Complete Genome Sequence of Pseudomonas denitrificans ATCC 13867.

Pseudomonas denitrificans ATCC 13867, a Gram-negative facultative anaerobic bacterium, is known to produce vitamin B12 under aerobic conditions. This paper reports the annotated whole-genome sequence of the circular chromosome of this organism.

GENOME ANNOUNCEMENT

Vitamin B12 is one of the most complex nonpolymeric biological molecules (1). It is essential for the proper function of the animal nervous system (2) and is used as a coenzyme for many microbial enzymes, including ethanolamine ammonia lyase, methionine synthase, diol dehydratase, and glycerol dehydratase (3–5). However, only a few microorganisms, such as Pseudomonas denitrificans, Klebsiella pneumoniae, Salmonella enterica subsp. enterica serovar Typhimurium, Propionibacterium freudenreichii, and Bacillus megaterium, can produce this important vitamin (6–8). Most microorganisms produce vitamin B12 under anaerobic conditions, while P. denitrificans produces B12 under aerobic conditions. P. denitrificans has been exclusively used for the industrial production of this vitamin (9). This paper reports the whole-genome sequence of P. denitrificans ATCC 13867, with an emphasis on its synthesis of vitamin B12.

The complete genome of P. denitrificans ATCC 13867 was sequenced using the 454 GS FLX Titanium system (Roche Diagnostics, Switzerland), with a combination of the 8-kb mate-pair library (623,018 reads; length, 231,129,016 bp) and the shotgun single-read library (653,245 reads; length, 262,508,692 bp) (10). The sequences obtained from these combined approaches were assembled using a Newbler assembler (Roche Diagnostics, Switzerland), which showed 45.78-fold coverage. The genome size of P. denitrificans ATCC 13867 is 5,696,307 bp with 65.2% G+C content. Gene prediction and annotations were carried out using the Basic Local Alignment Search Tool and an automated annotation system, Ensoltek (11). The P. denitrificans ATCC 13867 genome has 2,567 operons and 5,059 protein-encoding genes. It has genes for the 62 transfer RNAs (tRNAs), which represent all 20 amino acids. The ribosome-binding sites (RBS) and transcriptional terminators were predicted using the RBS finder and TransTerm tools, respectively (12). The prediction suggested 1,279 RBS and 816 transcription terminators. The protein-coding genes were subjected to the PSORTb protein tool to identify the subcellular localization of the proteins (13). Among the 5,059 proteins, 3,013 (59.56%) proteins were expected to be cytoplasmic and 982 proteins (19.41%) were noncytoplasmic. The locations of the remaining 1,045 (21.02%) proteins could not be identified.

The essential amino acid l-methionine is synthesized by two different pathways, coenzyme B12-independent (metE, cobalamin-independent homocysteine transmethylase) and coenzyme B12-dependent (metH, methionine synthase) (14). The P. denitrificans genome had only metH, indicating that coenzyme B12 is essential for the synthesis of l-methionine (15). The genome of P. denitrificans contains all the genes (encoding 26 enzymes) necessary for oxygen-dependent coenzyme B12 synthesis. The genes are distributed in two different clusters on the chromosome (clusters I and II). Cluster I consists of 5 operons that encode nine enzymes required for the formation of hydrogenobyrinic acid, an important intermediate of coenzyme B12, from uroporphyrinogen III. Operon 1 of this cluster contains cobGHIJ, operon 2 contains cobLFK, and operon 3 contains chlLD and xre/pbsX. On the other hand, operon 4 contains cobWN, and operon 5 contains cbtBA and cobEM. The genes cobOBRDCQPUV, bpgM (encoding bisphosphoglycerate mutase), and tonB, which are involved in the conversion of hydrogenobyrinic acid to adenosylcobalamine (16), were located separately as a single operon on cluster II.

Nucleotide sequence accession number.

The P. denitrificans ATCC 13867 whole-genome sequence assembly and its annotation were deposited in GenBank under the project accession no. CP004143.