Complete Genome Sequence of Bacillus amyloliquefaciens TA208, a Strain for Industrial Production of Guanosine and Ribavirin

Abstract

Here, we report the complete genome sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and synthesis of ribavirin by assimilation of formamide. Comparison of its genome sequence with those of strains DSM7 and FZB42 revealed horizontal gene transfer represented by unique prophages and restriction-modification systems and indicated significant accumulation of guanosine.

TEXT

With the development of next-generation sequencing technology, microbial genome data have increased explosively (11). However, knowledge on the industrially valuable strains is not yet sufficient, especially at the genome level. Here we report the complete genome sequence of Bacillus amyloliquefaciens TA208, which has been used in production of guanosine and also employed in microbial synthesis of the antiviral drug ribavirin by assimilation of formamide in fermentation. Strain TA208 is an adenine auxotrophic, 8-azaguanine- and methionine sulfoxide-resistant strain generated by conventional mutagenesis (15).

The genome was sequenced using the Illumina HiSeq 2000 at Beijing Genomics Institute (BGI; Shenzhen, China). A library containing 500-bp inserts was constructed. Sequencing was performed with the pair-end strategy of 90-bp reads to produce 419.04 Mb of filtered sequences, representing a 106.4-fold coverage of the genome. The sequences were assembled into 162 contigs and 18 scaffolds using the SOAPdenovo package (8). Gaps were closed by PCR and sequencing of the amplicons by primer walking. The closed genome is a circular chromosome of 3,937,511 bp without plasmids.

Genome annotation was performed at the NCBI Prokaryotic Genomes Automatic Annotation Pipeline, where 4,135 open reading frames (ORFs), six 16S-23S-5S rRNA operons, and 71 tRNAs were identified using Glimmer (4) and Genemark (10), BLAST (13) against the Rfam database (5), and tRNAscan-SE (9), respectively. A 1,259,096-bp fragment (position 1,057,022 to 2,316,118) was found to be inversed in the chromosome of strain TA208 compared to that in strains DSM7 (14) and FZB42 (2) by the Mauve package (3), presumably due to phage infection, since two novel potential prophages flanking this region were identified using Prophage Finder (1). Moreover, six other prophage and prophage remnants were found, providing evidence for horizontal gene transfer. Preliminary analysis revealed sets of restriction-modification systems in strain TA208, including M.BamHI, M.BamHII, R.BamHI, M.H2I (7), and a novel type II system composed of the ORFs BAMTA208_19835 and BAMTA208_19845. Strain TA208 harbors a complete set of genes required for natural competence but does not readily incorporate exogenous DNA (15), which might be caused by the frameshift mutation in comS (6) and the complex restriction-modification systems.

The most noticeable change in the genes for purine de novo synthesis is the presence of a truncated copy of purA in strain TA208, revealing the loss of activity for adenylosuccinate synthetase. Mutations potentially elevating the corresponding activity or abolishing the end product feedback inhibition were also observed in the enzymes encoded by purL (D624N), purM (Q322K, H334Q), and purC (N6S, I89V, H166L) compared to the reference strains. The nonsense mutation in pbuX caused the absence of guanine permease in strain TA208, corresponding to the phenotype of 8-azaguanine resistance. However, most of the other genes encoding the enzymes in de novo purine synthesis pathway, the nucleoside efflux pumps, and their riboswitch containing promoter regions (12) remain unchanged, indicating the potential for further strain improvement.

Nucleotide sequence accession number.

The complete genome sequence of B. amyloliquefaciens TA208 has been deposited in GenBank (accession number CP002627).