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

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%.


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 [5][6][7]. 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 skLN1 T is a facultative anaerobic, Grampositive bacterium isolated from freshwater lake sediment. Here, we descibe draft genome sequence of the type strain of this genus, Effusibacillus lacus strain skLN1 T . In this study, we summarize the features of E. lacus strain skLN1 T and show an overview of draft genome sequence and annotation of this strain.

Organism information
Classification and features E. lacus strain skLN1 T 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 mesodiaminopimelic 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 skLN1 T 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.
The phylogenetic position of E. lacus strain skLN1 T among the members of the family Alicyclobacillaceae is shown in the phylogenetic tree based on the 16S rRNA gene sequence (Fig. 2). Strain skLN1 T , E. consociatus and E. pohliae are classified into an independent cluster in the family Alicyclobacillaceae.

Genome sequencing information
Genome project history E. lacus strain skLN1 T 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.
Growth conditions and genomic DNA preparation E. lacus strain skLN1 T (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 skLN1 T 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 skLN1 T 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  Fig. 1 Photomicrograph of cells of Effusibacillus lacus strain skLN1 T . Cells were grown on aerobic R2A liquid medium at 50°C for 1 day 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 skLN1 T and the MIxS mandatory information were show in Table 1.

Genome properties
The total genome of E. lacus strain skLN1 T 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.
Insights from the genome sequence E. lacus strain skLN1 T 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 nitrousoxide reductase (NosZ), although the region was not annotated as nosZ gene because of the internal assembly gaps. Genome of E. lacus strain skLN1 T contains the genes for complete denitrification to N 2 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 tusciae 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 skLN1 T , 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 skLN1 T consists of 3683 protein-coding genes and 50 RNA genes. Genes involved in dissimilatory nitrate reduction were identified in the genome of this organism.