Standards in Genomic Sciences (2010) 3:174-182 DOI:10.4056/sigs.1161239

Ferrimonas balearica Rossello-Mora et al. 1996 is the type species of the genus Ferrimonas, which belongs to the family Ferrimonadaceae within the Gammaproteobacteria. The species is a Gram-negative, motile, facultatively anaerobic, non spore-forming bacterium, which is of special interest because it is a chemoorganotroph and has a strictly respiratory metabolism with oxygen, nitrate, Fe(III)-oxyhydroxide, Fe(III)-citrate, MnO2, selenate, selenite and thiosulfate as electron acceptors. This is the first completed genome sequence of a member of the genus Ferrimonas and also the first sequence from a member of the family Ferrimonadaceae. The 4,279,159 bp long genome with its 3,803 protein-coding and 144 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain PAT T (= DSM 9799 = CCM 4581) is the type strain of the species Ferrimonas balearica, which is the type species of its genus Ferrimonas [1,2]. Currently, there are five species in the genus Ferrimonas [3]. The generic name derives from the Latin word 'ferrum' meaning 'iron' and the Greek word 'monas' meaning 'unit', referring to an iron(III)-reducing cell. The species epithet is also derived from the Latin word 'balearica' meaning 'of the Balearic Islands', referring to the place where the strain was isolated [1]. Ferrimonas is the type genus of the family Ferrimonadaceae and one of two genera in the family Ferrimonadaceae [4]. Strain PAT T was described in 1995 by Rossello-Mora et al. [1] who isolated the strain from the upper few centimeters of marine sediment of the Palma de Mallorca harbor, Spain [1,5]. Here we present a summary classification and a set of features for F. balearica PAT T , together with the description of the complete genomic sequencing and annotation.

Classification and features
The 16S rRNA gene sequence of PAT T is 99% identical to four culturable strains, which are reported in GenBank [6]. Two strains, A2A-18 (AB193752) and A3B-47-3 (AB193753), were isolated from marine sand [7]. The culturable strain S8-05 (EU620413) was isolated from Palk Bay sediment in Thondi, India and another strain with accession number AY158002 was isolated from Ala Wai Canal sediment in Honolulu, USA. The 16S rRNA gene of strain PAT T shares 93.5-97.4% sequence identity with the sequences of the type strains from the other members of the family Ferrimonadaceae [8]. The environmental samples database (env_nt) contains the marine metagenome clone 1096626783183 (96% sequence identity, AA-CY020355234). The genomic survey sequences database (gss) contains the uncultured bacterium clone BYUP987.b1 (92%, EF996742), isolated from a fecal sample of adult woman who gave birth after 11 months [9]. Altogether, strains belonging to the species F. balearica or the genus Ferrimonas are rather rare in the habitats screened so far (status September 2010). Figure 1 shows the phylogenetic neighborhood of F. balearica PAT T in a 16S rRNA based tree. The sequences of the seven 16S rRNA gene copies in the genome differ from each other by up to five nucleotides, and differ by up to four nucleotides from the previously published sequence (X93021), which contains two ambiguous base calls. Figure 1. Phylogenetic tree highlighting the position of F. balearica PAT T relative to the type strains of the other species within the family Ferrimonadaceae and to the type of the neighboring family Psychromonadaceae. The trees were inferred from 1,449 aligned characters [10,11] of the 16S rRNA gene sequence under the maximum likelihood criterion [12] and rooted with the type strain of the order Alteromonadaceae. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 650 bootstrap replicates [13] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [14] are shown in blue, published genomes in bold (CP000510) [15].
Strain PAT T is a Gram-negative, nonspore-forming, facultatively anaerobic bacterium [1]. The cells are straight rods (0.3-0.5 × 1.2-1.5 µm) with rounded ends ( Figure 2, Table 1) [1,5] and appear singly, occasionally in pairs or short chains and usually not encapsulated [1,5]. Strain PAT T is motile by means of monotrichous flagella (not visible in Figure 2, but 10% of the cells in the original liquid culture were highly motile) [1]. Colonies produce a black iron precipitate when the cells are grown on TSI agar [1]. Although initially isolated using TSI based media this strain grows better on Marine Broth. Colonies are often brown and mucous when the cells are grown under aerobic conditions [5]. Fresh isolates of this species may not form colonies on PYG agar medium, but the colonies are formed after several subcultivations in enrichment medium [1,5]. Resting stages of strain PAT T are not known [5]. Cells of the strain undergo autolysis within five days under aerobic conditions [1,5]. Strain PAT T does not contain polyhydroxybutyrate (PHB) or other intracellular inclu-sions [2]. The strain is chemoorganotrophic. Under anaerobic conditions, the reduction of Fe(III)oxyhydroxide is coupled to the utilization of lactate as the electron donor, which yields magnetite [1,5]. Strain PAT T uses oxygen, nitrate, Fe(III)oxyhydroxide, Fe(III)-citrate, MnO2, selenate, selenite and thiosulfate as electron acceptors [1,5,25]. Strain PAT T requires a minimum of 0.5% NaCl for growth, with a range of NaCl tolerance of 0.5%-7.5% [1]. It does not grow at 5°C or 44°C but does grow at 42°C [1]. The pH range for growth is 6-9 [1]. Enzymatic reactions are positive for catalase, oxidase, phenylalanine deaminase, DNAse and lipase (Tween 20 and Tween 80), but negative for amylase, arginine dihydrolase, gelatinase, lysine decarboxylase, Simmons citrate and urease [1,5]. The strain does not hydrolyze starch [1]. The genus Ferrimonas can be distinguished from other strictly respiratory Gram-negative genera of the Gammaproteobacteria based on its ability to reduce Fe(III), denitrification, growth at 42°C, presence of phenylalanine deaminase activity, inability to grow in NaCL-free media, lack of gelatinase, urease and a negative reaction of Simmons citrate test [5].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [31], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [32]. The genome project is deposited in the Genome OnLine Database [14] and the complete genome sequence is deposited in Gen-Bank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the standard protocol as recommended by the manufacturer, with modification st/L for cell lysis as described in Wu et al. [32].

Genome sequencing and assembly
The genome was sequenced using the Sanger sequencing platform (6 and 40 kb DNA libraries). All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible misassemblies were corrected with Dupfinisher or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI) [34]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 404 additional custom primer reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed genome sequence contains 48,554 reads, achieving an average of 9.8-fold sequence coverage with an error rate less than 1 in 100,000.

Genome annotation
Genes were identified using Prodigal [35] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [36]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [37].

Genome properties
The genome consists of a 4,279,159 bp long chromosome with a 60.2% GC content (Table 3 and Figure 3). Of the 3,947 genes predicted, 3,803 were protein-coding genes, and 144 RNAs; twenty one pseudogenes were also identified. The majority of the protein-coding genes (72.5%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4. Standards in Genomics Sciences