Complete Genome Sequence of Coprothermobacter proteolyticus DSM 5265.

Here we present the complete 1,424,912-bp genome sequence of Coprothermobacter proteolyticus DSM 5265, isolated from a thermophilic digester fermenting tannery wastes and cattle manure.

GENOME ANNOUNCEMENT

Coprothermobacter proteolyticus is a nonmotile, non-spore-forming, rod-shaped, Gram-negative anaerobic bacterium isolated from a thermophilic consortium fermenting tannery wastes and cattle manure (1). C. proteolyticus has increased utilization of fructose, mannose, glucose, maltose, and sucrose with the addition of yeast extract with either rumen fluid or Trypticase peptone compared to when it is grown without these additives (1). It was first considered a member of the genus Thermobacteroides but was latter reclassified as Coprothermobacter proteolyticus (2). C. proteolyticus was selected in 2002 as part of a National Science Foundation-funded “Assembling the Tree of Life” project at the Institute for Genomic Research (TIGR) to sequence the genomes of representatives of the seven phyla of bacteria that at the time had cultured representatives but no available genome sequence.

C. proteolyticus DSM 5265 was grown in DSM medium 481, and DNA was extracted using standard techniques. Sanger sequencing and genome assembly were performed as previously described for genomes sequenced by TIGR (3–5). Small and large insert plasmid libraries were constructed in pUC-derived vectors after random mechanical shearing (nebulization) of genomic DNA.

Sequencing resulted in 14,614 reads with an average read length of 1,039 bp and a coverage estimate of 10×. Sequences were assembled using Celera Assembler (6). The coverage criteria were that every position required at least double-clone coverage (or sequence from a PCR product amplified from genomic DNA) and either sequence from both strands or two different sequencing chemistries. The sequence was edited manually, and additional PCR and sequencing reactions were done to close gaps, improve coverage and resolve sequence ambiguities (7). All repeated DNA regions were verified by PCR amplification across the repeat and sequencing of the product. The full assembly consists of 1,424,912 bases and has a G+C content of 44.8%.

The replication origin was determined by colocalization of genes (dnaA, dnaN, recF, and gyrA) often found near the origin in prokaryotic genomes and G+C nucleotide skew (G·C/G+C) analysis (8). Completeness of the genome was assessed using the Phylosift software (9), which searches for 40 highly conserved, single copy marker genes (10). Thirty-nine of these 40 markers were found in this assembly and the missing marker (encoding porphobilinogen deaminase) was only found in 80% of the original 1,000 genomes used to generate the markers.

An initial set of open reading frames likely to encode proteins (coding sequences [CDSs]) were predicted as previously described (7). All predicted proteins larger than 30 amino acids were searched against a nonredundant protein database as previously described (7). Protein membrane-spanning domains were identified by TopPred (11). The 5′ regions of the CDSs were inspected to define initiation codons using similarity searches and to identify positions of ribosomal binding sites and transcriptional terminators. Two sets of hidden Markov models were used to determine CDS membership in families and superfamilies: Pfam v11.0 (12) and TIGRFAMs 3.0 (13). Pfam v11.0 hidden Markov models were also used with a constraint of a minimum of two hits to find repeated domains within proteins and mask them. This annotation was submitted with the genome in 2008, but in 2014 we requested an in-place update of the annotation from NCBI, using their integrated PGAP pipeline (14).

Nucleotide sequence accession numbers.

This genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession no. CP001145. The version described in this paper is version CP001145.1.