Genome assembly of Chryseobacterium sp. strain IHBB 10212 from glacier top-surface soil in the Indian trans-Himalayas with potential for hydrolytic enzymes.

The cold-active esterases are gaining importance due to their catalytic activities finding applications in chemical industry, food processes and detergent industry as additives, and organic synthesis of unstable compounds as catalysts. In the present study, the complete genome sequence of 4,843,645 bp with an average 34.08% G + C content and 4260 protein-coding genes are reported for the low temperature-active esterase-producing novel strain of Chrysobacterium isolated from the top-surface soil of a glacier in the cold deserts of the Indian trans-Himalayas. The genome contained two plasmids of 16,553 and 11,450 bp with 40.54 and 40.37% G + C contents, respectively. Several genes encoding the hydrolysis of ester linkages of triglycerides into fatty acids and glycerol were predicted in the genome. The annotation also predicted the genes encoding proteases, lipases, amylases, β-glucosidases, endoglucanases and xylanases involved in biotechnological processes. The complete genome sequence of Chryseobacterium sp. strain IHBB 10212 and two plasmids have been deposited vide accession numbers CP015199, CP015200 and CP015201 at DDBJ/EMBL/GenBank.

Direct link to deposited data

https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA313490

Introduction

The microorganisms inhabiting the cold environments have been explored for the industrially important enzymes [1], [2], [3]. The genus Chryseobacterium has been reported for the production of antifungal protease by C. aquaticum [4], mannanase by C. indologenes [5], α-amylase by C. taeanense [6], metallo-protease by C. gleum [7], [8], and proteases by C. polytrichastri [9]. The genomic analyses have also supported the hydrolytic enzyme activities by Chryseobacterium sp. strain P1–3 [10], Chryseobacterium sp. strain StRB126 [21], C. gallinarum strain DSM 27622 [11], and C. polytrichastri strain ERMR1:04 [9]. The strain IHBB 10212 exhibited the hydrolysis of p-nitrophenyl butyrate (pNPB), p-nitrophenyl deconate (pNPD) and p-nitrophenyl caprylate (pNPC) substrates of esterases in quantitative assay [12]. The present study reports the genes encoding hydrolytic enzymes by the functional annotation of complete genome sequence of Chryseobacterium sp. strain IHBB 10212.

Experimental design, materials and methods

The strain IHBB 10212 was grown in tryptone soya broth (Himedia, India) at 28 ± 0.1 °C for 48 h and 200 rpm. Genomic DNA was isolated using phenol-chloroform-isoamyl alcohol extraction by Marmur method, assessed for quality by NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and quantified using Qubit 2.0 fluorometer (Invitrogen, USA). The DNA was sheared using Covaris g-TUBE, assessed for quality using Bioanalyzer DNA 12000 chip (Agilent Technologies, USA) and employed for preparation of genomic library using PacBio SMRTbell library preparation kit v1.0 (Pacific Biosciences, USA). The library was quantified and sequenced on PacBio RS II system (Pacific Biosciences, USA) on one SMRT cell employing P5 polymerase-C3 sequencing chemistry with 180-min movie [13]. The subreads were assembled de novo using RS hierarchical genome assembly process (HGAP) protocol version 2.0 in SMRT analysis version 2.2.0 (Pacific Biosciences, USA). The functional annotations were performed using Rapid Annotation Subsystem Technology (RAST) server [14]. The circular genome map was generated using GView online server [15]. The cluster of orthologous groups (COG) functional categories were determined by using WebMGA online server [16]. The genomic islands (GIs) were predicted using IslandViewer 3 web-based interface by integrating IslandPath-DIMOB and SIGI-HMM prediction methods [17].

Genomic analysis

The genome sequencing produced 551,857,867 nucleotide bases generated through 74,645 reads (N50 size 13,222 bp and mean read length 7393 bp). The genome assembly resulted in a gapless and complete circular genome with total size of 4,871,648 bases with 90-fold coverage. The circular chromosome was 4,843,645 bp in size with an average G + C content of 34.08% (Fig. 1). Two plasmids of 16,553 and 11,450 bp were also identified with the average G + C content 40.54 and 40.37%. The genomic features included 4260 protein-coding sequences and 21 rRNAs and 75 tRNAs (Table 1). Among the protein-coding genes, 2927 genes (68.7%) were classified into 21 categories of COG families (Table 2). Nineteen putative GIs were predicted in the chromosome. The smallest GI was 4018 bp in size having 2 genes while the largest GI was 44,550 bp in size with 30 genes. Seven GIs contained the mobile genetic elements like transposases and integrases (Fig. 2).

Fig. 1

Circular genome map of Chryseobacterium sp. strain IHBB 10212 generated using GView server. From outside to the center: genes on forward strand, genes on reverse strand, GC content (%), GC skew, colors of cluster of orthologous groups (COG) categories and scale in kbp.

Table 1

Genomic features of Chryseobacterium sp. strain IHBB 10212.

Attributes	Values
Chromosome size (bp)	4,843,645
G + C content (%)	34.08
Plasmids	2
Total genes	4400
Protein-coding genes	4260
rRNA (5S, 16S, 23S)	21
tRNAs	75
Pseudogenes	41

Table 2

COG functional categories of Chryseobacterium sp. strain IHBB 10212.

COG	Functional category	Values
C	Energy production and conversion	140
D	Cell cycle control, cell division, chromosome partitioning	23
E	Amino acid transport and metabolism	218
F	Nucleotide transport and metabolism	70
G	Carbohydrate transport and metabolism	168
H	Coenzyme transport and metabolism	138
I	Lipid transport and metabolism	127
J	Translation, ribosomal structure and biogenesis	163
K	Transcription	251
L	Replication, recombination and repair	164
M	Cell wall/membrane/envelope biogenesis	239
N	Cell motility	8
O	Posttranslational modification, protein turnover, chaperones	116
P	Inorganic ion transport and metabolism	164
Q	Secondary metabolites biosynthesis, transport and catabolism	59
R	General function prediction only	377
S	Function unknown	242
T	Signal transduction mechanisms	154
U	Intracellular trafficking, secretion, and vesicular transport	49
V	Defense mechanisms	55
Z	Cytoskeleton	2
Total		2927

Fig. 2

Genomic islands of Chryseobacterium sp. strain IHBB 10212 predicted by using IslandViewer 3 online tool. Orange color represents genomic islands predicted based on SIGI-HMM, blue color represents genomic islands predicted based on IslandPath-DIMOB, and red color represents genomic islands predicted based on integrated results of the two methods. Black plot represents G + C content (%).

The functional annotations also predicted the gene clusters coding for esterases (26 genes), proteases (23 genes), lipases (1 gene), lipases/esterases (6 genes) amylases (4 genes), endoglucanases (1 gene), β-glucosidases (7 genes) and xylanase (5 genes). Among the gene clusters coding for esterases, 1 gene each for sialic acid-specific 9-O-acetylesterase, 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (EC 3.1.4.16), 3′,5′-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17), acyl carrier protein phosphodiesterase (EC 3.1.4.14), alkaline phosphatase (EC 3.1.3.1), arylesterase-like protein slr1119, CheB methylesterase (EC:3.1.1.61), esterase/lipase, esterase/lipase/thioesterase family protein, esterase/lipase-like protein, FIG006285: ICC-like protein phosphoesterase, glycerophosphoryl diester phosphodiesterase (EC 3.1.4.46), lipase/esterase, pectinesterase (EC 3.1.1.11), putative carboxylesterase, putative phosphoesterase, serine esterase, thioesterase family protein, and thioesterase superfamily, 2 genes each for alkaline phosphodiesterase I (EC 3.1.4.1)/nucleotide pyrophosphatase (EC 3.6.1.9), thioesterase, phospholipase/carboxylesterase, putative esterase, and 5 genes for rhamnogalacturonan acetylesterase were predicted. A gene encoding for lipase (EC 3.1.1.3) was also predicted. Several genes encoding for proteases were predicted including 1 gene each for alkaline serine protease, ATP-dependent Clp protease ATP-binding subunit ClpX, ATP-dependent Clp protease proteolytic subunit (EC 3.4.21.92), ATP-dependent protease La (EC 3.4.21.53) type I, CAAX amino terminal protease family protein, carboxyl-terminal protease, HtrA protease/chaperone protein, membrane protease family protein BA0301, membrane-associated zinc metalloprotease, metalloprotease MEP2, protease II (EC 3.4.21.83), protease IV, protease precursor, protease synthase and sporulation negative regulatory protein PAI 2, putative stomatin/prohibitin-family membrane protease subunit PA4582, putative stomatin/prohibitin-family membrane protease subunit YbbK, serine alkaline protease (subtilisin E), subtilisin-like serine proteases and zinc metalloprotease superfamily, and 4 genes for zinc protease. Additionally genome sequence also showed coding sequences for amylases including 1 gene for α-amylase (neopullulanase) SusA (EC 3.2.1.135) and 4 genes for cytoplasmic α-amylase (EC 3.2.1.1). Genes related to β-glucosidases were also predicted which included 1 gene each for glucan endo-1,6-β-glucosidase (EC 3.2.1.75) and periplasmic β-glucosidase (EC 3.2.1.21), and 4 genes for β-glucosidase (EC 3.2.1.21).The functional annotations also predicted genes for xylanases including 1 gene each for endo-1,4-β-xylanase B precursor, xylanase and 3 genes for endo-1,4-β-xylanase A precursor (EC 3.2.1.8).

The analysis also predicted in Chryseobacterium sp. strain IHBB 10212 genome the presence of 4 CspA and 1 CspG genes responsible for survival during the cold shock. The upregulation of cold-shock proteins have been reported for survival against the cold shock by the microorganisms inhabiting the cold environments [18]. The genome contained genes for the nucleotide excision repair system including genes for excinuclease ABC (UvrABC), transcription-repair coupling factor and UvrD helicases which are reported for repairing DNA damage by UV radiations [19], [20]. The genomic analysis has provided an understanding on production of hydrolytic enzymes and genes related to cold adaptation and repairing DNA damage by UV radiations in the cold deserts.

Nucleotide sequence accession numbers

Complete genome sequence of Chryseobacterium sp. strain IHBB 10212 has been deposited vide the accession number CP015199 at DDBJ/EMBL/GenBank. The two plasmids have been assigned the accession numbers CP015200 and CP015201.

Conflict of interest

There is no conflict of interest among the authors.

Specifications
Organism	Chryseobacterium sp. strain IHBB 10212
Strain	IHBB 10212
Sex	Not applicable
Sequencer or array type	PacBio RS II
Data format	Analyzed
Experimental factors	Microbial strain
Experimental features	Complete genome sequence
Consent	Not applicable
Sample source location	Top-surface soil of a glacier (32°23′ 53.05″ N 77°37′ 45.03″ E) near the Kunzum Pass located in the Spiti valley in the State of Himachal Pradesh in India