Draft Genome Sequence of Bacillus pumilus BA06, a Producer of Alkaline Serine Protease with Leather-Dehairing Function

Abstract

Bacillus pumilus BA06 was isolated from the proteinaceous soil and produced an extracellular alkaline protease with leather-dehairing function. The genome of BA06 was sequenced. The comparative genome analysis indicated that strain BA06 is different in genome from the other B. pumilus strains, with limited insertions, deletions, and rearrangements.

GENOME ANNOUNCEMENT

Alkaline serine protease is widely used in various industries (15). Representative subtilisins from Bacillus subtilis and the related species have been extensively studied for many years (3, 13). Recently, alkaline serine proteases from various strains of Bacillus pumilus were characterized with unique biochemical characteristics, such as higher thermostability and catalytic efficiency (6, 9, 10, 12, 16). B. pumilus BA06, isolated from the proteinaceous soil, is able secrete a major alkaline serine protease that shows good potential in leather dehairing (8, 17). The gene encoding this protease was cloned and expressed in B. subtilis (WB600) (11). In order to get deep insights into transcriptional regulation and for comparative genome analysis, we sequenced the genome of B. pumilus BA06 using Illumina HiSeq 2000 at Shenzhen Huada Genomics Institute (China).

The sequencing generated 5,555,556 paired-end reads that were 90 bp in length, with genome coverage of about 250×. The clean reads were assembled primarily using the software ABySS (14) and Edena (7). The resulting two sets of contigs (>200 bp, 23 each) were aligned using BLAST (1) to improve the assembly results from ABySS; for that, several in-house Python (www.python.org) scripts were used to merge contigs and replace the Ns and other ambiguous bases. Finally, 15 contigs, with an N₅₀ of 630,692 bp and a largest contig of 835,516 bp, were obtained. In total, the draft genome of BA06 contains 3,747,698 bp with a G+C content of 41.3%, about 0.43 Mb more than the genome of B. pumilus SARF-32. The genome phylogenetic trees inferred from the whole orthologs and constructed using PhyML (5) showed that BA06 was close to (in order from near to far) B. pumilus SAFR-32, B. pumilus ATCC 7061, and Bacillus licheniformis ATCC 14580.

The draft genome was annotated by submitting it to the RAST system (2). Consequently, 3,890 protein-coding sequences (CDSs) were identified, among which 2,773 CDSs were able to be assigned to one of the 461 RAST subsystems. Numbers of predicted genes belonging to metabolisms of DNA, nitrogen, protein, carbohydrates, phosphorous, and potassium did not show obvious differences from those for B. pumilus SARF-32 and even B. subtilis 168. In contrast, the numbers of CDSs in the BA06 genome belonging to the subsystems of the membrane transporter, sulfur metabolism, mobility and chemotaxis, regulation, and cell signaling were much more than those for SARF-32. Comparative genome analysis was also carried out with the closest strains of Bacillus using the software Mauve (4). Overall, the sequences of BA06 and SARF-32 were colinear, with only limited insertions, deletions, and rearrangements. Compared to the BA06 genome, a prophage, type I restriction modification system, and some hypothetical CDSs did not present in SARF-32. Several operons or their members that are involved in assimilation of sulfate and thiosulfate, alkanesulfonates, nitrate, and arsenate were different in or omitted from the strain SARF-32. All the differences between those strains may result from the fitness to various niches.

Nucleotide sequence accession number.

The sequence of B. pumilus BA06 from this whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number AMDH00000000.