Complete genome sequence of the gliding, heparinolytic Pedobacter saltans type strain (113T)

Pedobacter saltans Steyn et al. 1998 is one of currently 32 species in the genus Pedobacter within the family Sphingobacteriaceae. The species is of interest for its isolated location in the tree of life. Like other members of the genus P. saltans is heparinolytic. Cells of P. saltans show a peculiar gliding, dancing motility and can be distinguished from other Pedobacter strains by their ability to utilize glycerol and the inability to assimilate D-cellobiose. The genome presented here is only the second completed genome sequence of a type strain from a member of the family Sphingobacteriaceae to be published. The 4,635,236 bp long genome with its 3,854 protein-coding and 67 RNA genes consists of one chromosome, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 113 T (= DSM 12145 = LMG 10337 = NBRC 100064) is the type strain of the species Pedobacter saltans [1], one of currently 32 validly named species in the genus Pedobacter [2]. We prefer to use here the strain designation '113' as originally published by Steyn et al. in 1992 [3] and as also shown in the LMG online catalogue [4] and in the StrainInfo database [5] over the designation 'LMG 10337 T ' which was later used for the description of the species by the same authors [1]. The genus name is derived from the Latinized Greek word 'pedon' meaning 'the ground, earth' and the Neo-Latin word 'bacter' meaning 'rod', yielding 'Pedobacter', the 'rod from soil' [1]. The species epithet is derived from the Latin word 'saltare' meaning 'to dance', yielding 'saltans', referring to the gliding motility of the strain' [1]. P. saltans strain 113 T was isolated from soil in Iceland; several more strains belonging to the species were isolated from soil in Iceland, Belgium (Brussels) and Germany (Rüdesheim) [1]. Members of the genus Pedobacter were isolated from various environments including different soils [1,[6][7][8][9][10], water [11][12][13], a nitrifying inoculum [14], glaciers [15,16], fish [1] and compost [17]. Here we present a summary classification and a set of features for P. saltans strain 113 T , together with the description of the complete genome sequence and the genome annotation.

Classification and features
A representative genomic 16S rRNA sequence of strain 113 T was compared using NCBI BLAST [18,19] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [20] and the relative frequencies of taxa and keywords (reduced to their stem [21]) were determined, weighted by BLAST scores. The most frequently occurring genera were Pedobacter (53.4%), Sphingobacterium (33.3%), Mucilaginibacter (5.0%), Flavobacterium (4.1%) and 'Sphingoterrabacterium' (2.1%) (95 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 99.7%, whereas the average coverage by HSPs was 97.6%. Regarding the 20 hits to sequences from other members of the genus, the average identity within HSPs was 92.8%, whereas the average coverage by HSPs was 84.1%. Among all other species, the one yielding the highest score was Pedobacter lentus (EF446146), which corresponded to an identity of 93.2% and an HSP coverage of 93.4%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highestscoring environmental sequence was HM008274 ('anodic biomass air-cathode single chamber microbial fuel cell clone 9week.anode.2'), which showed an identity of 94.6% and an HSP coverage of 83.8%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were 'skin' (8.6%), 'fossa' (4.2%), 'poplit' (2.2%), 'soil' (2.2%) and 'forearm, volar' (2.0%) (152 hits in total). Interestingly, several of the most frequent keywords relate to a mammalian or clinical habitats, which may allude to some yet unknown ecological features of P. saltans, taking into account that all known isolates are from soil in different countries [1]. However, environmental samples which yielded hits of a higher score than the highest scoring species were not found. Figure 1 shows the phylogenetic neighborhood of P. saltans in a 16S rRNA based tree. The sequences of the four 16S rRNA gene copies in the genome differ from each other by up to one nucleotide, and differ by up to three nucleotides from the previously published 16S rRNA sequence (AJ438173). The cells of P. saltans are short rods (0.5 × 0.7-1.0 µm) with rounded or slightly tapering ends (Figure 2) [1]. Three of the four strains belonging to P. saltans were described as being motile via gliding [1]. P. saltans cells strain Gram-negative and are non-spore-forming (Table 1). Strain 113 T is strictly aerobic and chemoorganotrophic [1]. Colonies on modified TSA are smooth, light yellow to yellow, translucent, round, 2-5 mm in diameter, convex to slightly umbonate with entire margins [1]. On nutrient agar colonies are smooth, yellow, round, 2-4 mm in diameter, convex with entire to scalloped margins [1]. The temperature range for growth is normally between 5°C and 30°C [1]. The biochemical features and antibiotic resistance of P. saltans has been described previously [1]. Strain 113 T produces acetoin from sodium pyruvate, degrades chondroitin sulfate and hydrolyzes aesculin. It grows on heparin, which is degraded by inducible enzymes. Good growth occurs on nutrient agar or on regular or modified TSA. P. saltans does not produce H2S from thiosulfate and does not grow on MacConkey agar [1]. P. saltans can be differentiated phenotypically from other Pedobacter species by its inability to assimilate D-cellobiose and the ability to utilize glycerol. The organism does not reduce nitrate [1].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [37], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [38]. The genome project is deposited in the Genome OnLine Database [28] 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.

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [41]. Pyrosequencing reads were assembled using the Newbler assembler (Roche

Genome annotation
Genes were identified using Prodigal [46] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [47]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, Uni-Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [48].

Genome properties
The genome consists of a 4,635,236 bp long chromosome with a G + C content of 36.6% ( Figure 3 and Table 3). Of the 3,921 genes predicted, 3,854 were protein-coding genes, and 67 RNAs; 62 pseudogenes were also identified. The majority of the protein-coding genes (64.8%) 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.

Insights into the genome sequence
An estimate of the overall similarity between Pedobacter heparinus and P. saltans [1] was generated with the GGDC-Genome-to-Genome Distance Calculator [49,50]. This system calculates the distances by comparing the genomes to obtain highscoring segment pairs (HSPs) and interfering distances from a set of three formulae (1, HSP length / total length; 2, identities / HSP length; 3, identities / total length). The comparison of P. heparinus and P. saltans revealed that an average of only 4.7% of the two genomes are covered with HSPs. The identity within these HSPs was 82.3%, whereas the identity over the whole genome was 3.8%. The fraction of shared genes in the genomes of P. heparinus, P. saltans and Novosphingobium aromaticivorans [51] is shown in a Venn diagram ( Figure  4). The phyogentically distant reference genome of N. aromaticivorans was selected based on its similar genome size and due to a lack of complete reference type strain genomes from the Sphingobacteriaceae. The numbers of pairwise shared genes were calculated with the phylogenetic profiler function of the IMG ER platform [48]. The homologous genes within the genomes were detected with a maximum E-value of 10 -5 and a minimum identity of 30%. Only about one quarter of all genes (954 genes) are shared by all three genomes, whereas the two Pedobacter species share 2,732 genes, corresponding to 63.7% (P. heparinus) and 70.9% (P. saltans) of their genes. The pairwise comparison of N. aromaticivorans with the two Pedobacter species revealed only 154 (P. heparinus) and 65 (N. aromaticivorans) homologous genes (Figure 4). Among those genes that are shared by the three genomes, are those which might be responsible for the yellow color of the organisms. These genes encode enzymes that are involved in the synthesis of carotenoids. Biosynthesis of carotenoids starts with geranylgeranyl pyrophosphate synthases combining farnesyl pyrophosphate with C5 isoprenoid units to C20-molecules, geranylgeranyl pyrophosphate. The phytoene synthase catalyzes the condensation of two geranylgeranyl pyrophosphate molecules followed by the removal of diphosphate and a proton shift leading to the formation of phytoene. Sequential desaturation steps are catalyzed by phytoene desaturase followed by cyclization of the ends of the molecules catalyzed by the lycopene cyclase [52]. Genes encoding lycopene cyclases (Phep_2088, Pedsa_2222, Sa-ro_1817) and phytoene synthases (Phep_2092, Pedsa_2218, Saro_1814) were identified in the genomes. In the two Pedobacter species, genes coding for phytoene desaturases (Phep_2093, Pedsa_2217) were also identified. A carotene hydroxylase gene (Saro_1168) was only identified in the genome of N. aromaticivorans. As the two Pedobacter species are known for their ability to degrade heparin, it is not surprising that the genomes encode several heparinase encoding genes: seven (P. saltans) and five (P. heparinus) heparinases, were identified, whereas N. aromaticivorans encodes only one heparinase. Fucoidan degradation was not determined experimentally, but is assumed as both P. saltans and P. heparinus have genes for eleven and ten αfucosidases respectively. In addition, 12 (P. saltans) and 18 (P. heparinus) α-sulfatases genes were identified, whereas N. aromaticivorans contains only five α-sulfatases and no α-fucosidase genes. Experimental evidence for the fucoidan hydrolysis in Pedobacter has not been found, but for Mucilaginibacter paludis and M. gracilis, which are also members of the family Sphingobacteriaceae, have been experimentally confirmed to exhibit fucoidan degradation [53]. Moreover, Sakai et al. [54] reported the existence of intracellular α-Lfucosidases and sulfatases, which enable 'F. fucoidanolyticus' to degrade fucoidan.