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Non-contiguous finished genome sequence of Phocaeicola abscessus type strain 7401987T
Standards in Genomic Sciencesvolume 9, pages351–358 (2013)
Phocaeicola abscessus strain 7401987T is the sole member of the genus Phocaeicola. This bacterium is Gram-negative, non-spore-forming, coccoid to rod-shaped and motile by lophotrichous flagella. It was isolated from a human brain abscess sample. In this work, we describe a set of features of this organism, together with the complete genome sequence and annotation. The 2,530,616 bp long genome contains 2,090 protein-coding genes and 54 RNA genes, including 4 rRNA operons.
Phocaeicola abscessus strain 7401987T(CSUR P22T= DSM 21584T= CCUG 55929T) is the type strain of P. abscessus. This bacterium was isolated from a brain abscess sample from a 76-year-old patient who underwent neurosurgical intervention after cancer of the face . It is a Gram-negative strictly anaerobic coccoid to rod-shaped bacterium. Currently, the genus Phocaeicola contains only one species .
Here we present a summary classification and a set of features for P. abscessus, together with the description of the non-contiguous finished genomic sequencing and annotation.
Classification and features
The 16S rRNA gene sequence of P. abscessus strain 7401987T was compared with sequences deposited in the Genbank database, confirming the initial taxonomic classification. Figure 1 shows the phylogenetic neighborhood of P. abscessus in a 16S rRNA based tree. The bacterium was characterized in 2007. It was isolated in the Timone Hospital microbiology laboratory (Table 1).
Cells are coccoid (0.3–0.6 µm wide and 0.4–0.9 µm long) to rod-shaped (0.4–1.7 µm wide and 1.2–6.5 µm long) and motile by flagella in a lophotrichous arrangement. Optimal growth of strain 7401987T occurs at 37°C with range for growth between 30 and 37 °C. Surface colonies on chocolate agar after 7 days incubation at 37 °C under anaerobic conditions were white, circular, regular, smooth, shiny, convex and 1 mm in diameter. The isolate was asaccharolytic. Activities of acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-β-glucosaminidase, α-fucosidase, α-galactosidase, β-galactosidase, β-galactosidase 6-phosphate, α-glucosidase, N-acetyl-β-glucosaminidase, alkaline phosphatase, leucyl glycine arylamidase and alanine arylamidase were detected. The fatty acid profile was characterized by the predominance of anteiso-C15:0 (28.2%), C16:0 (18.0%), iso-C15:0 (12.3%) and iso-C17:03-OH (11.7%). The size and ultrastructure of cells were determined by negative staining transmission electron microscopy. (Figure 2). Cells are coccoid (0.3–0.6 µm wide and 0.4–0.9 µm long) to rod-shaped (0.4–1.7 µm wide and 1.2–6.5 µm long).
Genome sequencing and annotation
Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the order Bacteroidales and is part of study of the new species characterized in our laboratory. A summary of the project information is shown in Table 2. The EMBL accession number is CAKQ01000000 and consists of 39 contigs (≥ 500 bp) and 9 scaffolds. Table 2 shows the project information and its association with MIGS version 2.0 compliance.
Growth conditions and DNA isolation
P. abscessus strain 7401987T, was grown anaerobically on chocolate agar at 37°C. Ten petri dishes were spread and resuspended in 3 ml of TE buffer. Three hundred µl of 10% SDS and 150 µl of proteinase K were then added and incubation was performed overnight at 56°C. The DNA was then extracted using the phenol/chloroform method. The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 88 ng/µl.
Genome sequencing and assembly
Shotgun and 3-kb paired-end sequencing strategies were performed. The shotgun library was constructed with 500 ng of DNA with a GS Rapid library Prep kit (Roche). For the paired-end sequencing, 5 µg of DNA was mechanically fragmented on a Hydroshear device (Digilab) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized using a 2100 BioAnalyzer (Agilent) on a DNA labchip 7500 with an optimal size of 3.1 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimal size of 579 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was then quantified using a Genios fluorometer (Tecan) at 8,770 pg/µL. The library concentration equivalence was calculated as 1.39E+10 molecules/µL. The library was stored at −20°C until further use.
The shotgun and paired-end libraries were clonally-amplified with 0.5 cpb and 2 cpb in 3 and 2 SV-emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 9.63% and 10.3%, respectively, in the 5 to 20% range from the Roche procedure. Approximately 790,000 beads for the shotgun application and for the 3kb paired end were loaded on a GS Titanium PicoTiterPlate PTP Kit 70x75 and sequenced with a GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 311,276 passed filter wells were obtained and generated 35.9 Mb with a length average of 282 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 9 scaffolds and 39 contigs (>500 bp).
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they were spanning a sequencing GAP region. The predicted bacterial protein sequences were searched against the GenBank database  and the Clusters of Orthologous Groups (COG) databases  using BLASTP. The tRNAscan-SE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer . Transmembrane domains and signal peptides were predicted using TMHMM  and SignalP , respectively. ORFans of alignment length greater than 80 amino acids were identified if their BLASTp E-value was lower than 1e-03. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Such parameter thresholds have been used in previous works to define ORFans.
To estimate the mean level of nucleotide sequence similarity at the genome level between P. abscessus and Prevotella timonensis, Bacteroides thetaiotaomicron and Paraprevotella clara, we compared the ORFs using only comparison sequences in the RAST server  at a query coverage of ≥70% and a minimum nucleotide length of 100 bp.
The genome is 2,530,616 bp long with a 47.31% GC content (Table 3, Figure 3). Of the 2,144 predicted genes, 2,090 were protein-coding genes, and 54 were RNAs. A total of 1,464 genes (70.05%) were assigned a putative function. A total of 112 genes were identified as ORFans (5.39%). The remaining genes were annotated as hypothetical proteins (436 genes (20.86%)). The remaining genes were annotated as either hypothetical proteins or proteins of unknown functions. The distribution of genes into COGs functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 3 and 4. Two CRISPRs were found using CRISPERfinder program online . The first one on contig 1 includes at least 3 predicted spacer regions and the second one on contig 18 includes at least 53 predicted spacer regions.
Comparison with other genomes
Phocaeicola abscessus is the sole bacterium included in the genus Phocaeicola. We compared the genome of P. abscessus with those of Prevotella timonensis (CBQQ010000001) Paraprevotella clara (AFFY01000000) and Bacteroides thetaiotaomicron (AE015928.1). P. abscessus showed a mean nucleotide sequence similarity of 76.40%, 77.06% and 77.52% at the genome level (range 70–92.25%, 70.04–95.51% and 70.04–93.02%) with P. timonensis, P. clara and B. thetaiotaomicron, respectively. Presently, the family to which P. abscessus belongs is undetermined and the sole comparison based on nucleotide sequence similarity may not be sufficient to answer this question. In the future, further comparison of the genomes will allow us to find traits to classify the genus Phocaeicola in one of these 3 families or to create a new family, the family Phocaeicolaceae.
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The authors thank Mr. Julien Paganini at Xegen Company (www.xegen.fr) for automating the genomic annotation process and Laetitia Pizzo for her technical assistance.