Genomic Analysis of the Moderately Haloalkaliphilic Bacterium Oceanobacillus kimchii Strain X50^T with Improved High-Quality Draft Genome Sequences

Abstract

Oceanobacillus kimchii is a member of the genus Oceanobacillus within the family Bacillaceae. Species of the Oceanobacillus possess moderate haloalkaliphilic features and originate from various alkali or salty environments. The haloalkaliphilic characteristics of Oceanobacillus advocate they may have possible uses in biotechnological and industrial applications, such as alkaline enzyme production and biodegradation. This study presents the draft genome sequence of O. kimchii X50^T and its annotation. Furthermore, comparative genomic analysis of O. kimchii X50^T was performed with two previously reported Oceanobacillus genome sequences. The 3,822,411 base-pair genome contains 3,792 protein-coding genes and 80 RNA genes with an average G+C content of 35.18 mol%. The strain carried 67 and 13 predicted genes annotated with transport system and osmoregulation, respectively, which support the tolerance phenotype of the strain in high-alkali and high-salt environments.

Introduction

The genus Oceanobacillus, a member of family Bacillaceae, was first introduced by Lu et al. (2001) [18]. At the time of writing, the genus comprises 17 validated species and two subspecies. Members of the genus Oceanobacillus are gram-stain-positive, motile, and endospore-forming rods. Most species in the genus Oceanobacillus are characterized as moderate haloalkaliphilic organisms and have been found in salty or alkaline environments, such as fermented indigo [7,8], fermented food [20,30], and marine environments [6,15,18].

Moderately alkaliphilic bacteria can survive in alkali environment in the pH 9–10 range [11]. To adapt to high external pH, these bacteria have homeostasis mechanisms for neutralizing cytoplasmic pH, such as Na+/H+ antiporter-dependent pH homeostasis [12]. Moderately halophilic bacteria can grow in salty condition within the range of 5–20% (w/v) NaCl by regulating their osmotic concentrations [1]. As a consequence of these osmoregulation strategies, these bacteria can use osmolytes or compatible solutes, such as betaines, polyols, and ectoines, under high-salt environmental conditions [5,22]. Collectively, the haloalkaliphilic features of Oceanobacillus species imply that these organisms may have biotechnological applications such as for organic pollutants biodegradation and alternative energy production [14] and alkaline enzymes [25].

Oceanobacillus kimchii type strain X50T (= DSM 23341T = JCM 16803T = KCTC 14914T) was isolated from a traditional Korean fermented food known as “mustard kimchi” [30]. The strain X50T showed the same haloalkaliphilic features as those of other Oceanobacillus species and can grow in 0–15% (w/v) NaCl and pH 7.0–10, and shows optimal growth at pH 9 [30]. The present study summarizes the polyphasic features of O. kimchii X50T, and provides not only genomic information derived from its draft genome sequence but also compares the features of strain X50T with those of other Oceanobacillus species.

 

Materials and Methods

Phylogenetic Analysis Based on 16S rRNA Gene Sequences

The taxonomic position of O. kimchii X50T was confirmed, based on its sequence of 16S rRNA gene. Comparison of the 16S rRNA gene sequence between strain O. kimchii X50T and closely related type strains in the EzTaxon-e database [9] indicated that strain X50T is a member of the genus Oceanobacillus in the family Bacillaceae. The strain shares 98.86% sequence similarity with O. iheyensis HTE831T and 96.09% sequence similarity with invalid species O. massiliensis N’DiopT. A phylogenetic consensus tree was constructed to determine the phylogenetic relationships between strain X50T and other Oceanobacillus species. The 16S rRNA gene sequences of strain X50T and other Oceanobacillus species were aligned using the multiple sequence alignment program Clustal W [29]. Phylogenetic tree construction was performed using aligned sequences with maximum-likelihood [3], maximum-parsimony [10], and neighbor-joining [24] algorithms applying 1,000 bootstrap replicates by MEGA 6 [27].

Genomic DNA Extraction, Sequencing, and Sequence Assembly

The biomass of O. kimchii strain X50T was prepared at 30℃ for 2 days in 1% (w/v) NaCl-containing marine 2216 medium (Difco). Genomic DNA extraction was performed with a Wizard Genomic DNA Purification Kit (Promega A1120). Three platforms were used for DNA sequencing: an Illumina Hiseq system with a 150 base pair (bp) paired end library, a 454 Genome Sequencer FLX Titanium system (Roche Diagnostics) with an 8 kb paired end library, and a PacBio RS system (Pacific Biosciences) by ChunLab Inc., Korea. The sequencing reads assemblies were carried out using CLCbio CLC Genomics Workbench 5.0 (CLCbio) and Roche gsAssembler 2.6 (Roche Diagnostics). In the process of sequences assembly, sequencing reads acquired from the 454 Genome Sequencer FLX Titanium system and Illumina Hiseq system were primary integrated, and then sequencing reads acquired from PacBio RS system were used for gap filling. The genome project is deposited in the Genomes OnLine Database [16] and the genome sequence is deposited in GenBank. A summary of the project information is shown in Table 1.

Table 1.Summary of genome sequencing information.

Gene Prediction and Annotation

The open reading frames (ORFs) were predicted by the Integrated Microbial Genomes-Expert Review (IMG-ER) pipeline [19]. Gene annotation and functional comparisons of the predicted ORFs were conducted using the IMG-ER platform [19] with NCBI COG [28], NCBI Refseq [21], and Pfam [4] databases. GLIMMER 3.02 [2] was used for the gene calling method. IMG-ER platform [19], tRNAscan-SE 1.23 [17], and RNAmer 1.2 [13] were utilized to find tRNA genes and rRNA genes.

Genomic Sequence and Functional Profile Comparison

Average nucleotide identities (ANI) between the three Oceanobacillus genome sequences were calculated by using the Ez-Taxon-e server. Functional profile-based correlation values were calculated by using the IMG-ER platform with COG, Pfam, KO, and TIGRfam profiles.

 

Results

The phylogenetic analysis indicated that O. kimchii X50T fell into a clade in the genus Oceanobacillus and formed a cluster with O. iheyensis, which is the closest related species to O. kimchii (Fig. 1).

Fig. 1.Phylogenetic consensus tree based on 16S rRNA gene sequences showing the relationship between Oceanobacillus kimchii X50T and the related type strains of Oceanobacillus species.

A total of 6,523,431 sequencing reads (267.1-fold genome coverage) were obtained using a combination of the Ilumina Hiseq system (6,376,362 reads; 251.9-fold coverage), Roche 454 system (130,352 reads; 5.6-fold coverage), and PacBio RS system (16,717 reads; 9.6-fold coverage). The assembled genome sequence of O. kimchii strain X50T comprises a single scaffold that includes 20 contigs and contains 3,822,411 bp with 35.18 mol% G+C content.

The genome has the capacity to code for a total of 3,872 predicted genes. Of these, 3,792 were assigned to protein-coding genes (97.93%) and 80 were assigned to RNA genes (2.07%), including 48 tRNA genes and 10 rRNA genes (two 16S rRNA, four 5S rRNA, and four 23S rRNA genes). A total of 3,193 predicted genes (82.46%) were assigned to have putative functions, whereas the remaining genes (17.54%) were considered as hypothetical proteins. Moreover, 2,635 genes were classified under 23 COG functional categories. The overall genome statistics are summarized in Table 2 and visualized in Fig. 2. The gene distributions in the COG functional categories are shown in Table 3.

Table 2.The percent of total is based on either athe genome size (bp) or bthe total gene number in the annotated genome.

Fig. 2.Graphical circular genome map.

Table 3.aThe number of protein-coding genes in the annotated genome was considered as the total, which was used for proportion calculation.

The genome of O. kimchii X50T contains 19 predicted genes associated with antibiotic resistance, including genes coding for resistance to vancomycin, fosfomycin, and beta-lactams, such as vancomycin B-type resistance protein (VanW) and beta-lactamase class A. Strain X50T encodes 96 predicted genes associated with sporulation, such as spore germination protein (GerKA) and stage V sporulation protein (SpoVAB). Sixty-nine motility-associated genes were predicted, including 12 chemotaxis and 57 flagella genes. These genes are characteristic of bacteria that engage in sporulation and flagella motility, which is the case for O. kimchii. To identify prophages in O. kimchii X50T, PHAST [31] was used. One prophage was identified in a 20.8kb (with 36.5% G+C content) region containing 26 CDS. Fifteen of the 26 CDS were annotated with Bacillus phage protein. O. kimchii X50T encodes 13 predicted genes associated with osmotic stress regulation, such as choline-glycine betaine transporter (BetT) and periplasmic glycine betaine/choline-binding lipoprotein of the ABC-type transport system (OpuAA). Strain X50T possesses 67 predicted genes associated with membrane transporter systems, including ABC transporters, Na+/H+ antiporter, protein translocation systems, cation transporters, and TRAP transporters, such as the C4-dicarboxylate transport system. These genes could be key factors allowing O. kimchii X50T to adapt to high-salt and high-alkali environments via osmotic regulation and adjustment of cytoplasmic pH, respectively.

To date, two Oceanobacillus strains, O. iheyensis HTE831T and O. massiliensis N’diopT, have been sequenced and validated [23,26]. O. kimchii X50T has the largest genome and highest number of predicted genes, but has the lowest G+C content of the validated genomes of the two Oceanobacillus strains. O. kimchii X50T showed 88.86% (88.72% in reciprocal) and 69.51% (69.45% in reciprocal) ANI with O. iheyensis HTE831T and O. massiliensis N’diopT, respectively (Table 4). O. kimchii X50T has 0.94 to 0.98 correlation values (Pearson coefficient) with O. iheyensis HTE831T and 0.82 to 0.93 correlation values with O. massiliensis N’diopT. The results of genome sequence and functional profile analyses indicate that O. kimchii X50T shares more genomic and functional features with O. iheyensis HTE831T than with O. massiliensis N’diopT.

Table 4.aAverage nucleotide identity value (%). bPearson coefficient (Pfam/KO/TIGRfam/COG).

 

Discussion

At the time of writing, O. kimchii X50T is the third strain in the genus Oceanobacillus to be subjected to genome analysis; the other two are O. iheyensis and O. massiliensis. The comparative analyses, which were based on average nucleotide genomic sequence similarities and correlations of functional profiles, show that O. kimchii X50T is more closely related to O. iheyensis HTE831T than to O. massiliensis N’diopT, which is in line with the results of 16S rRNA gene sequence-based phylogenetic analysis. The O. kimchii X50T genome encodes sporulation, flagella motility, osmoregulation, and pH homeostasis genes in accordance with the previously reported characteristics of O. kimchii. Further studies are required to elucidate the mechanisms involved in the osmoregulation and pH homeostasis of this haloalkaliphilic bacterium. The results of such analyses could facilitate its use in biotechnological applications.