Genomic information of the arsenic-resistant bacterium Lysobacter arseniciresistens type strain ZS79^T and comparison of Lysobacter draft genomes

Abstract

Lysobacter arseniciresistens ZS79^T 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 ZS79^T and the comparative genomics results of the Lysobacter strains were described. The draft genome sequence of the strain ZS79^T 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.

Introduction

Lysobacter arseniciresistens type strain ZS79^T (=CGMCC 1.10752^T = KCTC 23365 T) belongs to family Xanthomonadaceae [1]. It is an arsenic-resistant bacterium isolated from subsurface soil of Tieshan iron mine, Daye City, P. R. China [1]. So far, there are 32 validly published species of Lysobacter [2]. Most of these Lysobacter strains were isolated from soil except that Lysobacter brunescens [3] and Lysobacter oligotrophicus [4] were isolated from water, and Lysobacter concretionis [5], Lysobacter daecheongensis [6] Lysobacter spongiicola [7] 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 [10]), 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 ZS79^T, Lysobacter conceretionis Ko07^T [5], Lysobacter daejeonensis GH1-9^T [11], and Lysobacter defluvii IMMIB APB-9^T [12]). In this study, the genome features of L. arseniciresistens ZS79^T is provided and the comparative results of five genomes of Lysobacter are presented.

Organism information

Classification and features

Members of genus Lysobacter are rod-shaped, aerobic, Gram-negative bacteria [3]. 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 [3].

Phylogenetic analyses of L. arseniciresistens ZS79^T 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 ZS79^T 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).

Fig. 1

Phylogenetic analyses indicating the position of L. arseniciresistens (in bold) in family Xanthomonadaceae. a The NJ tree based on aligned sequences of 16S rRNA of ten strains of family Xanthomonadaceae. b The NJ tree based on 831 conserved proteins among the ten Xanthomonadaceae strains. Phylogenetic analyses were performed using MEGA version 6 [33]. The trees were built using p-distance model and a bootstrap analysis of 1000 replicates. The GenBank numbers are listed after each strain

L. arseniciresistens ZS79^T 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 [1].

Table 1 Classification and general features of L. arseniciresistens ZS79^T according to the MIGS recommendations [27]

Fig. 2

Transmission electron microscopy of L. arseniciresistens ZS79^T

The major ubiquinone is Q-8, the major cellular fatty acids (>10 %) are iso-C_{15 : 0}, iso-C_{17 :1} ω9ϲ, iso-C_{16 :0}, iso-C_{11 :0} and iso-C_{11 :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) [1].

Genome sequencing and annotation

Genome project history

The genome of L. arseniciresistens ZS79^T 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.

Table 2 Project information

Growth conditions and genomic DNA preparation

L. arseniciresistens ZS79^T 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 ZS79^T 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 [13]. 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 [14] the reads were assembled into 109 contigs with a cumulative genome size of 3,086,721 bp.

Genome annotation

The draft sequence of L. arseniciresistens ZS79^T was annotated using the National Center for Biotechnology Information Prokaryotic Genomes Annotation Pipeline [15]. 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 [16]. The different features were predicted by WebMGA [17], TMHMM [18] and SignalP [19].

Genome properties

The whole genome sequence of L. arseniciresistens ZS79^T 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 [20]. 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.

Table 3 Genome statistics

Table 4 Number of genes associated with general COG functional categories

Fig. 3

Graphical circular map of L. arseniciresistens ZS79^T genome. From outer to inner, ring 1 shows the genomic islands (red bars) that were predicted by IslandViewer [34]; ring 3,4 show the predicted genes on forward/reverse strand; ring 2,5 show the genes assigned to COGs; ring 6-9 show the ORFs similarity between the genome of L. arseniciresistens ZS79^T and the genomes of L. conceretionis Ko07^T, L. daejeonensis GH1-9^T, L. capsici AZ78 and L. defluvii IMMIB APB-9^T; ring 10 shows the G+C% content plot

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 [21]. 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 [22] and then calculated the nucleotide diversity using DnaSP v5 [23]. 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 [23].

Table 5 General features of the five Lysobacter genomes^a

Fig. 4

The core-genome and the unique genes of the five Lysobacter genomes. The Venn diagram shows the number of orthologous gene families of the core-genome (in the center) and the numbers of unique genes of each genome

In the genome of L. arseniciresistens ZS79^T, 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 [24] and these regions are unique in the genome of L. arseniciresistens ZS79^T.

According to Kyoto Encyclopedia of Genes and Genomes [25] annotation result, all of the five Lysobacter genomes have a nearly complete type II secretion system which could secret cell wall degrading enzymes [26]. This result may correspond to the behavior of Lysobacter members that were able to lyse cells of many microorganisms [3]. In addition, the genomes of L. arseniciresistens ZS79^T, L. concretionis Ko07^T and L. defluvii IMMIB APB-9^T contain genes for flagellar assembly, whereas the genome of L. daejeonensis GH1-9^T 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 ZS79^T (Additional file 1: Table S3). This result well explained the arsenite resistance of this strain [1]. By contrast, fewer arsenic resistance were found in the genomes of L. concretionis Ko07^T, L. defluvii IMMIB APB-9^T, L. capsici AZ78, and L. daejeonensis GH1-9^T compared to strain ZS79^T.

Conclusions

The genomic information of L. arseniciresistens ZS79^T 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.