Draft Genome Sequence of Amphibacillus jilinensis Y1T, a Facultatively Anaerobic, Alkaliphilic and Halotolerant Bacterium

The genus Amphibacillus was established in 1990, and seven additional species were described in the past two decades. Amphibacillus jilinensis Y1T is a facultatively anaerobic and alkaliphilic bacterium isolated from a soda lake in China. Here we describe the structural and genetic features of the draft genome about the type strain Y1T (3,831,075 bp, with a G+C content of 37.27%). This is the first genome report of the Amphibacillus genus.


Classification and features
A sediment sample was collected from a soda lake (44°45'N, 123°34'E) in Jilin province, China, in November 2007. There is no freshwater river to flow into the lake. Atmospheric water and groundwater are the only water sources of this lake. The lake is rich in Na + (257.2 mg/l), CO 3 2-(50.7 mg/l), Cl -(10.1 mg/l), HCO 3 -(6.5 mg/l) and SO 4 2-(4.4 mg/l), with the pH of the water sample in the same geographical location being 10.0 [5]. The strain Y1 T was isolated from enrichment cultures of sediment sample by the Hungate roll-tube technique [10] under a gas phase of O 2 -free N 2 [1,5]. Comparative 16S rRNA gene sequence analysis by BLASTN [11,12] using the NCBI-NR/NT database revealed 93.4-98.8% sequence similarity to members of the genus Amphibacillus. Neighbor-Joining phylogenetic analysis based on Tamura-Nei model indicated the taxonomic status of strain Y1 T is clearly classified into the same branch with genus Standards in Genomic Sciences Amphibacillus, and the most closely related genus is Halolactibacillus (Figure 1). A. jilinensis Y1 T can tolerant high salinity but can also survive without Na + . Growth occurs under either aerobic or anaerobic conditions. The optimal growth condition of strain Y1 T occurs in medium JY with 0.5 M Na + (0.06 M NaHCO 3 and 0.44 M NaCl) [5]. The optimum pH is 9.0, with a growth range of pH 7.5-10.5. No growth was observed at pH 7.0 or 11.0. Strain Y1 T is mesophilic, with a temperature range of 15-45 ºC and optimum growth at 32 ºC [ Table  1]. Cell morphology, motility and sporulation were examined by using transmission electron (H-600, Hitachi) microscopy. Cells of strain Y1 T are straight rods with petritrichous flagella, which have a diameter ranging 0.4-0.6 µm and a length of 2.0-3.2 µm (Figure 2a). In the late-exponential and stationary phases of growth, the rods can form terminal endospores ( Figure 2b).

Genome project history
The genome of A. jilinensis was selected for nextgeneration sequencing on the consideration of its facultatively anaerobic characterization and as a new member in genus Amphibacillus. This is the first genome report for any of the eight Amphibacillus species. Two others are the subject of ongoing own genome projects. This Whole Genome Shotgun project of A. jilinensis was deposited at DDBJ/EMBL/GenBank under the accession AMWI00000000 and consists of 83 contigs (further assembling constructed these contigs into 30 scaffolds). Table 2 presents the project information and its association with MIGS version 2.0 compliance [16].

Growth conditions and DNA isolation
A. jilinensis Y1 T was cultivated aerobically in modified JY medium, which contains (per liter distilled water) 2.0 g yeast extract (Difco), 5.0 g sucrose, 0.2 g KCl, 0.2 g KH 2 PO 4 , 0.1 g MgCl 2 . 6H 2 O, 0.5 g NH 4 Cl, 0.1 g CaCl 2 , 0.06 M NaHCO 3 and 0.44 M NaCl, final pH 9.0 at 32°C for 3 days [5]. Genomic DNA was extracted using the method described by Marmur [28]. The yield, purity and the concentration of genomic DNA was judged by the 0.7% agarose gel electrophoresis with λ-Hind III digest DNA Marker (TaKaRa, Dalian, China) and measured by the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc., USA). About 736.6 μg genomic DNA at the concentration 744 ng/μl was obtained.  [8,13] listed here: PRJNA42371 of A. xylanus DSM 6626 T , complete; PRJNA171498 of A. jilinensis Y1 T , Draft; PRJDB405 of A. sediminis Shu-P-Ggiii25-2 T , in progress. The phylogenetic tree uses 16S rRNA gene sequences aligned by the CLUSTALW [14], and phylogenetic inferences were made using Neighbor-joining method based on Tamura-Nei model within the MEGA5 software [15]. Numbers at the branching nodes are percentages of bootstrap values based on 1,000 replications. The scale bar indicates a 1% substitution per nucleotide position. Bacillus subtilis DSM 10 T was used as an outgroup.   , 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 [26,27]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

Genome sequencing and assembly
Genomic DNA sequencing of A. jilinensis Y1 T was performed using Solexa paired-end sequencing technology (HiSeq2000 system, Illumina, Inc., USA) [29] with a whole-genome shotgun (WGS) strategy, with a 500 bp-span paired-end library (~500 Mb available reads, ~130-fold genome coverage) and a 2,000 bp-span paired-end library (~250 Mb available reads, ~65-fold genome coverage). All these clean reads were assembled into 83 contigs (the minimum length is 231 bp) and 30 scaffolds (the minimum length is 542 bp) using the SOAPdenovo v.1.05 [30,31,50]. The quality of the sequencing reads data was estimated by G+C content and sequencing depth correlation analysis.

Genome annotation
The tRNAs and rRNAs were identified using tRNAscan-SE [32], RNAmmer [33] and Rfam database [34]; The open reading frames (ORFs) and the functional annotation of translated ORFs were predicted and achieved by using the RAST server online [35,51]. Classification of some predicted genes and pathways were analyzed using COGs [36,37] and KEGG [38][39][40] databases. Meanwhile, we used the InterPro [41,42] to obtain the GO annotation with the database of Pfam [43].

Genome properties
The draft genome sequence of A. jilinensis Y1 T revealed a genome size of 3,836,603 bp (scaffold length) and a G+C content of 37.27%. These scaffolds contain 3,649 coding sequences (CDSs), 51 tRNAs (removed 3 Pseudo tRNAs) and incomplete rRNA operons (two 5 S rRNA and one 16 S rRNA). A total of 2,683 protein-coding genes (67.72%) were assigned a predicted function (Table 3) and genes have been categorized into COGs functional groups (Table 4). 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.
b) Includes 1,092 hypothetical proteins and 19 unknown functional proteins by RAST subsystem annotation.

Insights from the genome sequence
The genomic annotation results suggest that strain Y1 T can adapt to an extremely basic environments. A large number of genes related to carbohydrate metabolism can encode proteins that provide a stable energy supply to maintain the lower internal pH despite the high external pH [44]. Several cation/proton antiporters were found in the genome, which are also crucial for the maintenance of internal pH [45]. However, the lower number of these genes in Y1 T when compared to Bacillus pseudofirmus OF4 [44]  . For facultatively anaerobic strains, these superoxide dismutases (SODs) may be critical because the systems can help to regulate intracellular oxidative stress when the cells grow during aerobic respiration, and can also be used in the treatment of disease, study of pharmacological activity [46] and in the cosmetic industry. It also contains 34 two-component system genes that encode response regulators and sensor histidine kinases. The two-component systems appear to be used to respond to a wide variety of stimuli, including the presence of nutrients, antibiotics and chemoattractants in the environment, changes in osmolarity, temperature, pH, etc [47,48]. This is especially true in strain Y1 T , in which these systems are thought to be used for recognizing environmental pH, and regulating its internal osmotic stress to survive various environments [49]. According to the database Pfam [43], there are also 9 CRISPRs-associated (Cas) proteins or Cas protein families in this genome of A. jilinensis.

Conclusion
Strain Y1 T is the fifth member of the genus Amphibacillus to be described and is the first for which a genome sequence report is available. These data will provide a new perspective of how microorganisms adapt to anoxic and alkaline environments, and may also provide a pool of functional enzymes that work at higher pH.