Open Access

High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway

  • Gro Elin Kjæreng Bjerga
  • , Erik Hjerde
  • , Concetta De Santi,
  • , Adele Kim Williamson
  • , Arne Oskar Smalås
  • , Nils Peder Willassen
  • and Bjørn Altermark

DOI: 10.4056/sigs.5038901

Received: 01 March 2014

Accepted: 01 March 2014

Published: 15 June 2014

Abstract

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.

Keywords:

BioprospectingenzymesmetabolitesStreptomycesActinobacteria

Introduction

The filamentous and Gram-positive genus Streptomyces, belonging to the phylum Actinobacteria [1], are attractive organisms for bioprospecting being the largest antibiotic-producing genus discovered in the microbial world so far [2]. These species have also been exploited for heterologous expression of a variety of secondary metabolites [3]. 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 [6], 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.

Table 1

Classification and general features of Streptomyces sp. strain AW19M42 according to the MIGS recommendations [17]

MIGS ID

     Property

     Term

     Evidence code

     Domain Bacteria

     TAS [18]

     Phylum Actinobacteria

     TAS [1]

     Class Actinobacteria

     TAS [19]

     Subclass Actinobacteridae

     TAS [19,20]

     Current classification

     Order Actinomycetales

     TAS [19-22]

     Suborder Streptomycineae

     TAS [19,20]

     Family Streptomycetaceae

     TAS [19,20,22-24]

     Genus Streptomyces

     TAS [22,24-27]

     Species Streptomyces sp.

     NAS

     Strain AW19M42

     IDA

     Gram stain

     Gram positive

     NDA

     Cell shape

     Branched mycelia

     NDA

     Motility

     Dispersion of spores

     NDA

     Sporulation

     Sporulating

     NDA

     Temperature range

     Range not determined, grows at 15°C and 28°C

     IDA

MIGS-6.3

     Salinity

     Not determined, but survives 50% natural sea water

     IDA

MIGS-22

     Oxygen requirements

     Aerobic

     NDA

     Carbon source

     Not reported

     Energy source

     Not reported

MIGS-6

     Habitat

     Inner organs of sea squirt

     IDA

MIGS-15

     Biotic relationship

     Free-living

     IDA

MIGS-14

     Pathogenicity

     Non-pathogenic

     NDA

     Biosafety level

     1

MIGS-4

     Geographic location

     Hellmofjorden, Norway

     IDA

MIGS-5

     Sample collection time

     April 2010

     IDA

MIGS-4.1

     Latitude

     N67 49.24316

     IDA

MIGS-4.2

     Longitude

     E16 28.99465

     IDA

MIGS-4.3

     Depth

     77.35 m

     IDA

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [28]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert or mentioned in the acknowledgements.

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 [29]. The gene encoding16S rRNA was amplified by using two universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG) and 1492R (5′-GGTTACCTTGTTACGACTT) [31], 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) [32]. Using the 16S rRNA sequence data in a homology search by BLAST [33] 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 [34]. The evolutionary history was inferred using the UPGMA method [35] and the evolutionary distances were computed using the Maximum Composite Likelihood method [36]. 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 [7], 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.

Figure 1

Phylogenetic tree indicating the phylogenetic relationship of Streptomyces sp. strain AW19M42 relative to other Streptomyces species. The phylogenetic tree was made by comparing the 16S rDNA sequence of the Streptomyces sp. strain AW19M42 to the closest related sequences from both validated type strains and unidentified isolates. S. venezuelea is used as outgroup. All positions containing gaps and missing data were eliminated. There were a total of 1,389 positions in the final dataset. The bar shows the number of base substitutions per site.

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 [37]. Gene prediction was performed using Glimmer 3 [38] and gene functions were annotated using an in-house genome annotation pipeline.

Table 2

Genome sequencing project information

MIGS ID

    Property

     Term

MIGS-31

    Finishing quality

     Improved high quality draft

MIGS-28

    Libraries used

     One Illumina Paired-End library

MIGS-29

    Sequencing platforms

     Illumina HiSeq2000

MIGS-31.2

    Fold coverage

     350×

MIGS-30

    Assemblers

     CLC paired-end assembly

MIGS-32

    Gene calling method

     Glimmer 3

    Genbank ID

     CBRG000000000

    Genbank Date of Release

     September 11, 2013

    GOLD ID

     Gi0070794

    Project relevance

     Bioprospecting

Genome properties

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.

Table 3

Genome statistics, including nucleotide content and gene count levels

Attribute

   Value

     % of totala

Genome size (bp)

   8,008,851

     100

DNA coding region (bp)

   6,979,999

     87.2

DNA G+C content (bp)

   4,951,797

     70.6

Total genes

   7,813

     n/a

rRNA operons

   8

     n/a

tRNA genes

   62

     n/a

Protein-coding genes

   7,727

     100

Genes assigned to COGs

   6,400

     82.8

Genes with signal peptides

   987

     12.8

Genes with transmembrane helices

   1,660

     21.5

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

Table 4

Number of genes associated with the 25 general COG functional categories

Code

    Value

   %agea

     Description

J

    264

   3.4

     Translation

A

    1

   0.0

     RNA processing and modification

K

    836

   10.8

     Transcription

L

    330

   4.3

     Replication, recombination and repair

B

    5

   0.1

     Chromatin structure and dynamics

D

    71

   0.9

     Cell cycle control, mitosis and meiosis

Y

    0

   0.0

     Nuclear structure

V

    159

   2.1

     Defense mechanisms

T

    442

   5.7

     Signal transduction mechanisms

M

    338

   4.3

     Cell wall/membrane biogenesis

N

    28

   0.4

     Cell motility

Z

    6

   0.1

     Cytoskeleton

W

    0

   0.0

     Extracellular structures

U

    79

   1.0

     Intracellular trafficking and secretion

O

    200

   2.6

     Posttranslational modification, protein turnover, chaperones

C

    409

   5.3

     Energy production and conversion

G

    665

   8.6

     Carbohydrate transport and metabolism

E

    730

   9.4

     Amino acid transport and metabolism

F

    123

   1.6

     Nucleotide transport and metabolism

H

    262

   3.4

     Coenzyme transport and metabolism

I

    330

   4.3

     Lipid transport and metabolism

P

    435

   5.6

     Inorganic ion transport and metabolism

Q

    417

   5.4

     Secondary metabolites biosynthesis, transport and catabolism

R

    1,181

   15.3

     General function prediction only

S

    465

   6.0

     Function unknown

-

    1,327

   17.2

     Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome.

All putative protein coding sequences were assigned KEGG orthology [39], and mapped onto pathways using the KEGG Automatic Annotation Server (KAAS) server [40]. 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 2.7.7.24), dTDP-glucose 4,6-dehydratase (EC 4.2.1.46) and dTDP-4-dehydrorhamnose reductase (EC 1.1.1.133)). 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).

Figure 2

Degradation halos around colonies of Streptomyces sp. AW19M42 growing on agar plates supplemented with A, skim milk, B, gelatin, C, tributyrin, D, DNA, E, chitin and F, starch.

Table 5

Candidate genes coding for putative lipase, caseinase, gelatinase and DNase activities identified in Streptomyces sp. strain AW19M42 draft genome.

Putative gene

     Annotation

    Size (aa)

Lipase

STREP_0737

     Lipase

    273

STREP_1671

     Triacylglycerol lipase

    266

STREP_1821

     G-D-S-L family lipolytic protein

    281

STREP_2698

     Lipase class 2

    297

STREP_2704

     Triacylglycerol lipase

    269

STREP_4585

     Secreted hydrolase

    268

STREP_5662

     Lipase or acylhydrolase family protein

    367

STREP_6665

     Esterase/lipase

    259

STREP_6850

     Esterase/lipase

    429

STREP_7611

     Triacylglycerol lipase

    366

Gelatinase

STREP_5784

     Peptidase M4 thermolysin

    523

STREP_6038

     Peptidase M4 thermolysin

    680

STREP_3662

     Peptidase M4 thermolysin

    358

Caseinase

STREP_0198

     Putative secreted serine protease

    361

STREP_0258

     Protease

    278

STREP_0974

     Protease

    488

STREP_1078

     Serine protease

    388

STREP_1313

     M6 family metalloprotease domain-containing protein

    398

STREP_1389

     M6 family metalloprotease domain protein

    1,389

STREP_2216

     Putative secreted subtilisin-like serine protease

    511

STREP_2239

     metalloprotease

    296

STREP_3135

     Metalloprotease domain protein

    127

STREP_3964

     ATP-dependent protease La

    808

STREP_3975

     ATP-dependent metalloprotease FtsH

    673

STREP_4000

     Streptogrisin-B - Pronase enzyme B SGPB/Serine protease B

    299

STREP_5179

     ATP-dependent Clp protease proteolytic subunit

    222

STREP_5180

     ATP-dependent Clp protease, ATP-binding subunit ClpX

    432

STREP_5944

     Protease

    527

STREP_5945

     Protease

    534

STREP_6196

     Protease

    383

STREP_6570

     Protease

    701

STREP_6821

     Putative protease

    352

STREP_7179

     Serine protease

    635

STREP_7580

     Protease

    856

DNase

STREP_0436

     Exodeoxyribonuclease VII, large subunit

    403

STREP_0437

     Exodeoxyribonuclease VII small subunit

    91

STREP_1352

     Exodeoxyribonuclease III Xth

    268

STREP_1969

     TatD-related deoxyribonuclease

    1,969

STREP_2155

     Deoxyribonuclease V

    220

STREP_2430

     Deoxyribonuclease/rho motif-related TRAM

    452

STREP_4206

     Deoxyribonuclease

    776

STREP_6678

     Probable endonuclease 4 - Endodeoxyribonuclease

    275

Chitinase

STREP_2729

     Chitinase, glycosyl hydrolase 18 family

    628

STREP_5817

     Chitinase, glycosyl hydrolase 18 family

    424

STREP_5513

     Carbohydrate-binding CenC domain protein

    577

STREP_3508

     Glycoside hydrolase family protein

    609

STREP_4257

     Putative endochitinase

    350

STREP_6187

     Chitinase, glycosyl hydrolase 19 family

    297

STREP_6188

     Chitinase, glycosyl hydrolase 19 family

    291

Amylase

STREP_1696

     Glycoside hydrolase starch-binding protein

    573

STREP_5789

     Secreted alpha-amylase

    458

STREP_7405

     Malto-oligosyltrehalose synthase

    834

STREP_1697

     Alpha-1,6-glucosidase, pullulanase-type

    1,774

Conclusion

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.

Declarations

Acknowledgements

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 (Web Site), 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.


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