Non-contiguous finished genome sequence and description of Paenibacillus senegalensis sp. nov.
© The Author(s) 2012
Published: 10 October 2012
Paenibacillus senegalensis strain JC66T, is the type strain of Paenibacillus senegalensis sp. nov., a new species within the genus Paenibacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. P. senegalensis strain JC66T is a facultative Gram-negative anaerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,581,254 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 48.2% and contains 5,008 protein-coding and 51 RNA genes, including 9 rRNA genes.
Paenibacillus senegalensis strain JC66T (= CSUR P157 = DSM 25958) is the type strain of P. senegalensis sp. nov. This bacterium was isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aiming at cultivating all species within human feces, individually. It is a Gram-negative, facultative anaerobic, indole-negative rod.
We recently proposed to include genomic data among other criteria to describe new bacterial species, rather than relying on the poorly reproducible DNA-DNA hybridization and G+C content determination . This strategy creates a polyphasic approach by combining  the use of 16S rRNA sequence cutoff values  with the plethora of new information provided by high throughput genome sequencing and mass spectrometric analyses of bacteria .
Here we present a summary classification and a set of features for P. senegalensis sp. nov. strain JC66T together with the description of the complete genomic sequencing and annotation. These characteristics support the creation of the P. senegalensis species.
To date, the genus Paenibacillus (Ash et al. 1994) includes Gram-variable, facultative anaerobic, endospore-forming bacteria, originally classified within the genus Bacillus and then reclassified as a separate genus in 1993 . The genus consists of 134 described species and 4 subspecies that have been isolated from a variety of environments including soil, water, rhizosphere, vegetable matter, forage and insect larvae, as well as human specimens [6–9]. The bacteria belonging to this genus produce various extracellular enzymes such as polysaccharide-degrading enzymes and proteases, and have gained importance in agriculture, horticulture, industrial and medical applications . Various Paenibacillus spp. also produce antimicrobial substances that are active on a wide spectrum of microorganisms such as fungi, soil bacteria, plant pathogenic bacteria and even important anaerobic pathogens such as Clostridium botulinum . In addition, several Paenibacillus bacteria can form complex patterns on semi-solid surfaces that require self-organization and cooperative behavior of individual cells by employing sophisticated chemical communication . Pattern formation and self-organization of bacteria within this genus reflect their social behavior and might provide insights into the evolutionary development of the collective action of cells in higher organisms . To the best of our knowledge, this is the first report of isolation of Paenibacillus sp. from the normal fecal flora.
Classification and features
Classification and general features of Paenibacillus senegalensis strain JC66T
Species Paenibacillus senegalensis
Type strain JC66T
growth in BHI medium + 5% NaCl
Sample collection time
51 m above sea level
Strain JC66T exhibited catalase activity but was negative for indole production. Using API 50CH, positive reactions were observed for D-galactose, D-glucose, D-fructose, D-mannose, and D-sorbitol fermentation. Positive reactions were also observed for N-acteylglucosamine arbutine, esculine, salicine, D-maltose, D-lactose, D-saccharose, D-trehalose, inuline and D-tagatose. Using API ZYM, positive reactions were observed for leucine arylamidase and weak reactions were observed for alkaline phosphatase, esterase lipase, acid phosphatase and naphtol-AS-BI-phosphohydrolase. Using API Coryne, positive reactions were observed for β-glucuronidase, phosphatase alkaline, α-glucosidase, α-galactosidase, and N-acetyl-β-glucosaminidase activities. P. senegalensis is susceptible to amoxicillin, ceftriaxone, imipenem, trimethoprim/sulfamethoxazole, ciprofloxacin, rifampin and vancomycin, but resistant to metronidazole.
Genome sequencing information
Genome project history
One 454 paired end 3-kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
Genbank Date of Release
January 3, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
P. senegalensis sp. nov. strain JC66T, (= CSUR P157 = DSM 25958), was grown on blood agar medium at 37°C. Ten petri dishes were spread and resuspended in 5x100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system) from MP Biomedicals, USA for 40 seconds. DNA was then incubated for a lysozyme treatment (30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 40.2 ng/µl.
Approximately 340,000 beads for the 1/8 region for the shotgun and 790,000 beads on the 1/4 region for the paired-end were loaded on the GS Titanium PicoTiterPlates PTP kit 70×75 and sequenced with the GS Titanium Sequencing Kit XLR70 (Roche). The runs were performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 370,625 passed filter wells were obtained and generated 115.35Mb with an average length of 312 bp. These sequences were assembled using Newbler with 90% identity and 40bp as overlap. The final assembly identified 197 large contigs (>1500bp) arranged into 18 scaffolds, for a genome size of 5.58Mb, which corresponds to a 21.01 × coverage.
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 tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer  and BLASTn against the NR database. Lipoprotein signal peptides and transmembrane helices were predicted using SignalP  and TMHMM , respectively. ORFans were identified if their BLASTP E-value was lower than 1e-03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Such parameter thresholds have already been used in previous works to define ORFans. To estimate the mean level of nucleotide sequence similarity at the genome level between Paenibacillus species, we compared the ORFs only using BLASTN and the following parameters: a query coverage of ≥ 70% and a minimum nucleotide length of 100 bp. Artemis  was used for data management and DNA Plotter  was used for visualization of genomic features. Mauve alignment tool was used for multiple genomic sequence alignment .
Nucleotide content and gene count levels of the genome
% of Totala
G+C content (bp)
Coding region (bp)
Number of replicons
Genes with function prediction
Genes assigned to COGs
Genes with peptide signals
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Comparison with the genomes from other Paenibacillus species
P. senegalensis shares mean degrees of sequence similarity at the genome level of 81.5% (range 70.34–100%), 80.6% (range 70.46–100%) and 81.32% (range 70.34–100%) with P. polymyxa, P. mucilaginosus and P. terrae, respectively.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Paenibacillus senegalensis sp. nov. that contains the strain JC66T. This bacterium has been found in Senegal.
Description of Paenibacillus senegalensis sp. nov.
Paenibacillus senegalensis (se.ne.gal.e’n.sis. L. gen. masc. n. senegalensis, pertaining to Senegal, the country from which the specimen was obtained).
Colonies are 2 mm in diameter on blood-enriched Columbia agar. Cells are rod-shaped with a mean diameter of 0.66 µm. Optimal growth is achieved in aerobic condition with or without CO2. Weak growth is observed in microaerophilic and anaerobic conditions. Growth occurs between 30 and 45°C, with optimal growth observed at 37°C, on blood-enriched agar. Cells are Gram-negative, endospore-forming, and motile. Cells are catalase positive but negative for indole production. D-galactose, D-glucose, D-fructose, D-mannose, D-sorbitol, N-Acetylglucosamine arbutine, esculine, salicine, D-maltose, D-lactose, D-saccharose, D-trehalose, inuline, D-tagatose, β-glucuronidase, phosphatase alkaline, α-glucosidase, α-galactosidase, and N-acetyl-β-glucosaminidase metabolic activities are present. Weak alkaline phosphatase, esterase lipase, acid phosphatase and naphtol-AS-BI-phosphohydrolase activities are observed. Cells are susceptible to amoxicillin, ceftriaxone, imipenem, trimethoprim/sulfamethoxazole, ciprofloxacin, rifampin and vancomycin, but resistant to metronidazole. The G+C content of the genome is 48.2%. The 16S rRNA and genome sequences are deposited in Genbank under accession numbers JF824808 and CAES00000000, respectively. The type strain is JC66T (= CSUR P157 = DSM 25958) was isolated from the fecal flora of a healthy patient in Senegal.
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