Genome sequence and description of Paenibacillus ihuae strain GD6 sp. nov., isolated from the stool of a 62-year-old Frenchman.

Paenibacillus ihuae strain GD6 (=CSUR P892 = DSMZ 45751T) is the new type strain collected from the stool of a 69-year-old Frenchman admitted to an intensive care unit and receiving a 10-day course of imipenem at the time of stool collection. This is a Gram-positive, facultative anaerobic, rod-shaped bacterium. We describe here the features of this organism, together with its complete genome sequence and annotation. The genome size is 6 719 043 bp with 49.6% G+C content and contains 6211 protein-coding and 65 sRNA genes, including four 5S rRNA genes, one 16S rRNA gene and one 23S rRNA gene.

Paenibacillus is a genus of facultative anaerobic, endospore-forming Gram-positive bacteria, originally included within the genus Bacillus and then reclassified as a separate genus in 1993 [1]. Since this classification, additional transfer to the genus Paenibacillus and proposal for novel strains to be designated as Paenibacillus species have increased. Bacteria belonging to this genus are commonly found in the environment such as soil, water, rhizosphere, vegetable matter, forage and insect larvae, but few species have been linked to infections in humans [2], [3], and it has been shown to produce a wide range of peptide antibiotics [4].

Paenibacillus ihuae strain GD6 was isolated from the stool of a 69-year-old man admitted to the intensive care unit and receiving a 10-day course of imipenem at the time of stool collection as part of a culturomics study aiming to isolate all bacterial species present in the human gut [5].

Here we present a summary of the classification and set of features for Paenibacillus ihuae sp. nov. strain GD6 (=CSUR P892 = DSMZ 45751T), together with the description of the complete genomic sequencing and annotation. These characteristics support the description of Paenibacillus ihuae sp. nov.

A stool sample was collected from a 69-year-old man living in France. The study was approved by the ethics committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France, under agreement 09-002.

The faecal specimen was preserved at −80°C after collection. Strain GD6 was isolated on Columbia agar supplemented with 5% sheep's blood (bioMérieux, Marcy l’Etoile, France) in aerobic condition at 37°C. Strain GD6 exhibited a 97.4% 16S rRNA sequence identity with Paenibacillus typhae strain xj7 5 (NR_109462.1), the phylogenetically closest bacterial species with standing in nomenclature (Fig. 1). Its 16S rRNA sequence was deposited in GenBank under accession number JX424768. As recommended by Stackebrandt and Ebers [6], this value was lower than the 98.7% 16S rRNA gene sequence threshold to delineate a new species without carrying out DNA-DNA hybridization, and a new species was thus identified. The spectrum of strain GD6 was added to our matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) database.

Fig. 1

Phylogenetic tree highlighting position of Paenibacillus ihuae strain GD6 16S rRNA gene sequence relative to other type strains within Paenibacillus genus. Sequences were aligned using MUSCLE, and phylogenetic inferences were obtained using maximum-likelihood method within MEGA6. Numbers at nodes are percentages of bootstrap values obtained by repeating analysis to generate majority consensus tree 1000 times.

The stool sample was diluted in phosphate-buffered saline (Life Technologies, Carlsbad, CA, USA). Obtained inoculum (100 μL) was incubated for 24 to 48 hours on 5% sheep's blood–enriched Columbia agar (bioMérieux) at 37°C. Growth was tested under aerobic and anaerobic conditions using AnaeroGen Compact (bioMérieux). Gram staining and electron microscopy were performed with a TechnaiG2 Cryo device (FEI Company, Limeil-Brévannes, France) at an operating voltage of 200 keV (Fig. 2). Cells were grown on 5% sheep's blood–enriched agar for 24 hours. A bacterial suspension was prefixed in 5% (v/v) glutaraldehyde in phosphate-buffered saline (Thermo Fisher Scientific, Waltham, MA, USA) for at least 1 hour at room temperature, washed in the same buffer and then stained with 1% (w/v) ammonium molybdate 1%. Oxidase (Becton Dickinson, Le Pont-de-Claix, France) and catalase (bioMérieux) assays were performed separately. Biochemical tests were performed using an APIZYM strip (bioMérieux) and an API50CH strip (bioMérieux). In vitro susceptibility to antibiotics was determined using the disc diffusion method (i2a, Montpellier, France) on Muller-Hinton agar with 5% blood.

Fig. 2

Transmission electron microscopy of Paenibacillus ihuae strain GD6 using TechnaiG2 Cryo (FEI Company, Limeil-Brevannes, France) at operating voltage of 200 keV. Scale bar = 500 nm.

Colonies obtained were isolated on 5% sheep's blood–enriched Columbia agar and were identified by MALDI-TOF MS. MALDI-TOF MS identification, measurement and analysis were performed as previously described [7] using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). No significant MALDI-TOF MS score was obtained for strain GD6 against the Bruker database, suggesting that our isolate was not a member of a known species. We added the spectrum from strain GD6 to our database.

Genomic DNA of strain GD6 was sequenced using MiSeq Technology (Illumina, San Diego, CA, USA) with the mate-pair strategy. The assembly was performed using the gsAssembler from Roche (Basel, Switzerland) with 90% identity and 40 bp as overlap. It led to 11 scaffolds and 564 large contigs (>1500 bp), generating a genome size of 6.71 Mb.

Genome was annotated by RAST [8]. The predicted bacterial protein sequences were searched against the GenBank database and the Clusters of Orthologous Groups (COGs) databases using BLASTp (E value 1e-03, coverage 0.7, identity percentage 30%). The tRNAScanSE tool [9] was used to find tRNA genes, whereas ribosomal RNAs were found by RNAmmer [10]. The resistome was analysed with the ARG-ANNOT database [11]. The exhaustive bacteriocin database available in our laboratories (Bacteriocins of the Unité des Maladies Infectieuses et Tropicales Emergentes (URMITE); http://drissifatima.wixsite.com/bacteriocins) was performed by collecting all currently available sequences from the databases and from the National Center for Biotechnology Information. Protein sequences from this database allowed putative bacteriocins from human gut microbiota to be identified using BLASTp methodology [12].

The presence of polyketide synthases (PKS) and nonribosomal peptide synthetase (NRPS) was analysed by discriminating genes with large size using a database realized in our laboratory; predicted proteins were compared against nonredundant GenBank database using BLASTp and finally examined using antiSMASH [15]. PHAST was used to identify phage sequences [13].

Phylogenetic relationships with closely related species were determined by MEGA6. The evolutionary history was concluded by using the maximum likelihood method based on the JTT matrix-based model. We compared the genome sequence of Paenibacillus ihuae strain GD6 with those of Paenibacillus graminis strain RSA19T (NZ_ASSG00000000.1), Paenibacillus polymyxa strain DSM 365T (NZ_JMIQ00000000.1), Paenibacillus massiliensis strain DSM 16942T (NZ_ARIL00000000.1), Paenibacillus typhae strain CGMCC 1.11012T (NZ_FNDX00000000.1) and Paenibacillus borealis strain DSM 13188T (NZ_CP009285.1).

Paenibacillus ihuae growth was obtained either on aerobic and anaerobic conditions on 5% sheep's blood–enriched Columbia agar at 37°C. Gram staining showed elongated-shaped Gram positive bacilli. The motility test was positive. Cells grown in trypticase soy broth medium have flagellum, as observed by electron microscopy (Fig. 2). Strain GD6 exhibits positive catalase and negative oxidase activity. Acid production was also observed using an API 50 CH strip (bioMérieux). Differential phenotype characteristics between P. ihuae and other species are shown in Table 1. Paenibacillus ihuae strain GD6 was resistant to oxacillin and metronidazole but was susceptible to cephalosporins, carbapenems, vancomycin, teicoplanin, lincomycin, gentamycin, amikacin, trimethoprim/sulfamethoxazole, rifampicin and fosfomycin.

Table 1

Differential characteristics of Paenibacillus ihuae strain GD6 and phylogenetically close members of other Paenibacillus species

Test	P. ihuae GD6	P. graminis	P. polymyxa	P. massiliensis	P. borealis
Catalase	+	+	+	−	+
Haemolyis	−	NA	+	−	NA
Spore presence	+	+	+	+	+
Anaerobic growth	+	+	−	+	+
Growth in presence of:
NaCl 5%	−	+	−	−	−
Glycerol	+	+	+	−	+
d-Arabinose	+	NA	−	−	−
l-Arabinose	+	+	+	NA	+/−
d-Xylose	+	+	+	+	+
d-Ribose	−	−	+	−	−
d-Trehalose	+	+	+	NA	+
d-Galactose	+	+	NA	NA	+
Starch	+	NA	+	−	+/−
d-Glucose	+	+	+	−	+
d-Lactose	+	+	+	NA	+
d-Mannose	+	NA	+	−	+
l-Rhamnose	+	−	−	−	−
d-Mannitol	+	+	+	+	−
Inulin	+	+	−	+	+
d-Raffinose	+	NA	+	+	NA
d-Turanose	+	NA	+	−	NA
d-Melezitose	+	+	−	+	NA
Methyl α-d-gluopyranoside	+	+	+	+	NA
Methyl α-d-mannopyranoside	−	−	+	NA	NA

+, positive result; −, negative result; NA, data not available.

The genome of Paenibacillus ihuae strain GD6 is 6 719 043 bp long with 49.6% G+C content. It is composed of 13 scaffolds (CTED00000000.1) comprising 600 contigs (LN831198 to LN831210). The phylogenetic tree highlights the position based on 16S rDNA of Paenibacillus ihuae strain GD6 (Fig. 1). A total of 6211 protein-coding genes are annotated; 65 were RNAs (including four 5S, one 16S and one 23S). The properties of the genome and the comparison with other genomes are summarized in Table 2, Table 3, respectively. The distribution of genes into COGs functional categories is presented in Table 4.

Table 2

Genome features of Paenibacillus ihuae strain GD6

Attribute	Value
Size (bp)	6 719 043
G+C content (bp)	49.6
RNAs gene	65
5S rRNA	4
16S rRNA	1
23S rRNA	1
Protein-coding gene	6211
Genes with unknown function	445
Genes assigned to COGs	5284
Genes associated to PKS or NRPS	1
Genes associated to toxin/antitoxin	0
Genes associated to resistome	0

COGs, Clusters of Orthologous Groups database; G+C, guanine cytosine; NRPS, nonribosomal peptide synthetase; PKS, polyketide synthase.

Table 3

Genomic comparison of Paenibacillus ihuae strain GD6 with other Paenibacillus species

Species	Strain	Size (Mb)	G+C%	Gene content
P. ihuae	GD6	6.71	49.6	6211
P. borealis	DSM 13188	8.15	51.4	7007
P. graminis	RSA19	6.98	50.30	6379
P. polymyxa	DSM 365	5.78	45.5	5031
P. massiliensis	DSM 16942	6.39	48.5	5461

Table 4

Distribution of genes into COGs functional categories

Code	Value	% of total	Description
A	2	0.032	RNA processing and modification
B	1	0.016	Chromatin structure and dynamics
C	204	3.28	Energy production and conversion
D	50	0.8	Cell cycle control, cell division, chromosome partitioning
E	364	5.9	Amino acid transport and metabolism
F	129	2.07	Nucleotide transport and metabolism
G	591	9.51	Carbohydrate transport and metabolism
H	193	3.1	Coenzyme transport and metabolism
I	108	1.73	Lipid transport and metabolism
J	222	3.6	Translation, ribosomal structure and biogenesis
K	578	9.306	Transcription
L	195	3.13	Replication, recombination and repair
M	263	4.23	Cell wall/membrane/envelope biogenesis
N	97	1.56	Cell motility
O	136	2.2	Posttranslational modification, protein turnover, chaperones
P	290	4.67	Inorganic ion transport and metabolism
Q	91	1.46	Secondary metabolites biosynthesis, transport and catabolism
R	641	10.32	General function prediction only
S	445	7.16	Function unknown
T	484	7.8	Signal transduction mechanisms
U	65	1.04	Intracellular trafficking, secretion and vesicular transport
V	134	2.15	Defense mechanisms
W	0	0	Extracellular structures
X	0	0	Nuclear structure
Z	1	0.016	Cytoskeleton
—	927	14.92	Not in COGs

COGs, Clusters of Orthologous Groups database.

The analysis of the resistome shows the absence of resistance genes. In silico analysis for PKS and NRPS revealed the presence of a NRPS organized as a highly modular mode in a massive multidomain enzyme organized with upstream enzyme clustering of condensation (C), adenylation (A), thiolation (T) or peptidyl carrier. The nonribosomal polyketide synthase (NRPKs) had a size of 3369 bp and a G+C content of 48%. This cluster showed 88% similarity with the NRPKs of Paenibacillus sp. FSL R5-0912 (Fig. 3).

Fig. 3

Phylogenetic of cluster representative of nonribosomal peptide synthase Paenibacillus ihuae strain GD6. Comparison of nonribosomal peptide synthetase (NRPS) of Paenibacillus sp. GD6 with closer cluster in other species. Percentages of identity are indicated for homologs found in cluster of WP 054942807.1 nonribosomal peptide synthetase of Paenibacillus sp. GD6 and closer cluster in other species.

Here we compared the genome of Paenibacillus ihuae strain GD6 with those of Paenibacillus graminis RSA19T, Paenibacillus polymyxa DSM 365T, Paenibacillus massiliensis DSM 16942T, Paenibacillus typhae CGMCC 1.11012T and Paenibacillus borealis DSM 13188T. The draft genome of Paenibacillus ihuae strain GD6 is bigger in size than those of P. polymyxa DSM 365T and P. massiliensis DSM 16942T (6.71 vs. 5.78 and 6.39 Mb, respectively) but smaller than those of P. graminis RSA19T, P. typhae CGMCC 1.11012T and Paenibacillus borealis DSM 13188T (6.71 vs. 6.98 Mb, 6.74 Mb and 8.16 Mb, respectively). The G+C content of Paenibacillus ihuae strain GD6 is smaller than those of P. graminis RSA19T, P. typhae CGMCC 1.11012T and P. borealis DSM 13188T (49.6% vs. 50.30%, 51.6% and 51.4%, respectively) but larger than those of P. polymyxa DSM 365T and P. massiliensis DSM 16942T (49.6% vs. 45.5% and 48.5%, respectively).

Genome-to-Genome Distance Calculator (GGDC) analysis between the Paenibacillus species mentioned above was performed using the GGDC web server as previously reported [14]. Comparing the species, with the exception of strain GD6, digital DNA-DNA hybridization (dDDH) values ranged from 19.2% to 23.8%. dDDH values between strain GD6 and compared species ranged from 19.6% with P. massiliensis to 24.9% with P. borealis. These values were less than 70%, the cutoff (Table 5).

Table 5

Pairwise comparison of Paenibacillus ihuae strain GD6 with other Paenibacillus species

	P. ihuae GD6	P. graminis RSA19T	P. polymyxa DSM 365T	P. massiliensis DSM 16942T	P. typhae CGMCC 1.11012T	P. borealis DSM 13188T
P. ihuae GD6	100 ± 00%	23.2 ± 2.5%	21.1 ± 2.5%	19.6 ± 2.4%	20.9 ± 3.5%	24.9 ± 2.3
P. graminis RSA19T		100 ± 00%	23.8 ± 2.5%	21 ± 3.4%	22.1 ± 3.4%	23.2 ± 2.3
P. polymyxa DSM 365T			100 ± 00%	19.2 ± 2.4%	20.5 ± 2.2%	21.2 ± 2.3
P. massiliensis DSM 16942T				100 ± 00%	20.2 ± 2.4%	20.7 ± 2.3
P. typhae CGMCC 1.11012T					100 ± 00%	21.7 ± 2.3
P. borealis DSM 13188T						100 ± 00%

Pairwise comparison performed using GGDC, formula 2 (DDH estimates based on identities/HSP length). dDDH values are DDH estimates based on identities/HSP length. Confidence intervals indicate inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size).

dDDH, digital DNA-DNA hybridization; DDH, DNA-DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; HSP, high-scoring segment pairs.

On the basis of phenotypic, phylogenetic and genomic analyses, we propose the identification of Paenibacillus ihuae strain GD6 sp. nov. Paenibacillus ihuae strain GD6 (=CSUR P892 = DSMZ 45751T), a new type strain collected from the stool sample of a 69-year-old Frenchman admitted to an intensive care unit and receiving a 10-day course of imipenem at the time of stool collection during a culturomics study aiming to isolate all bacterial species present in the human gut. Paenibacillus ihuae is a Gram-positive, facultative anaerobic, rod-shaped bacterium that exhibits positive catalase and negative oxidase activity. Growth was obtained under aerobic and anaerobic conditions on 5% sheep's blood–enriched Columbia agar at 37°C. Using API ZYM and API 50CH, positive reactions were observed for invertase (test 1) and for chitinase and invertase (test 2), as seen in Table 1. Cells of P. ihuae strain GD6 were resistant to oxacillin and metronidazole but were susceptible to other antibiotics. The analysis of the genome showed the absence of antibiotic resistance genes but the presence of a NRPS.

The genome of Paenibacillus ihuae strain GD6 has been submitted to the European Bioinformatics Institute (EBI) database under bioproject ID PRJEB549 with GenBank accession number CTED01000000 and 16S RNA accession number JX424768.

Conflict of interest

None declared.