- Short genome report
- Open Access
Genomic information of the arsenic-resistant bacterium Lysobacter arseniciresistens type strain ZS79T and comparison of Lysobacter draft genomes
Standards in Genomic Sciences volume 10, Article number: 88 (2015)
Lysobacter arseniciresistens ZS79T is a highly arsenic-resistant,rod-shaped, motile, non-spore-forming, aerobic, Gram-negative bacterium. In this study, four Lysobacter type strains were sequenced and the genomic information of L. arseniciresistens ZS79T and the comparative genomics results of the Lysobacter strains were described. The draft genome sequence of the strain ZS79T consists of 3,086,721 bp and is distributed in 109 contigs. It has a G+C content of 69.5 % and contains 2,363 protein-coding genes including eight arsenic resistant genes.
Lysobacter arseniciresistens type strain ZS79T (=CGMCC 1.10752T = KCTC 23365 T) belongs to family Xanthomonadaceae . It is an arsenic-resistant bacterium isolated from subsurface soil of Tieshan iron mine, Daye City, P. R. China . So far, there are 32 validly published species of Lysobacter . Most of these Lysobacter strains were isolated from soil except that Lysobacter brunescens  and Lysobacter oligotrophicus  were isolated from water, and Lysobacter concretionis , Lysobacter daecheongensis  Lysobacter spongiicola  were isolated from sludge, sediment and deep-sea sponge, respectively.
So far, the genomic sequences of two Lysobacter strains have been published ( Lysobacter capsici AZ78 [8, 9] and Lysobacter antibioticus 13-6 ), but the annotation of L. antibioticus 13-6 was not completed. In order to provide genome information of genus Lysobacter , we performed whole genome sequencing of four strains of Lysobacter ( L. arseniciresistens ZS79T, Lysobacter conceretionis Ko07T , Lysobacter daejeonensis GH1-9T , and Lysobacter defluvii IMMIB APB-9T ). In this study, the genome features of L. arseniciresistens ZS79T is provided and the comparative results of five genomes of Lysobacter are presented.
Classification and features
Members of genus Lysobacter are rod-shaped, aerobic, Gram-negative bacteria . Their G+C contents are 65.4–70.1 %. They use NO3 −, NH4 +, glutamate, asparaginate as sole nitrogen sources, Q-8 as the major respiratory quinone, and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyl-N-methylethanolamine as the major polar lipids [3, 8]. In addition, they could lyse cells of many creatures including bacteria, filamentous fungi, yeasts, algae and nematodes .
Phylogenetic analyses of L. arseniciresistens ZS79T and its related strains of family Xanthomonadaceae were performed based on 16S rRNA genes (Fig. 1a) and 831 conserved proteins (Fig. 1b). In both trees, strain ZS79T is clustered with the other four strains of genus Lysobacter . The phylogenies of the two trees are similar but genomic based tree is more stable than the 16S rRNA gene one (Fig. 1b vs 1a).
L. arseniciresistens ZS79T is aerobic, motile, and Gram-negative bacterium with a Minimum Inhibitory Concentration of 14 mM arsenite in R2A medium (Table 1). The cells are rod-shaped with one flagellum and non-spore-forming (Fig. 2). Colonies of this strain are yellow, nontransparent, convex, circular, and, smooth .
The major ubiquinone is Q-8, the major cellular fatty acids (>10 %) are iso-C15 : 0, iso-C17 :1 ω9ϲ, iso-C16 :0, iso-C11 :0 and iso-C11 :0 3-OH. The polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and a kind of unknown phospholipid The C + G content is was 70.7 mol% (HPLC) .
Genome sequencing and annotation
Genome project history
The genome of L. arseniciresistens ZS79T was sequenced in April, 2013 and finished within two months. The high-quality draft genome sequence is available in GenBank database under accession number AVPT00000000. The genome sequencing project information is summarized in Table 2.
Growth conditions and genomic DNA preparation
L. arseniciresistens ZS79T was cultured in 50 ml of LB (Luria–Bertani) medium at 28 °C for 3 days with 160 160 r/min shaking. About 10 mg cells were harvested by centrifugation and suspended in normal saline, and then lysed using lysozyme. DNA was isolated using cells were harvested by centrifugation and suspended in normal saline, and then lysed using lysozyme. The DNA was extracted and purified using the QiAamp kit according to the manufacturer’s instruction (Qiagen, Germany).
Genome sequencing and assembly
The whole genome sequencing of L. arseniciresistens ZS79T was performed on Illumina Hiseq2000 with Paired-End library strategy (300 bp insert size) at Majorbio Biomedical Science and Technology Co. Ltd. DNA libraries with insert sizes from 300 to 500 bp was constructed using the established protocol . The obtained high quality data contains 4,528,542 × 2 pared reads and 194,996 single reads with an average read length of 91 bp. The sequencing depth was 272.6×. Using SOAPdenovo v1.05  the reads were assembled into 109 contigs with a cumulative genome size of 3,086,721 bp.
The draft sequence of L. arseniciresistens ZS79T was annotated using the National Center for Biotechnology Information Prokaryotic Genomes Annotation Pipeline . The functions of the predicted genes were determined through blast alignment against the NCBI protein database. Genes were identified using the gene caller GeneMarkS+ with the similarity-based gene detection approach . The different features were predicted by WebMGA , TMHMM  and SignalP .
The whole genome sequence of L. arseniciresistens ZS79T is 3,086,721 bp long with a G+C content of 69.6 % and is distributed into 109 contigs. It has 2,422 predicted genes including 2,363 (97.6 %) protein coding genes, 50 (2.1 %) RNA genes, and 9 (0.4 %) pseudo genes. A total of 1633 (67.4 %) genes have functional prediction, and 1,858 (76.7 %) genes could be assigned to Clusters of Orthologous Groups . More detailed information of the genome statistics is showed in Table 3. The protein functional classification according to COGs is showed in Table 4. The genome map is showed in Fig. 3.
Insights from the genome sequences
To obtain features of Lysobacter genomes, we sequenced four genomes of genus Lysobacter and performed comparative genomic analysis among the five available genomes of this genus. The general features of these five genomes are summarized in Table 5. To calculate the pan-genome and core-genome of these five genomes, we performed orthologs clustering analysis using OrthoMCL . The pan-genome has 6,409 orthologs families and the core-genome has 1,207 orthologs. The numbers of unique genes of each genome are showed in Fig. 4. To evaluate the genome variation of these five genomes, we first performed multiple alignments among these genome sequences using MAUVE  and then calculated the nucleotide diversity using DnaSP v5 . These five genomes shared 0.73 Mb co-linear sequences. The π value of these sequences among these five genomes is 0.173 which means that the approximate nucleotide sequence homology is 83 % among genomes of Lysobacter .
In the genome of L. arseniciresistens ZS79T, we found that the genomic island distributions are consistent with the genome C + G content anomaly areas (Fig. 3). In addition, few gene sequences from the other four Lysobacter genomes could be aligned with these genomic island regions (Fig. 3, ring 6 to ring 9). These results indicated that the genes within the genomic islands were most probably acquired by horizontal transfer  and these regions are unique in the genome of L. arseniciresistens ZS79T.
According to Kyoto Encyclopedia of Genes and Genomes  annotation result, all of the five Lysobacter genomes have a nearly complete type II secretion system which could secret cell wall degrading enzymes . This result may correspond to the behavior of Lysobacter members that were able to lyse cells of many microorganisms . In addition, the genomes of L. arseniciresistens ZS79T, L. concretionis Ko07T and L. defluvii IMMIB APB-9T contain genes for flagellar assembly, whereas the genome of L. daejeonensis GH1-9T does not contain any genes for flagellar assembly and L. capsici AZ78 does not contain genes for flagellar filament (Additional file 1: Table S2). These genotypes correspond to the phenotype descriptions that L. daejeonensis and L. capsici are non-motile [8, 11].
Genomic analysis showed eight genes corresponding to arsenic resistance in the genomes of L. arseniciresistens ZS79T (Additional file 1: Table S3). This result well explained the arsenite resistance of this strain . By contrast, fewer arsenic resistance were found in the genomes of L. concretionis Ko07T, L. defluvii IMMIB APB-9T, L. capsici AZ78, and L. daejeonensis GH1-9T compared to strain ZS79T.
The genomic information of L. arseniciresistens ZS79T and the comparative genomics analysis of the five Lysobacter strains are obtained. The genomic based phylogeny is in agreement with the 16S rRNA gene based one indicating the usefulness of genomic information for bacterial taxonomic classification. Analysis of the genomes show certain correlation between the genotypes and the phenotypes.
Luo G, Shi Z, Wang G. Lysobacter arseniciresistens sp. nov., an arsenite-resistant bacterium isolated from iron-mined soil. Int J Syst Evol Microbiol. 2012;62:1659–65. PubMed http://www.ncbi.nlm.nih.gov/pubmed/21890727.
NCBI Taxonomy Browser http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi
Christensen P, Cook FD. Lysobacter, a New Genus of Nonfruiting, Gliding Bacteria with a High Base Ratio. Int J Syst Bacteriol. 1978;28:27. http://ijs.sgmjournals.org/cgi/content/abstract/28/3/367.
Fukuda W, Kimura T, Araki S, Miyoshi Y, Atomi H, Imanaka T. Lysobacter oligotrophicus sp. nov., isolated from an Antarctic freshwater lake in Antarctica. Int J Syst Evol Microbiol. 2013;63:3313–8. PubMed http://www.ncbi.nlm.nih.gov/pubmed/23475347.
Bae HS, Im WT, Lee ST. Lysobacter concretionis sp. nov., isolated from anaerobic granules in an upflow anaerobic sludge blanket reactor. Int J Syst Evol Microbiol. 2005;55:1155–61. PubMed http://www.ncbi.nlm.nih.gov/pubmed/15879248.
Ten LN, Jung HM, Im WT, Yoo SA, Lee ST. Lysobacter daecheongensis sp. nov., isolated from sediment of stream near the Daechung dam in South Korea. J Microbiol. 2008;46:519–24. PubMed http://www.ncbi.nlm.nih.gov/pubmed/18974952.
Romanenko LA, Uchino M, Tanaka N, Frolova GM, Mikhailov VV. Lysobacter spongiicola sp. nov., isolated from a deep-sea sponge. Int J Syst Evol Microbiol. 2008;58:370–4. PubMed http://www.ncbi.nlm.nih.gov/pubmed/18218933.
Park JH, Kim R, Aslam Z, Jeon CO, Chung YR. Lysobacter capsici sp. nov., with antimicrobial activity, isolated from the rhizosphere of pepper, and emended description of the genus Lysobacter. Int J Syst Evol Microbiol. 2008;58:387–92. PubMed http://www.ncbi.nlm.nih.gov/pubmed/18218936.
Puopolo G, Sonego P, Engelen K, Pertot I. Draft Genome Sequence of Lysobacter capsici AZ78, a Bacterium Antagonistic to Plant-Pathogenic Oomycetes. Genome Announc. 2014;2. PubMed http://www.ncbi.nlm.nih.gov/pubmed/24762937.
Zhou L, Li M, Yang J, Wei L, Ji G. Draft Genome Sequence of Antagonistic Agent Lysobacter antibioticus 13-6. Genome Announc. 2014;2. PubMed http://www.ncbi.nlm.nih.gov/pubmed/25301638.
Weon HY, Kim BY, Baek YK, Yoo SH, Kwon SW, Stackebrandt E, et al. Two novel species, Lysobacter daejeonensis sp. nov. and Lysobacter yangpyeongensis sp. nov., isolated from Korean greenhouse soils. Int J Syst Evol Microbiol. 2006;56:947–51. PubMed http://www.ncbi.nlm.nih.gov/pubmed/16627636.
Yassin AF, Chen WM, Hupfer H, Siering C, Kroppenstedt RM, Arun AB, et al. Lysobacter defluvii sp. nov., isolated from municipal solid waste. Int J Syst Evol Microbiol. 2007;57:1131–6. PubMed http://www.ncbi.nlm.nih.gov/pubmed/17473271.
Illumina official website http://www.illumina.com
Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience. 2012;1:18. PubMed http://www.ncbi.nlm.nih.gov/pubmed/23587118.
Prokaryotic Genome Annotation Pipeline http://www.ncbi.nlm.nih.gov/genome/annotation_prok.
Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 2001;29(12):2607–18. PubMed http://www.ncbi.nlm.nih.gov/pubmed/11410670.
Wu S, Zhu Z, Fu L, Niu B, Li W. WebMGA: a customizable web server for fast metagenomic sequence analysis. BMC Genomics. 2011;12:444. PubMed http://www.ncbi.nlm.nih.gov/pubmed/21899761.
Krogh A, Larsson BÈ, Von Heijne G, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305(3):567–80. PubMed http://www.ncbi.nlm.nih.gov/pubmed/11152613.
Dyrlov Bendtsen J, Nielsen H, von Heijne G. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004;340(4):783–95. PubMed http://www.ncbi.nlm.nih.gov/pubmed/15223320.
Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:41. PubMed http://www.ncbi.nlm.nih.gov/pubmed/12969510.
Li L, Stoeckert Jr CJ, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003;13:2178–89. PubMed http://www.ncbi.nlm.nih.gov/pubmed/12952885.
Darling AC, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–403. PubMed http://www.ncbi.nlm.nih.gov/pubmed/15231754.
Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25:1451–2. PubMed http://www.ncbi.nlm.nih.gov/pubmed/19346325.
Langille MG, Hsiao WW, Brinkman FS. Detecting genomic islands using bioinformatics approaches. Nat Rev Microbiol. 2010;8:373–82. PubMed http://www.ncbi.nlm.nih.gov/pubmed/20395967.
Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M. Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 2014;42:D199–205. PubMed http://www.ncbi.nlm.nih.gov/pubmed/24214961.
Cianciotto NP. Type II secretion: a protein secretion system for all seasons. Trends Microbiol. 2005;13:581–8. PubMed http://www.ncbi.nlm.nih.gov/pubmed/16216510.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7. PubMed http://www.ncbi.nlm.nih.gov/pubmed/18464787.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9. PubMed http://www.ncbi.nlm.nih.gov/pubmed/2112744.
Garrity G, Bell J, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity G, Brenner D, Krieg N, Staley J, editors. Bergey’s Manual of Systematic Bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 1.
Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int J Syst Evol Microbiol. 2005; 55:1743–5. PubMed [http://www.ncbi.nlm.nih.gov/pubmed/16166658].
Saddler G, Bradbury J. Order III. Xanthomonadales ord. nov. In: Garrity G, Brenner D, Krieg N, Staley J, editors. Bergey’s Manual of Systematic Bacteriology, vol. 2. 2nd ed. New York: Springer; 2005. p. 63.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9. PubMed http://www.ncbi.nlm.nih.gov/pubmed/10802651.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30:2725–9. PubMed http://www.ncbi.nlm.nih.gov/pubmed/24132122.
Langille MG, Brinkman FS. IslandViewer: an integrated interface for computational identification and visualization of genomic islands. Bioinformatics. 2009;25:664–5. PubMed http://www.ncbi.nlm.nih.gov/pubmed/19151094.
This work was supported by the National High Technology Research and Development Program of China (2012AA101402) and the National Natural Science Foundation of China (31470226).
The authors declare that they have no competing interests.
LL carried out sequence alignments and drafted the manuscript. SZ performed the genome annotation and genome comparison. ML and GW coordinated the study, participated in the design and corrected the manuscript. All authors read and approved the final manuscript.
Table S1. The proteins for Type II secretion in Lysobacter genomes. Table S2. The proteins for flagellar assembly in Lysobacter genomes. Table S3. The arsenic resistances genes found in five Lysobacter genomes. (XLSX 17 kb)