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Complete Genome Sequences of Francisella Noatunensis Subsp Gonçalves et al. Standards in Genomic Sciences (2016) 11:30 DOI 10.1186/s40793-016-0151-0 SHORT GENOME REPORT Open Access Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior Lucas Amorim Gonçalves1,2, Siomar de Castro Soares1, Felipe Luiz Pereira1, Fernanda Alves Dorella1, Alex Fiorini de Carvalho1, Gabriel Magno de Freitas Almeida1, Carlos Augusto Gomes Leal1, Vasco Azevedo2 and Henrique César Pereira Figueiredo1* Abstract The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp.orientalisis responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the genome sequences of F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190. The genomes include one circular chromosome of 1,859,720 bp, consisting of 32 % GC content, 1538 coded proteins and 363 pseudogenes for FNO12; one circular chromosome of 1,862,322 bp, consisting of 32 % GC content, 1537 coded proteins and 365 pseudogenes for FNO24; and one circular chromosome of 1,859,595 bp, consisting of 32 % GC content, 1539 coded proteins and 362 pseudogenes for FNO190. All genomes have similar genetic content, implicating a clonal-like behavior for this species. Keywords: Complete genome sequencing, Fish pathogen, Genetic clonal behavior Introduction been responsible for a large number of deaths of tilapia In 1922, Edward Francis (1872–1957), an American bac- and other freshwater species cultured in the United States, teriologist, described the bacterium that causes tularemia the United Kingdom, Japan, Taiwan, Jamaica, Costa Rica, in humans, Francisella tularensis. This bacterium is the Brazil and some other Latin American regions [4–6]. most studied of its genus [1, 2]. Until recently, the genus Nevertheless, besides infecting important cultivable species Francisella consisted of only two species: F. tularensis and such as tilapia, threeline grunt (Parapristipoma trilineatum) F. philomiragia; however, new species and new strains and hybrid striped bass (Morone chrysops X Morone saxati- were isolated, such as F. noatunensis and the subspecies F. lis), this bacterium is also capable of infecting wild fish such noatunensis subsp. orientalis [1], the latter being recog- as guapote tigre (Parachromis managuensis)[4,5]. nized as one of the most important pathogens of cultured Although the disease caused by this species presents with tilapia (Oreochromis spp.) [3]. a high mortality rate during outbreaks and has been re- F. noatunensis subsp. orientalis is the etiologic agent of ported in several countries, the phylogenomic relationships pyogranulomatous and granulomatous infections in fish. among isolates from different countries and the evolution- In the last few years, F. noatunensis subsp. orientalishas ary history of this pathogen are still poorly characterized. Therefore, the strains presented herein were isolated from * Correspondence: [email protected] three different regions and outbreaks to characterize the 1National Reference Laboratory for Aquatic Animal Diseases (AQUACEN), genetic diversity of the microorganism F. noatunensis Ministry of Fisheries and Aquaculture, Federal University of Minas Gerais, Belo Horizonte, Brazil subsp. orientalis strains FNO12, FNO24 and FNO190. Full list of author information is available at the end of the article © 2016 Gonçalves et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gonçalves et al. Standards in Genomic Sciences (2016) 11:30 Page 2 of 8 Table 1 Classification and general features of Francisella Table 1 Classification and general features of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190 noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190 according to the MIGS recommendations [9] according to the MIGS recommendations [9] (Continued) MIGS ID Property Term Evidence codea MIGS-4.4 Altitude FNO12 – ~1,006 NAS Classification Domain Bacteria TAS [26] FNO24 – ~848 Phylum Proteobacteria TAS [27] FNO190 – 370 Class Gammaproteobacteria TAS [28] aEvidence codes - IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS, Non-traceable Order Thiotrichales TAS [29] Author Statement (i.e., not directly observed for the living, isolated sample, Family Francisellaceae TAS [30] but based on a generally accepted property for the species, or an anecdotal evidence). These evidence codes are from the Gene Ontology project [11] Genus Francisella TAS [31, 32] Species Francisella TAS [33] Organism information noatunensis subsp. orientalis Classification and features Type strain FNO12, FNO24 IDA This Francisella genus, from phylum Proteobacteria,class and FNO190 Gammaproteobacteria,orderThiotrichales,andfamily Gram stain Gram-negative TAS [33] Francisellaceae, is a strictly aerobic, non-motile, pleo- Cell shape pleomorphic TAS [33] morphic, and Gram-negative bacteria of 0.5–1.5 μm Motility Non-motile TAS [33] (Table 1 and Fig. 1). It is negative for nitrate reduction as Sporulation Not reported NAS well as adonitol, arabinose, cellobiose, esculin, galacturo- nate, glucuronate, malonate, mannitol, melibiose, raffinose, Temperature Mesophilic (15–34 °C) TAS [33] range rhamnose, palatinose, and 5-ketogluconate fermentation. In contrast, it has C14 lipase, cystine arylamidase, para- Optimum <25 °C TAS [33] temperature phenylalanine deaminase, tetrathionate reductase, trypsin, α α pH range; Not reported NAS urease, valine arylamidase, -chymotrypsin, -fucosidase, Optimum α-galactosidase, α-mannosidase, and β-glucuronidase activ- Carbon Not reported NAS ity, as well as acid production from lactose. Additionally, it source is positive for acid phosphatase, alkaline phosphatase, C4 MIGS-6 Habitat FNO12 – Nile tilapia kidney NAS and C8 esterase, lipase, naphtol-AS-BI-phosphohydrolase, β FNO24 – Nile tilapia spleen -lactamase activity, and acid production from maltose [7]. Using the 16S RNA sequences with 1516 bp of FNO12, FNO190 – Nile tilapia spleen FNO24, and FNO190 with the neighbor-joining method MIGS-6.3 Salinity Not reported NAS based on 1000 randomly selected bootstrap replicates of MIGS-22 Oxygen Strictly aerobic TAS [33] alignments using Mega6 software [8], a phylogenetic tree requirement showing these strains positioned in a species-specific clade MIGS-15 Biotic Intracellular facultative TAS [7] was constructed (Fig. 2). relationship pathogen MIGS-14 Pathogenicity Pathogenic for fish TAS [7] Genome sequencing information MIGS-4 Geographic FNO12 – Brazil/State of Minas NAS Genome project history location Gerais/Areado city In the present study, the nucleotide sequence of the F. noa- FNO24 – Brazil/State of Minas tunensis subsp. orientalis FNO12, FNO24 and FNO190 Gerais/Alterosa city complete genomes was determined. Sequencing and assem- – FNO190 Brazil/State of São bly were performed by the National Reference Laboratory Paulo/Santa fé do Sul city for Aquatic Animal Diseases, and annotation was per- MIGS-5 Sample FNO12– Mai 5, 2012 NAS collection formed by the Laboratory of Cellular and Molecular Genet- FNO24 – Mai 5, 2012 ics. Both laboratories are located at the Federal University FNO190 – Nov 10, 2013 of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. MIGS-4.1 Latitude FNO12 – 21° 21′ S NAS Source DNA of these three strains are available at culture FNO24 – 21° 14′ S collection of AQUACEN. Table 2 presents the project information and its association with MIGS version 2.0 FNO190 – 20° 12′ S compliance [9]. MIGS-4.2 Longitude FNO12 – 46° 08′ W NAS FNO24 – 46° 08′ W Growth conditions and genomic DNA preparation FNO190 – 50° 55′ W F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190 were isolated from three different outbreaks Gonçalves et al. Standards in Genomic Sciences (2016) 11:30 Page 3 of 8 Fig. 1 Photomicrograph of the F. noatunensis subsp. orientalis strains. The strains FNO12, FNO24 and FNO190 are represented, respectively, by sections a, b and c from Nile tilapia fish farms. Swabs of kidney (FNO12) and chaotropic agent and a detergent, (ii) nucleic acids spleen (FNO24 and FNO190) tissues from each fish were were bound to silica magnetic particles, (iii) bound sampled aseptically, streaked onto cysteine heart agar particles were washed and isolated from other cell supplemented with 2 % bovine hemoglobin (BD Bio- components, and (iv) nucleic acids were eluted into a sciences, USA) and incubated at 28 °C for 4–7 days formulation for sequencing. Genomic DNAs were [7]. The isolates were stored at -80 °C in Mueller- measured using Qubit 2.0 Fluorometer (Life Tech- Hinton cation-adjusted broth supplemented with 2 % nologies, Thermo Scientific,
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