Complete Genome Sequence of Solvent-Tolerant Clostridium beijerinckii Strain SA-1.

We report the complete genome sequence of Clostridium beijerinckii SA-1, derived by directed evolution from C. beijerinckii NCIMB 8052, selecting for enhanced solvent tolerance. This sequence allows for accurate placement of SA-1 as C. beijerinckii, permits functional analyses of mutant phenotypes, and suggests methods for distinguishing SA-1 from its parent.

GENOME ANNOUNCEMENT

Clostridium beijerinckii is a Gram-positive, obligately anaerobic solventogenic organism, of interest primarily for acetone-butanol-ethanol (ABE) fermentations. Taxonomic assignments of solventogenic species of Clostridium historically have experienced some turmoil, and strains originally classified as one species have often been reassigned to another (1).

In 1983, using stress-directed evolution with a series of increasing butanol concentrations, Lin and Blaschek isolated an especially butanol-resistant strain of Clostridium, originally designated an offspring of C. acetobutylicum ATCC 824 (2). This strain was named SA-1, deposited, and catalogued as ATCC 35702; later it was reclassified as a strain of Clostridium beijerinckii, an offspring of C. beijerinckii NCIMB 8052 (1). SA-1’s nutritional requirements were determined previously, and butanol resistance and delayed sporulation phenotypes have been observed (3). Other studies on SA-1 have evaluated its extracellular alpha-amylase and glucoamylase activity and the effects of butanol stress on its membrane fatty acid composition (4, 5). Reportedly, SA-1 is genetically malleable (6).

Sequencing the genome of C. beijerinckii SA-1 allows for definitive placement of this strain within the C. beijerinckii species. Identification of variations in SA-1’s sequence from that of its parent can guide functional physiological studies to ascertain the extent of SA-1’s butanol tolerance and solventogenic capacities, as well as other mutant phenotypes (3, 7); alternatively, such a project allows for the identification of errors in the original sequence of C. beijerinckii NCIMB 8052 (7). Finally, discovery of large variations between strains suggests a simple molecular method for distinguishing them from each other (7).

The genome sequence of C. beijerinckii SA-1 was determined using the Illumina Genome Analyzer IIx platform by the US Department of Energy Joint Genome Institute. Reads were assembled by comparison to the sequence of parent strain C. beijerinckii NCIMB 8052 (GenBank accession no. NC_009617), and algorithms MAQ (8) and BreakDancer (9) were used to find small and large variations from the reference sequence. PCR and Sanger dye-terminator sequencing (Eton Bioscience, Research Triangle Park, NC) were used to validate variations found in silico. Annotations were copied from the GenBank record of the genome of NCIMB 8052 and modified as necessary.

Nucleotide sequence accession number.

The genome sequence of C. beijerinckii strain SA-1 (ATCC 35702) has been deposited in DDBJ/EMBL/GenBank under the accession no. CP006777. The version described in this paper is the first version.