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Non-contiguous finished genome sequence and description of Paenibacillus gorillae sp. nov.
Standards in Genomic Sciences volume 9, pages1031–1045 (2014)
Strain G1T sp. nov. is the type strain of Paenibacillus gorillae a newly proposed species within the genus Paenibacillus. This strain, whose genome is described here, was isolated in France from the fecal sample of a wild western lowland gorilla from Cameroon. P. gorillae is a facultative anaerobic, Gram-negative, rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,257,967 bp long genome (one chromosome but no plasmid) contains 5,856 protein-coding and 62 RNAs genes, including 60 tRNA genes.
Strain G1T (= CSUR P205 = DSM 26181) is the type strain of Paenibacillus gorillae sp. nov. This bacterium is a Gram-negative, flagellated, facultative anaerobic, indole-negative bacillus that has rounded-ends. It was isolated from the stool sample of Gorilla gorilla subsp. gorilla as part of a culturomics study aiming at cultivating bacterial species found within gorilla feces. By applying large-scale culture conditions, culturomics has previously facilitated the isolation of many new bacterial species from human stool samples [1–3].
The genus Paenibacillus was created by Ash et al. about 20 years ago [4,5]. To date, this genus comprises 145 validly published species  of Gram-positive, Gram-negative or variable, mostly motile and spore-forming bacteria. Members of the genus Paenibacillus are ubiquitous bacteria isolated from various environments including soil, water, rhizosphere, food, insect larvae and normal human flora . Moreover, Paenibacillus species were also isolated from or involved in human infections including wound infections, bacteremia and endocarditis [8–13]. Currently, a polyphasic approach that combines proteomics by MALDI-TOF spectral analysis, genomic data and phenotypic characterization is being used as a new approach to describe bacterial species [7,14–25].
Here we present a summary classification and a set of features for P. gorillae sp. nov. strain G1T together with the description of the complete genome sequence and annotation. These characteristics support the circumscription of the species P. gorillae .
Classification and features
In July 2011, a fecal sample was collected from a wild western lowland gorilla near Minton, a village in the south-central part of the DJA FAUNAL Park (Cameroon). The collection of the stool sample was approved by the Ministry of Scientific Research and Innovation of Cameroon. No experimentation was conducted on this gorilla. The fecal specimen was preserved at −80°C after collection and sent to Marseille. Strain G1T (Table 1) was isolated in January 2012 by cultivation on Columbia agar with sheep blood 5% (BioMerieux, France). This strain exhibited a 98.28% 16S rRNA nucleotide sequence similarity with Paenibacillus xinjiangensis, the phylogenetically closest validly published Paenibacillus species (Figure 1). This value was lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization .
Different growth temperatures (25, 30, 37, 45°C) were tested. Growth occurred for the temperatures (25°C-37°C), but the optimal growth was observed at 25°C. Colonies were 2–8 mm in diameter on Columbia agar, appear whitish in color at 25°C and produce a clear liquid. Growth of the strain was tested under anaerobic and microaerophilic conditions using the GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and in aerobic conditions, with or without 5% CO2. Growth was achieved under aerobic (with and without CO2), microaerophilic and anaerobic conditions. Gram staining showed Gram-negative bacilli (Figure 2). A motility test was positive. Cells grown on agar sporulate and the rods have a length ranging from 2.5 to 3.97 µm (mean 3.2 µm) and a diameter ranging from 0.76 to 0.83 µm (mean 0.79 µm) as determined by negative staining transmission electron microscopy (Figure 3).
Strain G1T exhibited oxidase activity but not catalase activity. Using the API 50CH system (BioMerieux), a positive reaction was observed for D-mannose, amygdalin, L-arabinose, cellobiose, lactose, D-xylose, D-glucose, mannitol, arabinose, xylose, glycerol, D-galactose, N-acetylglucosamine, arbutin, aesculin, D-sorbitol, D-maltose, D-saccharose, D-trehalose, D-tagatose, L-rhamnose, salicin, adonitol, D-melibiose, D-raffinose, D-ribose, D-fructose and hydrolysis of starch. Negative reactions were observed for potassium gluconate, potassium 2-cetogluconate, inulin, D-melezitose, Glycogen, β-gentiobiose, D-turanose, methyl-αD-mannopyranoside and methyl-αD-glucopyranoside. Using the API ZYM system, negative reactions were observed for lipase (C14), a-chymotrypsin, esterase (C4), esterase lipase (C8), naphthyl-AS-BI-phosphohydrolase, phenylalanine arylamidase, leucine arylamidase, cystine arylamidase, valine arylamidase, glycine arylamidase, arginine arylamidase and β-glucosidase. Using the API Coryne system, positive reactions were observed for β-glucuronidase, alkaline phosphatase, α-glucosidase, α-galactosidase and N-acetyl-β-glucosaminidase activities. The urease reaction, nitrate reduction and indole production were negative. P. gorillae is susceptible to imipenem, rifampicin, gentamycin, nitrofurantoin and vancomycin, but resistant to metronidazole, trimethoprim/sulfamethoxazole, ceftriaxone, ciprofloxacin and amoxicillin.
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described  using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve distinct deposits were made for strain G1T from 12 isolated colonies. The 12 G1T spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against 6,252 bacterial spectra including 123 spectra from 67 Paenibacillus species, used as reference data, in the BioTyper database. Interpretation of scores was as follows: a score ≥ 2 to a validly published species enabled the identification at the species level, a score ≥ 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. For strain G1T, the obtained scores ranged from 1.177 to 1.343, thus suggesting that our isolate was not a member of a known species. We incremented our database with the spectrum from strain G1T (Figure 4). Spectrum differences with other of Paenibacillus species are shown in Figure 5.
Genome sequencing information
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 genus Paenibacillus, and is part of a “culturomics” study of the gorilla flora which aims to isolate all bacterial species within gorilla feces. It is the 44th genome of a Paenibacillus species sequenced and the first genome of Paenibacillus gorillae sp. nov. sequenced. A summary of the project information is shown in Table 3. The Genbank accession number is CBVJ000000000 and consists of 167 contigs (150 large contigs). Table 3 shows the project information and its association with MIGS version 2.0 compliance .
Growth conditions and DNA isolation
P. gorillae sp. nov. strain G1T, CSUR P205, DSM 26181, was grown aerobically on 5% sheep blood-enriched Columbia agar at 25°C. Four Petri dishes were spread and resuspended in 3×500µl of TE buffer and stored at 80°C. Then, 500µl of this suspension were thawed, centrifuged 3 minutes at 10,000 rpm and resuspended in 3×100µL 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, MP Biomedicals, USA) using 2×20 seconds cycles. DNA was then treated with 2.5µg/µL lysozyme (30 minutes at 37°C) and extracted using the BioRobot EZ1 Advanced XL (Qiagen). The DNA was then concentrated and purified using the Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 50ng/µl.
Genome sequencing and assembly
A shotgun and a 3 kb paired end library were pyrosequenced on the 454_Roche_Titanium. This project was loaded on a 1/4 region for each application on PTP Picotiterplates. The shotgun library was constructed with 500ng of DNA as describes by the manufacturer Roche with the Rapid library Preparation kit for XL+. The concentration of the shotgun library was measured with a TBS fluorometer and determined to be 2.89E+09 molecules/µL. The paired-end library was prepared with 5 µg of bacterial DNA using the DNA fragmentation on a Covaris S-Series (S2) instrument (Woburn, Massachusetts, USA) with an enrichment size at 3.2kb. The DNA fragmentation was visualized with an Agilent 2100 BioAnalyzer on a DNA labchip 7500. The library was constructed according to the 454 GS FLX Titanium paired-end protocol (Roche). Circularization and nebulization were performed and generated a pattern with an optimum at 591bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was quantified using the Quant-it Ribogreen kit (Invitrogen) on a Genios Tecan fluorometer at 691 pg/µL. The library concentration equivalence was calculated as 1.07E+10 molecules/µL. The library was stored at −20°C until further use.
The shotgun XL+ library was clonally amplified with 6 cpb in 2 emPCR reactions. The paired-end library was clonally amplified with 0.5 cpb in 3 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 16.9% and 8.61% respectively, and within the expected yield range of 5 to 20%, as recommended by the Roche procedure.
A total of 790,000 beads for each ¼ region per application were loaded on the GS Titanium PicoTiterPlate PTP Kit 70×75 and sequenced with the 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 339,189 passed filter wells were obtained and generated 108.46 Mb of sequences with a length average of 330 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40-bp as overlap. The final assembly identified 11 scaffolds with 150 large contigs (>1.5kb) generating a genome size of 6.22 Mb corresponding to a genome coverage of 17.2×.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they spanned 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 GenBank database. 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.
To estimate the mean level of nucleotide sequence similarity at the genome level between P. gorillae sp nov. strain G1T and other Paenibacillaceae species, we use the Average Genomic Identity of orthologous gene Sequences (AGIOS) program. Briefly, this software combines the Proteinortho software  to detect orthologous proteins between genomes compared on a pair-wise basis, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm.
The genome 6,257,967 bp long (1 chromosome, but no plasmid) with a 48,80% G+C content (Figure 6 and Table 4). It is composed of 167 contigs (150 large contigs, 11 scaffolds). Of the 5,918 predicted genes, 5,856 were protein-coding genes and 62 were RNAs (1 gene is 16S rRNA, 1 gene is 23S rRNA and 60 are tRNA genes). A total of 4,296 genes (73.36%) were assigned a putative function (by COGS or by NR blast) and 304 genes were identified as ORFans (5.19%). The remaining genes were annotated as hypothetical proteins (917 genes, 15.66%). The distribution of genes into COGs functional categories is presented in Table 5. The properties and statistics of the genome are summarized in Tables 4 and 5.
Genomic comparison of P. gorillae and other members of the family Paenibacillaceae.
Here, we compared the genome of P. gorillae strain G1T with those of “G. massiliense” strain G5T, P. elgii strain B69, P. alvei strain DSM 29, P. massiliensis strain DSM 16942 and B. brevis strain NBRC 100599 (Table 6). The draft genome of P. gorillae is larger in size than that of “G. massiliense” (6.25 vs 5.54 Mb) and smaller in size than those of P. elgii, P. alvei, P. massiliensis and B. brevis (6.25 vs 7.96, 6.83, 6.39 and 6.3 Mb respectively). P. gorillae has a lower G+C content than those of “G. massiliense” and P. elgii (48.8% vs 50.39% and 52.6% respectively) but higher than those of P. alvei and B. brevis (48.8% vs 45.9% and 47.3% respectively) and slightly higher than P. massiliensis (48.8% vs 48.5%). The protein content of P. gorillae is lower than that of P. elgii, P. alvei and B. brevis (5,856 vs 7,597, 6,823 and 5,946 respectively) but higher than that of “G. massiliense” and P. massiliensis (5,856 vs 5,146 and 5,496 respectively) (Table 6). In addition, P. gorillae shares 1,987, 2,380, 2,055, 2,121 and 1,935 orthologous genes with “G. massiliense”,P. elgii, P. alvei,P. massiliensis and B. brevis, respectively (Table 7). The nucleotide sequence identity of orthologous genes ranges from 66.3 to 68.7% among previously published genomes, and from 65.7 to 68.6% between P. gorillae and the other studied genomes (Table 7), thus confirming its status as a new species. Table 7 summarizes the number of orthologous genes and the average percentage of nucleotide sequence identity between the different genomes studied.
On the basis of phenotypic (Table 2), phylogenetic and genomic analyses (taxonogenomics) (Table 7), we formally propose the creation of Paenibacillus gorillae sp. nov. that contains the strain G1T. This strain has been found in gorilla stool sample collected from Cameroon.
Description of Paenibacillus gorillae sp. nov.
Paenibacillus gorillae (gor.il.lae, N.L. gen fem, of the gorilla from which the stool sample was obtained).
P. gorillae is a facultative aerobic Gram-negative. Optimal growth is achieved aerobically. A weak growth is observed in microaerophilic or anaerobic conditions. Growth occurs between 25 and 37°C, with optimal growth observed at 25°C. Cells stain Gram-negative, are rod-shaped, endospore-forming and motile with a mean diameter of 0.79 µm (range 0.76 to 0.83 µm) and a mean length of 3.2 µm (range 2.5 to 3.97 µm). Colonies are whitish and 2–8 mm in diameter on blood-enriched Columbia agar.
Catalase negative, oxidase positive. Using the API 50CH system (BioMerieux), a positive reaction is obtained for D-mannose, amygdalin, L-arabinose, cellobiose, lactose, D-xylose, D-glucose, mannitol, arabinose, xylose, glycerol, D-galactose, N-acetylglucosamine, arbutin, aesculin, D-sorbitol, D-maltose, D-saccharose, D-trehalose, D-tagatose, L-rhamnose, salicin, adonitol, D-melibiose, D-raffinose, D-ribose, D-fructose and hydrolysis of starch. Negative reactions are obtained for potassium gluconate, potassium 2-cetogluconate, inulin, D-melezitose, Glycogen, β-gentiobiose, D-turanose, Methyl-αD-mannopyranoside and Methyl-αD-glucopyranoside. Using the API ZYM system, negative reactions are obtained for lipase (C14), a-chymotrypsin, esterase (C4), esterase lipase (C8), naphthyl-AS-BI-phosphohydrolase, phenylalanine arylamidase, leucine arylamidase, cystine arylamidase, valine arylamidase, glycine arylamidase, arginine arylamidase and β-glucosidase. Using the API Coryne system, positive reactions are observed for β-glucuronidase, alkaline phosphatase, α-glucosidase, α-galactosidase and N-acetyl-β-glucosaminidase activities. The urease reaction, nitrate reduction and indole production were negative. P. gorillae is susceptible to imipenem, rifampicin, gentamycin, nitrofurantoin and vancomycin, but resistant to metronidazole, trimethoprim/sulfamethoxazole, ceftriaxone, ciprofloxacin and amoxicillin.
The G+C content of the genome is 48.8%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JX650054 and CBVJ000000000, respectively. The type strain G1T (= CSUR P205 = DSM 26181) was isolated from the fecal flora of a Gorilla gorilla gorilla from Cameroon.
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Fadi Bittar was supported by a Chair of Excellence IRD provided by the Institut de Recherche pour le Développement / Méditerranée-Infection Foundation. Mamadou Bhoye Keita was funded by the Méditerranée-Infection foundation. The authors thank the Xegen company for automating the genome annotation process.