Complete Genome Sequence of Corynebacterium falsenii DSM 44353 To Study the Evolution of Corynebacterium Cluster 3 Species

Corynebacterium falsenii is a member of the natural microflora of wild and domesticated birds and is rarely detected in human clinical specimens. The chromosomal sequence of the type strain C. falsenii DSM 44353 comprises 2,677,607 bp and provides detailed insights into the evolution of Corynebacterium species assigned to the highly diverse cluster 3.

in a polyphasic taxonomic study of four coryneform strains that were isolated from human blood cultures and a cerebrospinal fluid sample between 1991 and 1995. Based on the alignments of rpoB gene sequences, C. falsenii was grouped into the cluster 3 of the genus Corynebacterium, with Corynebacterium jeikeium being the closest phylogenetic relative (2). The species C. falsenii is represented by the type strain DSM 44353, which was isolated in 1994 from a blood culture of a 4-year-old boy suffering from acute lymphatic leukemia (1). However, the clinical significance of C. falsenii remains largely unknown, as it has since been recovered very rarely from human clinical material (3,4). On the other hand, C. falsenii was isolated from the respiratory tracts of eagles and black storks (5, 6) and from bioaerosols sampled in duck houses (7). C. falsenii was also detected by 16S rRNA gene sequencing in the cloacal microbial community of black-winged stilts (8). Hence, C. falsenii may represent a member of the natural microflora of wild and domesticated birds.
To get insights into the genetic organization of this rarely recovered corynebacterium, we sequenced the genome of the C. falsenii type strain (1). C. falsenii DSM 44353 (BL 8171, CCUG 33651) was obtained from the Leibniz Institute DSMZ (Braunschweig) and grown in brain heart infusion broth-yeast extract at 37°C (9). Genomic DNA was purified with the Genomic-tip 500/G system and the Genomic DNA buffer set (Qiagen), and it was used as starting material to prepare a standard sequencing library according to the workflow of the Nextera DNA sample preparation kit (Illumina). The genomic library was sequenced in a 2 ϫ 250 nucleotide (nt) paired-end run using the MiSeq reagent kit version 2 and the MiSeq desktop sequencer (Illumina), resulting in 1,077,270 reads and an 81-fold genome coverage. The reads were preprocessed by quality trimming in such a way that the terminal five nucleotides had a Phred quality value of Ն30 (10). Preprocessed reads were assembled with the GS de novo assembler software (release 2.8) to yield 44 contigs in 12 scaffolds. The software r2cat (11) supported the ordering of the scaffolds according to alignments with the chromosomal sequence of C. jeikeium K411 (12). The remaining gaps in the genome sequence were closed in silico with the Consed software (version 24) (13).
The genome sequence of C. falsenii DSM 44353 includes a circular chromosome of 2,677,607 bp (63.18% GϩC content) and the circular corynephage ⌽CFAL8171I genome of 42,009 bp (61.74% GϩC content). An identical linear copy of ⌽CFAL8171I is present in the chromosome as a prophage, suggesting that this corynephage had entered a lytic cycle in a subpopulation of the culture used to prepare the genomic DNA. The automatic annotation of the genome sequence with the NCBI Prokaryotic Genome Annotation Pipeline and the GeneMarkSϩ software (version 2.3) revealed 2,248 protein-coding regions, 35 pseudogenes, 50 tRNA genes, 1 noncoding RNA (ncRNA) gene, and 3 rRNA operons in the chromosome of C. falsenii DSM 44353 and 58 protein-coding regions in the circular genome of ⌽CFAL8171I.
Nucleotide sequence accession numbers. This genome project has been deposited in the GenBank database under accession no. CP007156 (chromosome) and CP007157 (⌽CFAL8171I).

ACKNOWLEDGMENT
The C. falsenii genome project was supported by the Medical Microbiology and Genomics fund for practical training (eKVV 200937).