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High-quality-draft genome sequence of the heavy metal resistant and exopolysaccharides producing bacterium Mucilaginibacter pedocola TBZ30T

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Abstract

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.

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 TBZ30T (= CCTCC AB 2015301T = KCTC 42833T) was isolated from a heavy metal contaminated paddy field in Hunan Province, P. R. China [5]. 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.

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 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).

Fig. 1
figure1

A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain TBZ30T and the related species. The bootstrap value less than 50% are not shown. Bar, 0.005 substitutions per nucleotide position

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) [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 TBZ30T can produce EPS testing by aniline blue staining method [9] (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.

Fig. 2
figure2

A scanning electron microscope (SEM) image of Mucilaginibacter pedocola TBZ30T cells. The bar scale represents 0.5 μm

Table 1 Classification and general features of Mucilaginibacter pedocola TBZ30T [39]
Fig. 3
figure3

EPS detection using the aniline blue staining method [9]. a, b and c strain TBZ30T, positive control Mucilaginibacter litoreus BR-18T and negative control Nocardioides albus KCTC 9186T cultivated in LB plates, respectively; (d, e and f) the above three strains cultivated in LB-aniline blue plates, respectively

Fig. 4
figure4

Zn2+ and Cd2+ removal by strain TBZ30T in R2A liquid media. a Zn2+ removal by strain TBZ30T; (b) Cd2+ removal by strain TBZ30T. The control represents R2A liquid medium with 0.3 mM Zn2+ or 0.25 mM Cd2+ without the inoculation of strain TBZ30T. Data are shown as the mean of three replicates

Genome information

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.

Table 2 Project information

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 [12]. 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 [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 TBZ30T 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 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.

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
figure5

A graphical circular map of Mucilaginibacter pedocola TBZ30T. 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 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).

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

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 [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 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 [3]. Therefore, it is possible that the EPS of strain TBZ30T 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 TBZ30T and the related Mucilaginibacter members.

Abbreviations

EPS:

Exopolysaccharides

MIC:

Minimal inhibition concentration

References

  1. 1.

    Pankratov TA, Tindall BJ, Liesack W, Dedysh SN. Mucilaginibacter paludis gen. nov., sp. nov. and Mucilaginibacter gracilis sp. nov., pectin-, xylan- and laminarin-degrading members of the family Sphingobacteriaceae from acidic Sphagnum peat bog. Int J Syst Evol Microbiol. 2007;57(10):2349–54.

  2. 2.

    Baik KS, Park SC, Kim EM, Lim CH, Seong CN. Mucilaginibacter rigui sp. nov., isolated from wetland freshwater, and emended description of the genus Mucilaginibacter. Int J Syst Evol Microbiol. 2010;60(1):134–9.

  3. 3.

    Cui Y, Xu T, Qu X, Hu T, Jiang X, Zhao C. New insights into various production characteristics of Streptococcus thermophilus strains. Int J Mol Sci. 2016;17(10):1701.

  4. 4.

    More TT, Yadav JS, Yan S, Tyagi RD, Surampalli RY. Extracellular polymeric substances of bacteria and their potential environmental applications. J Environ Manag. 2014;144:1–25.

  5. 5.

    Tang J, Huang J, Qiao Z, Wang R, Wang G. Mucilaginibacter pedocola sp. nov., isolated from a heavy-metal-contaminated paddy field. Int J Syst Evol Microbiol. 2016;66(10):4033–8.

  6. 6.

    Khan H, Chung EJ, Jeon CO, Chung YR. Mucilaginibacter gynuensis sp. nov., isolated from rotten wood. Int J Syst Evol Microbiol. 2013;63(9):3225–31.

  7. 7.

    Mannisto MK, Tiirola M, McConnell J, Haggblom MM. Mucilaginibacter frigoritolerans sp. nov., Mucilaginibacter lappiensis sp. nov. and Mucilaginibacter mallensis sp. nov., isolated from soil and lichen samples. Int J Syst Evol Microbiol. 2010;60(12):2849–56.

  8. 8.

    Yoon JH, Kang SJ, Park S, Oh TK. Mucilaginibacter litoreus sp. nov., isolated from marine sand. Int J Syst Evol Microbiol. 2012;62(12):2822–7.

  9. 9.

    K N, Devasya RP, Bhagwath AA. Exopolysaccharide produced by Enterobacter sp. YG4 reduces uranium induced nephrotoxicity. Int J Biol Macromol. 2016;82:557–61.

  10. 10.

    Prauser H. Nocardioides, a new genus of the order actinomycetales. Int J Syst Evol Microbiol. 1976;26:58–65.

  11. 11.

    Skerman VBD, McGowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Bacteriol. 1980;30:225–420.

  12. 12.

    Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5(4):433–8.

  13. 13.

    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(1):18.

  14. 14.

    Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 2010;20(2):265–72.

  15. 15.

    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.

  16. 16.

    Li L, Stoeckert CJ Jr, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003;13(9):2178–89.

  17. 17.

    Fischer S, Brunk BP, Chen F, Gao X, Harb OS, Iodice JB, et al. Using OrthoMCL to assign proteins to OrthoMCL-DB groups or to cluster proteomes into new ortholog groups. Curr Protoc Bioinformatics. 2011;6:1–19.

  18. 18.

    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.

  19. 19.

    Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. Pfam: the protein families database. Nucleic Acids Res. 2014;42:222–30.

  20. 20.

    Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004;32:277–80.

  21. 21.

    Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305(3):567–80.

  22. 22.

    Petersen TN, Brunak S, Von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8(10):785–6.

  23. 23.

    Nies DH. CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus. J Bacteriol. 1992;174(24):8102–10.

  24. 24.

    Nies DH. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev. 2003;27(2–3):313–39.

  25. 25.

    Xiong J, Li D, Li H, Susan J, Miller LY, et al. Genome analysis and characterization of zinc efflux systems of a highly zinc-resistant bacterium, Comamonas testosteroni S44. Res Microbiol. 2011;162(7):671–9.

  26. 26.

    Rakesh S, Christopher R, Barry PR, Bharati M. The ATP hydrolytic activity of purified ZntA, a Pb(II)/cd(II)/Zn(II)-translocating ATPase from Escherichia coli. J Biol Chem. 2000;275:3873–8.

  27. 27.

    Solioz M, Vulpe C. CPx-type ATPases: a class of P-type ATPases that pump heavy metals. Trends Biochem Sci. 1996;21(7):237–41.

  28. 28.

    Li S, Zhou X, Huang Y, Zhu L, Zhang S, Zhao Y, et al. Identification and characterization of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) gene family in maize. BMC Plant Biol. 2013;13:114.

  29. 29.

    Potocki S, Valensin D, Kozlowski H. The specificity of interaction of Zn(2+), Ni(2+) and cu(2+) ions with the histidine-rich domain of the TjZNT1 ZIP family transporter. Dalton Trans. 2014;43(26):10215–23.

  30. 30.

    Guan G, Pinochet-Barros A, Gaballa A, Patel SJ, Argüello JM, Helmann JD. PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication. Mol Microbiol. 2015;98(4):787–803.

  31. 31.

    Yong-Qun Z, De-Yu Z, Hong-Xia L, Na Y, Gen-Pei L, Da-Cheng W. Purification and preliminary crystallographic studies of CutC, a novel copper homeostasis protein from Shigella flexneri. Protein Pept Lett. 2005;12:823–82.

  32. 32.

    Mauricio L, Felipe O, Reyes-Jara A, Guadalupe L, Mauricio G. CutC is induced late during copper exposure and can modify intracellular copper content in Enterococcus faecalis. Biochem Biophys Res Commun. 2011;406:633–7.

  33. 33.

    Liu G, Liu M, Kim EH, Maaty WS, Bothner B, Lei B, et al. A periplasmic arsenite-binding protein involved in regulating arsenite oxidation. Environ Microbiol. 2012;14(7):1624–34.

  34. 34.

    Li X, Zhang L, Wang G. Genomic evidence reveals the extreme diversity and wide distribution of arsenic-related genes in Burkholderides. PLoS One. 2014;9(3):e92236.

  35. 35.

    Qin J, Zhang Y, Barry R, Wang G, Franke S, Rensing C. Arsenic detoxification and evolution of trimethylarsine gas by microbial arsenite S-adenosylmethionine methyltransferase. Proc Natl Acad Sci. 2006;103(7):2075–80.

  36. 36.

    Schmid J, Sieber V, Rehm B. Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies. Front Microbiol. 2015;6:496.

  37. 37.

    Cuthbertson L, Mainprize IL, Naismith JH, Whitfield C. Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in gram-negative bacteria. Microbiol Mol Biol Rev. 2009;73(1):155–77.

  38. 38.

    Willis LM, Whitfield C. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways. Carbohydr Res. 2013;378:35–44.

  39. 39.

    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.

  40. 40.

    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.

  41. 41.

    Goodfellow M. Phylum XXVI. Actinobacteria phyl. nov. Bergey’s Manual of Systematic Bacteriology 2012;5; Part A:33.

  42. 42.

    Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Evol Microbiol. 1997;47:479–91.

  43. 43.

    Smith C. The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. Int J Syst Evol Microbiol. 2002;52:7–76.

  44. 44.

    Euzéby J. Validation list no. 143. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2012;62:1–4.

  45. 45.

    Kämpfer P. Class III. Sphingobacteriia class. nov. Bergey’s Manual of Systematic Bacteriology, vol. 4; 2011. p. 330.

  46. 46.

    Kämpfer P. Order I. Sphingobacteriales ord nov Bergey’s Manual of Systematic Bacteriology, vol. 4; 2011. p. 330.

  47. 47.

    Steyn PL, Segers P, Vancanneyt M, Sandra P, Kersters K, Joubert JJ. Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. Proposal of the family Sphingobacteriaceae fam. nov. Int J Syst Bacteriol. 1998;48:165–77.

  48. 48.

    Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Geneontology: tool for the unification of biology. The gene ontology consortium. Nat Genet. 2000;25:25–9.

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Funding

This study was supported by National key research and development program of China (2016YFD0800702).

Author information

XF and JT performed the phenotypic characterization, the data analysis and wrote the manuscript. LN participated in phenotypic experiments. JH participated in data analysis. GW was responsible for research design and revised the manuscript. All authors read and approved the final manuscript.

Correspondence to Gejiao Wang.

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Keywords

  • Mucilaginibacter pedocola
  • Genome sequence
  • Heavy metal resistance
  • Exopolysaccharides