Whole genome sequencing and annotation of halophilic Salinicoccus sp. BAB 3246 isolated from the coastal region of Gujarat.

Salinicoccus sp. BAB 3246 is a halophilic bacterium isolated from a marine water sample collected from the coastal region of Gujarat, India, from a surface water stream. Based on 16sRNA sequencing, the organism was identified as Salinicoccus sp. BAB 3246 (Genebank ID: KF889285). The present work was performed to determine the whole genome sequence of the organism using Ion Torrent PGM platform followed by assembly using the CLC genomics workbench and genome annotation using RAST, BASys and MaGe. The complete genome sequence was 713,204 bp identified by with second largest size for Salinicoccus sp. reported in the NCBI genome database. A total of 652 degradative pathways were identified by KEGG map analysis. Comparative genomic analysis revealed Salinicoccus sp. BAB 3246 as most highly related to Salinicoccus halodurans H3B36. Data mining identified stress response genes and operator pathway for degradation of various environmental pollutants. Annotation data and analysis indicate potential use in pollution control in industrial influent and saline environment.

Direct link to deposited data

BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA342322.

Introduction

The genus Salinicoccus, belonging to family Staphylococcaceae was first proposed by Ventosa et al., (1990) and is defined as moderately halophilic, aerobic, Gram-positive, non-motile, non-sporulating, and heterotrophic cocci [1]. The genomic DNA G + C content of the species in this genus lies within the range of 46–51 mol%. Most species in genus Salinicoccus including Salinicoccus albus, Salinicoccus carnicancri, Salinicoccus roseu, Salinicoccus halodurans, Salinicoccus luteus have been found in salty environments, such as fermented foods, solar salterns, salt mines, salt lakes, and saline soils [1], [2], [3], [4], [5], [6], [7]. Alongside, genus Salinicoccus is also reported for production of Amylase, Protease, Gelatinase like enzymes in hyper saline environments [8].

The members of the Salinicoccus genus are abundant in the marine environments suggesting that they play important roles in marine ecosystems, such as the degradation of aromatic compounds and the biogeochemical cycles of carbon and sulfur [5]. S. roseus has been reported to exhibit high salinity and high lactate resistance [9]. Salinicocci have much importance in biotechnology applications such as serine-metabolism strategies to adapt to lactate stress [10]. In order to understand the genetic variability and industrial applications of those genes, genome sequencing and annotation of strain Salinicoccus sp. BAB 3246 was executed. The prime interest was to identify presence of distinctive enzymes for potential industrial applications.

Experimental design, materials and methods

The halophilic organism was isolated from marine water collected from surface streams of coastal region near Bhavnagar, Gujarat, India (latitude, longitude: 21.67 N, 72.27E). The isolation was performed by providing 15% Sodium Chloride containing Medium. The identification of Salinococcus sp. BAB 3246 was validated by 16 s rRNA sequencing and submitted to Genebank (accession no: KF889285.1). Furthermore, the DNA was extracted using Hi-Media Kit for Genomic DNA isolation Kit. The genome sequencing was performed using Ion Torrent PGM generating 15,26,815 sequencing reads. Initially all reads were subjected to preprocessing and conversion of BAM to fasta file format using Galaxy NGS: BamTools, online server using default parameters provided by the developer [11]. The genome data were assembled using CLC Genomic Workbench 5. The final whole genome assembly size was reported is 7,13,204 bp. The genome annotation was performed using RAST (Rapid Annotation using Subsystem Technology) [12], BASys (a web server for automated bacterial genome annotation) [13] and MaGe (Microscope Genome Annotation) [14]. The RAST analysis revealed total 1691 coding sequences (Table 1). A total of 1009 subsystems were identified, including Stress Response (42), Sulfur Metabolism (4), Potassium metabolism (4) and Iron metabolism (1). However, the highest numbers of subsystems were observed for Amino Acids and Derivatives (159), Protein Metabolism (153) and Carbohydrate synthesis (150) (Fig. 1). KEGG pathway analysis was performing using seed viewer system of RAST. The KEGG map analysis revealed 652 pathways associated with only degradation of metabolites (Table 2).

Table 1

Summary of RAST annotation.

Genome	Salinicoccus sp. BAB 3246
Size (bp)	7,13,204
G + C content	49.1
Number of coding sequences	1691
Number of features	1762
Number of subsystems	1009
Number of RNAs	71
Number of contigs	1

Fig. 1

Subsystem category distribution.

Table 2

KEGG map analysis for degradation pathway.

No	Name of derivative	KEGG map	Salinicoccus sp. BAB-3246
1	1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) degradation	Tyrosine metabolism	4
2	1,2-Dichloroethane degradation	1,2-Dichloroethane degradation	1
		Glyoxylate and dicarboxylate metabolism	9
3	1,4-Dichlorobenzene degradation	Benzoate degradation via hydroxylation	2
		Glycolysis/gluconeogenesis	17
		Glyoxylate and dicarboxylate metabolism	9
		Pyruvate metabolism	14
4	1- and 2-Methylnaphthalene degradation	1- and 2-Methylnaphthalene degradation	2
		Naphthalene and anthracene degradation	1
5	2,4-Dichlorobenzoate degradation	Benzoate degradation via hydroxylation	2
		Naphthalene and anthracene degradation	1
6	3-Chloroacrylic acid degradation	3-Chloroacrylic acid degradation	1
		Pyruvate metabolism	14
7	Atrazine degradation	Atrazine degradation	1
		Folate biosynthesis	5
8	Benzoate degradation via CoA ligation	Benzoate degradation via CoA ligation	4
		Benzoate degradation via hydroxylation	2
		Butanoate metabolism	9
		Ethylbenzene degradation	1
		Phenylalanine metabolism	1
		Pyruvate metabolism	14
9	Benzoate degradation via hydroxylation	Benzoate degradation via CoA ligation	4
		Benzoate degradation via hydroxylation	2
		Caprolactam degradation	2
		Glycolysis/gluconeogenesis	17
		Naphthalene and anthracene degradation	1
		Phenylalanine metabolism	1
		Pyruvate metabolism	14
		Tryptophan metabolism	9
		Tyrosine metabolism	4
10	Biphenyl degradation	Benzoate degradation via CoA ligation	4
		Benzoate degradation via hydroxylation	2
		Glycolysis/gluconeogenesis	17
		Pyruvate metabolism	14
11	Bisphenol A degradation	Benzoate degradation via hydroxylation	2
12	Caprolactam degradation	Benzoate degradation via hydroxylation	2
		Caprolactam degradation	2
13	Carbazole degradation	Benzoate degradation via CoA ligation	4
		Benzoate degradation via hydroxylation	2
		Glycolysis/gluconeogenesis	17
		Pyruvate metabolism	14
		Tryptophan metabolism	9
14	Ethylbenzene degradation	Benzoate degradation via CoA ligation	4
		Ethylbenzene degradation	1
		Glycolysis/gluconeogenesis	17
		Propanoate metabolism	6
		Pyruvate metabolism	14
15	Fluorene degradation	Benzoate degradation via hydroxylation	2
		Glycolysis/gluconeogenesis	17
		Pyruvate metabolism	14
16	Fluorobenzoate degradation	Benzoate degradation via hydroxylation	2
17	Geraniol degradation	Geraniol degradation	3
		Valine, leucine and isoleucine degradation	9
18	Limonene and pinene degradation	Limonene and pinene degradation	3
19	Lysine degradation	Biotin metabolism	1
		Citrate cycle (TCA cycle)	14
		Lysine biosynthesis	5
		Lysine degradation	6
20	Naphthalene and anthracene degradation	Benzoate degradation via hydroxylation	2
		Naphthalene and anthracene degradation	1
		Pyruvate metabolism	14
		Tryptophan metabolism	9
		Tyrosine metabolism	4
21	Other glycan degradation	Glycosphingolipid biosynthesis - ganglio series	1
22	Styrene degradation	Citrate cycle (TCA cycle)	14
		Ethylbenzene degradation	1
		Glycolysis/gluconeogenesis	17
		Propanoate metabolism	6
		Pyruvate metabolism	14
23	Synthesis and degradation of ketone bodies	Butanoate metabolism	9
		Fatty acid metabolism	5
		Glycolysis/gluconeogenesis	17
		Pyruvate metabolism	14
24	Tetrachloroethene degradation	Glyoxylate and dicarboxylate metabolism	9
		Pyruvate metabolism	14
25	Toluene and xylene degradation	Benzoate degradation via CoA ligation	4
		Benzoate degradation via hydroxylation	2
		Glycerolipid metabolism	3
		Glycolysis/gluconeogenesis	17
		Pyruvate metabolism	14
26	Trinitrotoluene degradation	Trinitrotoluene degradation	1
27	Valine, leucine and isoleucine degradation	Biosynthesis of type II polyketide backbone	1
		Citrate cycle (TCA cycle)	14
		Propanoate metabolism	6
		Pyrimidine metabolism	17
		Valine, leucine and isoleucine biosynthesis	12
		Valine, leucine and isoleucine degradation	9
28	Gamma-Hexachlorocyclohexane degradation	Benzoate degradation via hydroxylation	2
		Citrate cycle (TCA cycle)	14
		Glyoxylate and dicarboxylate metabolism	9
		Naphthalene and anthracene degradation	1

The genome annotation using BASys annotate 955 genes amongst total 2330 genes reported in and automated mode. The amino acid composition was also examined using BASys (Fig. 2). The highest amino acid residue content was predicted for Leucine followed by Glycine, Glutamic acid and Alanine. Annotated data were displayed in the form of circular DNA as a genome browser map for easy representation of genome data (Fig. 3). The genome annotation using Microscope Genome Annotation identified 1772 Genomic Objects (without artifacts): CDS, 1326; fCDS, 358; misc_RNA, 16; rRNA, 12; tRNA, 60.

Fig. 2

Amino acid composition of Salinicoccus sp. BAB 3246.

Fig. 3

Genome browser map for Salinicoccussp. BAB 3246.

Quantitative comparison of coding sequences, rna and subsystem

The comparison of genome size for six different strains available in NCBI genome database revealed that, S. halodurans strain had the largest genome size of 2,778,379 bp followed by 873,136 bp, 713,204 bp, 679,606 bp, 461,933 bp and 342,819 bp respectively for S. carnicancri Crm, Salinicoccus sp. BAB 3246, S. luteus DSM 17002, S. roseus and S. albus DSM 19776 strain. A maximum of 2839 coding sequences was reported for S. halodurans followed by 1691, 863, 668, 449 and 334 respectively for Salinicoccus sp. BAB 3246, S. carnicancri Crm, S. luteus DSM 17002, S. roseus and S. albus DSM 19776 strain (Table 3).

Table 3

Quantitative comparison of coding sequence, RNA and subsystem.

Genome	Size (bp)	G + C content	Coding sequences	Features	RNAs	Subsystems	BioProject
Salinicoccus sp. BAB_3246	713,204	49.1	1691	1762	71	202	PRJNA342322
Salinicoccus roseus	461,933	49.9	449	459	10	80	PRJNA272357
Salinicoccus carnicancri Crm	873,136	47.6	863	909	46	138	PRJNA175941
Salinicoccus albus DSM 19776	342,819	45.2	334	334	0	77	PRJNA185242
Salinicoccus luteus DSM 17002	679,606	49.7	668	669	1	114	PRJNA235106
Salinicoccus halodurans	2,778,379	44.5	2839	2912	73	388	PRJNA282445

Nucleotide sequence accession number

The complete sequence of Salinicoccus sp. BAB 3246 genome can be accessed under the NCBI BioProject: PRJNA342322.

Specifications
Organism/cell line/tissue	Salinicoccus sp. BAB 3246
Sex	Not applicable
Sequencer or array type	Ion Torrent PGM platform
Data format	Fasta complete genome
Experimental factors	Marine water sample
Experimental features	Shotgun whole genome sequencing followed by genome annotation using RAST, BASys and MaGe.
Sample source location	Gujarat, India (21.672439 N 72.275925 E)
Data submission	BioProject: PRJNA342322RAST: genome ID 1437774.4 - Salinicoccus sp. BAB-3246