- Short genome report
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Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior
Standards in Genomic Sciencesvolume 11, Article number: 30 (2016)
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. orientalis is 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.
In 1922, Edward Francis (1872–1957), an American bacteriologist, described the bacterium that causes tularemia in humans, Francisella tularensis . This bacterium is the most studied of its genus [1, 2]. Until recently, the genus Francisella consisted of only two species: F. tularensis and F. philomiragia ; however, new species and new strains were isolated, such as F. noatunensis and the subspecies F. noatunensis subsp. orientalis , the latter being recognized as one of the most important pathogens of cultured tilapia ( Oreochromis spp.) .
F. noatunensis subsp. orientalis is the etiologic agent of pyogranulomatous and granulomatous infections in fish. In the last few years, F. noatunensis subsp. orientalishas been responsible for a large number of deaths of tilapia and other freshwater species cultured in the United States, the United Kingdom, Japan, Taiwan, Jamaica, Costa Rica, Brazil and some other Latin American regions [4–6]. Nevertheless, besides infecting important cultivable species such as tilapia, threeline grunt ( Parapristipoma trilineatum ) and hybrid striped bass ( Morone chrysops X Morone saxatilis ), this bacterium is also capable of infecting wild fish such as guapote tigre ( Parachromis managuensis ) [4, 5].
Although the disease caused by this species presents with a high mortality rate during outbreaks and has been reported in several countries, the phylogenomic relationships among isolates from different countries and the evolutionary history of this pathogen are still poorly characterized. Therefore, the strains presented herein were isolated from three different regions and outbreaks to characterize the genetic diversity of the microorganism F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190.
Classification and features
This Francisella genus, from phylum Proteobacteria , class Gammaproteobacteria , order Thiotrichales , and family Francisellaceae , is a strictly aerobic, non-motile, pleomorphic, and Gram-negative bacteria of 0.5–1.5 μm (Table 1 and Fig. 1). It is negative for nitrate reduction as well as adonitol, arabinose, cellobiose, esculin, galacturonate, glucuronate, malonate, mannitol, melibiose, raffinose, rhamnose, palatinose, and 5-ketogluconate fermentation. In contrast, it has C14 lipase, cystine arylamidase, para-phenylalanine deaminase, tetrathionate reductase, trypsin, urease, valine arylamidase, α-chymotrypsin, α-fucosidase, α-galactosidase, α-mannosidase, and β-glucuronidase activity, as well as acid production from lactose. Additionally, it is positive for acid phosphatase, alkaline phosphatase, C4 and C8 esterase, lipase, naphtol-AS-BI-phosphohydrolase, β-lactamase activity, and acid production from maltose . Using the 16S RNA sequences with 1516 bp of FNO12, FNO24, and FNO190 with the neighbor-joining method based on 1000 randomly selected bootstrap replicates of alignments using Mega6 software , a phylogenetic tree showing these strains positioned in a species-specific clade was constructed (Fig. 2).
Genome sequencing information
Genome project history
In the present study, the nucleotide sequence of the F. noatunensis subsp. orientalis FNO12, FNO24 and FNO190 complete genomes was determined. Sequencing and assembly were performed by the National Reference Laboratory for Aquatic Animal Diseases, and annotation was performed by the Laboratory of Cellular and Molecular Genetics. Both laboratories are located at the Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Source DNA of these three strains are available at culture collection of AQUACEN. Table 2 presents the project information and its association with MIGS version 2.0 compliance .
Growth conditions and genomic DNA preparation
F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190 were isolated from three different outbreaks from Nile tilapia fish farms. Swabs of kidney (FNO12) and spleen (FNO24 and FNO190) tissues from each fish were sampled aseptically, streaked onto cysteine heart agar supplemented with 2 % bovine hemoglobin (BD Biosciences, USA) and incubated at 28 °C for 4–7 days . The isolates were stored at -80 °C in Mueller-Hinton cation-adjusted broth supplemented with 2 % VX supplement (Laborclin, Brazil), 0.1 % glucose, and 15 % glycerol. The isolates were thawed, streaked onto CHAH and incubated at 28 °C for 48–72 h. Genomic DNA was extracted by the use of the Maxwell 16® Research Instrument (Promega, USA) according to the manufacturer’s recommendations. Briefly, (i) 2 x 109 cells were lysed in the presence of a chaotropic agent and a detergent, (ii) nucleic acids were bound to silica magnetic particles, (iii) bound particles were washed and isolated from other cell components, and (iv) nucleic acids were eluted into a formulation for sequencing. Genomic DNAs were measured using Qubit 2.0 Fluorometer (Life Technologies, Thermo Scientific, USA) and yield of DNA were 64.8 ng/μL (FNO12), 58.0 ng/μL (FNO24) and 54.4 ng/μL (FNO190). Purity of DNAs (UV A260/A280) was accessed by NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA). Ratios for each sample were 1.89, 1.95, and 1.96 for FNO12, FNO24 and FNO190, respectively. The extracted DNA was stored at -80 °C until use.
Genome sequencing and assembly
The genome sequencing of the FNO12 strain was performed with the MiSEQ platform (Illumina®, USA), while the genome sequencing of the FNO24 and FNO190 strains was performed with the Ion Torrent Personal Genome Machine™ (Life Technologies, USA). MiSEQ used the Nextera DNA Library Preparation Kit while PGM used the Ion PGM 200 bp Sequencing Kit. The quality of the raw data was analyzed using FastQC , and the assembly was performed using the Edena 2.9 , Mira 3.9  and Newbler 2.9 (Roche, USA) as the applied ab initio strategy. The assemblies of FNO12, FNO24 and FNO190 produced a total of 15, 57 and 16 contigs, respectively. The first strain resulted in ~1382-fold, coverage, the second had a value of ~79-fold, coverage, and the third had a value of ~203-fold coverage,. Additionally, the strains FNO12, FNO24 and FNO190 presented an N50 value of 275,043 bp, 87,100 bp, and 237,022 bp, respectively. A super scaffold for FNO12 was produced with an optical map as a reference using restriction enzyme NheI, on MapSolver software (OpGen Technologies, USA). The remaining gaps were filled through the use of CLC Genomics Workbench 7 (Qiagen, USA) by mapping the raw data in gap flank repeated times until the overlap was found. For FNO24 and FNO190, the complete genome of FNO12 was used as a reference to construct the super scaffolds on CONTIGuator 2.0 software , and gap filling was conducted as described for strain FNO12. All the raw sequencing data were mapped onto the each final genome and the lack of contamination with other genomes were confirmed by the coverage and the low number of unmapped reads.
Automatic annotation was performed using the RAST software ; tRNA and rRNA predictions were conducted using the tRNAscan-SE Search Server  and the RNAmmer , respectively. Manual curation of the annotation was done using Artemis software  and the UniProt database . All putative frameshifts were manually curated based on the raw data coverage in CLC Genomics Workbench 7 software (Qiagen, USA), which was used to correct indel errors in regions of homopolymers.
The genomes are each comprised of a circular chromosome with sizes of 1,859,720 bp, 1,862,322 bp, and 1,859,595 bp for FNO12, FNO24, and FNO190, respectively (Table 3). The GC content in the three strains is 32 %, and the number of pseudogenes is relatively high (363 on average). Strain FNO24 had more protein coding genes, and one RNA-coding gene fewer than the other two strains. For the FNO12 and FNO190 strains, 1280 genes were annotated with functional prediction, whereas for strain FNO24, 1282 genes were annotated. Each genome contained 621 CDSs classified as hypothetical proteins by the COG database . Table 4 summarizes the number of genes associated with general COG functional categories. Figure 3 shows the comparison of FNO12 with FNO24, FNO190 (presented in this study) with the other two strains deposited in GenBank ( F. noatunensis subsp. orientalis strains LADL-07-285A and Toba04, accession numbers: CP006875 and CP003402, respectively).
Insights from the genome sequence
A high similarity in the genetic content of these genomes was seen in Fig. 3. Additionally, Additional file 1 shows the only eight protein coding sequences with less than 99 % identity between the three sequenced genomes (six hypothetical proteins, one Type IV pili, and one secreted protein). Also, this high intraspecies similarity (100.00 ± 0 %) may be viewed in Additional file 2 and Additional file 3 using Gegenees  with threshold of 30 % and Mauve  with progessiveMauve algorithm, respectively. These analyses include the three strains of this work and other three deposited at GenBank (FNO01, Toba04, and LADL--07-285A, GenBank nos. CP012153, CP003402, and CP006875, respectively) belonging to the same species. In contrast, the similarity with the subspecies F. noatunensis subsp. noatunensis is reduced to 84.09 ± 0.40 % (Additional file 2). Furthermore, the orthoMCL software  was used to predict the cluster of orthologous genes. CDSs shared by all species were considered to be part of the core genome, whereas CDSs harbored by only species were considered to be species-specific genes. There are 891 CDSs shared by all Francisella species (Fig. 4). Interestingly, the F. tularensis subsp. mediasiatica shows only 2 singleton CDSs, that because this species shared 1380 of yours 1385 CDSs with F. tularensis subsp. tularensis , whereas the F. noatunensis subsp. orientalis had 296 species-specific CDSs (Additional file 4 shows COG functional categories found of each CDS). Finally, the GIPSy software  was used to predict genomic islands present on F. noatunensis subsp. orientalis . FNO12 strain was chosen as query, whereas three strains of close related species was used as references ( F. philomiragia subsp. philomiragia ATCC 25017, F. tularensis subsp. novicida U112, and Thiomicrospira crunogena XCL-2, GenBank nos. CP000937, CP000439, CP000109, respectively). Ten genomics islands were predicted by GIPSy, including 2 putative pathogenic islands (PAI1 and PAI2) and 1 putative resistance island (REI1), and plotted using BRIG software  (Additional file 5). GEI3 is, apparently, exclusive of F. noatunensis subsp. orientalis , and GEI4 is shared only with F. noatunensis subsp. noatunesis species, another species of marine environment. REI1 and PAI1 are partially shared by all species of Francisella genus. PAI2 is partially shared with all species of Francisella genus and totally shared with F. philomiragia and F. philomiragia subsp. philomiragia species. GEI6, predicted only as genomic island by GIPSy, contains the genes mltA, rplM, rpsI, mglA, mglB, rnhB, yfhQ, ptsN, mnmE, cysK, pdpA, pdpB, iglD, iglC, iglB, iglA, pdpD, anmK, related with the Francisella Pathogenicity Island, a previously described pathogenic island for the Francisella genus . Further studies are required to characterize these genomic islands, since the GIPSy analysis suggests a greater number of Horizontal Gene Transfer than previously described for this species.
Three genomes of an important fish pathogen are presented in this work. Despite being isolated from different outbreaks and from different host organs, they are very similar considering the brief analysis of this work. All analyses suggest the clonality of the strains with minor differences in the quantity of pseudogenes and the number of CDSs and RNAs. Furthermore, the high number of pseudogenes present in all sequenced strains corroborate that this species is undergoing genome decay .
cysteine heart agar supplemented with hemoglobin
personal genome machine
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This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Pesca e Aquicultura and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). We also acknowledge support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
The authors declare that they have no competing interests.
LAG, SCS and FLP drafted the manuscript. FAD, AFC and GMFA performed the laboratory experiments. LAG, SCS, FLP, FAD and AFC sequenced, assembled and annotated the genome. CAGL, VACA and HCPF worked on the conception, design, and coordination of this study and helped to write the manuscript. All authors read and approved the final manuscript.
Alignment of proteins coding sequences with less than 99 % identity between the three sequenced genomes. (TXT 20 kb)
Heat map showing high similarity between the sequenced genemes performed in Gegenees software with threshold of 30 %. (TIF 984 kb)
Synteny analysis of Francisella noatunensis subsp. orientalis FNO01, FNO12, FNO24, FNO190, Toba04 and LADL--07-285A strains performed with Mauve software with progessiveMauve algorithm. (TIF 381 kb)
COG functional categories found of each species-specific CDS of Francisella noatunensis subsp. orientalis. (TXT 16 kb)
The genomic islands predicted by GIPSy software (2 putative pathogenic islands, 1 putative resistance island, and 7 uncharacterized genomic island), plotted using BRIG software. (TIF 2066 kb)