Open Access

Non-contiguous finished genome sequence and description of Kallipyga massiliensis gen. nov., sp. nov., a new member of the family Clostridiales Incertae Sedis XI

  • Perrine Hugon
  • , Dhamodharan Ramasamy
  • , Catherine Robert
  • , Carine Couderc
  • , Didier Raoult
  • and Pierre-Edouard Fournier
Corresponding author

DOI: 10.4056/sigs.4047997

Received: 30 July 2013

Accepted: 30 July 2013

Published: 30 July 2013

Abstract

Kallipyga massiliensis strain ph2T is the type strain of Kallipyga massiliensis gen. nov., sp. nov., the type species of the new genus Kallipyga within the family Clostridiales Incertae Sedis XI. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. K. massiliensis is an obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,770,679 bp long genome (1 chromosome but no plasmid) contains 1,575 protein-coding and 50 RNA genes, including 4 rRNA genes.

Keywords:

Kallipyga massiliensisgenomeculturomicstaxonogenomics

Introduction

Kallipyga massiliensis strain ph2T (CSUR=P241, DSM=26229) is the type strain of K. massiliensis gen. nov., sp. nov. This bacterium was isolated from the stool sample of an obese French patient as part of a study aiming at individually cultivating all species occurring within human feces [1-3]. It is a Gram-positive, anaerobic, indole-negative coccus. Defining the taxonomic status of bacterial isolates remains a challenging task. The taxonomic molecular tools currently available, including 16S rRNA sequence similarity, G + C content and DNA–DNA hybridization (DDH) [4,5], although considered as gold standards, have limitations [6,7]. The 16S rRNA sequence similarity and G+C content thresholds do not apply uniformly to all species or genera, and the DDH method lacks intra- and inter-laboratory reproducibility [5]. The advent of high-throughput genome sequencing and proteomic analysis [8] has granted unprecedented access to exhaustive genetic and protein information for bacterial isolates. We recently proposed a polyphasic approach to describe new bacterial species in which genome sequences and MALDI-TOF spectra are used along with phenotypic characteristics [9-30].

The family Clostridiales Incertae Sedis XI (Garrity and Holt 2001) was created in 2001 [31] and currently includes the 11 following genera: Anaerococcus (Ezaki et al. 2001) [32], Dethiosulfatibacter (Takii et al. 2007) [33], Finegoldia (Murdoch and Shah 2000) [34], Gallicola (Ezaki et al. 2001) [32], Helcococcus (Collins et al. 1993) [35], Parvimonas (Tindall and Euzéby 2006) [36], Peptoniphilus (Ezaki et al. 2001) [32], Sedimentibacter (Breitenstein et al. 2002) [37], Soehngenia (Parshina et al. 2003) [38], Sporanaerobacter (Hernandez-Eugenio et al. 2002) [39] and Tissierella (Collins and Shah 1986) [40]. Currently, 31 species with validly published names are reported in this family [41]. The species listed in the Clostridiales Incertae Sedis XI are mostly comprised of Gram-positive, obligate anaerobic cocci. Members belonging to this family were identified as pathogens in both humans and animals. In humans, they were often isolated from patients with septic arthritis, necrotizing pneumonia, prosthetic joint infection and other clinical conditions associated with vaginal discharges and ovarian, peritoneal and sacral abscesses [42-46].

Here we present a summary classification and a set of features for K. massiliensis gen. nov., sp. nov., strain ph2T (CSUR=P241, DSM=26229) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the genus Kallipyga and its type species, K. massiliensis within the Clostridiales Incertae Sedis XI family.

Classification and features

A stool sample was collected from a 26-year-old woman living in Marseille (France). She suffered from morbid obesity and had a body mass index of 48.2 (118.8 kg, 1.57 meter). At the time of stool sample collection she did not take any medication and was not on a diet. The patient gave an informed and signed consent, and the agreement of the ethics committee of the Institut Fédératif de Recherche (IFR48, Faculty of Medicine, Marseille, France) was obtained under reference 09-022. Another four new bacterial species, Alistipes obesi, Peptoniphilus grossensis, P. obesi and Enorma massiliensis [25-27,33], were also isolated from this specimen using various culture conditions. The fecal specimen was preserved at -80°C after collection. Strain ph2 T (Table 1) was isolated in 2011 by anaerobic culture on 5% sheep blood-enriched agar in anaerobic atmosphere at 37°C, following 26 days in a blood culture bottle with rumen and sheep blood. The 16S rRNA nucleotide sequence (GenBank accession number JN837487) of Kallipyga massiliensis strain ph2T was 86.09% similar to Helcococcus sueciensis, the phylogenetically closest species (Figure 1). This value was lower than the 95.0% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers (2006) to delineate a new genus without carrying out DNA-DNA hybridization [5].

Table 1

Classification and general features of Kallipyga massiliensis strain ph2T according to the MIGS recommendations [45].

MIGS ID

      Property

      Term

     Evidence codea

      Domain Bacteria

     TAS [47]

      Phylum Firmicutes

     TAS [48-50]

      Class Clostridia

     TAS [51,52]

      Current classification

      Order Clostridiales

     TAS [53,54]

      Family Clostridiales Incertae Sedis XI

     TAS [55]

      Genus Kallipyga

     IDA

      Species Kallipyga massiliensis

     IDA

      Type strain ph2T

     IDA

      Gram stain

      Positive

     IDA

      Cell shape

      Cocci

     IDA

      Motility

      Non-motile

     IDA

      Sporulation

      Non-sporulating

     IDA

      Temperature range

      Mesophile

     IDA

      Optimum temperature

      37°C

     IDA

MIGS-6.3

      Salinity

      unknown

     IDA

MIGS-22

      Oxygen requirement

      Anaerobic

     IDA

      Carbon source

      Unknown

     NAS

      Energy source

      Unknown

     NAS

MIGS-6

      Habitat

      Human gut

     IDA

MIGS-15

      Biotic relationship

      Free living

     IDA

MIGS-14

      Pathogenicity      Biosafety level      Isolation

      Unknown      2      Human feces

     NAS

MIGS-4

      Geographic location

      France

     IDA

MIGS-5

      Sample collection time

      January 2011

     IDA

MIGS-4.1

      Latitude

      43.296482

     IDA

MIGS-4.1

      Longitude

      5.36978

     IDA

MIGS-4.3

      Depth

      Surface

     IDA

MIGS-4.4

      Altitude

      0 m above sea level

     IDA

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 [56]. 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.

Figure 1

Phylogenetic tree highlighting the position of Kallipyga massiliensis strain ph2T relative to other type strains within the Clostridiales Incertae Sedis XI family. Genbank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method in MEGA software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 500 times to generate a majority consensus tree. Eubacterium cylindroides was used as outgroup. The scale bar represents a 2% nucleotide sequence divergence.

By comparison to the Genbank database [57], strain ph2T also exhibited a nucleotide sequence similarity greater than 98.7% with 16 sequences from uncultured bacteria from the human skin microbiome [58]. These bacteria are most likely classified within the same species as strain ph2T.

Different growth temperatures (25, 30, 37, 45°C) were tested; no growth occurred at 25°C and 30°C, growth occurred between 37°C and 45°C, and optimal growth was observed at 37°C. Colonies were bright grey with a diameter of 1.0 mm on 5% blood-enriched Columbia agar. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and in the presence of air, with or without 5% CO2. Optimal growth was obtained anaerobically. No growth was observed under aerobic and microaerophilic conditions. Gram staining showed Gram-positive cocci (Figure 2). A motility test was negative. Cells grown on agar are Gram-positive, have a diameter in electron microscopy ranging from 0.57µm to 0.78µm (mean, 0.67 µm, Figure 3) and are mostly grouped in pairs, short chains or small clumps.

Figure 2

Gram staining of K massiliensis strain ph2T

Figure 3

Transmission electron microscopy of K. massiliensis strain ph2T using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 200 nm.

Strain ph2T exhibited neither catalase or oxidase activities. Using API 32A (BioMerieux), nitrate reduction, indole formation and urease production were negative. A positive reaction was obtained for α-galactosidase, arginine dihydrolase and arginine arylamidase, α-glucosidase and β-glucosidase. Strain ph2T did not ferment mannose or raffinose. Negative reactions were observed for β-galactosidase, β-galactosidase-6-phosphate, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Using an API Zym (BioMerieux), positive reactions were observed for esterase lipase, leucine arylamidase, α-glucosidase, β-glucosidase and acid phosphatase. Negative reactions were obtained for esterase, lipase, valine and cysteine arylamidase, trypsine, α-chymotrypsine, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using an API 50CH (BioMerieux), K. massiliensis weakly fermented D-ribose, D-glucose, D-fructose and aesculin. By comparison with its closest phylogenetic neighbors, K. massiliensis differed from Finegoldia magna in α-galactosidase and α-glucosidase production, D-ribose and D-fructose fermentation. It also differed from Helcococcus kunzii in oxygen requirement, α-galactosidase and leucine arylamidase production, D-ribose, D-glucose and esculin utilization. It differed from Parvimonas micra in alkaline phosphatase, glutamyl glutamic acid arylamidase, β-glucosidase, phenylalanine arylamidase and histidine arylamidase production and D-glucose fermentation. It differed from Peptoniphilus indolicus in α-galactosidase, indole, α-glucosidase, β-glucosidase, dihydrolase phenylalanine, phenylalanine arylamidase and histidine arylamidase production, and D-glucose fermentation (Table 2).

Table 2

Differential characteristics of Kallipyga massiliensis gen. nov., sp. nov., strain ph2T, Finegoldia magna strain ATCC 29328, Helcococcus kunzii strain ATCC 51366, Parvimonas micra strain ATCC 33270 and Peptoniphilus indolicus strain ATCC 29427T.

Properties

     K. massiliensis

    F. magna

     H. kunzii

      P. micra

      P. indolicus

Cell diameter (µm)

     0.67

    na

     na

      0.3-0.7

      na

Oxygen requirement

     anaerobic

    anaerobic

     facultative anaerobic

      anaerobic

      anaerobic

Colony color

     bright gray

    var

      na

      na

Gram stain

     +

    +

     +

      +

      +

Salt requirement

     -

    na

     +/-

      na

      -

Motility

     -

    -

     -

      na

      -

Endospore formation

     -

    -

     na

      na

      -

Production of

Alkaline Phosphatase

     -

    +/-

     -

      +

      -

Catalase

     -

    +/-

     -

      na

      na

Oxidase

     -

    na

     na

      na

      na

Nitrate reductase

     -

    -

     -

      na

      na

Urease

     -

    -

     na

      na

      -

α-galactosidase

     +

    -

     -

      na

      -

β-galactosidase

     -

    -

     -

      na

      -

Indole

     -

    -

     na

      -

      +

Arginine arylamidase

     +

    +

     na

      +

      +

Glutamyl glutamic acidarylamidase

     -

    na

     na

      +

      na

Arginine dihydrolase

     +

    +/-

     na

      na

      -

α-glucosidase

     +

    -

     na

      na

      -

β-glucosidase

     +

    na

     na

      -

      -

β-glucuronidase

     -

    -

     -

      na

      na

Phenylalanine arylamidase

     -

    -

     na

      +

      +

Esterase lipase

     +

    na

     na

      na

      na

Leucine arylamidase

     +

    +

     -

      na

      +

Cystine arylamidase

     -

    na

     na

      na

      na

Histidine arylamidase

     -

    -/w

     na

      +

      +

Fermentation of

D-mannose

     -

    -

     na

      na

      -

D-ribose

     w

    -

     -

      na

      na

D-glucose

     w

    -/w

     -

      -

      -

D-fructose

     w

    +

     na

      na

      na

Esculin

     w

    na

     +

      na

      na

Isolated from

     human gut

    human

     human

      human

      mastitis of cattle

na = data not available; var = variable; w = weak

K. massiliensis is susceptible to amoxicillin, amoxicillin-clavulanic acid, gentamicin 500, penicillin, imipenem, vancomycin, rifampicin and nitrofurantoin, but resistant to ciprofloxacin, metronidazole, gentamicin 10, trimethoprim/sulfamethoxazole, ceftriaxon, erythromycin and doxycycline.

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [59] using a Microflex spectrometer (Bruker Daltonics, Germany). Twelve distinct deposits were done for strain ph2T from 12 isolated colonies. The twelve ph2T spectra were imported into our database and compared to spectra from 3,769 bacteria using the MALDI BioTyper software (version 2.0, Bruker). A score enabled the presumptive identification and discrimination of the tested species from those in a database: a score > 2 with 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 ph2T, no significant score was obtained, suggesting that our isolate was not a member of any known species or genus (Figures 4 and 5). A broader study incorporating MALDI-TOF and 16S rDNA and genomic DNA identity data may be conducted to define taxonomic criteria at the family level.

Figure 4

Reference mass spectrum from K. massiliensis strain ph2T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Figure 5

Gel view comparing Kallipyga massiliensis sp. nov strain ph2T to other phylogenetically close species. The gel view displays the raw spectra of 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. Displayed species are indicated on the left.

Genome sequencing information

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to members of the family Clostridiales Incertae Sedis XI and is part of a study of the human digestive flora aiming at isolating all bacterial species within human feces [1-3]. It was the thirty-sixth genome from the family Clostridiales Incertae Sedis XI to be sequenced and the first genome of K. massiliensis gen. nov., sp. nov. The GenBank accession number is CAHC00000000 and consists of 22 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance [60].

Table 3

Project information

MIGS ID

  Property

   Term

MIGS-31

  Finishing quality

   High-quality draft

MIGS-28

  Libraries used

   454 GS paired-end 3- kb libraries

MIGS-29

  Sequencing platform

   454 GS FLX Titanium

MIGS-31.2

  Sequencing coverage

   51.23 ×

MIGS-30

  Assemblers

   Newbler

MIGS-32

  Gene calling method

   Prodigal

  Genbank Date of Release

   May 30, 2012

  NCBI project ID

   CAHC00000000

MIGS-13

  Project relevance

   Study of the human gut microbiome

Growth conditions and DNA isolation

Kallipyga massiliensis gen. nov., sp. nov., strain ph2T (CSUR= P241, DSM=26229) was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Three petri dishes were spread and the bacteria cultivated were 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 (MP Biomedicals, USA) using 2 × 20 seconds cycles. DNA was then treated with 2.5µg/µL lysozyme for 30 minutes at 37°C and extracted using the BioRobot EZ1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a QIAamp kit (Qiagen). The yield and concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 78.2ng/µl.

Genome sequencing and assembly

DNA (5 µg) was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3-4kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.179kb. A 3kb paired-end 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 optimal at 600 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was quantified on the Quant-it Ribogreen kit (Invitrogen) on the Genios Tecan fluorometer at 58 pg/µL. The library concentration equivalence was calculated as 1.77E+08 molecules/µL. The library was stored at -20°C until further use.

The paired-end library was clonally amplified with 0.5 cpb and 1 cbp in 2 SV-emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were essentially the same at 12.3 and 12%, in the range of 5 to 20% recommended by the Roche procedure.

Approximately 790,000 beads were loaded on 1/4 region of a GS Titanium PicoTiterPlate PTP Kit 70x75 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 gsAssembler (Roche). A total, of 261,794 passed filter wells were obtained and generated 90.68 Mb with a length average of 346 bp. The global passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 3 scaffolds and 22 large contigs (> 1,500 bp) generating a genome size of 1.77 Mb which corresponds to a coverage of 51.23× genome equivalent.

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [61] with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank [57] and Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan-SE [62] and RNAmmer [63] tools, respectively. Lipoprotein signal peptides and numbers of transmembrane helices were predicted using SignalP [64] and TMHMM [65], 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 [66] and DNA Plotter [67] 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 [68]. To estimate the mean level of nucleotide sequence similarity at the genome level between K. massiliensis and four other members of the family Clostridiales Incertae Sedis XI (Table 6), orthologous proteins were detected using the Proteinortho software [69] and genomes compared two by two. For each pair of genomes, we determined the mean percentage of nucleotide sequence identity among orthologous ORFs using BLASTn.

Genome properties

The genome is 1,770,679 bp long (one chromosome, no plasmid) with a G+C content of 51.40% (Figure 6 and Table 4). Of the 1,625 predicted chromosomal genes, 1,575 were protein-coding genes and 50 were RNAs. A total of 1,238 genes (76.18%) were assigned a putative function. Forty-two genes were identified as ORFans (2.66%) and the remaining genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Tables 4 and 5. The distribution of genes into COGs functional categories is presented in Table 5.

Figure 6

Graphical circular map of the chromosome. From the outside in, the outer two circles show the genes on the forward and reverse directions (colored by COG categories). The third circle marks the rRNA operon in red and tRNA genes in green. The fourth circle shows the G+C% content plot. The inner-most circle shows GC skew, with purple and olive indicating negative and positive values, respectively.

Table 4

Nucleotide content and gene count levels of the chromosome

Attribute

   Value

      % of totala

Genome size (bp)

   1,770,679

DNA coding region (bp)

   1,590,528

      89.82

DNA G+C content (bp)

   910,129

      51.40

Total genes

   1,625

      100

RNA genes

   50

      3.07

Protein-coding genes

   1,575

      96.92

Genes with function prediction

   1,238

      76.18

Genes assigned to COGs

   1,165

      71.69

Genes with peptide signals

   90

      5.53

Genes with transmembrane helices

   405

      24.92

a The 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

Table 5

Number of genes associated with the 25 general COG functional categories

Code

   Value

    % agea

    Description

J

   131

    4.86

    Translation

A

   0

    0.032

    RNA processing and modification

K

   75

    5.31

    Transcription

L

   107

    5.74

    Replication, recombination and repair

B

   0

    0

    Chromatin structure and dynamics

D

   15

    0.78

    Cell cycle control, mitosis and meiosis

Y

   0

    0

    Nuclear structure

V

   51

    1.53

    Defense mechanisms

T

   22

    1.69

    Signal transduction mechanisms

M

   72

    3.42

    Cell wall/membrane biogenesis

N

   1

    0

    Cell motility

Z

   0

    0

    Cytoskeleton

W

   0

    0

    Extracellular structures

U

   13

    0.84

    Intracellular trafficking and secretion

O

   45

    2.47

    Posttranslational modification, protein turnover, chaperones

C

   66

    4.53

    Energy production and conversion

G

   88

    2.87

    Carbohydrate transport and metabolism

E

   67

    6.16

    Amino acid transport and metabolism

F

   47

    2.05

    Nucleotide transport and metabolism

H

   34

    2.34

    Coenzyme transport and metabolism

I

   28

    4.01

    Lipid transport and metabolism

P

   59

    4.14

    Inorganic ion transport and metabolism

Q

   4

    0.81

    Secondary metabolites biosynthesis, transport and catabolism

R

   127

    8.15

    General function prediction only

S

   113

    5.93

    Function unknown

-

   410

    26.03

    Not in COGs

a The total is based on the total number of protein-coding genes in the annotated genome

Genomic comparison of K. massiliensis and other members of the family Clostridiales Incertae Sedis XI.

Currently, 35 genomes are available for members of the family Clostridiales Incertae Sedis XI. Here, we compared the genome sequence of K. massiliensis strain ph2T with those of Finegoldia magna strain ATCC 29328, Helcococcus kunzii strain ATCC 51366, Peptoniphilus indolicus strain ATCC 29427 and Parvimonas micra strain ATCC 33270. The draft genome of K. massiliensis (1.77Mb) is smaller than all other genomes except P. micra (1.70Mb) and exhibits a higher G+C content (51.40) (Table 6A). The gene content of K. massiliensis is also lower than the other four genomes used for comparison (Table 6B). In addition, K. massiliensis shared 653, 549, 592 and 548 orthologous genes with F.magna, H. kunzii,P. indolicus and P.micra respectively. The average nucleotide sequence identity ranged from 58.61 to 69.17% among Clostridiales Incertae Sedis XI family species, and from 58.61 to 59.97% between K. massiliensis and other species, thus confirming its new genus status (Table 6B).

Table 6A

Genomic comparison of K. massiliensis gen. nov., sp. nov., strain ph2T with four other members of the family Clostridiales Incertae Sedis XI

Species

    Strain

   Genome accession number

    Genome size (Mb)

   G+C content

K. massiliensis

    ph2T

   CAHC00000000

    1,770,679

   51.40

F. magna

    ATCC 29328

   NC_010376

    1,797,577

   32.1

H. kunzii

    ATCC 51366

   AGEI01000000

    2,083,191

   29.40

P. indolicus

    ATCC 29427

   AGBB01000000

    2,101,630

   31.70

P. micra

    ATCC 33270

   ABEE02000000

    1,703,772

   28.70

Species and strain names, GenBank genome accession numbers, sizes and G+C contents.

Table 6B

Genomic comparison of K. massiliensis gen. nov., sp. nov., strain ph2T with four other members of the family Clostridiales Incertae Sedis XI

   K. massiliensis

   F. magna

   H. kunzii

   P. indolicus

   P. micra

K. massiliensis

   1,568

   635

   549

   592

   548

F. magna

   59.22

   1,656

   629

   687

   665

H. kunzii

   59.06

   68.20

   1,878

   561

   560

P. indolicus

   59.97

   67.98

   67.57

   2,205

   615

P. micra

   58.61

   69.17

   68.52

   68.64

   1,597

Numbers of orthologous proteins shared between genomes (upper right), average percentage of nucleotide similarity of orthologous proteins shared between genomes (lower left). Bold numbers indicate numbers of proteins per genome.

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Kallipya massiliensis gen. nov., sp. nov., that contains the strain ph2T. This bacterium has been found in France.

Description of Kallipyga gen. nov.

Kallipyga (cal.li.pi’ga N.L. fem. N. Kallipyga of the Greek epithet kallipygos, said of a statue of Aphrodite having beautifully proportioned buttocks).

Gram-positive cocci. Strictly anaerobic. Mesophilic. Non-Motile. Does not exhibit catalase, oxidase and indole production nor nitrate reduction. Positive for α-galactosidase, arginine dihydrolase, arginine arylamidase, and α- and β-glucosidase. Habitat: human digestive tract. Type species: Kallipyga massiliensis.

Description of Kallipyga massiliensis gen. nov., sp. nov.

Kallipyga massiliensis (mas.il’ien’sis. L. gen. fem. n. massiliensis, of Massilia, the Latin name of Marseille where was cultivated strain ph2T). It has been isolated from the feces of an obese French patient.

Gram-positive cocci. Strictly anaerobic. Mesophilic. Optimal growth at 37°C. Non-motile and non-sporulating. Colonies are bright grey with 1.0 mm in diameter on blood-enriched Columbia agar. Cells are cocci with a diameter ranging from 0.57 µm to 0.78 µm with a mean diameter of 0.67. Catalase and oxidase activities are negative. Nitrate reduction and indole production are absent. Negative reactions were observed for β-galactosidase, β-galactosidase-6-phosphate, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase, mannose and raffinose fermentation, esterase, lipase, valine and cystine arylamidase, trypsine, α-chymotrypsine, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-Glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Positive reactions were observed for α-galactosidase, arginine dihydrolase and arginine arylamidase, α and β-glucosidase, esterase lipase, leucine arylamidase and acid phosphatase. Cells weakly oxidized D-ribose, D-glucose, D-fructose and aesculin. Cells are susceptible to amoxicillin, amoxicillin-clavulanic acid, gentamicin 500, penicillin, imipenem, vancomycin, rifampin and nitrofurantoine. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JN837487 and CAHC00000000, respectively. The G+C content of the genome is 51.40%. The type strain ph2T (= CSUR P241 = DSM 26229) was isolated from the fecal flora of an obese patient in France.

Declarations

Acknowledgements

The authors thank the Xegen Company (Web Site) for automating the genomic annotation process. This study was funded by the Mediterranee-Infection Foundation.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, Bittar F, Fournous G, Gimenez G and Maraninchi M. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect. 2012; 18:1185-1193PubMed
  2. Dubourg G, Lagier JC, Armougom F, Robert C, Hamad I and Brouqui P. 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; 32:637-645 View ArticlePubMed
  3. Pfleiderer A, Lagier JC, Armougom F, Robert C, Vialettes B and Raoult D. Culturomics identified 11 new bacterial species from a single anorexia nervosa stool sample. [Epub ahead of print]. Eur J Clin Microbiol Infect Dis. 2013 View ArticlePubMed
  4. Tindall BJ, Rossello-Mora R, Busse HJ, Ludwig W and Kampfer P. Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol. 2010; 60:249-266 View ArticlePubMed
  5. Stackebrandt E and Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today. 2006; 33:152-155
  6. Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE and Stackebrandt E. Report of the ad hoc committee on reconciliation of approaches to bacterial systematic. Int J Syst Bacteriol. 1987; 37:463-464 View Article
  7. Rossello-Mora R. DNA-DNA Reassociation Methods Applied to Microbial Taxonomy and Their Critical Evaluation. In: Stackebrandt E (ed), Molecular Identification, Systematics, and population Structure of Prokaryotes. Springer, Berlin, 2006; p. 23-50.
  8. Welker M and Moore ER. Applications of whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry in systematic microbiology. Syst Appl Microbiol. 2011; 34:2-11 View ArticlePubMed
  9. Kokcha S, Mishra AK, Lagier JC, Million M, Leroy Q, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Bacillus timonensis sp. nov. Stand Genomic Sci. 2012; 6:346-355 View ArticlePubMed
  10. Lagier JC, El Karkouri K, Nguyen TT, Armougom F, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov. Stand Genomic Sci. 2012; 6:116-125 View ArticlePubMed
  11. Mishra AK, Gimenez G, Lagier JC, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Alistipes senegalensis sp. nov. Stand Genomic Sci. 2012; 6:304-314 View Article
  12. Lagier JC, Armougom F, Mishra AK, Ngyuen TT, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Alistipes timonensis sp. nov. Stand Genomic Sci. 2012; 6:315-324PubMed
  13. Mishra AK, Lagier JC, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Clostridium senegalense sp. nov. Stand Genomic Sci. 2012; 6:386-395PubMed
  14. Mishra AK, Lagier JC, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Peptoniphilus timonensis sp. nov. Stand Genomic Sci. 2012; 7:1-11 View ArticlePubMed
  15. Mishra AK, Lagier JC, Rivet R, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Paenibacillus senegalensis sp. nov. Stand Genomic Sci. 2012; 7:70-81PubMed
  16. Lagier JC, Gimenez G, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Herbaspirillum massiliense sp. nov. Stand Genomic Sci. 2012; 7:200-209PubMed
  17. Roux V, El Karkouri K, Lagier JC, Robert C and Raoult D. Non-contiguous finished genome sequence and description of Kurthia massiliensis sp. nov. Stand Genomic Sci. 2012; 7:221-232 View ArticlePubMed
  18. Kokcha S, Ramasamy D, Lagier JC, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Brevibacterium senegalense sp. nov. Stand Genomic Sci. 2012; 7:233-245 View ArticlePubMed
  19. Ramasamy D, Kokcha S, Lagier JC, N’Guyen TT, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Aeromicrobium massilense sp. nov. Stand Genomic Sci. 2012; 7:246-257 View ArticlePubMed
  20. Lagier JC, Ramasamy D, Rivet R, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Cellulomonas massiliensis sp. nov. Stand Genomic Sci. 2012; 7:258-270 View ArticlePubMed
  21. Lagier JC, El Karkouri K, Rivet R, Couderc C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Senegalemassilia anaerobia sp. nov. Stand Genomic Sci. 2013; 7:343-356 View ArticlePubMed
  22. Mishra AK, Hugon P, Lagier JC, Nguyen TT, Robert C, Couderc C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Peptoniphilus obesi sp. nov. Stand Genomic Sci. 2013; 7:357-369 View ArticlePubMed
  23. Mishra AK, Lagier JC, Nguyen TT, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Peptoniphilus senegalensis sp. nov. Stand Genomic Sci. 2013; 7:370-381 View ArticlePubMed
  24. Lagier JC, El Karkouri K, Mishra AK, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Enterobacter massiliensis sp. nov. Stand Genomic Sci. 2013; 7:399-412 View ArticlePubMed
  25. Hugon P, Ramasamy D, Lagier JC, Rivet R, Couderc C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Alistipes obesi sp. nov. Stand Genomic Sci. 2013; 7:427-439 View ArticlePubMed
  26. Mishra AK, Hugon P, Robert C, Couderc C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Peptoniphilus grossensis sp. nov. Stand Genomic Sci. 2012; 7:320-330PubMed
  27. Mishra AK, Hugon P, Lagier JC, Nguyen TT, Couderc C, Raoult D and Fournier PE. 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 View ArticlePubMed
  28. Ramasamy D, Lagier JC, Gorlas A, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Bacillus massiliosenegalensis sp. nov. Stand Genomic Sci. 2013; 8:264-278 View ArticlePubMed
  29. Ramasamy D, Lagier JC, Nguyen TT, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of of Dielma fastidiosa gen. nov., sp. nov., a new member of the Family Erysipelotrichaceae. Stand Genomic Sci. 2013; 8:336-351 View ArticlePubMed
  30. Mishra AK, Lagier JC, Robert C, Raoult D and Fournier PE. Genome sequence and description of Timonella senegalensis gen. nov., sp. nov., a new member of the suborder Micrococcinae. Stand Genomic Sci. 2013; 8:318-335 View ArticlePubMed
  31. Garrity GM, Holt JG. Taxonomic outline of the Archae and Bacteria In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, second edition, Volume, Springer-Verlag, New York, 2001; p. 155-166.
  32. Ezaki T, Kawamura Y, Li N, Li ZY, Zhao L and Shu S. Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus. Int J Syst Evol Microbiol. 2001; 51:1521-1528PubMed
  33. Takii S, Hanada S, Tamaki H, Ueno Y, Sekiguchi Y, Ibe A and Matsuura K. Dethiosulfatibacter aminovorans gen. nov., sp. nov., a novel thiosulfate-reducing bacterium isolated from coastal marine sediment via sulfate-reducing enrichment with Casamino acids. Int J Syst Evol Microbiol. 2007; 57:2320-2326 View ArticlePubMed
  34. Murdoch DA and Shah HN. Reclassification of Peptostreptococcus magnus (Prevot 1933) Holdeman and Moore 1972 as Finegoldia magna comb. nov. and Peptostreptococcus micros (Prevot 1933) Smith 1957 as Micromonas micros comb. nov. Anaerobe. 1999; 5:555-559 View Article
  35. Collins MD, Facklam RR, Rodrigues UM and Ruoff KL. Phylogenetic analysis of some Aerococcus-like organisms from clinical sources: description of Helcococcus kunzii gen. nov., sp. nov. Int J Syst Bacteriol. 1993; 43:425-429 View ArticlePubMed
  36. Tindall BJ and Euzeby JP. Proposal of Parvimonas gen. nov. and Quatrionicoccus gen. nov. as replacements for the illegitimate, prokaryotic, generic names Micromonas Murdoch and Shah 2000 and Quadricoccus Maszenan et al. 2002, respectively. Int J Syst Evol Microbiol. 2006; 56:2711-2713 View ArticlePubMed
  37. Breitenstein A, Wiegel J, Haertig C, Weiss N, Andreesen JR and Lechner U. Reclassification of Clostridium hydroxybenzoicum as Sedimentibacter hydroxybenzoicus gen. nov., comb. nov., and description of Sedimentibacter saalensis sp. nov. Int J Syst Evol Microbiol. 2002; 52:801-807 View ArticlePubMed
  38. Parshina SN, Kleerebezem R, Sanz JL, Lettinga G, Nozhevnikova AN, Kostrikina NA, Lysenko AM and Stams AJ. Soehngenia saccharolytica gen. nov., sp. nov. and Clostridium amygdalinum sp. nov., two novel anaerobic, benzaldehyde-converting bacteria. Int J Syst Evol Microbiol. 2003; 53:1791-1799 View ArticlePubMed
  39. Hernandez-Eugenio G, Fardeau ML, Cayol JL, Patel BK, Thomas P, Macarie H, Garcia JL and Ollivier B. Sporanaerobacter acetigenes gen. nov., sp. nov., a novel acetogenic, facultatively sulfur-reducing bacterium. Int J Syst Evol Microbiol. 2002; 52:1217-1223 View ArticlePubMed
  40. Collins MD and Shah HN. Reclassification of Bacteroides praeacutus Tissier (Holdeman and Moore) in a new genus, Tissierella, as Tissierella praeacuta comb. nov. Int J Syst Bacteriol. 1986; 36:461-463 View Article
  41. List of Prokaryotic names with standing nomenclature (LPSN). Web Site
  42. Jain S, Bui V, Spencer C and Yee L. Septic arthritis in a native joint due to Anaerococcus prevotii. J Clin Pathol. 2007; 61:775-776 View ArticlePubMed
  43. Ulger-Toprak N, Liu C, Summanen PH and Finegold SM. Murdochiella asaccharolytica gen. nov., sp. nov., a Gram-stain-positive, anaerobic coccus isolated from human wound specimens. Int J Syst Evol Microbiol. 2010; 60:1013-1016 View ArticlePubMed
  44. Labutti K, Pukall R, Steenblock K, Glavina Del Rio T, Tice H, Copeland A, Cheng JF, Lucas S, Chen F and Nolan M. Complete genome sequence of Anaerococcus prevotii type strain (PC1). Stand Genomic Sci. 2009; 1:159-165 View ArticlePubMed
  45. Veloo AC, Erhard M, Welker M, Welling GW and Degener JE. Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry. Syst Appl Microbiol. 2011; 34:58-62 View ArticlePubMed
  46. Pépin J, Deslandes S, Giroux G, Sobela F, Khonde N, Diakite S, Demeule S, Labbe AC, Carrier N and Frost E. The complex vaginal flora of West African women with bacterial vaginosis. PLoS ONE. 2011; 6:e25082 View ArticlePubMed
  47. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the domains Archae, Bacteria, and Eukarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  48. Gibbons NE and Murray RGE. Proposals Concerning the Higher Taxa of Bacteria. Int J Syst Bacteriol. 1978; 28:1-6 View Article
  49. Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119-169.
  50. Murray RGE. The Higher Taxa, or, a Place for Everything...? In: Holt JG (ed), Bergey's Manual of Systematic Bacteriology, First Edition, Volume 1, The Williams and Wilkins Co., Baltimore, 1984, p. 31-34.
  51. 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 View Article
  52. Rainey FA. Class II. Clostridia class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 3, Springer-Verlag, New York, 2009, p. 736
  53. Skerman VBD and Sneath PHA. Approved list of bacterial names. Int J Syst Bact. 1980; 30:225-420 View Article
  54. Prevot AR. In: Hauduroy P, Ehringer G, Guillot G, et al.(eds.), Dictionnaire des bactéries pathogènes, Second Edition, Masson, Paris, 1953, p. 1-692.
  55. Garrity GM, Holt J. Taxonomic outline of the Archaea and Bacteria In: Garrity GM, Boone DR, Castenholz RW (eds.), Bergey's Manual of Systematic Bacteriology, Second Edition, Springer-Verlag, New York, 2003, p.155-166.
  56. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  57. Benson DA, Karsch-Mizrachi I, Clark K, Lipman DJ, Ostell J and Sayers EW. GenBank. Nucleic Acids Res. 2012; 40:D48-D53 View ArticlePubMed
  58. Kong HH, Grice EA, Conlan S, Deming CB, Freeman AF, Beatson M, Nomicos E, Young AC, Bouffard GG and Blakesley RW. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res. 2012; 22:850-859 View ArticlePubMed
  59. Seng P, Drancourt M, Gouriet F, La SB, Fournier PE and Rolain JM. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 2009; 49:543-551 View ArticlePubMed
  60. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  61. Prodigal. Web Site
  62. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964PubMed
  63. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T and Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007; 35:3100-3108 View ArticlePubMed
  64. Bendtsen JD, Nielsen H, von Heijne G and Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004; 340:783-795 View ArticlePubMed
  65. Krogh A, Larsson B, von Heijne G and Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001; 305:567-580 View ArticlePubMed
  66. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA and Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000; 16:944-945 View ArticlePubMed
  67. Carver T, Thomson N, Bleasby A, Berriman M and Parkhill J. DNAPlotter: circular and linear interactive genome visualization. Bioinformatics. 2009; 25:119-120 View ArticlePubMed
  68. Darling AC, Mau B, Blattner FR and Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004; 14:1394-1403 View ArticlePubMed
  69. Lechner M, Findeib S, Steiner L, Marz M, Stadler PF and Prohaska SJ. Proteinortho: Detection of (Co-)orthologs in large-scale analysis. BMC Bioinformatics. 2011; 12:124 View ArticlePubMed