genomics Workbench CLC. 5.0 software package. http://www.clcbio.com.
Delcher AL, Bratke KA, Powers EC, Salzberg SL. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 2007; 23:673–679. PubMed http://dx.doi.org/10.1093/bioinformatics/btm009
Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 2012; 40:D109–D114. PubMed http://dx.doi.org/10.1093/nar/gkr988
Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 2007; 35:W182–W185; PubMed. PubMed http://dx.doi.org/10.1093/nar/gkm321
Dang HY, Zhu H, Wang J, Li TG. Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough. World J Microbiol Biotechnol 2009; 25:71–79. http://dx.doi.org/10.1007/s11274-008-9865-5
Lee DG, Jeon JH, Jang MK, Kim NY, Lee JH, Lee JH, Kim SJ, Kim GD, Lee SH. Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library. Biotechnol Lett 2007; 29:465–472. PubMed http://dx.doi.org/10.1007/s10529-006-9263-8
Vermelho AB, Meirelles MN, Lopes A, Petinate SD, Chaia AA, Branquinha MH. Detection of extracellular proteases from microorganisms on agar plates. Mem Inst Oswaldo Cruz 1996; 91:755–760. PubMed http://dx.doi.org/10.1590/S0074-02761996000600020
Usharani TR, Gowda TKS. Cloning of chitinase gene from Bacillus thuringiensis. Indian J Biotechnol 2011; 10:264–269.
Mishra S, Behera N. Amylase activity of a starch degrading bacteria isolated from soil receiving kitchen wastes. Afr J Biotechnol 2008; 7:3326–3331.
Henne A, Schmitz RA, Bömeke M, Gottschalk G, Daniel R. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia col. Appl Environ Microbiol 2000; 66:3113–3116. PubMed http://dx.doi.org/10.1128/AEM.66.7.3113-3116.2000
This work was supported by the Research Council of Norway (Grant no. 192123). We would like to acknowledge Kristin E. Hansen and Seila Pandur for technical assistance during bacterial isolation and nucleic acid extraction. The sequencing service was provided by the Norwegian Sequencing Centre (www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the “Functional Genomics” and “Infrastructure” programs of the Research Council of Norway and the Southeastern Regional Health Authorities.