Open Access

Genome sequence of the moderately halophilic bacterium Salinicoccus carnicancri type strain CrmT (= DSM 23852T)

  • Dong-Wook Hyun
  • , Tae Woong Whon
  • , Yong-Joon Cho
  • , Jongsik Chun
  • , Min-Soo Kim
  • , Mi-Ja Jung
  • , Na-Ri Shin
  • , Joon-Yong Kim
  • , Pil Soo Kim
  • , Ji-Hyun Yun
  • , Jina Lee
  • , Sei Joon Oh
  • and Jin-Woo Bae
Corresponding author

DOI: 10.4056/sigs.3967649

Received: 03 June 2013

Accepted: 03 June 2013

Published: 15 June 2013


Salinicoccus carnicancri Jung et al. 2010 belongs to the genus Salinicoccus in the family Staphylococcaceae. Members of the Salinicoccus are moderately halophilic and originate from various salty environments. The halophilic features of the Salinicoccus suggest their possible uses in biotechnological applications, such as biodegradation and fermented food production. However, the genus Salinicoccus is poorly characterized at the genome level, despite its potential importance. This study presents the draft genome sequence of S. carnicancri strain CrmT and its annotation. The 2,673,309 base pair genome contained 2,700 protein-coding genes and 78 RNA genes with an average G+C content of 47.93 mol%. It was notable that the strain carried 72 predicted genes associated with osmoregulation, which suggests the presence of beneficial functions that facilitate growth in high-salt environments.


moderately halophilicSalinicoccus carnicancriStaphylococcaceae


The genus Salinicoccus in the 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 genus name is derived from the Latin adjective salinus, saline, and the Greek masculine noun kokkos, meaning a grain or berry, i.e., saline coccus [2]. Most species in the genus Salinicoccus have been found in salty environments, such as fermented foods [3-5], solar salterns [1,6], salt mines [7,8], a salt lake [9], and saline soils [10,11]. All type strains of Salinicoccus species were characterized as halotolerant organisms, where NaCl concentrations of 2–20% (wt/vol) were suitable for growth [12-14].

These moderately halophilic bacteria can survive in salt-rich environments and grow optimally at 5–20% (wt/vol) NaCl [15]. These bacteria can utilize compatible solutes or osmolytes, such as carbohydrates, amino acid, polyols, betaines, and ectoines, by regulating their osmotic concentrations in high-salt content environmental conditions [16,17]. Therefore, these organisms may have biotechnological importance with possible applications in food biotechnology for the production of fermented food [18], in environmental biotechnology for the biodegradation of organic pollutants and the production of alternative energy [19].

Strain CrmT (= DSM 23852 = JCM 15796 = KCTC 13301) is the type strain of the species Salinicoccus carnicancri. This strain was isolated from a traditional Korean fermented seafood, known as ‘ganjang-gejang,’ which is made from raw crabs preserved in soy sauce [20]. The species name was derived from the Latin nouns caro carnis, flesh, and cancer -cri, a crab, i.e., the flesh of a crab [2]. The strain can grow in 0–20% (wt/vol) NaCl with optimal growth at 12% (wt/vol) NaCl [20]. The present study summarizes the features of S. carnicancri strain CrmT and provides an analysis of its draft genome sequence, which is the first reported genome sequence of a species in the genus Salinicoccus.

Classification and features

A taxonomic analysis was conducted based on the 16S rRNA gene sequence. The representative 16S rRNA gene sequence of strain S. carnicancri CrmT was compared with the most recent release of the EzTaxon-e database [21]. The multiple sequence alignment program CLUSTAL W [22] was used to generate alignments with other gene sequences collected from databases. The alignments were trimmed and converted to the MEGA format before phylogenetic analysis. Phylogenetic consensus trees were constructed based on the aligned gene sequences using the neighbor-joining [23], maximum-parsimony [24], and maximum-likelihood [25] methods with 1,000 randomly selected bootstrap replicates using MEGA version 5 [26]. The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain CrmT was most closely related to Salinicoccus halodurans W24T with 96.99% similarity. The phylogenetic consensus tree based on the 16S rRNA gene sequences indicated that strain CrmT was clustered within a branch containing other species in the genus Salinicoccus (Figure 1).

Figure 1

Phylogenetic consensus tree based on 16S rRNA gene sequences showing the relationship between Salinicoccus carnicancri strain CrmT and the type strains of other species in the genus Salinicoccus. The type strain of Staphylococcus aureus was used as an outgroup. The GenBank accession numbers for the 16S rRNA genes of each strain are shown in parentheses. Filled diamonds indicate identical branches present in the phylogenetic consensus trees constructed using the neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) algorithms. The numbers at the nodes represent the bootstrap values as percentages of 1,000 replicates and values <70% are not shown at the branch points. The scale bar represents 0.01 nucleotide change per nucleotide position.

Strain CrmT (Table 1) was isolated from the fermented seafood ganjang-gejang during a project that investigated microbial communities in fermented foods, i.e., the Next-Generation BioGreen 21 Program (No. PJ008208) in Korea. Ganjang-gejang, with a NaCl (w/v) concentration of 24.5%, was produced by preserving scabbard crabs in soy sauce, garlic, and onions at –5°C for 4–5 days.

Table 1

Classification and general features of Salinicoccus carnicancri strain CrmT according to the MIGS recommendations [27].




   Evidence code

    Domain Bacteria

   TAS [28]

    Phylum Firmicutes

   TAS [29-31]

    Class Bacilli

   TAS [32,33]

   Current classification

    Order Bacillales

   TAS [34,35]

    Family Staphylococcaceae

   TAS [36,37]

    Genus Salinicoccus

   TAS [1,38]

    Species Salinicoccus carnicancri

   TAS [20]

    Type strain CrmT

   TAS [20]

   Gram stain


   TAS [20]

   Cell shape


   TAS [20]



   TAS [20]



   TAS [20]

   Temperature range


   TAS [20]

   Optimum temperature


   TAS [20]

   Salinity range

    0–20% (w/v)

   TAS [20]

   Optimum salinity

    12% (w/v)

   TAS [20]

   pH range


   TAS [20]

   Optimum pH


   TAS [20]

   Carbon source


   TAS [20]

   Energy source

    Not reported



    Fermented seafood (marinated crab)

   TAS [20]



    –5 to 5°C









   TAS [20]


   Biotic relationship


   TAS [20]




   Biosafety level




    The traditional Korean fermented seafood ‘ganjang-gejang’ (Crabs preserved in soy sauce)

   TAS [20]


   Geographic location

    Republic of Korea

   TAS [20]


   Sample collection time

    August, 2010




    Not reported



    Not reported



    Not reported



    Not reported

Evidence codes, as follows: IDA: inferred from direct assay; TAS: traceable author statement (i.e., a direct report exists in the literature); NAS: non-traceable author statement (i.e., not observed directly in a living, isolated sample, but based on a generally accepted property of the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [39].

S. carnicancri strain CrmT is a Gram-positive, moderately halophilic, non-motile, non-sporulating, and aerobic heterotrophic coccus with a diameter of 1.0–2.5 μm [20]. Figure 2 shows the morphological features of strain CrmT, which were obtained by scanning electron microscopy (SEM). Colonies were ivory-colored [20]. Growth occurred at 4–45°C, with an optimum of 30–37°C, and at pH values of 6.0–11.0, with an optimum of 7.0–8.0. The salinity range suitable for growth was 0–20% (w/v) NaCl, with an optimum of 12% (w/v) NaCl [20]. Strain CrmT contains menaquinone MK-6 as the predominant respiratory quinone [20]. The major fatty acids (>10% of total fatty acid) are anteiso-C15:0 (40.61%), iso-C15:0 (22.0%), and anteiso-C17:0 (12.12%) [20]. The major cellular polar lipids are phosphatidylglycerol and diphosphatidylglycerol [20]. Glycine and lysine are the major amino acid constituents of the cell-wall hydrolysate [20].

Figure 2

Scanning electron microscopy images of S. carnicancri CrmT obtained using a SUPRA VP55 (Carl Zeiss) at an operating voltage of 15kV. The scale bars represents 200 nm (left) and 1 μm (right), respectively.

Genome sequencing and annotation

Genome project history

S. carnicancri strain CrmT was selected for sequencing because of its environmental potential as part of the Next-Generation BioGreen 21 Program (No.PJ008208). The genome project is deposited in the Genomes OnLine Database [40] and the genome sequence is deposited in GenBank. Sequencing and annotation were performed by ChunLab Inc., South Korea. A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information.





    Finishing quality

    Improved high-quality draft


    Libraries used

    454 PE library (8 kb insert size) and Illumina library


    Number of reads

    7,434,400 sequencing reads


    Sequencing platforms

    454 GS FLX Titanium, Illumina Hiseq, and PacBio RS system


    Sequencing coverage

    443.60-fold coverage (12.1 × 454 pyrosequencing, 408.4 × Illumina, and 23.1 × PacBio)



    gsAssembler 2.6, CLC Genomics Workbench 5.0


    Gene calling method

    GLIMMER 3.02

    Genbank ID


    Genbank Date of Release

    January 2, 2013



    NCBI project ID


    Database: IMG-ER


    Source material identifier

    DSM 23852, JCM 15796, KCTC 13301

    Project relevance

    Environmental and biotechnological

Growth conditions and DNA isolation

S. carnicancri strain CrmT was grown aerobically in marine 2216 (Marine medium, BBL), supplemented with 10% (w/v) NaCl at 30°C. Genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega A1120), according to the manufacturer’s instructions.

Genome sequencing and assembly

The genome of S. carnicancri CrmT was sequenced using a combination of a 454 Genome Sequencer FLX Titanium system (Roche Diagnostics) with an 8 kb paired end library, an Illumina Hiseq system with a 150 base pair (bp) paired end library, and a PacBio RS system (Pacific Biosciences). A total of 7,434,400 sequencing reads (443.6-fold genome coverage) were obtained using the Roche 454 system (187,030 reads; 12.1-fold coverage), Ilumina Hiseq system (7,219,019 reads; 408.4-fold coverage), and PacBio RS system (28,351 reads; 23.1-fold coverage) combined. The Roche 454 pyrosequencing and Illumina sequencing reads were assembled using Roche gsAssembler 2.6 (Roche Diagnostics) and CLCbio CLC Genomics Workbench 5.0 (CLCbio), respectively. Table 2 shows the project information and its associated MIGS version 2.0 compliance levels [27].

Genome annotation

The open reading frames (ORFs) of the assembled genome were predicted using a combination of the Rapid Annotation using Subsystem Technology (RAST) pipeline [41] and the GLIMMER 3.02 modeling software package [42]. Comparisons of the predicted ORFs using the SEED [43], NCBI COG [44], NCBI Refseq [45], CatFam [46], Ez-Taxon-e [21], and Pfam [47] databases were conducted during gene annotation. RNAmmer 1.2 [48] and tRNAscan-SE 1.23 [49] were used to find rRNA genes and tRNA genes, respectively. Additional gene prediction analyses and functional annotation were performed using the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [50].

Genome properties

The draft genome sequence of S. carnicancri CrmT was 2,673,309 bp, which comprised three scaffolds that included 12 contigs. The G+C content was 47.93 mol% (Figure 3 and Table 3). RAST and GLIMMER predicted 2,778 coding sequences (CDSs) in the genome. Of the predicted ORFs, 2,700 ORFs were assigned to protein-coding genes. A total of 2,298 genes (82.72%) were assigned putative functions, whereas the remaining genes were annotated as hypothetical proteins. The genome contained 78 ORFs assigned to RNA genes, including 61 predicted tRNA genes, nine rRNA genes (three 5S rRNA, three 16S rRNA, and three 23S rRNA genes), and eight other RNA genes. The distributions of genes in the COG functional categories are presented in Table 4.

Figure 3

Graphical map of the largest scaffold, C792_Scaffold00001.1, which represented >99.6% of the chromosome. The smaller scaffolds of the chromosome are not shown. From bottom to top: genes on the forward strand (colored according to COG categories), genes on the reverse strand (colored according to COG categories), RNA genes (tRNAs = green, rRNAs = red, and other RNAs = black), GC content, and GC skew.

Table 3

Genome statistics.



     % of totala

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Total genes



RNA genes



rRNA operons



Protein-coding genes



Genes with predicted functions



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


aThe total is based on either the size of the genome (bp) or the total number of protein-coding genes in the annotated genome.

Table 4

Numbers of genes associated with the 25 general COG functional categories.












    RNA processing and modification








    Replication, recombination, and repair




    Chromatin structure and dynamics




    Cell cycle control, mitosis, and meiosis




    Nuclear structure




    Defense mechanisms




    Signal transduction mechanisms




    Cell-wall/membrane biogenesis




    Cell motility








    Extracellular structures




    Intracellular trafficking and secretion




    Posttranslational modification, protein turnover, and chaperones




    Energy production and conversion




    Carbohydrate transport and metabolism




    Amino acid transport and metabolism




    Nucleotide transport and metabolism




    Coenzyme transport and metabolism




    Lipid transport and metabolism




    Inorganic ion transport and metabolism




    Secondary metabolites biosynthesis, transport, and catabolism




    General function prediction only




    Function unknown




    Not in COGs

aThe total is based on the total number of protein-coding genes in the annotated genome.

Insights from the genome sequence

S. carnicancri CrmT encoded 72 predicted genes associated with the biosynthesis of compatible solutes and the transport of osmolytes, such as choline-glycine betaine transporter (BetT) and periplasmic glycine betaine/choline-binding lipoprotein of an ABC-type transport system (OpuBC). Potentially, these genes are key factors that allow S. carnicancri to adapt to high-salt environments (e.g., salt-fermented food) by regulating the osmotic concentration. Further studies are required to elucidate the osmoregulation mechanism, which could facilitate biotechnological applications of this halophilic bacterium.



We gratefully acknowledge the help of Dr. Seong Woon Roh and Mr. Hae-Won Lee during SEM analysis (Jeju Center, Korea Basic Science Institute, Korea). This work was supported by a grant from the Next-Generation BioGreen 21 Program (No.PJ008208), Rural Development Administration, Republic of Korea.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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