Genome Sequences of Actinobacteria from Extreme Environments in Colombia.

We sequenced six actinobacterial genomes isolated from a salt mine and from soil in a high-mountain Páramo ecosystem. The strains belonged to the genera Streptomyces, Nesterenkonia, and Isoptericola and were sequenced due to their antimicrobial and cytotoxic activities.

ANNOUNCEMENT

Actinobacteria produce a broad range of natural products, most of which have been obtained from actinomycetes belonging to the genus Streptomyces (1). Given the limitation of phenotypic screening and the rediscovery of previously isolated compounds, genome mining of microorganisms isolated from unique environments is attractive for the identification of novel biosynthetic gene clusters (BGCs) (2). Genomic analysis has shown that actinobacteria can harbor multiple BGCs that are not expressed in laboratory assays but may still have biological activities and industrial potential (3). The strains sequenced here were isolated from two locations of extreme environmental conditions in Colombia to expand the actinobacterial collection for biological activity assays (3). Samples were collected from a salt mine (Zipaquirá, 5°01′06.18″N, 74°0′13.63″W) (4), located at 2,656 m above sea level (masl), pH 6.6, and temperature of 17.3°C, and from an Andean Páramo ecosystem (5) soil in the Nevados Natural National Park (04°50′55.7″N, 75°21′51.3″W), located at 4,141 masl, with pH 5.6 and temperature of 7.2°C. We isolated three soil Streptomyces strains on Difco Actinomycete medium and three Nesterenkonia and Isoptericola halotolerant strains from the salt mine by growth on tryptic soy agar (Merck) supplemented with 8% NaCl, as described previously (4). The strains were genome sequenced to characterize and compare their metabolic potential, based on their previously characterized capacity to inhibit growth of Gram-positive and Gram-negative bacteria, eukaryotic microbes, and their cytotoxic activity against cell lines 4T1 (mouse mammary tumor) and MCF-7 (human mammary adenocarcinoma) (4) (Table 1).

TABLE 1

Strain and genome characteristics

Strain (GenBank accession no.)	Biological activity			No. of reads	No. of contigs/N50 (bp)	No. of proteins	No. of RNAs	No. of genes	GC content (%)	Length (Mb)	Coverage (×)	No. of BGCs
	Bacterial	Fungal	Cytotoxic
Streptomyces avidinii CG 885 (QLTM00000000)	+ (G+, G−)	+	−	10,452,432	45/588,328	6,783	90	6,873	71.08	7.65	204	55
Streptomyces microflavus CG 893 (OAOR00000000)	+ (G+, G−)	−	−	9,842,212	45/623,933	6,551	93	6,644	71.12	7.37	199	51
Streptomyces sp. CG 926 (QGGZ00000000)	G+	+	ND	12,394,618	46/470,107	7,552	108	7,660	71.83	8.51	176	72
Isoptericola sp. strain CG 20/1183(PVTW00000000)	−	−	+4T1	11,274,090	26/263,847	3,501	68	3,569	73.10	3.87	378	17
Isoptericola halotolerans CG 23/1184 (PVTX00000000)	−	−	+4T1, MCF7	11,400,520	22/280,589	3,486	69	3,555	73.11	3.85	382	18
Nesterenkonia sandarakina CG35/1185 (PVTY00000000)	−	−	+4T1, MCF7	11,167,940	58/180,192	2,983	62	3,045	67.46	3.22	454	13

Growth inhibition was absent (−) or present (+) when assayed against Gram-positive (G+) or Gram-negative (G−) bacteria or fungi. Cytotoxic activity according to reference 4. ND, not determined.

DNAs were extracted as reported previously (6) by centrifuging an overnight culture, lysing cells with 200-µm glass beads in the presence of cetyltrimethylammonium bromide (CTAB)-NaCl in a FastPrep (2 × 30 s) (MP Biomedical, Santa Ana, CA), followed by phenol-chloroform extraction and DNA precipitation with isopropanol. Draft genomes were obtained at the DOE Joint Genome Institute by constructing 300-bp-insert shotgun libraries that were barcoded and sequenced as pools of three libraries on the Illumina MiSeq platform (2 × 150-bp paired ends). All raw sequence data were filtered using BBDuk version 35.83 (7), which eliminates known Illumina artifacts and PhiX. Reads with more than one “N” or with quality scores averaging less than 8 (before trimming) or reads shorter than 51 bp (after trimming) were discarded. The remaining reads were mapped using BBMap version 35.83 (7) to human, cat, and dog references masked using BBMask version 35.83 (7). Assembly was carried out as follows: (i) artifact-filtered Illumina reads were assembled using Velvet (version 1.2.07) (8), (ii) 1 to 3-kbp simulated paired-end reads were created from the Velvet contigs using Wgsim (version 0.3.0) (9), and (iii) Illumina reads were assembled with simulated read pairs using AllPaths-LG (version r46652) (10). Parameters for the assembly steps were (i) Velvet (velveth, 63 –shortPaired; velvetg, –very clean yes –exportFiltered yes –min contig lgth 500 –scaffolding no –cov cutoff 10), (ii) wgsim (–e 0 –1 100 –2 100 –r 0 –R 0 –X 0), and (iii) AllPaths-LG (PrepareAllpathsInputs, PHRED 64 = 0 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50; RunAllpathsLG, THREADS = 8 RUN=std shredpairs TARGETS=standard VAPI WARN ONLY=True OVERWRITE=True). Annotation was performed using the DOE-JGI annotation pipeline version 4.10.5 (11), BGCs were identified using antiSMASH version 2.0.2 (12) and the IMG-ABC database (13), using default settings in both cases, and taxonomic identities were assigned based on 16S rRNA gene analysis.

The number of reads per genome ranged between 9,842,212 and 12,394,618. The N50 values ranged from 180,192 to 623,933 bp (Table 1). All genomes had a high GC content (67 to 71%) but differed in size, with the soil Streptomyces strains having genomes more than twice the size of those of the three halotolerant strains. The number of genes varied from 3,045 in Nesterenkonia sandarakina to 7,660 for Streptomyces sp. strain CG 926. The three Streptomyces genomes also had more BGCs (Table 1) predicted to encode various specialized metabolites. The differences in genome length and coding features for the two Isoptericola strains and three Streptomyces strains also indicate distinct metabolic potential in isolates of the same genus.

Data availability.

These draft genome sequences have been deposited in GenBank under the accession numbers QLTM00000000, OAOR00000000, QGGZ00000000, PVTW00000000, PVTX00000000, and PVTY00000000. The data are available in the NCBI Sequence Read Archive under accession numbers SRX2947591, SRX3047884, SRX3047887, SRX2947595, SRX2947597, and SRX2947593.