The Antarctic Ocean is the world’s southernmost ocean. The Ross Sea is a deep bay in Antarctica, located between Victoria Land and Marie Byrd Land at approximately 75°S, 175°W. It has unique geological features. Its continental shelf is extensive and its accelerated current causes substantial vertical and horizontal exchanges of nutrient-rich water making it a diverse habitat (Smith et al., 2007). The Ross Sea is one of the most productive regions of the Antarctic Ocean and is home to a wide array of organisms (Fabiano and Pusceddu, 1998). The benthos such as starfish, oysters, and sea cucumbers live at the bottom of seas and lakes . Some microorganisms are known to live on surfaces or inside of benthos and produce proteolytic enzymes, which help their hosts digest food (Burnett et al., 1997; Russell, 1998; Ravenschlag et al., 2001; Li et al., 2009; Cristóbal et al., 2011; Lee et al., 2014). This study was initiated to gain insights into such symbiotic bacteria.

The intestines of starfish (a member of genus Pterocirrus) caught from the Terra Nova Bay of the Ross Sea were homogenized using a tissue homogenizer and spreaded on Marine Broth (MB) agar medium containing 1% skim milk to differentiate bacteria with extracellular proteolytic activities. Initially isolated as protease positive, strain SiA1 produced a clear zone of 37 mm in diameter surrounding a 1.0-mm diameter colony in 24 h at 25°C.

In order to taxonomically identify strain SiA1, its 16S rRNA gene was amplified via colony PCR 27F (5'-AGAGTTTGAT CCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACG ACTT-3') primers and the PCR product was directly sequenced with same primer pairs. Analysis of the nucleotide sequence on BLAST showed that SiA1 16S rRNA sequence has high levels of identity (ca. 99%) with those from several different Pseudoalteromonas species. SiA1 was thus positively identified as a Pseudoalteromonas sp.

In an effort to better understand the proteolytic potential of SiA1, its genome was sequenced using PacBio RSII and Illumina HiSeq (Macrogen, Inc.). The resulting sequences were de novo assembled using HGAP v.3.0 and Pilon v.1.21. There were a total of 839,170,695 subread bases and the genome coverage was 206x. Then, the genome was annotated using the NCBI prokaryotic genome annotation pipeline. Annotation method and software revision were GenMarks-2+ and v.5.2, respectively.

The features of SiA1’s complete genome are summarized in Table 1. SiA1’s whole genome sequence was composed of 4,070,638 bp across two chromosomes. Chromosomes 1 and 2 were determined to contain 3,355,071 bp and 715,567 bp, respectively. A total of 3,597 coding sequences (CDSs) were identified (3,001 in chromosome 1 and 596 in chromosome 2). G + C content of each chromosome was calculated to be 40.44% and 39.79%, respectively. Also, average nucleotide identity (ANI) analysis revealed that SiA1 was most closely related to Pseudoalteromonas tetraodonis (Fig. 1).

Table 1

	Chromosome 1	Chromosome 2
Genome size (bp)	3,355,071	715,567
Number of contigs	1	1
G + C contents (%)	40.44	39.79
CDS	3,001	596
tRNA	100	0
rRNA	25	0

Fig. 1. Dendrogram and heatmap of ANI values of genomes from four closely related Pseudoalteromonas spp. ANI value was calculated using OrthoANI (Lee et al., 2016).

The annotation results revealed the presence of a total of 98 CDSs related to proteolytic activities. Among the 98 CDSs, eighty three are present on chromosome 1 while chromosome 2 harbors the remaining fifteen. Subsequently, all the 98 CDSs were carefully examined for the presence of signal peptide sequences to select extracellular enzyme candidates. Twenty five CDSs from chromosome 1 and eleven ORFs from chromosome 2 were found to have signal peptide sequences, respectively. The whole genome sequences were deposited in GenBank under accession numbers CP076084 (chromosome 1) and CP076085 (chromosome 2).

남극해에 서식하는 불가사리의 내장에서 분리한 Pseudoalteromonas sp. strain SiA1은 총 2개의 염색체(염색체 1, 염색체 2)를 가지고 있는 것으로 밝혀졌다. 그 크기는 각각 3,355,071 bp와 715,567 bp이며, 유전체의 G + C 비율은 염색체 1에서 40.44%, 염색체 2에서 39.79%로 나타났다. 컴퓨터 분석 결과 3,597개의 코딩서열이 확인되었는데, 이 가운데 최소 98개(약 2.7%)가 세포 외 단백질 분해 효소 단백질을 암호화 하는 것으로 추정된다. 이는 분리 과정에서 확인된 SiA1의 탁월한 단백질 분해 능력을 유전자 차원에서 입증하는 결과라고 추측된다.

This work was supported by the National Research Foundation of Korea (NRF-2019S1A5C2A04083293).

The authors have no conflict of interest to report.