Complete genome sequence of Paludibacter propionicigenes type strain (WB4T)

Paludibacter propionicigenes Ueki et al. 2006 is the type species of the genus Paludibacter, which belongs to the family Porphyromonadaceae. The species is of interest because of the position it occupies in the tree of life where it can be found in close proximity to members of the genus Dysgonomonas. This is the first completed genome sequence of a member of the genus Paludibacter and the third sequence from the family Porphyromonadaceae. The 3,685,504 bp long genome with its 3,054 protein-coding and 64 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain WB4 T (= DSM 17365 = CCUG 53888 = JCM 13257) is the type strain of P. propionicigenes, which is the type species of the genus Paludibacter [1,2]. Currently, there is only one species placed in the genus Paludibacter [1]. The generic name derives from the Latin noun palus -udis meaning swamp or marsh and the Neo-Latin word bacter meaning a rod, referring to a rod living in swamps [2]. The species epithet is derived from the Neo-Latin word acidum propionicum meaning propionic acid and the Greek verb gennao meaning to produce, referring to the metabolic property of the species [2]. P. propionicigenes strain WB4 T was isolated together with a number of other strains from rice plant residues in an anoxic rice-field soil in Yamagata, Japan, and described for the first time by Akasaka et al. in 2003 [3]. In 2006 the species was formally described by Ueki et al. and the genus Paludibacter was introduced [2]. No further isolates have been obtained for P. propionicigenes, however, cultivation-independent 16S rRNA-dependent molecular investigations showed the presence of P. propionicigenes in the rumen of sheep [4]. Here we present a summary classification and a set of features for P. propionicigenes WB4 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of strain WB4 T was compared using NCBI BLAST under default values (e.g., considering only the best 250 hits) with the most recent release of the Greengenes database [5] and the relative frequencies, of taxa and keywords (reduced to their stems [6]) were determined, weighted by BLAST scores. The most frequently occurring genus was Dysgonomonas (100%) (8 hits in total). Among all other species, the one yielding the highest score was Dysgonomonas capnocytophagoides, which corresponded to an identity of 91.9% and a HSP coverage of 83.6%. The highest-scoring environmental sequence was AY212569 ('water 10 m down-stream manure clone 118ds10'), which showed an identity of 99.6% and a HSP coverage of 100.1%. The five most frequent keywords within the labels of environmental samples which yielded hits were 'digest' (11.7%), 'anaerob' (6.2%), 'sludge' (6.1%), 'wastewater' (6.0%) and 'mesophile' (5.9%) (241 hits in total). The single most frequent keyword within the labels of environmental samples which yielded hits of a higher score than the highest scoring species was 'downstream/manure/water' (33.3%) (1 hit in total). Figure 1 shows the phylogenetic neighborhood of P. propionicigenes WB4 T in a 16S rRNA based tree. The three identical 16S rRNA sequences in the genome differ by one nucleotide from the previously published 16S rRNA sequence (AB078842). propionicigenes relative to the other type strains within the family Porphyromonadaceae. The tree was inferred from 1,400 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood criterion [9] and rooted in accordance with the current taxonomy. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 300 bootstrap replicates [10] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [11] are shown in blue, published genomes in bold [12,13].
The cells of P. propionicigenes are generally rodshaped (0.5-0.6 μm × 1.3 -1.7 µm) with ends that are round or slightly tapered [2]. Elongated cells can also be seen, either as single cells or in short chains ( Figure 2). P. propionicigenes is a Gramnegative and non spore-forming bacterium ( Table  1). The organism is described to be nonmotile; only eight genes associated with motility were identified in the genome. The organism is strictly anaerobic and chemoorganotrophic [2,3]. The temperature range for growth is between 15°C and 35°C, with an optimum at 30°C [2]. The organism does not grow at 37°C [2]. The pH range for growth is 5.0-7.6 with an optimum at pH 6.6 [2]. NaCl concentrations from 0-0.5% (w/v) are tolerated. P. propionicigenes is able to utilize arabinose, glucose, fructose, xylose, cellobiose, galactose, mannose, maltose, melibiose, glycogen and soluble starch as growth substrates [2]. The organism does not utilize ribose, lactose, sucrose, melezitose, raffinose, sorbose, rhamnose, trehalose, cellulose, xylan, salicin, dulcitol, inositol, mannitol, sorbitol, ethanol, glycerol, fumarate, ma-late, lactate, succinate or pyruvate [2]. Glucose is fermented to propionate and acetate in a molar ratio of 2:1 as major products and succinate as a minor product [2]. The organism does not reduce nitrate, it does not hydrolyze gelatin or urea and does not produce indole or hydrogen sulfide [2]. P. propionicigenes does not grow in the presence of bile salts. Catalase and oxidase are not present in the organism [2].

MIGS ID Property Term Evidence code
Current classification Domain Bacteria TAS [15] Phylum Bacteroidetes TAS [16] Class Bacteroidia TAS [16,17] Order Bacteroidales TAS [16] Family Porphyromonadaceae TAS [16] Genus Paludibacter TAS [2] Species Paludibacter propionicigenes TAS [2] Type strain WB4 TAS [2] Gram stain negative TAS [3] Cell shape rod-shaped TAS [3] Motility 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 of the Gene Ontology project [19]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [20], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [21]. The genome project is deposited in the Genomes OnLine Database [11] and the complete genome sequence is deposited in GenBank. 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 [23]. Pyrosequencing reads were assembled using the Newbler assembler version 2.3-PreRelease-10-21-2009-gcc-4.1.2-threads (Roche). The initial Newbler assembly consisting of 26 contigs in one scaffold which was converted into a phrap assembly by [24] making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (967 Mb) was assembled with Velvet [25] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 93.4 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package was used for sequence assem-bly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [23], Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [26]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 124 additional reactions and one shatter library were necessary to close the gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [27]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 365.7 × coverage of the genome. The final assembly contained 333,397 pyrosequence and 34,564,373 Illumina reads.

Genome annotation
Genes were identified using Prodigal [28] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [29]. The predicted CDSs were translated and used to search the National Center for Biotechnology In-formation (NCBI) nonredundant 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 [30].

Genome properties
The genome consists of a 3,685,504 bp long chromosome with a GC content of 38.9% (Figure 3 and Table 3). Of the 3,118 genes predicted, 3,054 were protein-coding genes, and 64 RNAs; 34 pseudogenes were also identified. The majority of the protein-coding genes (65.8%) were assigned with 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.