Non-contiguous finished genome sequence of the opportunistic oral pathogen Prevotella multisaccharivorax type strain (PPPA20T)

Prevotella multisaccharivorax Sakamoto et al. 2005 is a species of the large genus Prevotella, which belongs to the family Prevotellaceae. The species is of medical interest because its members are able to cause diseases in the human oral cavity such as periodontitis, root caries and others. Although 77 Prevotella genomes have already been sequenced or are targeted for sequencing, this is only the second completed genome sequence of a type strain of a species within the genus Prevotella to be published. The 3,388,644 bp long genome is assembled in three non-contiguous contigs, harbors 2,876 protein-coding and 75 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain PPPA20 T (= DSM 17128 = JCM 12954) is the type strain of Prevotella multisaccharivorax [1]. Currently, there are about 50 species placed in the genus Prevotella [1]. The species epithet is derived from the Latin adjective multus meaning 'many/much', the Latin noun saccharum meaning 'sugar' and the Latin adjective vorax meaning 'liking to eat' referring to the metabolic properties of the species to digest a variety of carbohydrates [2]. P. multisaccharivorax strain PPPA20 T is considered to be an opportunistic pathogen and was isolated from subgingival plaque from a patient with chronic periodontitis. Additionally, five more strains isolated from the human oral cavity were placed in the species P. multisaccharivorax [2]. Using non-culture techniques on sites affected by endodontic and periodontal diseases, a large number of sequences have been found that belong to Prevotella and Prevotella-like bacteria. Many of those species have never been isolated or described [3]. The complex microbial community living in the rich ecological niche of the human oral cavity and its interaction with consumed food will be of lasting interest for medical and ecological reasons [4,5]. Here we present a summary classification and a set of features for P. multisaccharivorax PPPA20 T , together with the description of the non-contiguous finished genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of P. multisaccharivorax PPPA20 T was compared using NCBI BLAST [6] 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 [7] and the relative frequencies of taxa and keywords (reduced to their stem [8]) were determined, weighted by BLAST scores. The most frequently occurring genus was Prevotella (100.0%) (14 hits in total). Regarding the single hit to sequences from members of the species, the average identity within HSPs was 100.0%, whereas the average coverage by HSPs was 98.0%. Regarding the nine hits to sequences from other members of the genus, the average identity within HSPs was 90.3%, whereas the average coverage by HSPs was 66.5%. Among all other species, the one yielding the highest score was Prevotella ruminicola (AF218618), which corresponded to an identity of 91.5% and an HSP coverage of 66.3%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was AY550995 ('human carious dentine clone IDR-CEC-0032'), which showed an identity of 99.8% and an HSP coverage of 94.5%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were 'fecal' (4.4%), 'beef, cattl' (4.1%), 'anim, coli, escherichia, feedlot, habitat, marc, pen, primari, secondari, stec, synecolog' (4.0%), 'neg' (2.5%) and 'fece' (2.4%) (236 hits in total). The most frequently occurring keywords within the labels of environmental samples which yielded hits of a higher score than the highest scoring species were 'fece' (7.9%), 'goeldi, marmoset' (4.8%), 'microbiom' (4.3%), 'aspect, canal, oral, root' (3.9%) and 'rumen' (3.8%) (54 hits in total). While some of these keywords correspond to the well known habitat of P. multisaccharivorax, others indicate additional habitats related to animals. Figure 1 shows the phylogenetic neighborhood of P. multisaccharivorax 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 two nucleotides, and differ by up to two nucleotides from the previously published 16S rRNA sequence AB200414. The cells of P. multisaccharivorax generally have the shape of rods (0.8 × 2.5-8.3 µm) and occur singly or in pairs ( Figure 2). They can also form longer filaments. P. multisaccharivorax is a Gram-negative, non spore-forming bacterium ( Table 1). The organism is described as non-motile and only four genes associated with motility were identified in the genome (see below). P. multisaccharivorax grows well at 37°C, is strictly anaerobic, chemoorganotrophic and is able to ferment cellobiose, glucose, glycerol, lactose, maltose, mannose, melezitose, raffinose, rhamnose, sorbitol, sucrose, trehalose and xylose [2]. Acid production from arabinose and salicin is variable. The organism does not reduce nitrate or produce indole from tryptophan but it hydrolyzes esculin and digests gelatin [2]. Growth of P. multisaccharivorax is inhibited by the addition of 20% bile. Major fermentation products are succinic and acetic acid, isovaleric acid is produced in small amounts [2]. Activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6GPDH) were not detected in isolates of this species, whereas malate dehydrogenase and glutamate dehydrogenase activities were detected in all strains. P. multisaccharivorax produces acid and alkaline phosphatase, β-galactosidase, αand β-glucosidase, N-acetyl-β-glucosaminidase, αaminofuranosidase and alanine aminopeptidase. The organism has no demonstrable esterase (C4), esterase lipase (C4), lipase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, pyroglutamic acid arylamidase, trypsin, chymotrypsin, β-glucuronidase, α-mannosidase, α-fucosidase, arginine aminopeptidase, leucine aminopeptidase, proline aminopeptidase, tyrosine aminopeptidase, phenylalanine aminopeptidase, urease or catalase activity [2].

Chemotaxonomy
In contrast to other Prevotella species all strains of P. multisaccharivorax harbor the menaquinones MK-12 (40-55%) and MK-13 (40-45%) in large amounts, whereas MK-10 (1-3%) and MK-11 (8-10%) were found only in small amounts [2]. The fatty acid pattern for all strains of P. multisaccharivorax revealed C18:1 ω9c (21.7%) and C16:0 (12.9%) as major compounds as well as iso-C17:0 3-OH (9.2%), anteiso-C15:0 (7.8%), C18:2 ω6,9c (7.5%) and iso-C15:0 (6.4%) in smaller amounts [2]. Additionally, the unusual dimethyl acetals were found with C16:0 dimethyl aldehyde in the highest amount of 8.2%. This clearly distinguishes the species of P. multisaccharivorax from other related Prevotella species [2]. Phylogenetic tree highlighting the position of P. multisaccharivorax relative to the type strains of the other species within the family. The tree was inferred from 1,425 aligned characters [9,10] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [11]. Rooting was done initially using the midpoint method [12] and then checked for its agreement with the current classification ( Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 600 ML bootstrap replicates [13] (left) and from 1,000 maximum parsimony bootstrap replicates [14] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [15] are labeled with one asterisk, those also listed as 'Complete and Published' should be labeled with two asterisks: P. ruminicola [16] and P. melaninogenica (CP002122/CP002123) Standards in Genomic Sciences  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 of the Gene Ontology project [25]. If the evidence code is IDA, 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 [26], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [27]. The genome project is deposited in the Genomes On Line Database [15] 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.

Growth conditions and DNA isolation
P. multisaccharivorax PPPA20 T , DSM 17128, was grown anaerobically in DSMZ medium 104 (PYGmedium) [28] at 37°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/DL for cell lysis as described in Wu et al. 2009 [27]. DNA is available through the DNA Bank Network [29].

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 [30]. Pyrosequencing reads were assembled using the Newbler assembler (Roche

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) 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 [37].

Genome properties
The assembled genome sequence consists of three non-contiguous contigs with a length of 3,334,154 bp, 47,474 bp and 7,016 bp with a G+C content of 48.3% (Figure 3 and Table 3). Of the 2,951 genes predicted, 2,876 were protein-coding genes, and 75 RNAs; 166 pseudogenes were also identified. The majority of the protein-coding genes (60.5%) 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.