Complete Genome Sequence of Raoultella ornithinolytica Strain B6, a 2,3-Butanediol-Producing Bacterium Isolated from Oil-Contaminated Soil

Abstract

Here we report the full genome sequence of Raoultella ornithinolytica strain B6, a Gram-negative aerobic bacillus belonging to the family Enterobacteriaceae. This 2,3-butanediol-producing bacterium was isolated from oil-contaminated soil on Backwoon Mountain in South Korea. Strain B6 contains 5,398,151 bp with 4,909 protein-coding genes, 104 structural RNAs, and 55.88% G+C content.

GENOME ANNOUNCEMENT

2,3-Butanediol is a very useful chemical, which is used as a solvent and as a precursor to various chemicals and synthetic polymers (1, 2). A 2,3-butanediol-producing bacterium, strain B6, was isolated from an oil-contaminated soil sample collected from the Backwoon Mountain in South Korea for screening of organic compound-tolerant bacteria. This strain was identified as Raoultella ornithinolytica by 16S rRNA gene sequencing and physiological analysis (3). The results showed the highest 16S rRNA gene sequence identity, 99.9%, to strain JCM6096^T (NCBI accession number AJ251467) (4). R. ornithinolytica (formerly known as Klebsiella ornithinolytica) is a Gram-negative aerobic bacillus which belongs to the family Enterobacteriaceae. R. ornithinolytica B6 can be grown using glycerol, hexoses (glucose, fructose, galactose), pentose (xylose), or disaccharide (sucrose) as a sole carbon source. When sugars are supplied, the main product of the bacterium is 2,3-butanediol, even at a relatively low temperature (25°C). Hence, R. ornithinolytica B6 has energy-saving advantages for industrial production of 2,3-butanediol.

The genomic DNA was isolated from an overnight culture of strain B6 using a DNeasy blood and tissue kit (Qiagen) and was sequenced by 454 GS FLX titanium pyrosequencing (Roche), following the manufacturer’s instructions, with 25× coverage (5). Subsequently, the 398,549 reads generated, with a total length of 126,825,580 bp, were assembled using a GS de novo assembler (version 2.3; Roche). The contig N₅₀ was 145,738 bp, and the largest contig assembled was 438,809 bp. This assembly generated 86 large contigs (>500 bp), with a length of 5,365,416 bp. The 385,542 reads (96.73% of the total) were assembled into 9 scaffolds, which were composed of 67 contigs, with a length of 5,379,360 bp. The scaffold N₅₀ was 3,097,984 bp, and the average length of the scaffolds was 3,097,984 bp.

The order of scaffolds was determined by a similarity search among published references and confirmed by a PCR size check. The gaps both within and between scaffolds were closed by long-range PCR (using MG Taq-HF DNA polymerase; Macrogen, Seoul, South Korea) and subsequent Sanger sequencing using an ABI 3730XL capillary sequencer. The sequences from ABI 3730XL sequencing and scaffolds were completely assembled into one circular genome using Phred/Phrap/Consed software (6) and annotated with the Prokaryote Genomes Automatic Annotation Pipeline (PGAAP) (7).

The complete genome of B6 is composed of a circular chromosome of 5,398,151 bp (55.9% GC content), which includes 4,909 coding genes, 79 tRNAs, and 25 rRNAs. Overall, 4,070 coding genes (82.90% of the total coding genes) have putative functions assigned on the basis of annotation.

A transcriptome analysis of B6 using the Roche/FLX system was performed to improve its genome annotation. Although small amounts of non-rRNA sequences were obtained from transcriptome analysis, 6,195 reads (91%) of the 6,746 mapped non-rRNA reads were mapped to 1,169 predicted coding genes.

We found that acetoin production increases rapidly in stationary phase in producing 2,3-butanediol. Also through transcriptome analysis, we found that the expression level of acetoin reductase increased sharply after 2,3-butanediol production. In producing 2,3-butanediol, it is necessary to block the route of succinic acid, lactic acid, and fumaric acid, as well as inactivate acetoin reductase.

Nucleotide sequence accession number.

The complete genome sequence of Raoultella ornithinolytica B6 has been deposited in GenBank under accession number CP004142.