Skip to main content
NCBI home page
Genome Announcements logoLink to Genome Announcements
. 2015 Feb 12;3(1):e01565-14. doi: 10.1128/genomeA.01565-14

Draft Genome Sequence of Cellulophaga sp. E6, a Marine Algal Epibiont That Produces a Quorum-Sensing Inhibitory Compound Active against Pseudomonas aeruginosa

J E Lafleur a,b, S K Costa a,*, A S Bitzer a, M W Silby a,✉
PMCID: PMC4333669  PMID: 25676769

Abstract

The genus Cellulophaga is composed of obligate aerobic Gram-negative bacteria commonly found in association with marine algae. We report the approximately 4.42-Mbp draft genome sequence of Cellulophaga sp. E6, which inhibits N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL)–mediated quorum sensing (QS), lasB transcription, and biofilm formation by Pseudomonas aeruginosa.

GENOME ANNOUNCEMENT

We isolated Cellulophaga sp. E6 (1, 2) from the surface of a coastal seaweed as part of a bioprospecting search for marine bacteria which have the potential to control P. aeruginosa virulence through inhibiting acylhomoserine lactone (AHL)-based quorum sensing (QS). Cellulophaga spp. are obligate aerobes which hydrolyze agar and carrageenan. Most Cellulophaga spp. display gliding motility (3, 4), and are strongly proteolytic (5), and like many Flavobacteriaceae, Cellulophaga can be algalytic (6–9). Algalysis is achieved both through direct attack, and through indirect chemical means (8). Recent reports have focused on potential applications for novel enzymes from Cellulophaga species that break down carrageenan (3, 10, 11).

Using a luciferase-based reporter construct (PlasB-luxCDABE) specific for 3-oxo-C12-HSL (12), we have shown that Cellulophaga sp. E6 inhibits 3-oxo-C12-HSL QS in P. aeruginosa which uses this system and another AHL-based QS system (C4-HSL) to control virulence gene expression and biofilm formation (13). Supernatant from Cellulophaga sp. E6 culture reduced expression of the 3-oxo-C12-HSL-dependent virulence-associated gene lasB, and reduced biofilm formation in a dose-dependent manner. Activity-guided purification of the QS inhibitory activity has shown that the target molecule is smaller than 1000 Da, water-soluble, and stable to temperatures of 50°C.

Genomic DNA was isolated from Cellulophaga E6 using the Wizard Genomic DNA purification kit (Promega). The sequencing library was prepared using Nextera XT sequencing library preparation kit (Illumina). Sequencing was carried out using a MiSeq Genome Sequencer (Illumina) at Tufts University Genomics Core, which generated 4,425,292 2 × 250 bp paired-end reads. de novo assembly was done using CLC Genomics Workbench v7.0.4 (CLC Bio, Denmark) with the minimum contig size set to 250 bp, resulting in 72 contigs. The contigs range in size from 528 to 692,305 bp. The assembled genome had 300-fold coverage, with an N50 scaffold size of 203,379 bp. Annotation was added by the NCBI Prokaryotic Genome Annotation Pipeline. The total size for the combined contigs is 4,420,065 bp, leading us to estimate the genome as approximately 4.42 Mb; it is predicted to contain 3,630 protein-coding genes, 36 tRNA genes, and 2 rRNA genes. 16S and 23S rRNA genes were detected, but since the assembly is based on short reads, the numbers and locations of multiple copies could not be determined. The DNA G+C content was 34.6%, which is consistent with those of other Cellulophaga genomes.

Some Cellulophaga spp. have been reported to quorum sense with AI-2 (8). A potential mechanism for Cellulophaga spp. to inhibit P. aeruginosa QS is via 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a by-product of in vitro LuxS-mediated synthesis of the QS molecule AI-2 (4,5-dihydroxy-2,3-pentanedione) (14, 15), which has been reported to inhibit P. aeruginosa QS, and biofilm formation (16). However, we have not detected sequences related to luxS in the Cellulophaga sp. E6 genome, suggesting that Cellulophaga sp. E6 does not employ AI-2 QS and thus may inhibit QS using a novel small molecule. The genome sequence of Cellulophaga sp. E6 will facilitate the search for the genetic basis of the QS inhibiting molecule, complementing the biochemical analysis of its synthesis.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. JQCT00000000. The version described in this paper is version JQCT01000000.

ACKNOWLEDGMENTS

This work was supported through a Junior faculty Research Development grant from the Department of Emergency Medicine, Alpert School of Medicine, Brown University (to J.E.L.), and in part by the UMass Dartmouth Chancellor’s Research Fund and the UMass Joseph Healey Endowment (to M.W.S.), and a UMass Dartmouth Office of Undergraduate Research award (to S.K.C.).

Footnotes

Citation Lafleur JE, Costa SK, Bitzer AS, Silby MW. 2015. Draft genome sequence of Cellulophaga sp. E6, a marine algal epibiont that produces a quorum-sensing inhibitory compound active against Pseudomonas aeruginosa. Genome Announc 3(1):e01565-14. doi:10.1128/genomeA.01565-14.

REFERENCES

  • 1.Lewin RA. 1969. A classification of flexibacteria. J Gen Microbiol 58:189–206. doi: 10.1099/00221287-58-2-189. [DOI] [PubMed] [Google Scholar]
  • 2.Johansen JE, Nielsen P, Sjøholm C. 1999. Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov. Int J Syst Bacteriol 49:1231–1240. doi: 10.1099/00207713-49-3-1231. [DOI] [PubMed] [Google Scholar]
  • 3.Shan D, Ying J, Li X, Gao Z, Wei G, Shao Z. 2014. Draft genome sequence of the carrageenan-degrading bacterium Cellulophaga sp. strain KL-A, isolated from decaying marine algae. Genome Announc 2(2):e00145-14. doi: 10.1128/genomeA.00145-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bowman J. 2011. Genus IX. Cellulophaga Johansen, Nielsen and Sjøholm 1999, 1238VP. p 176–180 In Krieg NR, et al. (ed), Bergey’s manual of systematic bacteriology, 2nd ed., vol 4 Springer, New York, NY. [Google Scholar]
  • 5.Bowman JP. 2000. Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50:1861–1868. [DOI] [PubMed] [Google Scholar]
  • 6.Imai I, Ishida Y, Hata Y. 1993. Killing of marine phytoplankton by a gliding bacterium Cytophaga sp., isolated from the coastal sea of Japan. Mar Biol 116:527–532. doi: 10.1007/BF00355470. [DOI] [Google Scholar]
  • 7.Crump BC, Armbrust EV, Baross JA. 1999. Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia River, its estuary, and the adjacent coastal ocean. Appl Environ Microbiol 65:3192–3103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Skerratt J, Bowman J, Hallegraeff G, James S, Nichols P. 2002. Algicidal bacteria associated with blooms of a toxic dinoflagellate in a temperate Australian estuary. Mar Ecol Prog Ser 244:1–15. doi: 10.3354/meps244001. [DOI] [Google Scholar]
  • 9.Mayali X, Azam F. 2004. Algicidal bacteria in the sea and their impact on algal blooms. J Eukaryot Microbiol 51:139–144. doi: 10.1111/j.1550-7408.2004.tb00538.x. [DOI] [PubMed] [Google Scholar]
  • 10.Ma S, Duan G, Chai W, Geng C, Tan Y, Wang L, Le Sourd F, Michel G, Yu W, Han F. 2013. Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3. PLoS One 8:e64666. doi: 10.1371/journal.pone.0064666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Yao Z, Wang F, Gao Z, Jin L, Wu H. 2013. Characterization of a κ-carrageenase from marine Cellulophaga lytica strain N5–2 and analysis of its degradation products. Int J Mol Sci 14:24592–24602. doi: 10.3390/ijms141224592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Romero M, Diggle SP, Heeb S, Cámara M, Otero A. 2008. Quorum quenching activity in Anabaena sp. PCC 7120: identification of AiiC, a novel AHL-acylase. FEMS Microbiol Lett 280:73–80. doi: 10.1111/j.1574-6968.2007.01046.x. [DOI] [PubMed] [Google Scholar]
  • 13.Winson MK, Camara M, Latifi A, Foglino M, Chhabra SR, Daykin M, Bally M, Chapon V, Salmond GP, Bycroft BW. 1995. Multiple N-acyl-l-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92:9427–9431. doi: 10.1073/pnas.92.20.9427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Winzer K, Hardie KR, Burgess N, Doherty N, Kirke D, Holden MTG, Linforth R, Cornell KA, Taylor AJ, Hill PJ, Williams P. 2002. LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone. Microbiology 148:909–922. [DOI] [PubMed] [Google Scholar]
  • 15.Camilli A, Bassler BL. 2006. Bacterial small-molecule signaling pathways. Science 311:1113–1116. doi: 10.1126/science.1121357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Choi S-C, Zhang C, Moon S, Oh Y-S. 2014. Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1. J Microbiol 52:734–742. doi: 10.1007/s12275-014-4060-x. [DOI] [PubMed] [Google Scholar]

Articles from Genome Announcements are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES