Open Access

Whole genome sequence and comparative genomic analysis of multidrug-resistant Staphylococcus capitis subsp. urealyticus strain LNZR-1

  • Xiaoxia Li1,
  • Min Lei2,
  • Yanli Song2,
  • Kunwei Gong2,
  • Ling Li2,
  • Hongyan Liang1 and
  • Xiaofeng Jiang1, 3Email author
Contributed equally
Gut Pathogens20146:45

DOI: 10.1186/s13099-014-0045-x

Received: 27 September 2014

Accepted: 11 November 2014

Published: 20 December 2014

Abstract

Background

Staphylococcus capitis is an emerging opportunistic pathogen of humans, and found as a colonizer of the human gut. Here, we report a case of S. capitis subsp. urealyticus infection. The strain LNZR-1 was isolated from the blood culture of a patient with sigmoid colon cancer. It was found to be resistant to some important antibiotics, such as linezolid, a highly effective antimicrobial against clinically important Staphylococci pathogens. However, data on the genetic resistance mechanisms in S. capitis subsp. urealyticus are only sparsely available.

Results

The draft genome of S. capitis subsp. urealyticus strain LNZR-1 was sequenced by using next-generation sequencing technologies. Sequence data assembly revealed a genome size of 2,595,865 bp with a G + C content of 32.67%. Genome annotation revealed the presence of antibiotic resistance genes conferring resistance against some of the tested antibiotics as well as non-tested antibiotics. The genome also possesses a lot of genes that may be related to multidrug resistance. Whole genome comparison of the LNZR-1 with five other S. capitis strains showed that some functional regions are highly homologous between the six assemblies made herein. The LNZR-1 genome has high similarity with the genomes of the strains VCU116 and CR01, although some short stretches present in the genomes of strains VCU116 and CR01 were absent in the strain LNZR-1.

Conclusions

The presence of a plethora of genes responsible for antibiotic resistance suggests that strain LNZR-1 could present a potential threat to human health. The comparative genomic analysis of S. capitis strains presented in this study is important for better understanding of multidrug resistance in S. capitis.

Keywords

Staphylococcus capitis subsp. urealyticus Multidrug-resistant Genome sequencing Comparative genomic analysis

Background

Staphylococcus capitis are Gram positive cocci belonging to the Coagulase-Negative Staphylococci group (CoNS) that is frequently found on the human skin and mucosa [1],[2] and even in the human gut [3]. Although infection caused by this species is rare compared with S. aureus, infection cases associated with S. capitis increase gradually [4]. Recent reports indicate its emergence as a significant pathogen causing nosocomial and bloodstream infections, meningitis, prosthetic valve endocarditis, and late-onset sepsis [4]-[7]. This bacterium is a subtype of CoNS and the pathogenesis of S. capitis is mainly due to its ability to produce a slimy biofilm, enabling it to adhere to the medical devices such as prosthetic valves and catheters; this makes them difficult to be controlled or cleared by immune responses or antibiotic therapy [7].

The S. capitis subsp. urealyticus strain LNZR-1 described herein was isolated from the blood culture of a patient with sigmoid colon cancer. Antimicrobial susceptibility assay revealed that it was resistant to some important antibiotics, such as linezolid. In order to elucidate the molecular mechanisms behind the multidrug resistance of S. capitis subsp. urealyticus LNZR-1 clone, here, we report the sequencing and annotation of its genome, together with a functional level genomic comparison with other important S. capitis strains, namely QN1 [8], CR01 [9], VCU116, C87 and SK14.

Methods

Strain information and growth conditions

The blood samples were collected from a patient with sigmoid colon cancer from the Fourth Affiliated Hospital of Harbin Medical University, in March 2013. S. capitis subsp. urealyticus strain LNZR-1 was isolated after cultivation. It is a Gram positive, coccus-shaped bacterium growing on 5% sheep blood enriched Columbia agar (BioMérieux, Marcyl’Etoile, France) at 37°C. Cell morphology, motility and sporulation were examined by using scanning electron microscopy.

Genomic DNA extraction and 16S rRNA gene PCR

Late log-phase cells were harvested and lysed with EDTA and lysozyme, followed by proteinase K and RNase digestion. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s recommended protocol. Agarose gel (0.7%) electrophoresis was used to evaluate the genomic DNA purity and the concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA). The genomic DNA was stored at -20°C. Strain LNZR-1 was identified by 16S rRNA gene sequencing as described earlier [6]. PCR amplification was performed by using primers 27 F (5′-AGAGTTTGATCCTG GCTCAG-3′) and 1500R (5′-AGAAAGGAGGTGATCCAGGC-3′). Agarose gel (1%) electrophoresis was used to separate amplified PCR fragments which were subjected to sequencing of the 16 s rRNA gene. Phylogenetic analysis was conducted based on the 16S rRNA nucleotide sequence. The representative 16S rRNA nucleotide sequence of strain LNZR-1 was compared against the most recent release of the EzTaxon-e database [10]. Phylogenetic inferences were made using Neighbor-joining method based on Tamura-Nei model within the MEGA 6.06 [11].

Antimicrobial susceptibility testing

Antimicrobial susceptibility was determined by the disk diffusion method on Mueller-Hinton agar recommended by the Clinical and Laboratory Standards Institute guidelines [12]. The following antimicrobial agents were tested: sulfamethoxazole, gentamicin, oxacillin, tetracycline, linezolid, clindamycin, ciprofloxacin, cefoxitin, cefazolin, cefuroxime and vancomycin. The other reference strains used for this study were Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603.

Genome sequencing, assembly and annotation

The genome of S. capitis subsp. urealyticus strain LNZR-1 was sequenced using a standard run of Illumina HiSeq 2000 sequencing technology which generated paired-end libraries (500-bp insert size) according to the manufacturer’s instructions. Clean reads were assembled into scaffolds using Velvet version 1.2.07 [13], and Post-Assembly Genome Improvement Toolkit (PAGIT) was used to extend the initial contiguous sequences (contigs) and to correct sequencing errors [14]. Open reading frames (ORFs) were identified using Glimmer version 3.0 [15]. Transfer RNAs and ribosomal RNA genes rRNAs were detected by tRNAscan-SE [16] and RNAmmer 1.2 software [17], respectively. The genome was annotated using the RAST (Rapid Annotation using Subsystem Technology) server [18]. The classification of some predicted genes and pathways was analyzed using the Clusters of Orthologous Groups of proteins (COGs) [19] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [20] databases. Functional annotation was also performed by using public database of National Centre for Biotechnology Information (NCBI).

Initial comparative genomic analysis

For comparative analysis, we downloaded the reference genome sequences of the closest genetic relatives of strain LNZR-1 and representative strains from the NCBI database: S. capitis C87 (ACRH00000000), S. capitis SK14 (ACFR00000000), S. capitis CR01 (CBUB000000000), S. capitis VCU116 (AFTX00000000) and S. capitis QN1 (AJTH00000000). Mauve in the progressive mode was used for whole genome comparison [21].

Orthology identification was performed by using NCBI blastp 2.2.25+ with default parameters. Then, bidirectional best hits (BBHs) among proteins from different strains were identified. Furthermore, an identity threshold over a given alignment length to define orthologous genes was applied. Score Ratio Values (SRVs) of BBHs between two genes was calculated with the following formula:
SRVs = Bits AB + Bits BA Bits AA + Bits B B

Bits (AB) means bits score when using gene A as query while B as database; Bits (BA) means bits score when using gene B as query while A as database; Bits (AA) or Bits (BB) are bits score when using gene A or B to align with itself, respectively. BBHs with SRVs no less than 0.3 was considered as one candidate orthology group (SRVs Table in Additional file 1: Table S1).

Quality assurance

Biochemical features were tested by using Vitek2 Compact (bioMérieux, Marcy l’Etoile, France). Positive reactions were observed for arginine dihydrolase 1, L-lactate alkalinization, bacitracin resistance, mannose, growth in 6.5% NaCl, O/129 resistance (comp.Vibrio.) and optochin resistance. Negative reactions were obtained for D-amygdalin, phosphatidylinositol phospholipase c, D-xylose, beta-galactose, Ala-Phe-Pro arylamidase, alpha-galactosidase, cyclodextrin, L-aspartate, beta- galactopyranosidase, alpha-mannosidase, phosphatase, leucine arylamidase, L-proline arylamidase, beta-glucuronidase, L-pyrrolidonyl-arylamidase, beta-glucuronidase, alpha-galactosidase, alanine arylamidase, tyrosine arylamidase, D-sorbitol, urease, D-galactose, D-ribose, lactose, N-acetyl-D-glucosamine, methyl-B-D-glucopyranoside, D-maltose, novobiocin resistance, D-mannitol, pullulan, D-raffinose, salicin, D-trehalose and sucrose. Based on the morphological and biochemical characterization, the strain LNZR-1 was identified as S. capitis. Bioinformatics assessment of potential contamination of the genomic library by allochthonous microorganisms was achieved using the BLAST non-redundant database.

Initial findings

Identification of strain LNZR-1

Cells of strain LNZR-1 are cocci, 0.7 to 1.2 μm in diameter, occurring predominantly singly or in pairs (Additional file 2: Figure S1). To assess the purity of strain LNZR-1, the 16S rRNA gene sequence of strain LNZR-1 was aligned with sequences of other members of the genus Staphylococcus retrieved from the EzTaxon database. Phylogenetic tree indicated the taxonomic status of strain LNZR-1 clearly classified into the same branch with species S. capitis subsp. urealyticus GTC 727T (Figure 1).
Figure 1

Phylogenetic tree highlighting the position of S. capitis subsp. urealyticus strain LNZR-1 relative to other type strains within the genus Staphylococcus. The strains and their corresponding GenBank accession numbers for 16S rRNA genes are shown following the organism names. Numbers at the branching nodes are percentages of bootstrap values based on 1,000 replications. Bootstrap values greater than 50% are shown at the branch points. Macrococcus caseolyticus ATCC 13548T was used as an out group. The scale bar represents 0.005 substitutions per nucleotide position.

Genomic features of S. capitis subsp. urealyticus LNZR-1

A total of 2,214,438 available reads were filtered from 5,619,015 raw reads. Quality control was performed with following criteria: reads that contained more than one N bases were removed. Reads that contained more than 50 bases with low quality (Q30) were removed. Reads with more than 10 bases with low quality (Q30) or N bases in the tail of the reads were trimmed. For sequences which lost their mated reads were considered as single reads and were not used in the downstream analysis (Additional file 1: Table S1). 177 initial contigs (best kmer length 57) was assembled by Velvet; then 90 prolonged contigs were assembled based on PAGIT flow (PAGIT is just a flow and it actually does not provide any practically available scripts or programs). The assembled genome of S. capitis subsp. urealyticus revealed a genome size of 2,595,865 bp and a G + C content of 32.67% (90 scaffolds). The largest contig consisted of 319,806 bp and the length of N50 contig was 66,677 bp. These scaffolds contain 2430 coding sequences (CDSs), 8 tRNAs (excluding 1 Pseudo tRNA) and 2 incomplete rRNA operons (1 small subunit rRNA and 1 large subunit rRNA). The properties and the statistics of the genome are summarized in Table 1. RAST server based annotation of the whole genome describes the subsystem distribution of strain LNZR-1 (Figure 2). Genes responsible for amino acids and derivatives (264 ORFs), carbohydrates (209 ORFs), and protein metabolism (175 ORFs) were abundant among the subsystem categories. 1934 genes were categorized into COGs functional groups (including putative or hypothetical genes, Figure 3). For COGs distribution, R (general function prediction only; 427 ORFs), E (amino acid transport and metabolism; 300 ORFs), P (inorganic ion transport and metabolism; 220 ORFs), S (function unknown; 207 ORFs), and G (carbohydrate metabolism and transport; 191 ORFs) were abundant categories (>10% of total COGs matched counts).
Table 1

Summary of the annotated genome

Attribute

Genome (total)

G + C content (bp)

848,103

Coding region (bp)

2,226,123

Total genes

2,461

RNA genes

10

Protein-coding genes

2,430

Genes assigned to COGs

1,934

Genes with signal peptides

124

Genes with transmembrane helices

638

Figure 2

Subsystems distribution statistic of S. capitis subsp. urealyticus strain LNZR-1 based on genome annotations performed according to RAST server. The pie chart presents the abundance of each subsystem category and the count of each subsystem feature is listed in parentheses at the chart legend.

Figure 3

COGs distribution of S. capitis subsp. urealyticus strain LNZR-1. Statistics of annotated genes for LNZR-1 were based on COG database.

Antibiotic resistance profile

The in vitro antibiotic sensitivity tests demonstrated that this strain is susceptible to vancomycin and sulfamethoxazole, and resistant to tetracycline, gentamicin, ampicillin, methicillin, linezolid, clindamycin, cefoxitin, cefazolin, cefuroxime and ciprofloxacin. To gain insights into the genomic basis for the observed antibiotic resistance traits, the genome was searched for specific genes known to confer antibiotic resistance. The results revealed the presence of antibiotic resistance genes conferring resistance against some of the tested antibiotics as well as non-tested antibiotics (Table 2). MDR-type ABC transporters, multidrug and toxin extrusion (MATE) family efflux pumps and multidrug major facilitator superfamily (MFS) transporters were also detected in the genome. Furthermore, 10 putative MarR family transcriptional regulators were found in the genome, which are recognized as a widely conserved group of multiple antibiotic resistance regulators that respond to diverse antibiotics [22].
Table 2

Summary of putative genes in response to antibiotic resistance in the genome of LNZR-1

Start

Stop

Protein product

Length

Protein name

187729

188952

WP_030065164.1

407

Methicillin resistance protein FmtA

819346

819768

WP_002454275.1

140

Fosmidomycin resistance protein

2236269

2238524

WP_030058872.1

751

Daunorubicin resistance protein DrrC

1011427

1012635

WP_023351187.1

402

Bicyclomycin resistance protein TcaB

1024323

1025573

WP_030063338.1

416

Methicillin resistance protein

1074692

1076092

WP_030063319.1

466

Quinolone resistance protein

1378210

1379967

WP_000952923.1

585

Methicillin resistance protein

1848720

1850102

WP_030059174.1

460

Quinolone resistance protein

1979421

1980605

WP_030059066.1

394

Tetracycline resistance protein

985388

987451

WP_023351245.1

687

Drug resistance transporter, EmrB/QacA family

927892

928221

WP_002432921.1

109

Multidrug resistance protein SMR

2584152

2584619

WP_002432814.1

155

Multidrug resistance protein SepA

726004

727740

WP_002435897.1

578

Multidrug ABC transporter ATP-binding protein

845428

846168

WP_002453567.1

246

Multidrug ABC transporter ATP-binding protein

927892

928221

WP_002432921.1

109

Multidrug resistance protein SMR

985388

987451

WP_023351245.1

687

Drug resistance transporter, EmrB/QacA family

987464

988111

WP_002432737.1

215

Multidrug efflux protein

1062539

1063714

WP_023351172.1

391

Putative drug transporter

1523272

1524450

WP_030061422.1

392

Multidrug MFS transporter

1549535

1550377

WP_030061365.1

280

Multidrug ABC transporter ATP-binding protein

1923738

1924907

WP_030059115.1

389

Multidrug MFS transporter

2192933

2194378

WP_030058818.1

481

Multidrug MFS transporter

2195936

2197183

WP_030058823.1

415

Multidrug MFS transporter

2515785

2516984

WP_002432900.1

399

Multidrug MFS transporter

2520095

2523274

WP_030056889.1

1059

Multidrug transporter

2582688

2584034

WP_023351326.1

448

Multidrug transporter

2584152

2584619

WP_002432814.1

155

Multidrug resistance protein SepA

2584703

2586145

WP_002453755.1

480

Multidrug MFS transporter

1182425

1183750

WP_023350278.1

441

MATE efflux family protein

177910

178329

WP_002435477.1

139

MarR family transcriptional regulator

991886

992341

WP_002453883.1

151

MarR family transcriptional regulator

1009611

1009976

WP_030063362.1

122

MarR family transcriptional regulator

1399165

1399611

WP_002432941.1

148

MarR family transcriptional regulator

1894346

1894801

WP_002436214.1

151

MarR family transcripitonal regulator

1932506

1932946

WP_002433445.1

146

MarR family transcriptional regulator

1948086

1948445

WP_002433103.1

119

MarR family transcriptional regulator

2424049

2424513

WP_030058661.1

154

MarR family transcriptional regulator

2515376

2515726

WP_002432752.1

116

MarR family transcriptional regulator

2516984

2517424

WP_030056887.1

146

MarR family transcriptional regulator

Comparative analysis with other S. capitis strains

Whole genome comparison of the LNZR-1 with S. capitis C87, S. capitis SK14, S. capitis CR01, S. capitis VCU116 and S. capitis QN1 showed that some functional regions are highly homologous between the six assemblies (Figure 4). The LNZR-1 genome has high similarity with VCU116 and CR01, although some short stretches present in the genome of VCU116 and CR01 were absent in LNZR-1. Furthermore, LNZR-1, C87, CR01, VCU116 and QN1 revealed a large number of orthologous genes (Figure 5). Venn diagram indicates the presence of a large core-genome. These five S. capitis strains shared 2042 CDS in the genome. A particular overlap between C87 and QN1 became evident; these two chromosomes shared 121 orthologous CDS exclusively. The chromosome of LNZR-1 overlapped less with the C87 and QN1, which shared 5 and 4 exclusively orthologous CDS, respectively. In addition, 244 CDS from the LNZR-1 genome were classified as unique.
Figure 4

MAUVE alignment of the genomes of S. capitis subsp. urealyticus LNZR-1, S. capitis C87, S. capitis SK14, S. capitis CR01, S. capitis VCU116 and S. capitis QN1. MAUVE identifies and aligns regions of local collinearity called locally collinear blocks (LCBs), a region without rearrangement of homologous backbone sequence. LCBs below a genome’s center line are in the reverse complement orientation relative to the reference genome. Lines between genomes trace each orthologous LCB through every genome.

Figure 5

Venn diagram representing the pan-genome of S. capitis subsp. urealyticus LNZR-1, S. capitis C87, S. capitis CR01, S. capitis VCU116 and S. capitis QN1. Numbers inside the Venn diagram indicate the number of genes found to be shared among the indicated genomes.

Future directions

In recent times, the alarming spread of antibiotic resistance has severely limited the treatment options for nosocomial infections. Currently, the antibiotics linezolid, vancomycin and daptomycin form an empirical therapy towards the control of serious infections caused by Staphylococci. However, the overuse of these antibiotics could have resulted in the emergence of multidrug resistant bacteria. Despite untiring efforts directed at the analyses of intermediately resistant clinical isolates, the explicit mode of the development of resistance to these antimicrobials remains obscure. Further studies by using high-throughput mRNA sequencing (RNA-Seq) experiments to explore differential RNA expression levels under selective antibiotic pressures are warranted. Further studies are required to elucidate the resistance mechanisms, and information on these mechanisms could potentially aid in antibiotic development.

Ethics approval

This research was approved by the Research Ethics Committee of the Fourth affiliated Hospital of Harbin Medical University, and informed consent was obtained from the patient.

Availability of supporting data

This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JGYJ00000000. The version described in this paper is version JGYJ01000000.

Notes

Declarations

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81171657) and Specialized Research Fund for the Doctoral Program of Higher Education (No.20112307110021).

Authors’ Affiliations

(1)
Department of Clinical Biochemistry Laboratory, The Fourth affiliated Hospital of Harbin Medical University
(2)
Department of Clinical Medical Laboratory, The Fifth Hospital of Harbin
(3)
Department of Clinical Biochemistry Laboratory, The Fourth affiliated Hospital of Harbin Medical University

References

  1. Schleifer KH, Kloos WE: Isolation and Characterization of Staphylococci from Human Skin I. Amended Descriptions of Staphylococcus epidermidis and Staphylococcus saprophyticus and Descriptions of Three New Species: Staphylococcus cohnii, Staphylococcus haemolyticus, and Staphylococcus xylosus. Int J Syst Bacteriol. 1975, 25: 50-61. 10.1099/00207713-25-1-50.View ArticleGoogle Scholar
  2. Bannerman TL, Kloos WE: Staphylococcus capitis subsp. ureolyticus subsp. nov. from human skin. Int J Syst Bacteriol. 1991, 41: 144-147. 10.1099/00207713-41-1-144.View ArticlePubMedGoogle Scholar
  3. Vitali LA, Petrelli D, Lamikanra A, Prenna M, Akinkunmi EO: Diversity of antibiotic resistance genes and staphylococcal cassette chromosome mec elements in faecal isolates of coagulase-negative staphylococci from Nigeria. BMC Microbiol. 2014, 14: 106-10.1186/1471-2180-14-106.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Martins Simoes P, Rasigade JP, Lemriss H, Butin M, Ginevra C, Lemriss S, Goering RV, Ibrahimi A, Picaud JC, El Kabbaj S, Laurent F: Characterization of a novel composite staphylococcal cassette chromosome mec (SCCmec-SCCcad/ars/cop) in the neonatal sepsis-associated Staphylococcus capitis pulsotype NRCS-A. Antimicrob Agents Chemother. 2013, 57: 6354-6357. 10.1128/AAC.01576-13.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Oud L: Community-acquired meningitis due to Staphylococcus capitis in the absence of neurologic trauma, surgery, or implants. Heart Lung. 2011, 40: 467-471. 10.1016/j.hrtlng.2010.09.002.View ArticlePubMedGoogle Scholar
  6. Takano T, Ohtsu Y, Terasaki T, Wada Y, Amano J: Prosthetic valve endocarditis caused by Staphylococcus capitis: report of 4 cases. J Cardiothorac Surg. 2011, 6: 131-10.1186/1749-8090-6-131.PubMed CentralView ArticlePubMedGoogle Scholar
  7. Rasigade JP, Raulin O, Picaud JC, Tellini C, Bes M, Grando J, Ben Said M, Claris O, Etienne J, Tigaud S, Laurent F: Methicillin-resistant Staphylococcus capitis with reduced vancomycin susceptibility causes late-onset sepsis in intensive care neonates. PLoS One. 2012, 7: e31548-10.1371/journal.pone.0031548.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Qin N, Ding W, Yao J, Su K, Wu L, Li L: Genome sequence of Staphylococcus capitis QN1, which causes infective endocarditis. J Bacteriol. 2012, 194: 4469-4470. 10.1128/JB.00827-12.PubMed CentralView ArticlePubMedGoogle Scholar
  9. Lemriss H, Martins Simoes P, Lemriss S, Butin M, Ibrahimi A, El Kabbaj S, Rasigade J, Laurent F: Non-contiguous finished genome sequence of Staphylococcus capitis CR01 (pulsetype NRCS-A). Stand Genomic Sci. 2014, 9: 1118-1127. 10.4056/sigs.5491045.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J: Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012, 62: 716-721. 10.1099/ijs.0.038075-0.View ArticlePubMedGoogle Scholar
  11. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S: MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013, 30: 2725-2729. 10.1093/molbev/mst197.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Susceptibility Testing. 21th Informational Supplement. Document M100-S21s. 2011, CLSI, Wayne P, USAGoogle Scholar
  13. Zerbino DR, Birney E: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008, 18: 821-829. 10.1101/gr.074492.107.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Swain MT, Tsai IJ, Assefa SA, Newbold C, Berriman M, Otto TD: A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs. Nat Protoc. 2012, 7: 1260-1284. 10.1038/nprot.2012.068.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007, 23: 673-679. 10.1093/bioinformatics/btm009.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25: 0955-0964. 10.1093/nar/25.5.0955.View ArticleGoogle Scholar
  17. Lagesen K, Hallin P, Rødland EA, Stærfeldt H-H, Rognes T, Ussery DW: RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007, 35: 3100-3108. 10.1093/nar/gkm160.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O: The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008, 9: 75-10.1186/1471-2164-9-75.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, Rao BS, Smirnov S, Sverdlov AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA: The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003, 4: 41-10.1186/1471-2105-4-41.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Mitra S, Rupek P, Richter DC, Urich T, Gilbert JA, Meyer F, Wilke A, Huson DH: Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG. BMC Bioinformatics. 2011, 12 Suppl 1: S21-10.1186/1471-2105-12-S1-S21.View ArticlePubMedGoogle Scholar
  21. Darling AE, Mau B, Perna NT: ProgressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One. 2010, 5: e11147-10.1371/journal.pone.0011147.PubMed CentralView ArticlePubMedGoogle Scholar
  22. Hao Z, Lou H, Zhu R, Zhu J, Zhang D, Zhao BS, Zeng S, Chen X, Chan J, He C, Chen PR: The multiple antibiotic resistance regulator MarR is a copper sensor in Escherichia coli. Nat Chem Biol. 2014, 10: 21-28. 10.1038/nchembio.1380.View ArticlePubMedGoogle Scholar

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