High-quality-draft genome sequence of the heavy metal resistant and exopolysaccharides producing bacterium Mucilaginibacter pedocola TBZ30^T

Abstract

Mucilaginibacter pedocola TBZ30^T (= CCTCC AB 2015301^T = KCTC 42833^T) is a Gram- negative, rod-shaped, non-motile and non-spore-forming bacterium isolated from a heavy metal contaminated paddy field. It shows resistance to multiple heavy metals and can adsorb/remove Zn²⁺ and Cd²⁺ during cultivation. In addition, strain TBZ30^T produces exopolysaccharides (EPS). These features make it a great potential to bioremediate heavy metal contamination and biotechnical application. Here we describe the genome sequence and annotation of strain TBZ30^T. The genome size is 7,035,113 bp, contains 3132 protein-coding genes (2736 with predicted functions), 50 tRNA encoding genes and 14 rRNA encoding genes. Putative heavy metal resistant genes and EPS associated genes are found in the genome.

Introduction

The genus Mucilaginibacter was first established by Pankratov et al. in 2007 and the type species is Mucilaginibacter paludis [1]. The common characteristics of this genus are Gram-negative, non-spore-forming, non-motile, rod-shaped and producing exopolysaccharides (EPS) [1, 2]. EPS are long-chain polysaccharides and consist of branched, repeating units of sugars or sugar derivatives [3]. EPS producing bacteria play an important role in environmental bioremediation such as water treatment, sludge dewatering and metal removal [4]. So far, genomic features of Mucilaginibacter strains are less studied.

Mucilaginibacter pedocola TBZ30^T (= CCTCC AB 2015301^T = KCTC 42833^T) was isolated from a heavy metal contaminated paddy field in Hunan Province, P. R. China [5]. Here we show that strain TBZ30^T is resistant to multiple heavy metals and remove Zn²⁺ and Cd²⁺. In addition, strain TBZ30^T is able to produce EPS. The genomic information of strain TBZ30^T are provided.

Organism information

Classification and features

Similarity analysis was performed using neighbor-joining method based on the 16S rRNA gene sequences and a phylogenetic tree was constructed using MEGA version 6.0 software (Fig. 1). Bootstrap analysis with 1000 replications was conducted to obtain confidence levels of the branches. Strain TBZ30^T showed the highest 16S rRNA gene sequence similarity with Mucilaginibacter gynuensis YC7003^T (95.8%), Mucilaginibacter mallensis MP1X4^T (95.4%) and Mucilaginibacter litoreus BR-18^T (95.4%) [6,7,8] and grouped together with M. gynuensis YC7003^T (95.8%) and M. mallensis MP1X4^T (Fig. 1).

Fig. 1

A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain TBZ30^T and the related species. The bootstrap value less than 50% are not shown. Bar, 0.005 substitutions per nucleotide position

Strain TBZ30^T is Gram-negative, non-motile, and non-spore-forming. Cells are rod-shaped (0.3–0.4 × 1.1–1.3 μm) (Fig. 2). Colonies are circular, pink, convex and smooth on R2A agar. Growth occurs aerobically at 4–28 °C (optimum, 25 °C), pH 5.0–8.5 (optimum, pH 7.0), and in the presence of 0–1.0 (w/v) NaCl (optimum, without NaCl) (Table 1) [5]. Oxidase- and catalase-positive [5]. It can use glucose, mannose, L-arabinose, maltose, melibiose, rhamnose and glycogen as the sole carbon sources [5]. Strain TBZ30^T can produce EPS testing by aniline blue staining method [9] (Fig. 3). The colonies of strain TBZ30^T and the known EPS producing strain M. litoreus BR-18^T are pink on LB plates (Fig. 3a and b), while the colonies are blue on LB-aniline blue plate (Fig. 3d and e). However, the colonies are always white for the negative control Nocardioides albus KCTC 9186^T [10, 11] on either LB or LB-aniline blue plates (Fig. 3c and f). All of the above strains were incubated at 28 °C for 7 days. In addition, strain TBZ30^T is resistant to multiple heavy metals. The minimal inhibition concentration (MIC) tests for different heavy metals were performed on R2A agar plates at 28 °C for 7 days. The MICs for ZnSO₄, CdCl₂, PbSO₄, CuSO₄ and NaAsO₂ are 3.5 mM, 1.5 mM, 0.4 mM, 1.2 mM and 0.35 mM, respectively. Furthermore, strain TBZ30^T could adsorb/remove nearly 60% of Zn²⁺ and 55% of Cd²⁺ in the R2A liquid medium (added with 0.3 mM ZnSO₄ and 0.25 mM CdCl₂, respectively) (Fig. 4). The amount of the heavy metals were detected by an atomic absorption spectrometer.

Fig. 2

A scanning electron microscope (SEM) image of Mucilaginibacter pedocola TBZ30^T cells. The bar scale represents 0.5 μm

Table 1 Classification and general features of Mucilaginibacter pedocola TBZ30^T [39]

Fig. 3

EPS detection using the aniline blue staining method [9]. a, b and c strain TBZ30^T, positive control Mucilaginibacter litoreus BR-18^T and negative control Nocardioides albus KCTC 9186^T cultivated in LB plates, respectively; (d, e and f) the above three strains cultivated in LB-aniline blue plates, respectively

Fig. 4

Zn²⁺ and Cd²⁺ removal by strain TBZ30^T in R2A liquid media. a Zn²⁺ removal by strain TBZ30^T; (b) Cd²⁺ removal by strain TBZ30^T. The control represents R2A liquid medium with 0.3 mM Zn²⁺ or 0.25 mM Cd²⁺ without the inoculation of strain TBZ30^T. Data are shown as the mean of three replicates

Genome information

Genome project history

M. pedocola TBZ30^T was sequenced on the basis of its abilities of heavy metals resistance and removal, which has a great potential for bioremediation. The draft genome was sequenced by Wuhan Bio-Broad Co., Ltd., Wuhan, China. The high-quality-draft genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession number MBTF00000000.1. The project information is shown in Table 2.

Table 2 Project information

Growth condition and DNA isolation

M. pedocola TBZ30^T was grown in R2A medium at 28 °C for 36 h with continuous shaking at 120 rpm. Bacterial cells were harvested through centrifugation (13,400×g for 5 min at 4 °C) and the total genomic DNA was extracted using the QiAamp kit (Qiagen, Germany). The quality and quantity of the DNA were determined using a spectrophotometer (NanoDrop 2000, Thermo).

Genome sequencing and assembly

Whole-genome DNA sequencing was performed in Bio-broad Co., Ltd., Wuhan, China using Illumina standard shotgun library and Hiseq2000 pair-end sequencing strategy [12]. For accuracy of assembly, low quality of the original sequence data reads were removed. The assembly of TBZ30^T genome is based on 16,967,512 quality reads totaling 2,523,391,653 bases with a 377.50× average genome coverage. The final reads were assembled into 39 contigs (> 200 bp) using SOAPdenovo v2.04 [13]. The part gaps of assembly were filled and the error bases were revised using GapCloser v1.12 [14].

Genome annotation

The genome of strain TBZ30^T was annotated through the NCBI PGAP, which combined the gene caller GeneMarkS⁺ with the similarity-based gene detection approach [15]. Pseudo genes were predicted using the NCBI PGAP. Internal gene clustering was performed by the OrthoMCL program using Match cutoff of 50% and E-value Exponent cutoff of 1-e5 [16, 17]. The COGs functional categories were assigned by the WebMGA server with E-value cutoff of 1-e10 [18]. The translations of the predicted CDSs were used to search against the Pfam protein family database and the KEGG database [19, 20]. The transmembrane helices and signal peptides were predicted by TMHMM v. 2.0 and SignalP 4.1, respectively [21, 22].

Genome properties

The genome size of strain TBZ30^T is 7,035,113 bp with an average G + C content of 46.1% (Table 3). It has 6072 genes including 5935 protein-coding genes, 70 pseudo genes and 14 rRNA, 50 tRNA, and 3 ncRNA genes. The information of the genome statistics is shown in Table 3 and the classification of genes into COGs functional categories is summarized in Table 4. The graphical genome map is provided in Fig. 5.

Table 3 Nucleotide content and gene count levels of the genome

Table 4 Number of genes associated with the 21 general COG functional categories

Fig. 5

A graphical circular map of Mucilaginibacter pedocola TBZ30^T. From outside to center, rings 1, 4 show protein-coding genes colored by COG categories on forward/reverse strand; rings 2, 3 denote genes on forward/reverse strand; rings 5 show G + C % content; ring 6 shows G + C % content plot and the innermost ring shows GC skew

Insights from the genome sequence

Strain TBZ30^T could be resistant to multiple heavy metals (Zn²⁺, Cd²⁺, Pb²⁺, Cu²⁺ and As³⁺) and adsorb/remove Zn²⁺ and Cd²⁺ during cultivation. Analyzing of its genome, various putative proteins related to multiple heavy metals resistance are found (Table 5). RND efflux systems (CzcABC), CDF efflux systems (CzcD and YieF) and P-type ATPases (HMA and ZntA) are responsible for the efflux of Zn²⁺, Cd²⁺ and Pb²⁺ [23,24,25,26,27]. Zip family metal transporter and P-type ATPase ZosA are associated with the efflux of Zn²⁺, Cd²⁺ or Cu²⁺ [28,29,30], and CutC is involved in Cu²⁺ homeostasis [30,31,32]. Moreover, As³⁺ resistant proteins including arsenite efflux pump ACR3, arsenate reductase ArsC, arsenite S-adenosylmethyltransferase ArsM and arsenic resistance repressor ArsR are also found [33,34,35] (Table 5).

Table 5 Putative protein involved in heavy metals resistance and EPS production

Strain TBZ30^T produces EPS during cultivation. According to KEGG analysis, the complete biosynthesis pathway of repeating units of nucleotide sugars are identified in the genome, including the biosynthesis of CDP-Glc, ADP-Glc and GDP-D-man (Table 5). Genes related to long-chain polysaccharide assembly are also found (Table 5). The EPS production pathway in strain TBZ30^T appears to belong to ABC transporter dependent pathway [36]. First, the 3-deoxy-D-manno-octulosonic-acid transferase (KdtA) is responsible for the synthesis of poly-Kdo linker using either diacyl or monoacyl phosphatidylglycerol as the substrate [36]; Then priming glycosyltransferase (CpsE) catalyzes the transformation of the first repeating unit to the poly-Kdo linker; Next, glycosyltransferases catalyze the synthesis of EPS repeat-unit; Finally, the polymerized repeat-units are exported through an envelope-spanning complex consisting of ABC transporter (KpsMT), polysaccharide co-polymerase protein (PCP) and outer membrane polysaccharide protein (OPX) [37, 38]. In addition, strain TBZ30^T genome owns a flippase (Wzx) which catalyzes the translocation of repeat-units crossing the cytoplasmic membrane. EPS have been reported to play an important role in metal removal [3]. Therefore, it is possible that the EPS of strain TBZ30^T participate in Zn²⁺ and Cd²⁺ removal by adsorption.

Conclusions

To the best of our knowledge, this study presents the first genomic information of a Mucilaginibacter type strain. The data reveal good correlation between genotypes and phenotypes. The genome information and the features provide insights for further theoretical and applied analysis of M. pedocola TBZ30^T and the related Mucilaginibacter members.