New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis

Michael G. Smith; Tara A. Gianoulis; Stefan Pukatzki; John J. Mekalanos; L. Nicholas Ornston; Mark Gerstein; Michael Snyder

doi:10.1101/gad.1510307

New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis

¹ Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA;
² Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, Connecticut 06520, USA;
³ Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut 06520, USA;
⁴ Harvard Medical School, Department of Microbiology and Molecular Genetics, Boston, Massachusetts 02115, USA

Abstract

Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicemia, and urinary tract infections. We explored the pathogenic content of this harmful pathogen using a combination of DNA sequencing and insertional mutagenesis. The genome of this organism was sequenced using a strategy involving high-density pyrosequencing, a novel, rapid method of high-throughput sequencing. Excluding the rDNA repeats, the assembled genome is 3,976,746 base pairs (bp) and has 3830 ORFs. A significant fraction of ORFs (17.2%) are located in 28 putative alien islands, indicating that the genome has acquired a large amount of foreign DNA. Consistent with its role in pathogenesis, a remarkable number of the islands (16) contain genes implicated in virulence, indicating the organism devotes a considerable portion of its genes to pathogenesis. The largest island contains elements homologous to the Legionella/Coxiella Type IV secretion apparatus. Type IV secretion systems have been demonstrated to be important for virulence in other organisms and thus are likely to help mediate pathogenesis of A. baumannii. Insertional mutagenesis generated avirulent isolates of A. baumannii and verified that six of the islands contain virulence genes, including two novel islands containing genes that lacked homology with others in the databases. The DNA sequencing approach described in this study allows the rapid elucidation of the DNA sequence of any microbe and, when combined with genetic screens, can identify many novel genes important for microbial pathogenesis.

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Footnotes

↵5 Present address: Invitrogen Corporation, Protein Array Center, 688 East Main Street, Branford, CT 06405, USA.
↵6 Corresponding author.

↵6 E-MAIL Michael.snyder{at}yale.edu; FAX (203) 432-6161.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1510307
- Received November 8, 2006.
- Accepted January 16, 2007.