- Short genome report
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High-quality-draft genome sequence of the heavy metal resistant and exopolysaccharides producing bacterium Mucilaginibacter pedocola TBZ30T
Standards in Genomic Sciencesvolume 13, Article number: 34 (2018)
Mucilaginibacter pedocola TBZ30T (= CCTCC AB 2015301T = KCTC 42833T) 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 TBZ30T 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 TBZ30T. 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.
The genus Mucilaginibacter was first established by Pankratov et al. in 2007 and the type species is Mucilaginibacter paludis . 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 . EPS producing bacteria play an important role in environmental bioremediation such as water treatment, sludge dewatering and metal removal . So far, genomic features of Mucilaginibacter strains are less studied.
Mucilaginibacter pedocola TBZ30T (= CCTCC AB 2015301T = KCTC 42833T) was isolated from a heavy metal contaminated paddy field in Hunan Province, P. R. China . Here we show that strain TBZ30T is resistant to multiple heavy metals and remove Zn2+ and Cd2+. In addition, strain TBZ30T is able to produce EPS. The genomic information of strain TBZ30T are provided.
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 TBZ30T showed the highest 16S rRNA gene sequence similarity with Mucilaginibacter gynuensis YC7003T (95.8%), Mucilaginibacter mallensis MP1X4T (95.4%) and Mucilaginibacter litoreus BR-18T (95.4%) [6,7,8] and grouped together with M. gynuensis YC7003T (95.8%) and M. mallensis MP1X4T (Fig. 1).
Strain TBZ30T 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) . Oxidase- and catalase-positive . It can use glucose, mannose, L-arabinose, maltose, melibiose, rhamnose and glycogen as the sole carbon sources . Strain TBZ30T can produce EPS testing by aniline blue staining method  (Fig. 3). The colonies of strain TBZ30T and the known EPS producing strain M. litoreus BR-18T 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 9186T [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 TBZ30T 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 TBZ30T 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.
Genome project history
M. pedocola TBZ30T 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.
Growth condition and DNA isolation
M. pedocola TBZ30T 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 . For accuracy of assembly, low quality of the original sequence data reads were removed. The assembly of TBZ30T 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 . The part gaps of assembly were filled and the error bases were revised using GapCloser v1.12 .
The genome of strain TBZ30T was annotated through the NCBI PGAP, which combined the gene caller GeneMarkS+ with the similarity-based gene detection approach . 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 . 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].
The genome size of strain TBZ30T 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.
Insights from the genome sequence
Strain TBZ30T 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).
Strain TBZ30T 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 TBZ30T appears to belong to ABC transporter dependent pathway . 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 ; 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 TBZ30T 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 . Therefore, it is possible that the EPS of strain TBZ30T participate in Zn2+ and Cd2+ removal by adsorption.
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 TBZ30T and the related Mucilaginibacter members.
Minimal inhibition concentration
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This study was supported by National key research and development program of China (2016YFD0800702).
The authors declare that they have no competing interests.
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