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J Bacteriol. Jan 2011; 193(1): 323–324.
Published online Oct 29, 2010. doi:  10.1128/JB.01211-10
PMCID: PMC3019927

Complete Genome Sequence of Corynebacterium pseudotuberculosis I19, a Strain Isolated from a Cow in Israel with Bovine Mastitis [down-pointing small open triangle]

Abstract

This work reports the completion and annotation of the genome sequence of Corynebacterium pseudotuberculosis I19, isolated from an Israeli dairy cow with severe clinical mastitis. To present the whole-genome sequence, a de novo assembly approach using 33 million short (25-bp) mate-paired SOLiD reads only was applied. Furthermore, the automatic, functional, and manual annotations were attained with the use of several algorithms in a multistep process.

Corynebacterium pseudotuberculosis is the etiology of common disease conditions in sheep, goats, South American camelids, and horses; however, infections in cattle and humans are sporadic and rare.

Based on nitrate reduction, C. pseudotuberculosis has two biovars: C. pseudotuberculosis bv. equi, infecting mainly bovines and equines, and C. pseudotuberculosis bv. ovis, infecting sheep and goats (1, 2, 8). The widespread occurrence and the economic importance of infection with this pathogen have prompted investigation of its pathogenesis. The use of whole-genome sequence analysis helps to understand the molecular and genetic bases of this bacterium's virulence. Genome sequencing of strains isolated from a human being, a goat, and a sheep was carried out by our team.

Israel is probably the only place in the world to experience large-scale outbreaks of bovine C. pseudotuberculosis infection. These outbreaks are also associated with cases of mastitis (9, 10). Strain I19 was isolated from a dairy cow with severe clinical mastitis in two quarters; milk samples from both quarters were positive for C. pseudotuberculosis. The cow was culled on the day of milk sampling. In the present research, the SOLiD system was used in sequencing the entire genome of C. pseudotuberculosis I19. The sequencing generated 33,368,273 mate-paired 25-nucleotide-long short reads, which is tantamount to 834,206,825 nucleotides of information, rendering a mean genome coverage depth of 321-fold given an expected genome size of 2.6 Mb. The de novo assembly strategy for the assembly of short reads in this work combines De Bruijn graph and overlap-layout-consensus methods with the use of a reference genome as a basis for orientation and ordering of the de novo-generated contigs (6). This strategy allowed closure of all gaps and an effective coverage of 35-fold.

The genome of C. pseudotuberculosis strain I19 consists of a 2,337,730-bp circular chromosome. The average G+C content of the chromosome is 52.84%. The annotation procedure involved the use of several algorithms in a multistep process. For structural annotation, the following software programs were employed: FgenesB, a gene predictor (http://www.softberry.com); RNAmmer, an rRNA predictor (4); tRNAscan-SE, a tRNA predictor (5); and Tandem Repeats Finder, a repetitive-DNA predictor (http://tandem.bu.edu/trf/trf.html). Functional annotation was performed by similarity analyses using public databases and by InterProScan analysis (11). Manual annotation was performed using Artemis (7). Identification and confirmation of putative pseudogenes in the genome were carried out using Consed. Manual analysis was performed based on the Phred quality of each base in the frameshift area (3). This analysis enabled the identification of erroneous insertions or deletions of bases in the genome information produced by the sequencing process and prevented identification of false-positive pseudogenes. The genome of C. pseudotuberculosis strain I19 was predicted to contain 2,124 coding sequences (CDSs), 4 rRNA operons, and 50 tRNAs, and 55 pseudogenes were found.

More detailed analysis of this genome and comparative analysis with other sequenced genomes of members of the genus and the same species will provide further insight for understanding virulence and may be useful for the development of new diagnostic methods and vaccines, contributing to the control of the different diseases caused by this pathogen.

Acknowledgments

This research work is the result of collaboration of reputable organizations, with the support of long-standing institutions which include the Rede Paraense de Genômica e Proteômica, supported by the Fundação de Amparo a Pesquisa do Estado do Pará, the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). M.P.C.S., V.A., and A.S. were supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We also acknolwedge support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 October 2010.

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