Complete genome sequence of Pedobacter heparinus type strain (HIM 762-3T)

Pedobacter heparinus (Payza and Korn 1956) Steyn et al. 1998 comb. nov. is the type species of the rapidly growing genus Pedobacter within the family Sphingobacteriaceae of the phylum ‘Bacteroidetes’. P. heparinus is of interest, because it was the first isolated strain shown to grow with heparin as sole carbon and nitrogen source and because it produces several enzymes involved in the degradation of mucopolysaccharides. All available data about this species are based on a sole strain that was isolated from dry soil. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first report on a complete genome sequence of a member of the genus Pedobacter, and the 5,167,383 bp long single replicon genome with its 4287 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Pedobacter heparinus strain HIM 762-3 (DSM 2366 = ATCC 13125 = JCM 7457 and other culture collections) is the type strain of the species, and was first described in 1956 by Payza and Korn as Flavobacterium heparinum (basonym) [1]. The authors of the original species description provided no type strain designation when depositing their isolate in the American Type Culture Collection (ATCC 13125 T ). In the Approved Lists of Bacterial Names (1980) the type strain of F heparinum ap-pears as ATCC 13125 T . Strain HIM 762-3 T was deposited in the DSMZ culture collection by Walter Mannheim (Marburg) in 1982, and ATCC is using the same strain designation for their ATCC 13125 T . Following successive transfers of this species to the genera Cytophaga [2] and Sphingobacterium [3] the present name P. heparinus was proposed by Steyn et al. in 1998 [4]. Enzymes produced by P. heparinus could be successfully used for the study of the structure of heparin and chondroitin, im-portant animal mucopolysaccharides with sulfate groups. Here we present a summary classification and a set of features for P. heparinus HIM 762-3 T (Table 1), together with the description of the complete genomic sequencing and annotation.

Classification and features
Until now the species P. heparinus has comprised only one strain, HIM 762-3 T . Two closely related strains, Gsoil 042 T and LMG 10353 T , were recently described and affiliated to the species P. panaciterrae [5] and P. africanus [4], respectively, based on low DNA-DNA binding values to the type strain of P. heparinus. Unclassified strains with significant (98%) 16S rRNA sequence similarity to these species were observed from Ginseng field soil (AM279216), dune grassland soil [6] and activated sludge samples [7]. Environmental genomic surveys indicated highly similar (96% 16S rRNA gene sequence identity) phylotypes in BAC libraries generated from Brassica rapa subsp. pekinensis (field mustard) and Sorghum bicolor (milo) (ED512136, DX082358, BZ779630). A draft genome sequence of the unclassified Pedobacter strain BAL39 isolated from the Baltic Sea was recently determined by the J. Craig Venter Institute (Genbank NZ_ABCM00000000). Figure 1 shows the phylogenetic neighborhood of P. heparinus strain HIM 762-3 T in a 16S rRNA based tree. The sequences of the three 16S rRNA gene copies in the genome are identical and differ by only one nucleotide from the previously published 16S rRNA gene sequence derived from DSM 2366 (AJ438172). Phylogenetic tree of P. heparinus strain HIM 762-3 T and the type strains of the genus Pedobacter, as well as all type strains of the other genera within the family Sphingobacteriaceae, inferred from 1373 aligned characters [8,9] of the 16S rRNA gene under the maximum likelihood criterion [10]. The tree was rooted with the type strains of the other families within the order 'Sphingobacteriales'. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [11] are shown in blue, published genomes in bold. P. heparinus cells are Gram-negative, nonflagellated, non-spore-forming, flexible rods with rounded or slightly tapered ends. Cell width is 0.4-0.5 µm and cell length can vary from 0.7 to 6 µm. Protrusions can be observed on the cell surface ( Figure 2). Some authors have reported a gliding motility [2]. Colonies are 1-4 mm in diameter and produce a yellowish, water insoluble, nonfluorescent pigment upon growth on nutrient agar [4]. Growth occurs at 10 and 35°C, but not above 37°C. The optimal temperature for growth is between 25 and 30°C [2]. The pH range for growth is 7-10 [2]. Strain HIM 762-3 T is strictly aerobic and prefers carbohydrates and sugars as carbon sources. Neither nitrate nor nitrite is reduced. The strain is catalase and oxidase positive. Acetoin is produced from pyruvate, but indole is not produced from tryptophan. HIM 762-3 T is negative for gelatinase, urease and DNase, but esculin and Tween 20-80 are hydrolyzed; acid and alkaline phosphatases are present [4]. The strain does not require vitamins, but L-histidine is essential for growth [12]. A special feature of strain HIM 762-3 T is its ability to degrade acidic sulfated mucoheteropolysaccharides, like heparin and chondroitin that are formed in various animal tissues. Enzymes involved in the degradation of heparin are only produced after induction by the substrate and are formed intracellularly [12]. Several different types of enzymes are involved in the complete degradation of heparin, including heparinases, glycuronidase, sulfoesterases and sulfamidases [13]. The first step in the degradation of heparin is catalyzed by heparinase (EC 4.2.2.7), an α1-4eliminase which acts specifically on the glycosidic linkage between N-sulfated D-glucosamine and sulfated D-glucuronic acid (or L-iduronic acid). The use of heparinase in the elucidation of the structure of heparin, blood deheparinization or enzymatic assay of heparin have been proposed [12]. The genetics of heparin and chondrotitin degradation in P. heparinus was studied extensively and a high-level expression system for glycosaminoglycan lyases in this species has been developed [14]. Three different genes encoding heparinases (hepA-C) and two different genes for chondroitinases (cslA and cslB) could be characterized [14]. The crystal structures of the chondroitinase B [15] and heparinase II [16] of P. heparinus were resolved at high resolution.

Chemotaxonomy
The peptidoglycan structure of strain HIM 762-3 T is still unknown. The cellular fatty acid pattern is dominated by saturated, iso branched and hydroxylated acids. The most abundant non-polar cellular fatty acids are iso-15:0, 16:1 ω7c, iso-17:0 (3-OH), and iso-15:0 (2-OH) [4]. Large amounts of long-chain bases are formed, one of which has been identified as dihydrosphingosin [3]. Strain HIM 762-3 T contains menaquinone MK-7. Altitude not reported Evidence codes -IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [20]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Genome sequencing and annotation
Genome project history A summary of the project information is shown in Table 2. Growth conditions and DNA isolation P. heparinus strain HIM 762-3 T , DSM 2366, was grown in DSMZ medium 1 (Nutrient Brot) at 28°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modified protocol for cell lysis, adding additonal 100 µl lsozyme; 500 µl chromopeptidase, lysostaphin, mutanolysin, each, to the standard lysis solution, but reducing proteinase K to 160µl, only. Lysis solution was incubated overnight at 35°C on a shaker.

Genome sequencing and assembly
The genome was sequenced using Sanger sequencing platform only. All general aspects of library construction and sequencing performed at the DOE JGI can be found on their website. 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 Soft ware, LLC). Possible mis-assemblies were corrected with Dupfinisher [21] or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 1,897 finishing reactions were produced to close gaps and to raise the quality of the finished sequence. The completed genome sequences of P. heparinus contains 45,821 Sanger reads, achieving an average of 7.5 x sequence coverage per base with an error rate less than 1 in 100,000.

Genome annotation
Genes were identified using Prodigal [22] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [23]. 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 (IMG-ER) platform [24].

Genome properties
The genome is 5,167,383 bp long and comprises one main circular chromosome with a 42.1% GC content (Table 3, Figure 3). Of the 4,341 genes predicted, 4,287 were protein coding genes, and 54 RNAs. Thirty-five pseudogenes were also identified. A minority of the genes (38.1%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4.