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Whole genome of strain RR4-35, a novel bacterium in the family Rhodobacteraceae
Korean J. Microbiol. 2021;57(1):58-61
Published online March 31, 2021
© 2021 The Microbiological Society of Korea.

Hyun-Kyoung Jung1,2†, Young-Sam Kim1,2†, and Kyoung-Ho Kim1,2*

1Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea
2School of Marine and Fisheries Life Science, Pukyong National University, Busan 48513, Republic of Korea
Correspondence to: E-mail:;
Tel.:+82-51-629-5611;Fax: +82-51-629-5619
These authors contributed equally to this work.
Received December 31, 2020; Revised January 25, 2021; Accepted January 28, 2021.
A bacterium strain RR4-35, belonging to the family Rhodobacteraceae was isolated from a biofilter of seawater recirculating aquaculture system (RAS) located in Busan, South Korea. The strain showed low 16S rRNA similarity (< 96.47%) against species with valid names in the family. PacBio RS II sequencing yielded one complete chromosome (3,833,345 bp with 59.3% G + C content) and six plasmids. A total of 4,487 genes, 4,436 CDSs, 47 tRNAs and 3 rRNAs were annotated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis detected gene clusters related to pathways such as denitrification and benzoate degradation. Genomic analysis might show the potential role of strain RR4-35 in the nitrogen cycle and biodegradation of organic compounds in the RAS.
Keywords : Rhodobacteraceae, RR4-35, complete genome sequence, recirculating aquaculture system

The family Rhodobacteraceae is one of the major bacterial groups in the class Alphaproteobacteria (Garrity et al., 2005). Rhodobacteraceae was first established by Garrity et al. (2005). At the time of writing, the family contained 182 genera which have a validly published and correct name including the type genus, Rhodobacter ( The family comprises photoheterotrophic and chemoorganotrophic bacteria in an aerobic environment and anaerobic non-sulfur bacteria involving degradation and mediation of various compounds in marine environments (Pujalte et al., 2014). Members of this family were found in diverse environments, especially in marine environments such as seawater (Wu et al., 2015), aquaculture farm (Rhee et al., 2018), brine-sea water interface (Zhang et al., 2017), and deep-sea vent (Takai et al., 2009).

A recirculating aquaculture system (RAS) was a closed aquaculture system that reused the rearing water by purifying water in the system. In the RAS, biodegradation and detoxification of waste organic matters, nitrogen compounds and feed debris are mainly carried out by bacterial activity (Zhang et al., 2011). A culture-independent study showed that Rhodobacteraceae was one of the major groups in the RAS (Lee et al., 2016). Rhodobacteraceae was also found to be one of the most predominant bacterial groups (30.2~49.6%) at family level in the bioreactor of RAS for shrimp aquaculture (Chen et al., 2019).

In this study, we obtained and analyzed the whole genome sequences of the strain RR4-35 that was isolated from a biofilter of a seawater RAS and was distantly related to known species. The analysis of genome will provide a basis for understanding the role of the Rhdobacteraceae bacterium in the RAS.

Strain RR4-35 was isolated from a biofilm sample obtained from the surface of a RAS-biofilter in Busan, South Korea. The sample was serially diluted using phosphate buffered saline solution (1X PBS; pH 7.4) and incubated on Marine Agar 2216 (MA, Difco) at 28°C for a week. Pure culture was obtained and phylogenetic analysis was conducted as described previously (Kim et al., 2019). The 16S rRNA gene sequence showed low similarities against the species with valid names in Ezbiocloud server (Yoon et al., 2017) such as Roseovarius litorisediminis CECT 8287T (96.47%), Roseovarius aestuarii CECT 7745T (96.18%), Roseovarius lutimaris DSM 28463T (95.62%), and Phaeobacter inhibens DSM 16374T (95.59%) that all belong to the family Rhodobacteraceae.

Pure isolate cultured on MA at 28°C for 3 days was used to extract genomic DNA using Genomic DNA Prep Kit (BIOFACT Corp.). The genomic DNA was then subjected to library construction and single-molecule real-time (SMRT) sequencing (Macrogen) (Ardui et al., 2018) using PacBio RS II system (Pacific Biosciences). A total of 152,071 subreads (1,358,239,045 total bases; N50, 12,284 bp; mean length, 8,931 bp) were generated using SMRT sequencing and subreads were de novo assembled by Hierarchical Genome Assembly Process (HGAP, Version 3.0, Pacific Biosciences). A total of seven contigs (total base, 4,557,782 bp) consisting of one chromosome (3,833,345 bp with 59.3 mol% G + C content) and six plasmids (168,707 bp, 59.31 mol%; 140,737 bp, 59.5 mol%; 136,456 bp, 59.6 mol%; 126,025 bp, 60.8 mol%; 90,429 bp, 57.0 mol%; 62,083 bp, 62.9 mol%) were obtained after assembly. All contigs are circular except for contig 6 (Table 1). The genomes were annotated by NCBI Prokaryotic Genome Annotation Pipeline (PGAP; Jan. 2020) (Tatusova et al., 2016) and a total of 4,487 genes, 4,436 CDSs, 47 tRNAs and 3 rRNAs were annotated from chromosome and plasmids (Table 1). Constructing graphical circular genome maps was performed using CGView Comparison Tool (Grant and Stothard, 2008) (Fig. 1). One CRISPR locus and two CRISPRs candidates were detected by CRISPRFinder ( (Grissa et al., 2007). It was detected that one incomplete prophage sequence coding viral components such as tail, head and phage-like proteins using Phage Search Tool Enhanced Release (PHASTER; (Arndt et al., 2016). All softwares were used as default parameters unless otherwise indicated.

Genomic features of strain RR4-35

Feature Chromosome Plasmid Total
Alias Contig 1 Contig 2 Contig 3 Contig 4 Contig 5 Contig 6 Contig 7
Genome size (bp) 3,833,345 168,707 140,737 136,456 126,025 90,429 62,083 4,5557,782
G + C contents (%) 59.28 59.31 59.47 59.59 60.8 57.03 62.89 59.34
Depth (X) 190 133 120 145 102 100 46 178
Circular Yes Yes Yes Yes Yes No Yes
Total gene 3,775 188 143 127 118 90 46 4,487
Total CDS 3,724 188 143 127 118 90 46 4,436
Genes assigned to COG 2,691 114 71 72 71 43 13
rRNA (5S, 16S, 23S) 1, 1, 1 0 0 0 0 0 0
tRNA 47 0 0 0 0 0 0
tmRNA 1 0 0 0 0 0 0

Fig. 1. Circular map of strain RR4-35 contigs. From the center to the outside: genome label, GC skew (green and purple), G + C content (black), CDSs colored by COG categories on the reverse strand, CDSs including RNAs on the reverse strand, CDSs including RNAs on the forward strand, CDSs colored by COG categories on the forward strand.

It was detected that genes for denitrification such as three subunits of respiratory nitrate reductase (narI; WP_165197213.1, narH; WP_165197215.1, and narG; WP_165197216.1), nitrate/nitrite transporter (narK; WP_165197217.1), nitrite reductase (nirK; WP_165193008.1), two subunits of nitric oxide reductase (norB; WP_165192992.1 and norC; WP_165192994.1) and nitric oxide reductase activation protein (norD; WP_019298187.1) from Kyoto Encyclopedia of Genes and Genomes (KEGG; (Kanehisa and Goto, 2000) analysis, while nitrous-oxide reductase (nosZ), the enzyme for final step of denitrification that reduces nitrous oxide and produces nitrogen gas, was not observed.

Annotation showed genes related to the benzoate degradation pathway such as genes for 4-hydroxybenzoate 3-monooxygenase (pobA; WP_165192748.1), two subunits protocatechuate 3, 4-dioxygenase (pcaH; WP_165194558.1 and pcaG; WP_ 165194560.1), 3-carboxy-cis,cis-muconate cycloisomerase (pcaB; WP_165194563.1), 4-carboxymuconolactone decarboxylase (pcaC; WP_165194557.1) and 3-oxoadipate enol-lactonase (pcaD; WP_165192750.1), in which we identified the presence of genes responsible for a degradation pathway from 3,4-dihydroxy-benzoate to 3-oxoadipate.

Benzoate biodegradation and nitrate reduction pathways were also detected in Phaeobacter gallaeciensis DSM 17395T (pcaG; WP_014878970.1, pcaH; WP_014878971.1, HpaR; WP_ 014881250.1, nirK; WP_014881756.1, norB; WP_014881752.1 and norC; WP_014881753.1) and some Roseovarius species, included in Roseovarius mucosus SMR3T (npcC; WP_008281990.1, nasA; WP_081507525.1, nasD; WP_081507524.1 and nosZ; WP_081508048.1) and Roseovarius indicus DSM 26383T (pcaH; WP_057822108.1, pcaG; WP_151175233.1, WP_151175215.1, nirK; WP_057816130.1, norB; WP_057816123.1 and norC; WP_057816125.1).

Degradation of organic compounds might be coupled with denitrification. A study reported that benzene degradation was observed in enrichment cultures under nitrate reduction condition (Burland and Edwards, 1999). The whole genome analysis of strain RR4-35 showed its potential role associated to nitrogen cycle and organic compound biodegradation.

Nucleotide sequence and strain accession numbers

The strain is available at the Korean Collection for Type Cultures (accession number KCTC 72134). The complete genome sequences including chromosome and plasmids of strain RR4-35 were deposited in DDBJ/EMBL/NCBI GenBank under accession numbers CP049037 (chromosome) and CP049038-CP0490343 (plasmids).

적 요

Rhodobacteraceae 과에 속하는 신종 균주 RR4-35는 해수 순환 여과 양식 시스템(RAS)의 바이오 필터에서 분리되었다. 이 균주는 그 과에 속한 유효명을 가진 종들과 낮은 16S rRNA 유전자 유사도를 보여주었다(< 96.47%). PacBio RS II 분석을 통해 하나의 염색체(3,833,345 bp 크기의 G + C 함량 59.3 mol%)와 여섯 개의 플라스미드 서열이 확인되었다. 이 균주의 유전체들은 총 4,487개의 유전자, 4,436개의 CDS, 47개의 tRNA와 3개의 rRNA 유전자를 포함한다. Kyoto Encyclopedia of Genes and Genomes 분석을 통해 탈질 과정과 벤조에이트 분해와 관련된 유전자 클러스터들이 확인되었다. 본 유전체 분석 결과는 RAS에서 질소 순환과 유기 물질의 분해에 대한 RR4-35 균주의 잠재적인 역할을 보여준다.


This work was supported by a Research Grant of Pukyong National University (2019).

  1. Ardui S, Ameur A, Vermeesch JR, and Hestand MS. 2018. Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res. 46, 2159-2168.
    Pubmed KoreaMed CrossRef
  2. Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y, and Wishart DS. 2016. PHASTER: A better, faster version of the PHAST phage search tool. Nucleic Acids Res. 44, W16-W21.
    Pubmed KoreaMed CrossRef
  3. Burland SM and Edwards EA. 1999. Anaerobic benzene biodegradation linked to nitrate reduction. Appl. Environ. Microbiol. 65, 529-533.
    Pubmed KoreaMed CrossRef
  4. Chen Z, Chang Z, Zhang L, Jiang Y, Ge H, Song X, Chen S, Zhao F, and Li J. 2019. Effects of water recirculation rate on the microbial community and water quality in relation to the growth and survival of white shrimp (Litopenaeus vannamei). BMC Microbiol. 19, 192.
    Pubmed KoreaMed CrossRef
  5. Garrity G, Brenner DJ, Krieg NR, and Staley JR. Bergey's manual of systematic bacteriology. Volume 2: The Proteobacteria. Springer US, Boston, Massachusetts, USA.
  6. Grant JR and Stothard PJ. 2008. The CGView server: A comparative genomics tool for circular genomes. Nucleic Acids Res. 36, W181-W184.
    Pubmed KoreaMed CrossRef
  7. Grissa I, Vergnaud G, and Pourcel CJ. 2007. CRISPERFinder: A web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35, W52-W57.
    Pubmed KoreaMed CrossRef
  8. Kanehisa M and Goto S. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27-30.
    Pubmed KoreaMed CrossRef
  9. Kim YS, Jeon YJ, and Kim KH. 2019. Salaquimonas pukyongi gen. nov., sp. nov., a novel bacterium within the family Phyllobacteriaceae. Int. J. Syst. Evol. Microbiol. 69, 3751-3756.
    Pubmed CrossRef
  10. Lee DE, Lee J, Kim YM, Myeong JI, and Kim KH. 2016. Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing. J. Microbiol. 54, 296-304.
    Pubmed CrossRef
  11. Pujalte MJ, Lucena T, Ruvira MA, Arahal DR, and Macián MC. 2014. The family Rhodobacteraceae, pp. 439-512. In Rosenberg E, De Long EF, Lory S, Stackebrandt E, and Thompson F (eds.). The Prokaryotes. Springer, Berlin, Heidelberg, Germany.
  12. Rhee C, Kim H, Emmanuel SA, Kim HG, Won S, Bae J, Bai SC, and Koh SC. 2018. Microbial community analysis of an eco-friendly recirculating aquaculture system for olive flounder (Paralichthys olivaceus) using complex microbial probiotics. Korean J. Microbiol. 54, 369-378.
  13. Takai K, Miyazaki M, Hirayama H, Nakagawa S, Querellou J, and Godfroy A. 2009. Isolation and physiological characterization of two novel, piezophilic, thermophilic chemolithoautotrophs from a deep-sea hydrothermal vent chimney. Environ. Microbiol. 11, 1983-1997.
    Pubmed CrossRef
  14. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, and Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614-6624.
    Pubmed KoreaMed CrossRef
  15. Wu YH, Xu L, Zhou P, Wang CS, Oren A, and Xu XW. 2015. Brevirhabdus pacifica gen. nov., sp. nov., isolated from deep-sea sediment in a hydrothermal vent field. Int. J. Syst. Evol. Microbiol. 65, 3645-3651.
    Pubmed CrossRef
  16. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, and Chun J. 2017. Introducing Ezbiocloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613-1617.
    Pubmed KoreaMed CrossRef
  17. Zhang G, Haroon MF, Zhang R, Dong X, Wang D, Liu Y, Xun W, Dong X, and Stingl U. 2017. Ruegeria profundi sp. nov. and Ruegeria marisrubri sp. nov., isolated from the brine-seawater interface at Erba deep in the Red Sea. Int. J. Syst. Evol. Microbiol. 67, 4624-4631.
    Pubmed CrossRef
  18. Zhang SY, Li G, Wu HB, Liu XG, Yao YH, Tao L, and Liu H. 2011. An integrated recirculating aquaculture system (RAS) for land-based fish farming: The effects on water quality and fish production. Aquac. Eng. 45, 93-102.

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