C. efficiens YS-314T(= NBRC 100395T) is very similar to the well known C. glutamicum that has been serving for the industrial production of L-amino acids, in particular L-glutamic acid (a million tons each year), for a long time. C. efficiens YS-314 can grow at temperatures above 40 ¡ëC in contrast to C. glutamicum that grows at 30 ¡ëC. This feature is quite beneficial for its industrial use, because fermenters to be used for its cultivation need to be cooled down only to a lesser extent for heat removal. Hence, it is said to be a Corynebacterium of the next generation.
The genomic sequence information of this bacterium would be useful for the understanding of thermostability of various enzymes. It has been reported that, despite the similarity between the structures of C. glutamicum and C. efficiens enzymes, they contain distinct amino acid differences such as lysine to arginine, serine to alanine, and serine to threonine substitutions, respectively, all of which contribute to the higher G+C content of the C. efficiens genome and at the same time might be responsible for higher heat-stability of C. efficiens enzymes.
Corynebacterium efficiens strain YS-314T (synonymous to AJ 12310T, JCM 11189T and DSM 44549T) is very similar to C. glutamicum, another Corynebacterial strain whose genome has been analyzed.
2009-05-09 ..... 1
Corynebacterium efficiens YS-314 were updated (We changed EC number of ORFs)
The entire genomic nucleotide sequence of C. efficiens K1 was determined by the whole genome shotgun sequencing method as in the case of other organisms analyzed at NITE-DOB.
Construction of a whole genome shotgun clone library
The genomic DNA prepared from C. efficiens YS-314 cells was sonified and fragments between 0.8 and 1.2 kb as well as those between 2.0 and 2.5 kb in length were isolated after agarose gel electrophoresis which were subsequently cloned into pUC118.
Plasmid DNA prepared by Autogen 740 was subjected to cycle-sequencing by using an ABI PRISM BigDye terminator or BigDye primer sequencing kit. The sequencing reaction mixtures were applied to ABI PRISM 377 DNA sequencers and analyzed.
Assembly of sequence data
The resultant raw sequencing data were assembled with the Phred/Phrap/Consed package (http://www.phrap.org/). Subsequently, each of the 30 kb regions of the consensus sequence data constructed by Phred/Phrap were separated, re-assembled, examined and edited with Sequencher (GeneCodes).
Validation of the assembled sequence data
From the final nucleotide sequence, PCR primer sequences were generated at appropriate intervals throughout the genome which were then used to amplify the corresponding genomic regions. The restriction enzyme digestion patterns of each of the PCR fragments thus obtained were accordingly compared with those deduced from the sequence data of the regions to validate the correctness of the assembled sequence data.
ORF Assignment and Annotation
ORFs were assigned by using Glimmer version 2.0 with its default settings.
The Shine-Dalgarno sequence was subsequently searched for to assign the start codon of each ORF.
The BLASTP similarity search of each ORF was performed against a non-redundant protein sequence database.
ORFs encoding proteins of 50 aa or shorter were selected only when they showed similarity to proteins encoded by genes of other organisms.