Open Access

Non contiguous-finished genome sequence and description of Bacillus jeddahensis sp. nov.

  • Fadi Bittar1Email author,
  • Fehmida Bibi2,
  • Dhamodharan Ramasamy1,
  • Jean-Christophe Lagier1,
  • Esam I. Azhar2, 3,
  • Asif A. Jiman-Fatani4,
  • Ahmed K. Al-Ghamdi3,
  • Ti Thien Nguyen1,
  • Muhammad Yasir2,
  • Pierre-Edouard Fournier1 and
  • Didier Raoult1, 2
Standards in Genomic Sciences201510:47

DOI: 10.1186/s40793-015-0024-y

Received: 23 May 2014

Accepted: 21 May 2015

Published: 1 August 2015

Abstract

Strain JCET was isolated from the fecal sample of a 24-year-old obese man living in Jeddah, Saudi Arabia. It is an aerobic, Gram-positive, rod-shaped bacterium. This strain exhibits a 16S rRNA nucleotide sequence similarity of 97.5 % with Bacillus niacini, the phylogenetically closest species with standing nomenclature. Moreover, the strain JCET presents many phenotypic differences, when it is compared to other Bacillus species, and shows a low MALDI-TOF Mass Spectrometry score that does not allow any identification. Thus, it is likely that this strain represents a new species. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,762,944 bp long genome (1 chromosome but no plasmid) contains 4,654 protein-coding and 98 RNAs genes, including 92 tRNA genes. The strain JCET differs from most of the other closely Bacillus species by more than 1 % in G + C content. In addition, digital DNA-DNA hybridization values for the genome of the strain JCET against the closest Bacillus genomes range between 19.5 to 28.1, that confirming again its new species status. On the basis of these polyphasic data made of phenotypic and genomic analyses, we propose the creation of Bacillus jeddahensis sp. nov. that contains the strain JCET.

Keywords

Bacillus jeddahensis Genome Taxono-genomics Culturomics Human feces

Introduction

Currently, a polyphasic approach that combines proteomic by MALDI-TOF spectra analysis, genomic data and phenotypic characterization is used widely to describe new bacterial species [113].

The genus Bacillus, described by Cohn [14] more than 140 years ago, includes actually 310 species names (296 validly and 14 not-validly published species) [15]. Species belonging to this genus are Gram-positive or variable and mostly motile and spore-forming bacteria. Bacillus spp. are ubiquitous bacteria isolated from various environmental sources but it could be involved in human infections [16].

Strain JCET (= CSUR P732 = DSM 28281) is the type strain of Bacillus jeddahensis sp. nov. This bacterium is a Gram-positive, flagellated, facultatively anaerobic, indole-negative bacillus that has rounded-ends. It was isolated from the stool sample of a 24-year-old obese man living in Jeddah, Saudi Arabia as part of a culturomics study aiming at cultivating bacterial species within human feces. By applying large scale of culture conditions, culturomics allowed previously the isolation of many new bacterial species from human stool samples [1719].

Here we present a summary classification and a set of features for B. jeddahensis sp. nov. strain JCET together with the description of the complete genome sequence and annotation. These characteristics support the circumscription of the species B. jeddahensis [20].

Organism information

Classification and features

In April 2013, a fecal sample was collected from a 24-year-old obese (body mass index 52 kg/m2) man living in Jeddah, Saudi Arabia (Table 1). Written assent was obtained from this individual. Both the study and the assent procedure were approved by Ethical Committee of the King Abdulaziz University, King Fahd medical Research Centre, Saudi Arabia (agreement number 014-CEGMR-2-ETH-P) and the Ethical Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medecine, Marseille, France (agreement numbers 09–022 and 11–017). The fecal specimen was preserved at −80 °C after collection and sent to Marseille. Strain JCET (Table 1) was isolated in July 2013 by cultivation on blood culture bottle (Becton Dickinson, Temse, Belgique) supplemented with rumen fluid and sheep blood. This strain exhibited a 97.5 % 16S rRNA nucleotide sequence similarity with Bacillus niacini, the phylogenetically closest validly published Bacillus species (Fig. 1), when it was compared against NCBI database and Ribosomal Database Project. This value was equal to the percentage of 16S rRNA gene sequence threshold recommended by Meier-Kolthoff et al. for Firmicutes to delineate a new species without carrying out DNA-DNA hybridization with maximum error probability of 0.01 % [21].
Table 1

Classification and general features of Bacillus jeddahensis strain JCET

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain: Bacteria

TAS [39]

Phylum: Firmicutes

TAS [4042]

Class: Bacilli

TAS [43, 44]

Order: Bacillales

TAS [45, 46]

Family: Bacillaceae

TAS [45, 47]

Genus: Bacillus

TAS [4, 45, 48]

Species: Bacillus jeddahensis

IDA

Type strain: JCET

IDA

 

Gram stain

Positive

IDA

 

Cell shape

Rod-shaped

IDA

 

Motility

Non-motile

IDA

 

Sporulation

Sporulating

IDA

 

Temperature range

Mesophile

IDA

 

Optimum temperature

37 °C

IDA

 

pH range; Optimum

Not determined

 

MIGS-6.3

Salinity

growth in BHI medium + 3 % NaCl

IDA

MIGS-22

Oxygen requirement

Facultative Anaerobic

IDA

 

Carbon source

varied (see Additional file 1: Table S1)

IDA

 

Energy source

chemoorganoheterotrophic

IDA

MIGS-6

Habitat

Human gut

IDA

MIGS-15

Biotic relationship

Free living

IDA

MIGS-14

Pathogenicity

Unknown

 

Biosafety level

2

NAS

Isolation

Human faeces

IDA

MIGS-4

Geographic location

Saudi Arabia

IDA

MIGS-5

Sample collection time

July 2013

IDA

MIGS-4.1

Latitude

21° 25' 20.953"

IDA

MIGS-4.1

Longitude

39° 49' 34.262"

IDA

MIGS-4.3

Depth

unknown

 

MIGS-4.4

Altitude

unknown

 

Evidence codes - IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [49]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements

Fig. 1

Phylogenetic tree highlighting the position of Bacillus jeddahensis strain JCET relative to other type strains within the Bacillus genus. GenBank accession numbers are indicated in parentheses. Sequences were aligned using MUSCLE, and phylogenetic inferences obtained using the maximum-likelihood method and Kimura 2-parameter model within the MEGA 6 software [50]. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Clostridium botulinum was used as outgroup. The scale bar represents a rate of substitution per site of 0.01. *indicates the strains used in the tree have a sequenced genome. # indicates that a sequenced genome is available for this species but not for the strain used to build the tree

Different growth temperatures (28, 30, 37, 45, 56 °C) were tested. Growth occurred for the temperatures (28–45 °C), but the optimal growth was observed at 37 °C. Colonies were 0.4–0.5 mm in diameter on Columbia agar, appear smooth and grey in color at 37 °C. Growth of the strain was tested under anaerobic and microaerophilic conditions using 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 positive bacilli (Fig. 2). A motility test was negative. Cells grown on agar sporulate and the rods have a length ranging from 3.83 to 4.71 μm (mean 4.14 μm) and a diameter ranging from 0.75 to 0.95 μm (mean 0.87 μm). Both the length and the diameter were determined by negative staining transmission electron microscopy (Fig. 3).
Fig. 2

Gram staining of B. jeddahensis strain JCET

Fig. 3

Transmission electron microscopy of B. jeddahensis strain JCET, using a Morgani 268D (Philips) at an operating voltage of 60 kV. The scale bar represents 1 μm

Strain JCET exhibited oxidase activity but not catalase activity. Using API 50CH system (BioMerieux), a positive reaction was observed for D-arabinose, L-arabinose, D-xylose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, esculin, D-maltose, D-trehalose, and weak reaction for D-melezitose. Negative reactions were observed for the remaining carbohydrate tests (i.e. glycerol, erythritol, D-ribose, L-xylose, D-adonitol, methyl-β-D-xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, D-saccharose, inulin, D-raffinose, amidon, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate). Using API ZYM, positive reactions were observed for esterase (C 4), esterase lipase (C 8), acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase. Negative reactions were observed for alkaline phosphatase, lipase (C 14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using API NE system, nitrates were reduced to nitrites, the urease reaction, indole production, arginine dihydrolase and gelatin hydrolysis were negative, the following carbon sources were assimilated: D-glucose, D-mannose, N-acetylglucosamine and D-maltose, and the following carbon sources were not assimilated: L-arabinose, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. B. jeddahensis is susceptible to imipenem, doxycyclin amoxicillin, amoxicillin-clavulanate and gentamycin, but resistant to metronidazole, trimethoprim/sulfamethoxazole, rifampicin, vancomycin, erythromycin, ceftriaxone, ciprofloxacin and benzylpenicillin.

When compared to other Bacillus species [18, 2224], B. jeddahensis sp. nov. strain JCET exhibited the phenotypic differences detailed in Additional file 1: Table S1.

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [2] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve distinct deposits were done for strain JCET from 12 isolated colonies. The twelve JCET spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against 6,335 bacterial spectra including 210 spectra from 110 Bacillus species, used as reference data, in the BioTyper database. Interpretation of scores was as follows: a score ≥ 2 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 (These scores were established by the manufacturer Bruker Daltonics). For strain JCET, the obtained scores ranged from 1.4 to 1.6, thus suggesting that our isolate was not a member of a known species. We incremented our database with the spectrum from strain JCET (Fig. 4). Spectrum differences with other of Bacillus species are shown in Fig. 5.
Fig. 4

Reference mass spectrum from B. jeddahensis strain JCET. Spectra from twelve individual colonies were compared and a reference spectrum was generated

Fig. 5

Gel view comparing Bacillus jeddahensis JCET spectra with other members of the Bacillus genus (B. niacini, B. drentensis, B. novalis, B. bataviensis, B. vireti, B. massilioanorexius, B. massiliosenegalensis, B. megaterium, B. timonensis, B. cereus and B. licheniformis). The Gel View displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a gray-scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units

Genome sequencing information

Genome project history

On the basis of phenotypic characteristics of this strain and because of the low16S rRNA similarity to other members of the genus Bacillus , it is likely that the strain represents a new species and thus it was chosen for genome sequencing. It was the 348th genome of a Bacillus species (Genomes Online Database) and the first genome of Bacillus jeddahensis sp. nov. sequenced. A summary of the project information is shown in Table 2. The Genbank accession number is CCAS00000000 (Table 2) and consists of 149 contigs. Table 2 shows the project information and its association with MIGS version 2.0 compliance [25].
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

Paired end and mate pair

MIGS-29

Sequencing platform

MiSeq Technology (Illumina Inc)

MIGS-31.2

Sequencing coverage

94.91x

MIGS-30

Assemblers

Newbler version 2.5.3

MIGS-32

Gene calling method

Prodigal

 

EMBL Date of Release

2014

 

EMBL ID

CCAS00000000

MIGS-13

Source material identifier

JCET

Project relevance

Study of the human gut microbiome

Growth conditions and genomic DNA preparation

B. jeddahensis sp. nov. strain JCET, CSUR P732, DSM 28281, was grown aerobically on 5 % sheep blood-enriched Columbia agar at 37 °C. Four Petri dishes were spread and resuspended in 3 × 500 μl of TE buffer and stored at 80 °C. Then, 500 μl of this suspension were thawed, centrifuged 3 min at 10,000 rpm and resuspended in 3 × 100 μL 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 s cycles. DNA was then treated with 2.5 μg/μL lysozyme (30 min 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 50 ng/μl.

Genome sequencing and assembly

Genomic DNA of B. jeddahensis was sequenced on the MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the 2 applications: paired end and mate pair. The paired end and the mate pair strategies were barcoded in order to be mixed respectively with 14 others genomic projects prepared with the Nextera XT DNA sample prep kit (Illumina) and eleven others projects with the Nextera Mate Pair sample prep kit (Illumina). The DNAg was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 16 ng/μl and dilution was performed to require 1ng of each genome as input to prepare the paired end library. The « tagmentation » step fragmented and tagged the DNA. Then limited cycle PCR amplification (twelve cycles) completed the tag adapters and introduced dual-index barcodes. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), the libraries were then normalized on specific beads according to the Nextera XT protocol (Illumina). Normalized libraries were pooled into a single library for sequencing on the MiSeq. The pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and paired end sequencing with dual index reads were performed in a single 39-h run in 2 × 250-bp. Total information of 5.3 Gb was obtained from a 574 K/mm2 cluster density with a cluster passing quality control filters of 95.4 % (11,188,000 clusters). Within this run, the index representation for B. jeddahensis was determined to 10.3 %. The 1,062,432 reads were filtered according to the read qualities. The mate pair library was prepared with 1 μg of genomic DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from 1 kb up to 11 kb with an optimal size at 5 kb. No size selection was performed and 600 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with an optimal at 692 bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA). The libraries were normalized at 2 nM and pooled. After a denaturation step and dilution at 10 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run were performed in a single 42-h run in a 2 × 250-bp. Total information of 3.9 Gb was obtained from a 399 K/mm2 cluster density with a cluster passing quality control filters of 97.9 % (7,840,000 clusters). Within this run, the index representation for B. jeddahensis was determined to 9.37 %. The 718,848 reads were filtered according to the read qualities. The passed filter sequences were assembled using Newbler with 90 % identity and 40-bp as overlap. The final assembly identified 149 large contigs (>1.5 kb) generating a genome size of 4.76 Mb which corresponds to a genome coverage of 94.91x.

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [26] 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 [27] and the Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [28] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [29] and BLASTn against the GenBank database. Signal peptides and numbers of transmembrane helices were predicted using SignalP [30] and TMHMM [31], 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. Artemis [32] and DNA Plotter [33] were used for data management and visualization of genomic features, respectively. Mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignment [34].

To estimate the mean level of nucleotide sequence similarity at the genome level between B. jeddahensis sp nov. strain JCET and nine other members of the genus Bacillus, we use the Average Genomic Identity of orthologous gene Sequences (AGIOS) program. Briefly, this software combines the Proteinortho software [35] 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. Moreover, we used Genome-to-Genome Distance Calculator (GGDC) web server available at (http://ggdc.dsmz.de) to estimate of the overall similarity among the compared genomes and to replace the wet-lab DNA-DNA hybridization (DDH) by a digital DDH (dDDH) [36, 37]. GGDC 2.0 BLAST+ was chosen as alignment method and the recommended formula 2 was taken into account to interpret the results.

Genome properties

The genome 4,762,944 bp long (1 chromosome, but no plasmid) with a 39.42 % G + C content (Fig. 6 and Table 3). It is composed of 149 contigs. Of the 4,741 predicted genes, 4,654 were protein-coding genes and 98 were RNAs including 6 rRNA (1 gene is 16S rRNA, 1 gene is 23S rRNA and 5 genes are 5S rRNA). A total of 3,410 genes (71.92 %) were assigned a putative function (by COGs or by NR blast) and 147 genes were identified as ORFans (3.17 %). The distribution of genes into COGs functional categories is presented in Table 4. The properties and statistics of the genome are summarized in Tables 3 and 4.
Fig. 6

Graphical circular map of the chromosome. From outside to the center: Genes on the forward strand colored by COG categories (only genes assigned to COG), genes on the reverse strand colored by COG categories (only gene assigned to COG), RNA genes (tRNAs green, rRNAs red), G + C content and GC skew. Purple and olive indicating negative and positive values, respectively

Table 3

Nucleotide content and gene count levels of the genome

Attribute

Genome (total)

 

Value

% of totala

Size (bp)

4,762,944

100

G + C content (bp)

1,876,599

39.42

Coding region (bp)

4,065,588

85.36

DNA scaffolds

ND

 

Total genes

4,741

100

RNA genes

98

2.07

Protein-coding genes

4,654

97.72

Pseudo genes

ND

 

Genes in internal clusters

ND

 

Genes with function prediction

3,410

71.92

Genes assigned to COGs

2,902

61.21

Genes with Pfam domains

ND

 

Genes with peptide signals

93

1.96

Genes with transmembrane helices

1,301

27.44

CRISPR repeats

ND

 

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome. ND not determined

Table 4

Number of genes associated with the 25 general COG functional categories

Code

Value

% of totala

Description

J

179

3.86

Translation

A

0

0

RNA processing and modification

K

342

7.38

Transcription

L

195

4.21

Replication, recombination and repair

B

1

0.02

Chromatin structure and dynamics

D

39

0.84

Cell cycle control, mitosis and meiosis

Y

0

0

Nuclear structure

V

81

1.75

Defense mechanisms

T

255

5.5

Signal transduction mechanisms

M

192

4.14

Cell wall/membrane biogenesis

N

57

1.23

Cell motility

Z

0

0

Cytoskeleton

W

0

0

Extracellular structures

U

48

1.04

Intracellular trafficking and secretion

O

119

2.57

Posttranslational modification, protein turnover, chaperones

C

224

4.83

Energy production and conversion

G

300

6.48

Carbohydrate transport and metabolism

E

425

9.17

Amino acid transport and metabolism

F

89

1.92

Nucleotide transport and metabolism

H

131

2.83

Coenzyme transport and metabolism

I

139

3

Lipid transport and metabolism

P

261

5.63

Inorganic ion transport and metabolism

Q

82

1.77

Secondary metabolites biosynthesis, transport and catabolism

R

500

10.79

General function prediction only

S

348

7.51

Function unknown

-

1223

26.4

Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome

Insights from the genome sequence

Here, we compared the genome of B. jeddahensis strain JCET with those of “Bacillus massiliosenegalensis” strain JC6T , “ Bacillus massilioanorexius strain AP8T , “ Bacillus timonensis” strain MM10403188, Bacillus cereus strain ATCC 14579, Bacillus megaterium strain DSM 319, Bacillus licheniformis strain ATCC 14580, Bacillus bataviensis strain DSM 15601T, Bacillus vireti DSM 15602T, Bacillus niacini JAM F8 and Bacillus niacini DSM 2923T (Tables 5 and 6). The draft genome sequence of B. jeddahensis strain is larger in size than those of “B. massilioanorexius”, “B. timonensis, B. licheniformis and B. niacini DSM 2923T (4.76 vs 4.59, 4.66, 4.22 and 2.2 Mb, respectively), but smaller than those of “B. massiliosenegalensis”, B. cereus, B. megaterium , B. bataviensis , B. vireti and B. niacini JAM F8 (4.76 vs 4.97, 5.43, 5.10, 5.37, 5.29 and 6.37 Mb, respectively) (Table 5). B. jeddahensis has a lower G + C content than those of B. licheniformis , B. bataviensis and B.vireti (39.42 vs 46.19, 39.6 and 39.74 %, respectively) and higher than those of “B. massiliosenegalensis,” B. massilioanorexius,” B. timonensis, B. cereus , B. megaterium , B. niacini JAM F8 and B. niacini DSM 2923T (39.42 vs 37.58, 34.10, 37.28, 35.29, 38.13, 37.83 and 38.30 %, respectively) (Table 5). As it was reported recently that the G + C content varies no more than 1 % within species [38] and because the strain JCET differs from most of the other closely strains by more than 1 % in G + C content, this might provide an additional argument for the new taxon described herein. The protein content of B. jeddahensis is higher than those of “B. massilioanorexius, “ B. timonensis, B. licheniformis and B. niacini DSM 2923T (4654 vs 4436, 4647, 4173 and 2184, respectively) but lower than those of “B. massiliosenegalensis, B. cereus , B. megaterium , B. bataviensis , B.vireti andB. niacini JAM F8 (4654 vs 4935, 5231, 5100, 5207, 5092 and 6103, respectively) (Table 6). The distribution of genes into COG categories was not entirely similar in all the nine compared genomes (Fig. 7). In addition, B. jeddahensis shares 2075, 1786, 1930, 1729, 1894, 1715, 2494, 2433, 2404 and 914 orthologous genes with those of “B. massiliosenegalensis, “ B. massilioanorexius, “ B. timonensis, B. cereus , B. megaterium , B. licheniformis , B. bataviensis , B. vireti , B. niacini JAM F8 and B. niacini DSM 2923T, respectively. Among compared genomes except B. jeddahensis, AGIOS values range from 64.44 between B.cereus and B. licheniformis to 83.91 % between B. niacini JAM F8 and B. niacini DSM 2923T. When B. jeddahensis was compared to other species, AGIOS values range from 65.50 with B. licheniformis to 78.49 % with B. bataviensis (Table 6). dDDH estimation of the strain JCET against the compared genomes ranged between 19.50 to 28.10. These values are very low and below the cutoff of 70 %, thus confirming again the new species status of the strain JCET. Table 5 summarizes the number of orthologous genes and the average percentage of nucleotide sequence identity between the different genomes studied.
Table 5

Genomic comparison of B. jeddahensis sp. nov., strain JCET with other Bacillus species. Species and strain names, GenBank genome accession numbers, sizes and G + C contents

Species

Strain

Genome accession number

Genome size (Mb)

G + C content

Bacillus jeddahensis

JCET

CCAS00000000

4.76

39.42

Bacillus massiliosenegalensis

JC6T

CAHJ00000000

4.97

37.58

Bacillus massilioanorexius

AP8T

CAPG00000000

4.59

34.10

Bacillus timonensis

MM10403188

CAET00000000

4.66

37.28

Bacillus cereus

ATCC 14579

NC_004722

5.43

35.29

Bacillus megaterium

DSM 319

NC_014103

5.10

38.13

Bacillus licheniformis

ATCC 14580

NC_006270

4.22

46.19

Bacillus bataviensis

DSM 15601T

AJLS00000000

5.37

39.60

Bacillus vireti

DSM 15602T

ALAN00000000

5.29

39.74

Bacillus niacini

JAM F8

BAWM00000000

6.37

37.83

Bacillus niacini

DSM 2923T

JRYQ00000000

2.20

38.30

Bold numbers indicate numbers of proteins per genome

Table 6

Genomic comparison of B. jeddahensis sp. nov., strain JCET with other Bacillus species. Numbers of orthologous proteins shared between genomes (upper right triangle), average percentage similarity of nucleotides corresponding to orthologous proteins shared between genomes (lower left triangle)

 

JCET

JC6T

AP8T

MM10403188

ATCC 14579

DSM 319

ATCC 14580

DSM 15601T

DSM 15602T

JAM F8

DSM 2923T

JCET

4654

2075

1786

1930

1729

1894

1715

2494

2433

2404

914

JC6T

69.86

4935

1858

1960

1669

1862

1746

2118

2132

2169

836

AP8T

68.13

68.67

4436

1726

1674

1778

1636

1848

1854

1911

711

MM10403188

68.48

68.73

68.49

4647

1631

1811

1691

2092

2047

2103

829

ATCC 14579

66.99

67.35

67.83

67.99

5231

1837

1678

1776

1792

1837

726

DSM 319

67.26

67.43

67.80

68.24

67.94

5100

1871

1978

1941

2033

805

ATCC 14580

65.50

65.33

64.32

65.72

64.44

66.06

4173

1761

1794

1784

716

DSM 15601T

78.49

69.51

67.94

68.53

66.69

67.18

65.54

5207

2699

2639

989

DSM 15602T

77.41

69.37

67.69

68.32

66.58

66.97

65.61

79.72

5092

2528

956

JAM F8

74.84

70.04

68.49

68.90

67.21

67.42

65.18

74.95

74.98

6103

1102

DSM 2923T

74.17

69.58

68.48

68.64

67.05

67.39

65.54

74.55

74.46

83.91

2184

Bold numbers indicate numbers of proteins per genome

Fig. 7

Distribution of predicted genes of B. jeddahensis and eight other Bacillus species into COG categories

Conclusions

On the basis of phenotypic characteristics (Additional file 1: Table S1), phylogenetic position (Fig. 1), genomic analyses (taxonogenomics) (Table 5) and GGDC results, we formally propose the creation of Bacillus jeddahensis sp. nov. that contains the strain JCET. This strain has been found in obese human feces collected from Jeddah, Saudi Arabia.

Description of Bacillus jeddahensis sp. nov. strain JCET

Bacillus jeddahensis (jed.dah. en′sis L. gen. neutr. n. jeddahensis, pertaining to, or originating from Jeddah, the capital of Saudi Arabia, where the type strain was isolated).

B. jeddahensis is a Gram-positive. Optimal growth is achieved aerobically. But growth is also observed in microaerophilic or anaerobic conditions. Growth occurs on axenic media between 28 and 45 °C, with optimal growth observed at 37 °C. Cells stain Gram-positive, are rod-shaped, endospore-forming and non-motile with a mean diameter of 0.87 μm (range 0.75 to 0.95 μm) and a mean length of 4.1 μm (range 3.8 to 4.7 μm). Colonies are smooth grey and 0.4–0.5 mm in diameter on blood-enriched Columbia agar.

Catalase negative, oxidase positive. A positive reaction is obtained for D-arabinose, L-arabinose, D-xylose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, esculin, D-maltose, D-trehalose, and weak reaction for D-melezitose. Negative reactions are obtained for the remaining carbohydrate tests (i.e. glycerol, erythritol, D-ribose, L-xylose, D-adonitol, methyl-β-D-xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, D-saccharose, inulin, D-raffinose, amidon, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate). Positive reactions are observed for esterase (C 4), esterase lipase (C 8), acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase. Negative reactions are obtained for alkaline phosphatase, lipase (C 14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Nitrates are reduced to nitrites, the urease reaction, indole production, arginine dihydrolase and gelatin hydrolysis are negative, the following carbon sources are assimilated: D-glucose, D-mannose, N-acetylglucosamine and D-maltose, and the following carbon sources were not assimilated: L-arabinose, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. B. jeddahensis is susceptible to imipenem, doxycyclin amoxicillin, amoxicillin-clavulanate and gentamycin, but resistant to metronidazole, trimethoprim/sulfamethoxazole, rifampicin, vancomycin, erythromycin, ceftriaxone, ciprofloxacin and benzylpenicillin.

The G + C content of the genome is 39.42 %. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers HG931339 and CCAS00000000, respectively. The type strain JCET (= CSUR P732 = DSM 28281) was isolated from the fecal flora of an obese man from Jeddah in Saudi Arabia.

Abbreviations

URMITE: 

Unité de Recherchesur les Maladies Infectieuses et Tropicales Emergentes

CSUR: 

Collection de Souches de l’Unité des Rickettsies

DSM: 

Deutsche Sammlung von Mikroorganismen

MALDI-TOF MS: 

Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry

TE buffer: 

Tris-EDTA buffer

GGDC: 

Genome-to-Genome Distance Calculator

dDDH: 

digital DNA-DNA hybridization

Declarations

Acknowledgements

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, under grant No. (1-141/1433 HiCi). The authors, therefore, acknowledge technical and financial support of KAU. Fadi Bittar was supported by a Chair of Excellence IRD provided by the Institut de Recherche pour le Développement / Méditerranée-Infection foundation.

Authors’ Affiliations

(1)
URMITE, Aix-Marseille Université, Faculté de médecine
(2)
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University
(3)
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University
(4)
Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University

References

  1. Mishra AK, Lagier JC, Rivet R, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of “Paenibacillus senegalensis” sp. nov. Stand Genomic Sci. 2012;7:70–81.PubMed CentralPubMedView ArticleGoogle Scholar
  2. Mamadou Bhoye K, Seydina D, Catherine R, Didier R, Pierre-Edouard F, Fadi B. Non-contiguous finished genome sequence and description of Bacillus massiliogorillae sp. nov. Stand Genomic Sci. 2013;9:93–105.View ArticleGoogle Scholar
  3. Mishra AK, Lagier JC, Robert C, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of Clostridium senegalense sp. nov. Stand Genomic Sci. 2012;6:386–95.PubMed CentralPubMedGoogle Scholar
  4. Lagier JC, Armougom F, Mishra AK, Ngyuen TT, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of Alistipes timonensis sp. nov. Stand Genomic Sci. 2012;6:315–24.PubMed CentralPubMedView ArticleGoogle Scholar
  5. Lagier JC, El Karkouri K, Nguyen TT, Armougom F, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov. Stand Genomic Sci. 2012;6:116–25.PubMed CentralPubMedView ArticleGoogle Scholar
  6. Roux V, El Karkouri K, Lagier JC, Robert C, Raoult D. Non-contiguous finished genome sequence and description of Kurthia massiliensis sp. nov. Stand Genomic Sci. 2012;7:221–32.PubMed CentralPubMedView ArticleGoogle Scholar
  7. Mishra AK, Lagier JC, Robert C, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of Peptoniphilus timonensis sp. nov. Stand Genomic Sci. 2012;7:1–11.PubMed CentralPubMedView ArticleGoogle Scholar
  8. Hugon P, Ramasamy D, Lagier JC, Rivet R, Couderc C, Raoult D, et al. Non contiguous-finished genome sequence and description of Alistipes obesi sp. nov. Stand Genomic Sci. 2013;7:427–39.PubMed CentralPubMedView ArticleGoogle Scholar
  9. Ramasamy D, Lagier JC, Nguyen TT, Raoult D, Fournier PE. Non contiguous-finished genome sequence and description of Dielma fastidiosa gen. nov., sp. nov., a new member of the Family Erysipelotrichaceae. Stand Genomic Sci. 2013;8:336–51.PubMed CentralPubMedView ArticleGoogle Scholar
  10. Mishra AK, Lagier JC, Robert C, Raoult D, Fournier PE. Genome sequence and description of Timonella senegalensis gen. nov., sp. nov., a new member of the suborder Micrococcinae. Stand Genomic Sci. 2013;8:318–35.PubMed CentralPubMedView ArticleGoogle Scholar
  11. Mishra AK, Hugon P, Lagier JC, Nguyen TT, Couderc C, Raoult D, et al. Non contiguous-finished genome sequence and description of Enorma massiliensis gen. nov., sp. nov., a new member of the Family Coriobacteriaceae. Stand Genomic Sci. 2013;8:290–305.PubMed CentralPubMedView ArticleGoogle Scholar
  12. Ramasamy D, Lagier JC, Gorlas A, Raoult D, Fournier PE. Non contiguous-finished genome sequence and description of Bacillus massiliosenegalensis sp. nov. Stand Genomic Sci. 2013;8:264–78.PubMed CentralPubMedView ArticleGoogle Scholar
  13. Hugon P, Mishra AK, Lagier JC, Nguyen TT, Couderc C, Raoult D, et al. Non-contiguous finished genome sequence and description of Brevibacillus massiliensis sp. nov. Stand Genomic Sci. 2013;8:1–14.PubMed CentralPubMedView ArticleGoogle Scholar
  14. Cohn F. Untersuchungen über Bakterien. Beitrage zur Biologie der Pflanzen Heft. 1872;1:127–224.Google Scholar
  15. Abstract for the genus Bacillus. NamesforLife, LLC. Retrieved January 30, 2014. http://doi.namesforlife.com/10.1601/tx.4857
  16. Mandell GL, Bennett JE, Dolin R. Principles and Practice of Infectious Diseases. Elsevier. 2010; 4320 p. CHURCHILL LIVINGSTONE, Pennsylvania.
  17. Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect. 2012;18:1185–93.PubMedView ArticleGoogle Scholar
  18. Dubourg G, Lagier JC, Armougom F, Robert C, Hamad I, Brouqui P, et al. The gut microbiota of a patient with resistant tuberculosis is more comprehensively studied by culturomics than by metagenomics. Eur J Clin Microbiol Infect Dis. 2013;32:637–45.PubMedView ArticleGoogle Scholar
  19. Pfleiderer A, Lagier JC, Armougom F, Robert C, Vialettes B, Raoult D. Culturomics identified 11 new bacterial species from a single anorexia nervosa stool sample. Eur J Clin Microbiol Infect Dis. 2013;32:1471–81.PubMedView ArticleGoogle Scholar
  20. Sentausa E, Fournier PE. Advantages and limitations of genomics in prokaryotic taxonomy. Clin Microbiol Infect. 2013;19:790-5.
  21. Meier-Kolthoff JP, Göker M, Spröer C, Klenk HP. When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol. 2013;6:413–8.View ArticleGoogle Scholar
  22. Heyrman J, Vanparys B, Logan NA, Balcaen A, Rodríguez-Díaz M, Felske A, et al. Bacillus novalis sp. nov., Bacillus vireti sp. nov., Bacillus soli sp. nov., Bacillus bataviensis sp. nov. and Bacillus drentensis sp. nov., from the Drentse A grasslands. Int J Syst Evol Microbiol. 2004;54:47–57.PubMedView ArticleGoogle Scholar
  23. Mishra AK, Pfleiderer A, Lagier JC, Robert C, Raoult D, Fournier PE. Non-contiguous finished genome sequence and description of Bacillus massilioanorexius sp. nov. Stand Genomic Sci. 2013;8:465–79.PubMed CentralPubMedView ArticleGoogle Scholar
  24. Nagel M, Andreesen JR. Bacillus niacini sp. nov. a nicotinate-metabolizing mesophile isolated from soil. Int J Syst Bacteriol. 1991;41:134–9.View ArticleGoogle Scholar
  25. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.PubMed CentralPubMedView ArticleGoogle Scholar
  26. Prodigal. http://prodigal.ornl.gov
  27. GenBank database. http://www.ncbi.nlm.nih.gov/genbank
  28. Lowe TM, Eddy SR. t-RNAscan-SE: a program for improved detection of transfer RNA gene in genomic sequence. Nucl Acids Res. 1997;25:955–64.PubMed CentralPubMedView ArticleGoogle Scholar
  29. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucl Acids Res. 2007;35:3100–8.PubMed CentralPubMedView ArticleGoogle Scholar
  30. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004;340:783–95.PubMedView ArticleGoogle Scholar
  31. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–80.PubMedView ArticleGoogle Scholar
  32. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, et al. Artemis: sequence visualization and annotation. Bioinformatics. 2000;16:944–5.PubMedView ArticleGoogle Scholar
  33. Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J. DNAPlotter: circular and linear interactive genome visualization. Bioinformatics. 2009;25:119–20.PubMed CentralPubMedView ArticleGoogle Scholar
  34. Darling AC, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–403.PubMed CentralPubMedView ArticleGoogle Scholar
  35. Lechner M, Findeib S, Steiner L, Marz M, Stadler PF, Prohaska SJ. Proteinortho: detection of (Co-) orthologs in large-scale analysis. BMC Bioinformatics. 2011;12:124.PubMed CentralPubMedView ArticleGoogle Scholar
  36. Auch AF, von Jan M, Klenk HP, Göker M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci. 2010;2:117–34.PubMed CentralPubMedView ArticleGoogle Scholar
  37. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14:60.PubMed CentralPubMedView ArticleGoogle Scholar
  38. Meier-Kolthoff JP, Klenk HP, Göker M. Taxonomic use of DNA G + C content and DNA-DNA hybridization in the genomic age. Int J Syst Evol Microbiol. 2014;64(Pt 2):352–6.PubMedView ArticleGoogle Scholar
  39. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archae, Bacteria, and Eukarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.PubMed CentralPubMedView ArticleGoogle Scholar
  40. Gibbons NE, Murray RGE. Proposals concerning the higher taxa of Bacteria. Int J Syst Bacteriol. 1978;28:1–6.View ArticleGoogle Scholar
  41. Garrity GM, Holt JG. The road map to the manual. In: Garrity GM, Boone DR, Castenholz RW, editors. Bergey’s Manual of Systematic Bacteriology, vol. 1. 2nd ed. New York: Springer; 2001. p. 119–69.View ArticleGoogle Scholar
  42. Murray RGE. The higher taxa, or, a place for everything…? In: Holt JG, editor. Bergey’s Manual of Systematic Bacteriology, vol. 1. 1st ed. Baltimore: The Williams and Wilkins Co.; 1984. p. 31–4.Google Scholar
  43. Ludwig W, Schleifer KH, Whitman WB. Class I. Bacilli class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB, editors. Bergey’s Manual of Systematic Bacteriology. Second ed. New York: Springer; 2009. p. 19–20.Google Scholar
  44. Euzéby J. List of new names and new combinations previously effectively, but not validly, published. List no. 132. Int J Syst Evol Microbiol 2010; 60:469–472.
  45. Skerman VBD, McGowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Bacteriol. 1980;30:225–420.View ArticleGoogle Scholar
  46. Prévot AR. In: Hauderoy P, Ehringer G, Guillot G, Magrou J, Prévot AR, Rosset D, Urbain A (eds), Dictionaire des Bactéries Pathogènes, Second Edition, Masson et Cie, Paris, 1953, p. 1–692
  47. Fischer A. Untersuchungen über bakterien. Jahrbücher für Wissenschaftliche Botanik. 1895;27:1–163.Google Scholar
  48. Gibson T, Gordon RE, Genus I. Bacillus Cohn 1872, 174; Nom. gen. cons. Nomencl. Comm. Intern. Soc. Microbiol. 1937, 28; Opin. A. Jud. Comm. 1955, 39. In: Buchanan RE, Gibbons NE, editors. Bergey’s Manual of Determinative Bacteriology. Eighthth ed. Baltimore: The Williams and Wilkins Co; 1974. p. 529–50.Google Scholar
  49. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet. 2000;25:25–9.PubMed CentralPubMedView ArticleGoogle Scholar
  50. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.PubMed CentralPubMedView ArticleGoogle Scholar

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© Bittar et al. 2015

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