Genome Sequence of the Solvent-Producing Bacterium Clostridium carboxidivorans Strain P7^T

Abstract

Clostridium carboxidivorans strain P7^T is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO₂ fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate.

Clostridium carboxidivorans strain P7^T (equivalent to ATCC BAA-624^T and DSM 15243^T) is an obligate anaerobe that can grow autotrophically with H₂ and CO₂ or CO (fixing carbon via the Wood-Ljungdahl pathway), or it can grow chemoorganotrophically with simple sugars (1). Acetate, ethanol, butanol, and butyrate are end products of metabolism.

For slow-growing strict anaerobes such as Clostridium carboxidivorans, genome sequencing provides a rapid theoretical characterization of its metabolism compared to traditional methods. We isolated and amplified genomic C. carboxidivorans DNA using the Wizard genomic DNA purification kit (Promega, Madison, WI) and the REPLI-g kit (Qiagen). A single shotgun pyrosequencing run using a Genome Sequencer FLX system (454 Life Sciences, Branford, CT) resulted in 429,680 high-quality reads (mean read length, 231.6 bp) that were assembled using Newbler software (454 Life Sciences) into 225 contigs >500 bp long. Paired-end sequencing produced 111,154 reads (mean read length, 256.3 bp). Assembly of the paired-end and shotgun reads produced 73 scaffolds containing 216 large contigs with a mean sequence depth of 16.33 reads. PCR amplification and Sanger sequencing were conducted, followed by scaffold assembly using Sequencher (Gene Codes, Ann Arbor, MI). The 4.4-Mb final assembly has 33 scaffolds containing 69 contigs with a Phred-equivalent quality score of 40 or above (accuracy, >99.99%) (GenBank accession no. ADEK00000000).

The sequence was annotated using Annotation Engine (J. Craig Venter Institute) and manually curated using Manatee (http://manatee.sourceforge.net/). The genome has 29.7% G+C content and contains 4,174 protein-coding sequences, 3 rRNA operons, 1 tmRNA (dual tRNA-like and mRNA-like nature), 6 noncoding RNAs (ncRNAs), and 48 tRNA genes. (6). Comparison of 16S rRNA genes showed that C. carboxidivorans is closely related to Clostridium scatologenes ATCC 25775^T (97% sequence identity) and Clostridium drakei type strain SL1^T (99% sequence identity). C. carboxidivorans shares 94% 16S rRNA sequence identity with Clostridium ljungdahlii (4.6 Mb), another solventogenic species.

Pathway analyses indicated that C. carboxidivorans is similar to other anaerobic acetogens, such as Moorella thermoacetica (8), in having an incomplete reductive tricarboxylic acid (TCA) cycle where fumarate reductase is absent. Like other acetogenic clostridia, C. carboxidivorans uses the Wood-Ljungdahl pathway for fixing carbon dioxide to organic carbon via acetyl coenzyme A (acetyl-CoA) (5). Two of these genes encode carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which form a complex to catalyze the carbonyl branch of the pathway for carbon fixation and acetyl-CoA production. C. carboxidivorans has genes that encode phosphotransacetylase and acetate kinase for converting acetyl-CoA into acetate, yielding ATP (2).

C. carboxidivorans is unique among other known acetogenic clostridia because it can fix carbon via the Wood-Ljungdahl pathway and convert acetyl-CoA into butanol, which is more energy dense than ethanol. Both C. carboxidivorans and Clostridium acetobutylicum encode NADPH-dependent butanol dehydrogenase (74% identity) to convert acetyl-CoA into butanol (3, 4), but C. acetobutylicum cannot fix CO₂ or CO into acetyl-CoA. Conversely, C. ljungdahlii can fix CO and CO₂, but it lacks butanol dehydrogenase and cannot convert acetyl-CoA into butanol. Therefore, P7 includes beneficial properties of both these industrially important strains. The genome sequence of C. carboxidivorans P7 could potentially accelerate research allowing its industrial application for biofuel production or to enable some of its pathways to be used directly in synthetic biology for biofuel production.

Nucleotide sequence accession number.

The genome sequence was deposited in GenBank under accession number ADEK00000000.