Complete Genome Sequence of Bacillus amyloliquefaciens LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-γ-Glutamic Acid

Abstract

Bacillus amyloliquefaciens is one of most prevalent Gram-positive aerobic spore-forming bacteria with the ability to synthesize polysaccharides and polypeptides. Here, we report the complete genome sequence of B. amyloliquefaciens LL3, which was isolated from fermented food and presents the glutamic acid-independent production of poly-γ-glutamic acid.

GENOME ANNOUNCEMENT

Poly-γ-glutamic acid (γ-PGA), which is a capsular component or extracellular secretion of Bacillus and a few other organisms, is a natural polyamide consisting of d- and l-glutamic acid units connected by γ-amide linkages (1, 3, 10, 13). With outstanding water solubility, biocompatibility, and degradability, γ-PGA is widely used in medicine, cosmetics, food, and wastewater treatment (14). The known γ-PGA producers with high productivity are mostly l-glutamic acid dependent; however, the strains not requiring glutamic acid are of great interest in the search for lower costs and a simplified process in industrial systems (5).

Bacillus amyloliquefaciens was originally described as a potent producer of liquefying amylase and other industrial ectoenzymes (14). The DSM and FZB series were discovered to be soilborne and plant associated, i.e., to possess the abilities to stimulate plant growth and suppress plant pathogens by promoting seedling emergence, plant biomass, and disease control (7). Previously, the genome sequences of B. amyloliquefaciens FZB42 and DSM7 were available from NCBI, which highlighted the potentials to synthesize lipopeptides and polyketides nonribosomally and to utilize specific plant-derived macromolecules (7, 14). However, little was known about the relationship between γ-PGA synthesis and genetic sequences. We report herein the genome sequence of glutamic acid-independent B. amyloliquefaciens LL3, which was isolated from fermented food (Korean bibimbap) and synthesizes medicinally applied γ-PGA with a molecular weight of 300,000 to 500,000 (6). The strain is currently maintained at the China Center for Type Culture Collection.

Whole-genome sequencing was done by using a shotgun strategy combining the Roche 454 GS-FLX system and a Solexa analyzer (Illumina) (9), which produced approximately 280,000 reads with 70-fold coverage. All of the paired reads were assembled using Newbler Assembler 2.3, and this produced 5 large contigs in one potential large scaffold. Annotation was done by merging the results obtained from RAST (rapid annotation using subsystem technology) (2), Glimmer 3.02 modeling (8), tRNAscan-SE 1.21 (12), and RNAmmer 1.2 (11).

The genome of B. amyloliquefaciens LL3, with a GC content of 45.7%, is composed of a 3,995,227-bp circular chromosome and a 6,758-bp cryptic plasmid. The chromosome contains 4,325 protein coding genes (CDSs), 72 tRNA genes, and 22 copies of the ribosomal operons, all of which holds about 89.57% of the whole genome. A comparative analysis of the chromosomal CDSs of LL3 and DSM7 revealed that 3,691 CDSs possessed specific functions, 211 CDSs were presented as hypothetical proteins, and the remaining 423 CDSs lacked matches. However, there were no tRNA genes and rRNA operons existing in the plasmid, while 9 CDSs possessed 72.63% of the base pairs. One CDS had demonstrable functions, two CDSs were correlated with hypothetical proteins, and the others are unknown. The sequencing of LL3 provided the deduction of functional genes for γ-PGA biosynthesis, including the synthetase genes pgsBCA, the hypothetical gene pgsE (4), the depolymerase gene pgdS, and the regulator genes comPA and degSU (16). Its availability will provide a better-defined genetic background for the metabolic pathways of the production of glutamic acid-independent γ-PGA and other significant peptides and polysaccharides.

Nucleotide sequence accession numbers.

The genome sequence of B. amyloliquefaciens LL3 has been deposited in GenBank, where the circular chromosome accession no. is CP002634 and the plasmid sequence accession no. is CP002635.