2. Key Laboratory of Extreme Environmental Microbial Resources and Engineering of Gansu Province, Lanzhou, Gansu 730000, China;
3. Xinjiang Institute of Ecology and Geography, Chinese Academy of Science, Urumqi, Xinjiang 830011, China;
4. School of Chemistry and Environment Science, Lanzhou City University; Lanzhou, Gansu 730000, China;
5. College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China;
6. School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China;
7. Institute of Life Science, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK
Methicillin-resistant Staphylococcus aureus (MRSA) has occurred in many countries since its discovery in 1961 (Rosenbach, 1884; Hiramatsu, 2001). However, in recent years, clinicians have been concerned by the increased frequency of MRSA infections (Hiramatsu et al., 1997 ; Chambers, 2001). This resurging MRSA problem seems to be based on the lack of potent therapeutic agents having an unequivocal cell-killing effect, and thus capable of eliminating MRSA from the patient's body. MRSA is now a problem in hospitals worldwide and is increasingly recovered from nursing homes and the community (Hussain et al., 2000 ; Enright et al., 2002 ). The methicillin-resistance gene (mecA) encodes a methicillin-resistant, penicillin-binding protein that is not present in susceptible strains and is believed to be acquired from a distantly related species (Hiramatsu et al., 2001 ). MecA is carried on a mobile genetic element, the staphylococcal cassette chromosome mec, of which four forms have been described that differ in size and genetic composition (Stefani et al., 2009 ). Many MRSA isolates are multiply resistant and a cause of great public health concern.
The Qinghai-Tibet Plateau in East Asia is a unique and important permafrost environment. However, its microbiology remains largely unexplored to date. Members of the genus Streptomyces, gram-positive filamentous actinomycetes, are an attractive source for bioactive secondary metabolites (Yabuuchi, 2001; Procopio et al., 2012 ). These species have also been exploited for heterologous expression of a variety of secondary metabolites (Baltz, 2010). Various new species of Streptomyces have been isolated from the Qinghai-Tibet Plateau (Zhang et al., 2016 ). Analysis the Streptomyces, which lived in the Qinghai-Tibet Plateau, will facilitate efforts to probe the potential of new antibiotics to kill multi-drugs-resistant MRSA.2 Materials and methods 2.1 Preparation of high-molecular-weight DNA
The Streptomyces agglomeratus 5-1-8 was cultured in the 2X YT medium at 30 °C for 3 days. The culture was divided into 5-mL aliquots, and the mycelium harvested by centrifugation at 4,000 rpm for 5 min at room temperature; the supernatant was discarded, and the mycelium washed with 5 mL of 20% glycerol; aliquots not to be immediately processed were stored in 20% glycerol at 20 °C. Washed mycelium was harvested by centrifugation at 4,000 rpm for 5 min at room temperature, providing about 0.5 mL of wet mycelial pellet that was used as starting material for the salting-out protocol ( Gomez-Escribano et al., 2015 ), using the same volume of solutions despite the lower amount of starting mycelium; incubation times with lysozyme and proteinase K were increased as required until a clear lysate was obtained.2.2 DNA sequencing and assembly with Illumina and Pacific Biosciences
A shotgun paired-end library (average size of 550 bp) was prepared using the Illumina Nextera kit; no PCR (polymerase chain reaction) amplification was performed. Sequencing was performed in an Illumina HiSeq device. 1.14 Gb from 8.2 million reads were assembled. The PacBio library was constructed with a 20-kb, size-selected protocol using DNA Template Prep Kit 2.0, purified, and further selected for long insert size with a 0.35X AMPure bead-size selection. The library was sequenced on a PacBio RSII device using the reagents DNA/Polymerase Binding Kit P4, DNA Sequencing Reagent 2.0 PN, SMRT Cell V3, and Magbead, loading at 200 pM on the plate. Data were collected with Stage Start with 180-minute movies. During the single 180-min run time, 1,018,308,244 read bases were generated, with 136,287 reads.2.3 General visualisation, analysis, and manipulation of DNA sequence data
Visualisation and manipulation of text files was performed with advanced text editors Notepad++ (http://notepad-plus-plus.org/) or TextWrangler (http://www.barebones.com/products/textwrangler/) under MacOSX 10.6.8. DNA sequence analysis and annotation were predicted by Prokaryotic Genome Annotation Pipeline, version 2.8. Pfam searches were performed at the Pfam web server (http://pfam. xfam.org/) (Finn et al., 2014 ). Automated searches for natural product biosynthetic gene clusters were performed at the antiSMASH server (Weber et al., 2015 ). COG (clusters of orthologous genes) func-tional categories were calculated with BASys (Tatusov et al., 1997 , Van Domselaar, et al., 2005 ).3 Results 3.1 Organism information
To screen new antibiotics, Streptomyces agglomeratus 5-1-8 was isolated from the frozen soil of Tibet in China. This strain grew well on IPS (International Streptomyces project) 4, ISP5, and MS agar. The color of aerial mycelia was white, and that of the reverse side was yellow on IPS4 agar. Diffusible pigments were not formed on any agar media that we examined. A scanning electron micrograph of this strain is shown in Figure 1. Growth occurred at 10~37 °C (optimum 28 °C) and pH 5~9 (optimum pH 7). Strain 5-1-8 exhibited growth with 0%~7% (w/v) NaCl (optimum 0.5% NaCl). Strain 5-1-8 utilized D-glucose, sucrose, inositol, L-rhamnose, D-mannitol, D-raffinose, D-fructose, L-arabinose, and D-xylose for growth ( Table 1). The genes encoding 16S rRNA were amplified by PCR using two universal primers, 8 F and 1492R. Homology search of the sequence indicated the highest similarity (1481/1481, 100%) to Streptomyces agglomeratus NBRC 13663 as the closest type strain. A phylogenetic tree was reconstructed on the basis of the 16S rRNA gene sequence (Perriere and Gouy, 1996; Thompson et al., 1997 ; Chen et al., 2015 ) (Figure 2). The phylogenetic analysis confirmed that the strain 5-1-8 belongs to the genus Streptomyces.
This organism was selected for sequencing on the basis of its strong ability to kill MRSA. The genome sequencing was performed in September 2015, and the Whole Genome Shotgun project was deposited in the DDBJ/EMBL/GenBank databases under the accession number MEHL00000000. The version described in this study is the first version, labeled MEHL00000000.1. The sequencing-project information and its association with the Minimum Information about a Genome Sequence version 2.0 compliance are described in Table 2.
The strain was cultured aerobically in a 250-mL Erlenmeyer flask containing 50 mL of 2XYT medium, on a rotary shaker at 200 rpm. and 30 °C. Genomic DNA was isolated from 50 mL of culture using the Omega Bacterial DNA Kit, following the standard protocol recommended by the manufacturer. The quantity and purity of the extracted genomic DNA were assessed with a Picodrop Microliter UV/Vis Spectrophotometer, respectively. Finally, a DNA concentration of 560.0 ng/μL and OD 260/OD 280 of 1.82 was determined.3.2.3 Genome sequencing and assembly
The genome of strain 5-1-8 was sequenced using the single-molecule, real-time DNA sequencing platform on the Pacific Biosciences RS II sequencer with P5 polymerase-C3 sequencing chemistry and Illumina Hiseq 4000 platform. A 20-kb insert SMRT-cell library was prepared from the sheared genomic DNA and loaded onto two SMRT cells. During the single 180-min run time, 1,018,308,244 read bases were generated with 136,287 reads. The reads of less than 100 bp or with low accuracy (below 0.8) were removed. A 300-bp insert library was prepared from the sheared genomic DNA. 1,146 M bp read bases were generated, with 8,187,498 reads by the Illumina Hiseq 4000 platform.
All post-filtered reads were assembled de novo using the RS hierarchical genome assembly process, version 3.3, in SMRT analysis software, version 2.3.0 (Pacific Bio-sciences), and resulted in 3 contigs, with 305-fold coverage. The maximal contig length and N50 contig length had the same size of 7,047,685 bp. The whole genome was found to be 7,087,990 bp long.3.2.4 Genome annotation
The protein-coding sequences were predicted by Prokaryotic Genome Annotation Pipeline, version 2.8, on the NCBI website (Tatusova et al., 2016 ). Additional gene prediction and functional annotation were performed on the Rapid Annotation using Subsystems Technology (RAST) server and antibiotics & Secondary Metabolite Analysis shell (antiSMASH), respectively.3.2.5 Genome properties
The genome size of strain 5-1-8 was found to be 7,087,990 bp, with the average G+C content of 70.81%. The genome was predicted to contain a total of 6,284 genes, which include 5,969 protein-coding genes with 89 RNA genes (24 rRNAs and 65 tRNAs). Of these, 4,301 genes were assigned to putative functions; and 4,416 genes (approximately 70.27%) were assigned to the COG functional categories. The genome statistics are presented in Table 3. The gene distribution within the COG functional categories is presented in Table 4.
When the strain 5-1-8 was cultured on the ISP4 medium over 1 week, the medium had a strong ability to kill the MRSA strain 337 (Foxr, Fusr, Eryr, Rifr, Dapr, Vanr, Tecr) and MRSA strain 414 (Foxr, Genr, Rifr, Lzdr, Cipr, Dapr), with the diameter of the inhibition zone being 25 mm and 25 mm, respectively. Using antiSMASH to analyze the antibiotics and secondary metabolite gene clusters in the strain of 5-1-8, it was found that the genome contains 12 gene clusters involved in antibiotics production and 1 gene cluster involved in anticancer-compounds production; 4 gene clusters belong to polyketides and nonribosomal peptides, 1 gene cluster belongs to the butyrolactone, 4 gene clusters belong to the bacteriocin or lantipeptide, and 3 gene clusters belong to the others (Table 5). These findings suggest the strain of 5-1-8 is a good candidate for discovery of a new antibiotic to kill MRSA or cancer.
The 7.1-Mb draft genome of Streptomyces agglomeratus 5-1-8, which has a strong ability to kill multi-drugs-resistant MRSA, has been deposited at GenBank/ENA/DDBJ under accession number MEHL00000000. We successfully identified 12 gene clusters involved in antibiotics production and 1 gene cluster involved in anticancer-compounds production; 4 gene clusters belong to polyketides and nonribosomal peptides, 1 gene cluster belongs to the butyrolactone, 4 gene clusters belong to the bacteriocin or lantipeptide, and 3 gene clusters belong to the others. The genome-sequence information disclosed in this study will be utilized for the investigation of additional new bioactive compounds from this strain and will also serve as a valuable reference for evaluation of the metabolic potential of frozen-soil-derived Streptomyces.Acknowledgments:
This study was supported by the National Natural Science Foundation of China (Grant No. 31400437), the international cooperation program of Gansu (1504WKCA097), the application transformation foundation of CAS (HHS-CGZH-16-02), and UK BBSRC China Partnering Grant (BB/J020419/1).
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