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Novo2 [13], and iterative optimization was used to obtain the optimal k-mer value through the use of 31?5 k-mers. The 500-bp libraries were used to build scaffolds, and the SOAPdenovo gap closer software was also used (http:// soap.genomics.org.cn/soapdenovo.html). To close the remaining gaps, reference-guided assemblies were carried out with the CLC Genomics Workbench v. 6.05 (CLC bio, Aarbus, Denmark). The combination of de novo assembly and reference-guided assembly was performed manually using the microbial genome-finishing module in the CLC genomics workbench (CLC Staurosporine bio, Aarbus, Denmark). The complete genome sequence of P. pentosaceus ATCC 25745 was used as the reference genome.Genome annotationMethodsDetermination of cultural, morphological and physiological propertiesGrowth was investigated under different temperature, pH and NaCl conditions. Cell morphologies, motilities and sporulation activities were examined using transmission electron (H-600, Hitachi Ltd., Tokyo, Japan) microscopy. Phenotypic identification was achieved with API CH50 strips and the API CHL medium system according to the manufacturer's instructions (BioM ieux SA, Marcy-l'Etoile, France). Other physiological and biochemical tests were conducted as described previously [11]. Phylogenetic analysis was conducted using the neighbor-joining method based on the 16S rRNA and housekeeping gene sequences [12].Cultural conditions and DNA isolationP. pentosaceus LI05 genes were identified using Glimmer [14] together with comparative gene prediction by the direct mapping of the ORFs of the P. pentosaceus ATCC reference strain from the NCBI Genome Database. After a round of manual curation, the unannotated predicted coding sequences (CDS) were translated into amino acid sequences for a query using the NCBI non-redundant database as well as the UniProt, Pfam, COG, and InterPro databases to identify the closest existing homology annotations. Transfer RNA (tRNA) genes were detected using tRNAScanSE [15]. Ribosomal RNAs (rRNAs) were identified using a BLASTn [16] search against the ribosomal RNA databases. Signal peptides were predicted using SignalP 4.0 [17], whereas transmembrane helices in proteins were predicted using TMHMM [18]. The Integrated Microbial Genomes (IMG) platform (http://img.jgi. doe.gov/) was used to support additional gene prediction analyses and manual functional annotations [19].Comparative genomicsAfter revival using standard methods, the P. pentosaceus LI05 strain (CGMCC 7049) was anaerobically cultured in DeMan-Rogosa-Sharpe (MRS; OXOID, Thermo Fisher Biochemicals Ltd., Beijing, China) broth at 37 for 24 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 h. Cells were obtained by centrifugation at 8,000 g for 10 min at 4 . DNA was extracted using the QIAampA comparative genomic analysis using BRIG [20] was conducted comparing P. pentosaceus LI05 from the human gastrointestinal tract with three food-borne strains with available genomic sequences, including P. pentosaceus ATCC 25745, SL4 and IE-3. The P. pentosaceus LI05 genome sequences sharing low identities (<50 ) with the other strains were designated as the P. pentosaceus LI05-unique regions. The proteins encoded by theLv et al. Gut Pathogens 2014, 6:36 http://www.gutpathogens.com/content/6/1/Page 3 ofgenes that only existed in P. pentosaceus LI05 or that possessed sequence similarities of less than 50 with the three food-borne strains were further analyzed by BLASTp.P. pentosaceus ATCC 25745, P. pentosaceus SL4 and P. pentosaceus IE-3.Genome of P.