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  • Open Access

High-quality draft genome sequence of Effusibacillus lacus strain skLN1T, facultative anaerobic spore-former isolated from freshwater lake sediment

Standards in Genomic Sciences201712:76

https://doi.org/10.1186/s40793-017-0302-y

  • Received: 15 September 2017
  • Accepted: 5 December 2017
  • Published:

Abstract

Effusibacillus lacus strain skLN1T is the type strain of the type species in the genus Effusibacillus which is the one of the genera in the family Alicyclobacillaceae within the phylum Firmicutes . Effusibacillus lacus strain skLN1T is a Gram-positive, spore-forming thermophilic neutrophile isolated from freshwater lake sediment. Here, we present the draft genome sequence of strain skLN1T, which consists of 3,902,380 bp with a G + C content of 50.38%.

Keywords

  • Draft genome sequence
  • Spore forming bacteria
  • The family Alicyclobacillaceae
  • The genus Effusibacillus

Background

The family Alicyclobacillaceae consists of four genera; Alicyclobacillus , Kyrpidia , Tumebacillus and Effusibacillus. Alicyclobacillus spp. are known as the significant causative microorganisms of fruit juice spoilage [1, 2] Kyrpidia tusciae , a sole characterized species of the genus Kyrpidia is a thermoacidophile which grows best under autotrophic conditions [3, 4]. Members of the genus Tumebacillus are mesoneutrophile which are derived from various environments, such as the Arctic permafrost, wastewater and and soil [57]. Genus Effusibacillus was established in this family together with the reclassification of Alicyclobacillus pohliae as Effusibacillus pohliae and Alicyclobacillus consociatus as Effusibacillus consociatus [8]. Effusibacillus lacus strain skLN1T is a facultative anaerobic, Gram-positive bacterium isolated from freshwater lake sediment. Here, we descibe draft genome sequence of the type strain of this genus, Effusibacillus lacus strain skLN1T. In this study, we summarize the features of E. lacus strain skLN1T and show an overview of draft genome sequence and annotation of this strain.

Organism information

Classification and features

E. lacus strain skLN1T was isolated from sediments of a freshwater lake, Lake Yamanashi, Japan [8]. Cell wall structure of this strain is Gram-positive type. Cells of this strain are spore-forming rods varied from 5 to 100 μm in length (Fig. 1, Table 1). The major cellualr fatty acids of this strain are iso-C 14 : 0, iso-C 15 : 0 and iso-C 16 : 0. Respiratory quinones of this strain are MK-7 (99.5%) and MK-8 (0.5%). The cell-wall peptidoglycan of this strain consists of meso-diaminopimelic acid, alanine and glutamic acid, indicating the presence of A1γ-type polymer. This bacterium is facultative anaerobe and is capable of respiration and fermentation. Sugars, organic acids, peptides and amino acids are used for fermentative growth of this strain. Strain skLN1T reduce nitrate to nitrite under anaerobic conditions in the presence of lactate. This strain cannot grow lithoautotrophically with elemental sulfur or thiosulfate under oxic/anoxic conditions in the presence nitrate.
Fig. 1
Fig. 1

Photomicrograph of cells of Effusibacillus lacus strain skLN1T. Cells were grown on aerobic R2A liquid medium at 50 °C for 1 day

Table 1

Classification and general features of Effusibacillus lacus strain skLN1T according to MIGS recommendations

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [9]

  

Phylum Firmicutes

TAS [18, 19]

  

Class Bacilli

TAS [20]

  

Order Bacillaceae

TAS [21, 22]

  

Family Alicyclobacillales

TAS [3, 23]

  

Genus Effusibacillus

TAS [8]

  

Species Effusibacillus lacus

TAS [8]

  

Type strain: skLN1T (BDUF00000000)

 
 

Gram stain

Variable

TAS [8]

 

Cell shape

Rod

TAS [8]

 

Motility

Motile

TAS [8]

 

Sporulation

Spore-forming

TAS [8]

 

Temperature range

28–60 °C

TAS [8]

 

Optimum temperature

50–52 °C

TAS [8]

 

pH range; Optimum

7.0–8.5; 7.25–7.5

TAS [8]

 

Carbon source

Organic acids, sugars, peptones, amino acids

TAS [8]

MIGS-6

Habitat

freshwater lake sediment

TAS [8]

MIGS-6.3

Salinity

0% NaCl (w/v)

TAS [8]

MIGS-22

Oxygen requirement

Facultatively anaerobic

TAS [8]

MIGS-15

Biotic relationship

Free-living

NAS

MIGS-14

Pathogenicity

None

NAS

MIGS-4

Geographic location

Yamanashi, Japan

TAS [8]

MIGS-5

Sample collection

March 2009

NAS

MIGS-4.1 MIGS-4.2

Latitude-Longitude

not reported

NAS

MIGS-4.4

Altitude

not reported

NAS

aEvidence codes - 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). NA not avairable

The phylogenetic position of E. lacus strain skLN1T among the members of the family Alicyclobacillaceae is shown in the phylogenetic tree based on the 16S rRNA gene sequence (Fig. 2). Strain skLN1T, E. consociatus and E. pohliae are classified into an independent cluster in the family Alicyclobacillaceae .
Fig. 2
Fig. 2

Phylogenetic tree showing the relationship of Effusibacillus lacus strain skLN1T and related representatives. The maximum-likelihood tree was constructed with MEGA version 7.0.20 [24] based on ClustalX version 2.1 [25] aligned sequences of 16S rRNA gene. Bootstrap values (percentages of 1000 replications) of ≥50% are shown at nodes

Genome sequencing information

Genome project history

E. lacus strain skLN1T was selected for genome sequencing on the basis of its 16S rRNA gene-based phylogenetic position in the family Alicyclobacillaceae (Fig. 2). Table 2 shows a summary of the genome sequencing project information and its association with MIGS version 2.0 compliance [9]. The genome consists of 127 contigs, which has been deposited at DDBJ/EMBL/GenBank under accession number BDUF01000000.
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

High-quality draft

MIGS-28

Libraries used

TruSeq Nano DNA library prep kit

MIGS 29

Sequencing platforms

Illumina Hiseq paired-end

MIGS 31.2

Fold coverage

282×

MIGS-30

Assemblers

Velvet version 1.2.08

MIGS 32

Gene calling method

MetaGene

Locus Tag

EFBL

Genbank ID

BDUF00000000

Genbank Date of Release

September 13, 2017

GOLD ID

NA

BIOPROJECT

PRJDB5819

MIGS 13

Source Material Identifier

SAMD00081395, DSM 27172

Project relevance

Ecology and evolution

Growth conditions and genomic DNA preparation

E. lacus strain skLN1T (DSM 27172) was grown aerobically on TSB liquid medium (Daigo) at 50 °C without shaking. Genomic DNA was extracted from collected cells using Wizard® genomic DNA purification kit (Promega).

Genome sequencing and assembly

The genome sequence of strain skLN1T was determined using paired-end Illumina sequencing at Hokkaido System Science Co., Ltd. (Japan). The 11,205,386 reads were generated from a library with 100 bp inserts. After trimming of the reads, a total of 11,009,340 high-quality filtered paired end reads with a hash length of 95 bp were obtained. Reads were assembled de novo using Velvet version 1.2.08 into 127 scaffolds.

Genome annotation

vhThe genome sequence of strain skLN1T was automatically annotated and analyzed through the MiGAP pipeline [10]. In this pipeline, RNAmmer [11] and tRNAscan-SE [12] were used to identify rRNA and tRNA genes, respectively. MetaGene Annotator [13] was used for prediction of open reading frames likely to encode proteins (coding sequences), and functional annotation was performed based on reference databases, including Reference Sequence, TrEMBL, and Clusters of Orthologous Groups. Manual annotation was performed using IMC-GE software (In Silico Biology; Yokohama, Japan). Putative CDSs possessing BLASTP matches with more than 70% coverage, 35% identity and E-values less than 1 × e−5 were considered potentially functional genes. The CDSs were annotated as hypothetical proteins when these standard values were not satisfied. Transcription start sites of predicted proteins were corrected based on multiple sequence alignments. The protein-coding genes in the genome were also subjected to analysis on WebMGA [14] for the COGs and Protein family annotations. Transmembrane helices and signal peptides were predicted by using Phobius [15]. CRISPR loci were distinguished using the CRISPR Recognition Tool [16]. General features of Effusibacillus lacus strain skLN1T and the MIxS mandatory information were show in Table 1.

Genome properties

The total genome of E. lacus strain skLN1T was 3,902,380 bp in size with a GC content of 50.38% (Table 3). It was predicted to contain 3733 genes including 3683 protein-coding genes and 50 RNA genes (for tRNA). Approximately 77.5% of the predicted genes were assigned to COG functional categories. The distribution of genes into COGs functional categories is presented in Table 4.
Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

3,902,380

100

DNA coding (bp)

3,237,729

82.97

DNA G + C (bp)

1,966,019

50.38

DNA scaffolds

127

Total genes

3733

100

Protein coding genes

3683

98.66

RNA genes

50

1.34

Pseudo genes

NA

NA

Genes in internal clusters

NA

NA

Genes with function prediction

2588

69.33

Genes assigned to COGs

2893

77.50

Genes with Pfam domains

3111

83.34

Genes with signal peptides

434

11.63

Genes with transmembrane helices

799

21.40

CRISPR repeats

2

NA not avairable

Table 4

Number of genes associated with general COG functional categories

Code

count

%age

description

J

165

4.42

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

243

6.51

Transcription

L

146

3.91

Replication, recombination and repair

B

1

0.03

Chromatin structure and dynamics

D

42

1.13

Cell cycle control, cell division, chromosome partitioning

V

30

0.80

Defense mechanisms

T

194

5.20

Signal transduction mechanisms

M

178

4.77

Cell wall/membrane/envelope biogenesis

N

76

2.04

Cell motility

U

69

1.85

Intracellular trafficking, secretion, and vesicular transport

O

125

3.35

Posttranslational modification, protein turnover, chaperones

C

241

6.46

Energy production and conversion

G

176

4.71

Carbohydrate transport and metabolism

E

341

9.13

Amino acid transport and metabolism

F

74

1.98

Nucleotide transport and metabolism

H

165

4.42

Coenzyme transport and metabolism

I

153

4.10

Lipid transport and metabolism

P

177

4.74

Inorganic ion transport and metabolism

Q

83

2.22

Secondary metabolites biosynthesis, transport and catabolism

R

402

10.77

General function prediction only

S

271

7.26

Function unknown

840

22.50

Not in COGs

Insights from the genome sequence

E. lacus strain skLN1T possesses genes of key enzymes for dissimilatory nitrate reduction, i.e. napA (locus tag: EFBL_1421), narGHJI (EFBL_3070–3073), nirK (EFBL_0113), norB (EFBL_3053), nrfA (EFBL_2499) and related genes. Both genes for membrane-bound and periplasmic nitrate reductases (narG and napA) were identified in the genome. A protein coded in the 61,298–63,379 bp region of contig095 showed high amino-acid sequence similarity (≤ 74%) to nitrous-oxide reductase (NosZ), although the region was not annotated as nosZ gene because of the internal assembly gaps. Genome of E. lacus strain skLN1T contains the genes for complete denitrification to N2 gas (nirK, norB and nosZ) and dissimilatory ammonification (nrfA), although end product of nitrate reduction identified in the previous study was nitrite [8]. The reduction of nitrate to nitrite was reported in several species in the family Alicylobacillaceae , but denitrifying organisms have not been reported in this family. Genetic components involved in dissimilatory nitrate reduction were not found in the genome of Effucibacillus pohliae strain DSM 22757 T. Kyrpidia tuscia e DSM 2912 T possesses norB gene, but genes for the other denitrification enzymes were not found in the genome of this strain [17]. Additionally, genes for dissimilatory sulfur oxidation were not identified in the genome of E. lacus strain skLN1T, although this organism was isolated from a sulfur-oxidizing enrichment culture [8].

Conclusions

This study contributed to the knowledge of genome sequences of the genus Effusibacillus within the family Alicyclobacillaceae . The genome of E. lacus strain skLN1T consists of 3683 protein-coding genes and 50 RNA genes. Genes involved in dissimilatory nitrate reduction were identified in the genome of this organism.

Abbreviations

CRISPR: 

Clustered regularly interspaced short palindromic repeat

MiGAP: 

Microbial genome annotation pipeline

nap: 

Periplasmic nitrate reductase

nar: 

Respiratory nitrate reductase

nir: 

Nitrite reductase

nor: 

Nitric oxide reductase

nos: 

Nitrous oxide reductase

nrf: 

Ammonia-forming cytochrome c nitrite reductase subunit c552

Declarations

Acknowledgements

This study was supported by a grant-in-aid for Research Fellow of Japan Society for the Promotion Science to MW and JSPS KAKENHI Grant Number 22370005 to MF.

Authors’ contributions

MF and HK designed and supervised the study. MW characterized the strain. RT and MW carried out all the bioinformatics analysis. MW and HK drafted the manuscript. All authors discussed the data and read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Institute of Low Temperature Science, Hokkaido University, Nishi 8, Kita 19, Kita-ku, Sapporo Hokkaido, 060-0819, Japan
(2)
Postdoctoral Research Fellow of the Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8471, Japan

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