Open Access

Non contiguous-finished genome sequence and description of Microbacterium gorillae sp. nov.

  • Linda Hadjadj1,
  • Jaishriram Rathored1,
  • Mamadou Bhoye Keita1,
  • Caroline Michelle1,
  • Anthony Levasseur1,
  • Didier Raoult1, 2,
  • Pierre-Edouard Fournier1,
  • Jean-Marc Rolain1 and
  • Fadi Bittar1Email author
Standards in Genomic Sciences201611:32

https://doi.org/10.1186/s40793-016-0152-z

Received: 4 June 2015

Accepted: 8 April 2016

Published: 14 April 2016

Abstract

Strain G3T (CSUR P207 = DSM 26203) was isolated from the fecal sample of a wild gorilla (Gorilla gorilla subsp gorilla) from Cameroon. It is a Gram-positive, facultative anaerobic short rod. This strain exhibits a 16S rRNA sequence similarity of 98.2 % with Microbacterium thalassium, the closest validly published Microbacterium species and member of the family Microbacteriaceae. Moreover, it shows a low MALDI-TOF-MS score (1.1 to 1.3) that does not allow any identification. Thus, it is likely that this strain represents a new species. Here we describe the phenotypic features of this organism, the complete genome sequence and annotation. The 3,692,770 bp long genome (one chromosome but no plasmid) contains 3,505 protein-coding and 61 RNA genes, including 4 rRNA genes. In addition, digital DNA-DNA hybridization values for the genome of the strain G3T against the closest Microbacterium genomes range between 19.7 to 20.5, once again confirming its new status as a new species. On the basis of these polyphasic data, consisting of phenotypic and genomic analyses, we propose the creation of Microbacterium gorillae sp. nov. that contains the strain G3T.

Keywords

Microbacterium gorillae Genome Culturomics Taxonomo-genomics Gorilla stool sample

Introduction

Strain G3T (= CSUR P207 = DSM 26203) is the type strain of Microbacterium gorillae sp. nov. This bacterium is a Gram-positive, non-spore-forming, indole-negative, facultative anaerobic rod shaped bacillus. It was isolated from the feces of western lowland gorilla in Cameroon as part of a culturomics study to describe the bacterial communities of the gorilla gut [1]. By applying a large variety of culture conditions, culturomics allowed previously the isolation of numerous new bacterial species from gorilla fecal samples [1].

Furthermore, since the creation of the genus Microbacterium by Orla-Jensenin (1919) [2] to date, 91 bacterial species belonging to this genus have been validly published [3]. These species are Gram-positive and non-endospore-forming bacteria. Many studies have described Microbactertium species in diverse origins including human clinical specimens, soil, sea sediments, plants and hairspray [47].

In this report, we present a summary classification, phenotypic features for M. gorillae sp. nov. strain G3T, together with the description of the complete genome sequence and annotation. These characteristics support the circumscription of the species M. gorillae [8].

Organism information

Classification and features

Information about the fecal sample collection and conservation are described previously [1]. Strain G3T (Table 1) was isolated in January 2012 as part of a culturomics study [1] by cultivation on Columbia agar supplemented with sheep blood (BioMérieux, Craponne, France).
Table 1

Classification and general features of Microbacterium gorillae strain G3T

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain: Bacteria

TAS [34]

Phylum: Actinobacteria

TAS [35]

Class: Actinobacteria

TAS [35]

Order: Actinomycetales

TAS [2]

Family: Microbacteriaceae

TAS [36]

Genus: Microbacterium

TAS [2]

Species: Microbacterium gorillae

IDA

Type strain: G3T

IDA

 

Gram stain

Positive

IDA

 

Cell shape

Rod-shaped

IDA

 

Motility

Non-motile

IDA

 

Sporulation

Non-sporulating

IDA

 

Temperature range

Mesophilic

IDA

 

Optimum temperature

25 °C

IDA

 

pH range; Optimum

Not determined

 
 

Carbon source

Varied (see Additional file 4)

IDA

MIGS-6

Habitat

Gorilla gut

IDA

MIGS-6.3

Salinity

2 % NaCl

IDA

MIGS-22

Oxygen requirement

Facultative anaerobic

IDA

MIGS-15

Biotic relationship

Free living

IDA

MIGS-14

Pathogenicity

Unknown

 

Biosafety level

2

NAS

Isolation

Gorilla feces

IDA

MIGS-4

Geographic location

Cameroon

IDA

MIGS-5

Sample collection time

July 2011

IDA

MIGS-4.1

Latitude

2° 47′ 2.1768″

IDA

MIGS-4.2

Longitude

13° 1′ 49.6986″

IDA

MIGS-4.4

Altitude

>600 m above sea level

IDA

a 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 [37]. 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

When compared to sequences available in GenBank, the 16S rRNA gene sequence of M. gorillae strain G3T (GenBank accession number JX650056) exhibited an identity of 98.2 % with Microbacterium thalassium , the closest validly published Microbacterium species. This value was equal to the percentage of 16S rRNA gene sequence threshold recommended by Meier-Kolthoff et al. for class Actinobacteria to delineate a new species without carrying out DNA-DNA hybridization with maximum error probability of 0.1 % [9]. Figure 1 presents the 16S rRNA based tree for the strain G3T and other Microbacterium species.
Fig. 1

Phylogenetic tree highlighting the position of Microbacterium gorillae strain G3T relative to other type strains within the Microbacterium genus using 16S rRNA gene. GenBank accession numbers are indicated in parentheses. Sequences were aligned using MUSCLE. Alignments were then cleaned from highly divergent blocks using Gblocks version 0.91b [38]. Maximum likelihood (ML) phylogenetic tree was generated using RAxML [39], employing the GTR GAMMA substitution model with 500 bootstraps. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 500 times to generate a majority consensus tree. Corynebacterium diphtheriae was used as outgroup. The scale bar represents a rate of substitution per nucleotide position of 0.02. (T) indicates that the sequence used in the tree is from the type strain of the species.* 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 (20, 25, 30, 37, 45 °C) were tested. Growth occurred between 25 °C and 37 °C, but the optimal growth was observed at 25 °C, 24 h after inoculation. No growth occurred at 20 and 45 °C. Colonies were 0.8 mm in diameter, appear as gray color on Columbia agar supplemented with sheep blood. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and under 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 short bacilli (Fig. 2, left panel). A motility test with API M medium (BioMérieux) produced a negative result. Cells grown on agar do not sporulate and the rods have a mean length of 1 μm and a mean width of 0.5 μm. Both the length and the diameter were determined by negative staining transmission electron microscopy (Fig. 2, right panel).
Fig. 2

Gram staining (left panel) and Transmission electron microscopy using a Morgani 268D (Philips) at an operating voltage of 60 kV (right panel) of M. gorillae strain G3T. The scale bar represents 500 nm

Strain G3T exhibited catalase activity but not oxidase activity using ID color catalase and oxidase reagent, respectively (BioMérieux). In assays with API 50CH system (BioMérieux), strain G3T produced acid from esculin, D-cellobiose, D-maltose, D-lactose, D-mannose, D-mannitol, D-saccharose, D-trehalose and gentiobiose. By contrast, acid production was not observed for glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl-αD-xylopyranoside, D-galactose, D-glucose, L-fructose, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl-αD-mannopyranoside, Methyl-αD-glucopyranoside, xylitol, D-tagatose, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-cetogluconate, potassium 5-cetogluconate, D-melezitose, D-raffinose, Glycogen, N-acetylglucosamin, amygdalin, arbutin, salicin and hydrolysis of starch. Using APIZYM, positive enzyme activities were observed for esterase (C4), esterase lipase (C8), leucine aramidase, phosphatase acid, naphtol-AS-BI-phosphohydrolase, α-mannosidase, α- glucosidase and N-acetyl-β-glucosaminidase. Negative results for lipase (C14), phosphatase alcalin, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β – galactosidase, β-glucosidase, β-glucuronidase, β-glucosidase, and α-fucosidase.

M. gorillae is susceptible to amoxicillin (25 μg), erythromycin (15UI), doxycyclin (30UI), rifampicin (30 μg), vancomycin (50 μg), amoxicillin-clavulanic acid (20 μg + 10 μg), trimethoprim-sulfamethoxazole (1.25 μg / 23.75 μg) and imipenem (10 μg) but resistant to ciprofloxacin (5 μg) and gentamycin (15 μg).

When compared to other Microbacterium species [1016], M. gorillae sp. nov. strain G3T exhibited the phenotypic differences detailed in Additional file 1: Table S1.

Extended feature descriptions

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [17] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve distinct deposits were done for strain G3T from 12 isolated colonies. Two microliters of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50 % acetronitrile and 2.5 % trifluoroacetic-acid were distributed on each smear and submitted at air drying for five minutes. Then, the spectra from the 12 different colonies were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against 5,626 bacterial spectra including 43 spectra from 33 Microbacterium species, used as reference data, in the BioTyper database. Briefly, a score ≥ 2 with a species with a validly published name provided allows the identification at the species level, a score ≥ 1.7 but < 2 allows the identification at the genus level; and a score < 1.7 does not allow any identification. For strain G3T, no good score was obtained, suggesting that our isolate was not a member of any known species. We incremented our database with the spectrum from strain G3T (Additional file 2: Figure S1). The gel view highlighted spectrum differences with other Microbacterium species (Additional file 3: Figure S2).

Genome sequencing information

Genome project history

According to phenotypic characteristics of this strain and MALDI-TOF result and because of the low16S rRNA similarity to other members of the genus Microbacterium , it is likely that the strain represents a new species and thus it was chosen for genome sequencing. It was the 20th genome of a Microbacterium species (Genomes Online Database) and the first genome of Microbacterium gorillae sp. nov. A summary of the project information is shown in Table 2. The GenBank accession number is CDAR00000000 and consists of 14 contigs. Table 2 shows the project information and its association with MIGS version 2.0 compliance [18].
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

Mate pair and paired end

MIGS-29

Sequencing platforms

MiSeq-Illumina

MIGS-31.2

Fold coverage

213X

MIGS-30

Assemblers

Spades

MIGS-32

Gene calling method

Prodigal

 

Locus Tag

BN1193

 

GenBank ID

CDAR00000000

 

GenBank Date of Release

November 04, 2014

 

GOLD ID

Gp0025154

 

BIOPROJECT

PRJEB7582

MIGS-13

Source Material Identifier

G3T

 

Project relevance

DSM 26203, CSUR P207

Growth conditions and genomic DNA preparation

Microbacterium gorillae sp.nov strain G3T (= CSUR P207 = DSM 26203) was grown aerobically on 5 % sheep blood-enriched Columbia agar (BioMérieux) at 25 °C. Bacteria grown on four Petri dishes were resuspended in 3x500μl 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 3x100μL 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 2x20 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 M. gorillae was sequenced on the MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the 2 applications: paired end and mate paired. The gDNA was barcoded in order to be mixed with 11 others projects with the Nextera Mate Pair sample prep kit (Illumina) and with 17 others projects with the Nextera XT DNA sample prep kit (Illumina).

gDNA was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 46.7 ng/μlTo prepare the paired end library, dilution was performed to require 1 ng of each genome as input. The « tagmentation » step fragmented and tagged the DNA. Then limited cycle PCR amplification (12 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 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 2x250-bp.

Total information of 7.6 Gb was obtained from a 931 K/mm2 cluster density with a cluster passing quality control filters of 82.8 % (17,658,000 clusters). Within this run, the index representation for M. gorillae was determined to 5.11 %. The 732,922 paired end reads were trimmed and filtered by Trimmomatic tool using the recommended parameters for Illumina sequence data [19].

Two mate pair libraries were prepared with 1 and 1.5 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 pattern 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 from 1 kb to 11 kb in size with the majority of fragments at 8.8 and 9.4 kb of size. No size selection was performed and 45 ng for the 1st library and 600 ng for the second library of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with the majority at 400 and 380 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) and the final concentration library was measured at 0.65 and 0.59 nmol/l respectively. The libraries were normalized at 2nM and pooled. After a denaturation step and dilution at 15 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 39-h run in a 2x251-bp. The first libray was loaded three times on a flowcell and the second once. Within these runs, the index representation for M. gorillae was determined as an average at 3.51 %. The 1,881,286 paired reads were filtered according to the read qualities. The global paired end and mate pair libraries lead to 2,614,208 paired reads which were trimmed by Trimmomatic [19] then assembled by Spades software using the recommended options “--careful” and “-k 127” to fix the kmer size to 127 [20]. The final assembly identified 14 scaffolds generating a genome size of 3.69 Mb which corresponds to genome coverage of 213X.

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [21] 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 [22] and the Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [23] was used to find tRNA genes, whereas ribosomal RNAs were found using RNAmmer [24] and BLASTn against the GenBank database. Lipoprotein signal peptides and the number of transmembrane helices were predicted using SignalP [25] and TMHMM [26] 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 [27] was used for data management and DNA Plotter [28] for visualization of genomic features. The Mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignment [29]. To estimate the mean level of nucleotide sequence similarity at the genome level between M. gorillae sp. nov. strain G3T and other members of the genus Microbacterium , we used the MAGI home-made software to calculate the average genomic identity of gene sequences (AGIOS) among compared genomes [30]. Briefly, this software combines the Proteinortho software [31] for detecting orthologous proteins in pairwise genomic comparisons, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm. Finally, 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) [32, 33]. 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 of M. gorillae strain G3T is 3,692,770 bp-long with a 69.3 % G+C content (Table 3, Fig. 3). Of the 3,566 predicted genes, 3,505 were protein-coding genes and 61 were RNA genes, including 4 complete rRNA operons (Additional file 4). A total of 2,412 genes (68.82 %) were assigned a putative function. A total of 6.33 % were identified as Pseudo-genes. The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4 and Additional file 4.
Table 3

Nucleotide content and gene count levels of the genome

Attribute

Value

% of totala

Genome size (bp)

3,692,770

100

DNA coding (bp)

3,396,745

92

DNA G + C (bp)

2,558,287

69.3

DNA scaffolds

14

 

Total genes

3,566

100

Protein coding genes

3,505

98.3

RNA genes

61

1.71

Pseudo genes

226

6.33

Genes in internal clusters

ND

ND

Genes with function prediction

2,412

68.8

Genes assigned to COGs

2,202

62.8

Genes with Pfam domains

0

0

Genes with signals peptides

365

10.4

Genes with transmembrane helices

843

24.1

CRISPR repeats

0

0

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

Fig. 3

Graphical circular map of the Microbacterium gorillae strain G3 T chromosome. The outer two circles show open reading frames oriented in the forward (colored by COG categories) and reverse (colored by COG categories) directions, respectively. The third circle shows the RNA genes (tRNAs green, rRNAs red). The fourth circle shows the G + C% content plot. The inner-most circle shows GC skew, purple indicating negative values whereas olive for positive values

Table 4

Number of genes associated with the 25 general COG functional categories

Code

Value

% of totala

Description

J

149

4.25

Translation

A

1

0.03

RNA processing and modification

K

269

7.67

Transcription

L

109

3.11

Replication, recombination and repair

B

0

0.00

Chromatin structure and dynamics

D

16

0.46

Cell cycle control, mitosis and meiosis

Y

0

0.00

Nuclear structure

V

41

1.17

Defense mechanisms

T

75

2.14

Signal transduction mechanisms

M

82

2.34

Cell wall/membrane biogenesis

N

1

0.03

Cell motility

Z

0

0.00

Cytoskeleton

W

0

0.00

Extracellular structures

U

24

0.68

Intracellular trafficking and secretion

O

66

1.88

Posttranslational modification, protein turnover, chaperones

C

150

4.28

Energy production and conversion

G

257

7.33

Carbohydrate transport and metabolism

E

325

9.27

Amino acid transport and metabolism

F

69

1.97

Nucleotide transport and metabolism

H

83

2.37

Coenzyme transport and metabolism

I

151

4.31

Lipid transport and metabolism

P

184

5.25

Inorganic ion transport and metabolism

Q

95

2.71

Secondary metabolites biosynthesis, transport and catabolism

R

410

11.70

General function prediction only

S

145

4.14

Function unknown

-

1303

37.17

Not in COGs

a The 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 sequences of M. gorillae strain G3T (CDAR00000000) with those of Microbacterium barkeri strain 2011-R4 (AKVP00000000), Microbacterium maritypicum strain MF109 (ATAO00000000), Microbacterium indicum strain DSM 19969 (AULR00000000), Microbacterium laevaniformans strain OR221 (AJGR00000000), Microbacterium luticocti strain DSM 19459 (AULS00000000), Microbacterium paraoxydans strain 77MFTsu3.2 (AQYI00000000), Microbacterium testaceum strain StLB037 (AP012052) and Microbacterium yannicii strain PS01 (CAJF00000000). The draft genome of M. gorillae has a larger size than those of M. indicum , M. luticocti , M. laevaniformans , M. paraoxydans and M. barkeri , (3.69 vs 2.81, 3.11, 3.43, 3.48 and 3.64 Mb respectively) but is smaller than those of M. maritypicum , M. testaceum and M. yannicii (3.69 vs 4.0, 3.98 and 3.95 Mb respectively). The G+C content of M. gorillae is higher than those of M. laevaniformans and M. maritypicum (69.3 vs 68.0 and 68.2 % respectively) but lower than those of M. indicum , M. luticocti , M. testaceum , M. yannicii , M. paraoxydans and M. barkeri (69.3 vs 71.4, 70.7, 70.3, 69.5, 69.5, 69.2 %, respectively). The gene content of M. gorillae is lower than those of M. maritypicum and M. testaceum , (3,505 vs 3,856 and 3,676 genes respectively) but higher than those of, M. paraoxydens, M. yannicii , M. laevaniformans , M. barkeri , M. luticocti and M. indicum (3,312, 3,279. 3,249, 3,099, 2,355, 2,183 genes respectively) (Table 5). However the distribution of genes into COG categories was similar in all compared genomes (Additional file 5: Figure S3). In addition, M. gorillae shares 1,593, 1,658, 1,269, 1,396, 1,390, 1,416, 1,498 and 1,497 orthologous genes with M. barkeri , M. maritypicum , M. indicum , M. laevaniformans , M. luticocti , M. paraoxydans , M. testaceum and M. yannici i respectively (Table 5). Among compared genomes except M. gorillae, AGIOS values range from 75.51 % between M. indicum and M. maritypicum to 85.33 % between M. maritypicum and M. barkeri . When M. gorillae was compared to other species, AGIOS values range from 75.22 % with M. maritypicum to 76.41 % with M. luticocti (Table 5). dDDH estimation of the strain G3T against the compared genomes ranged between 19.70 to 20.50. These values are very low and below the cutoff of 70 %, thus confirming again the new species status of the strain G3T.
Table 5

Genomic comparison of M. gorillae sp. nov., strain G3T with other Microbacterium species.

Species

M. gorillae

M. barkeri

M. maritypicum

M. indicum

M. laevaniformans

M. luticocti

M. paraoxydans

M. testaceum

M. yannicii

M. gorillae

3,505

1,593

1,658

1,269

1,396

1,390

1,416

1,498

1,497

M. barkeri

75.91

3,099

2,111

1,390

1,511

1,461

1,595

1,685

1,684

M. maritypicum

75.22

85.33

3,856

1,429

1,581

1,549

1,634

1,755

1,734

M. indicum

75.39

76.16

75.51

2,183

1,296

1,191

1,324

1446

1,349

M. laevaniformans

75.80

76.59

76.07

76.05

3,249

1414

1,602

1,638

1,580

M. luticocti

76.41

76.99

76.50

76.34

77.94

2,355

1,395

1,433

1,512

M. paraoxydans

75.66

76.36

75.90

76.43

78.49

77.34

3,312

1,710

1,632

M. testaceum

75.64

76.48

75.84

76.30

77.64

77.64

77.52

3,676

1,723

M. yannicii

75.85

76.89

76.34

76.53

78.06

78.60

77.82

78.10

3,279

The numbers of orthologous proteins shared between genomes (upper right triangle), average percentage similarity of nucleotides corresponding to orthologous protein shared between genomes (lower left triangle) and numbers of proteins per genome (bold)

Conclusions

On the basis of phenotypic characteristics, phylogenetic position, genomic analyses (taxonogenomics) and GGDC results, we formally propose the creation of Microbacterium gorillae sp. nov. that contains the strain G3T. This strain has been isolated from a gorilla stool sample collected from Cameroon.

Taxonomic and nomenclatural proposals

Description of Microbacterium gorillae sp. nov.

Microbacterium gorillae (go.ril’lae. NL neut. gen gorilla, pertaining to a gorilla from which the stool sample was obtained).

Cells stain Gram-positive, are small rod, non-endospore-forming, non-motile and have a diameter of 0.5 μm and a length of 1 μm. Colonies are gray and 2 mm in diameter on blood-enriched Columbia agar. Growth occurs between 25 and 37 °C, with optimal growth observed at 25 °C.

Strain G3T exhibited catalase activity but not oxidase activity. Strain produces acid from esculin, D-cellobiose, D-maltose, D-lactose, D-mannose, D-mannitol, D-saccharose, D-trehalose and gentiobiose but not from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl-αD-xylopyranoside, D-galactose, D-glucose, L-fructose, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl-αD-mannopyranoside, Methyl-αD-glucopyranoside, xylitol, D-tagatose, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-cetogluconate, potassium 5-cetogluconate, D-melezitose, D-raffinose, Glycogen, N-acetylglucosamin, amygdalin, arbutin, salicin and hydrolysis of starch.

Positive enzyme activities were observed for esterase (C4), esterase lipase (C8), leucine aramidase, phosphatase acid, naphtol-AS-BI-phosphohydrolase, α-mannosidase, α- glucosidase and N-acetyl-β-glucosaminidase. Negative results for lipase (C14), phosphatase alcalin, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β – galactosidase, β-glucosidase, β-glucuronidase, β-glucosidase, and α-fucosidase.

M. gorillae is susceptible to amoxicillin, erythromycin, doxycyclin, rifampicin, vancomycin, amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole and imipenem but resistant to ciprofloxacin and gentamycin.

The G+C content of the genome is 69.3 %. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JX650056 and CDAR00000000, respectively. The type strain G3T (= CSUR P207 = DSM 26203) was isolated from the fecal sample of a western lowland gorilla from Cameroon.

Abbreviations

CSUR: 

Collection de souches de l’Unité des Rickettsies

URMITE: 

Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes

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

Fadi Bittar was supported by a Chair of Excellence IRD provided by the Institut de Recherche pour le Développement / Méditerranée-Infection foundation. Mamadou Bhoye Keita was funded by the Méditerranée-Infection foundation. The authors thank Xegen company for automating the genome annotation process.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), UM63, CNRS7278, IRD 198, Inserm 1095, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille Université
(2)
King Fahad Medical Research Center, King Abdul Aziz University

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© Hadjadj et al. 2016