Published online 30 April 2004
Nucleic Acids Research, 2004, Vol. 32, No. 8 2353-2361
© 2004 Oxford University Press
Identification and functional analysis of ‘hypothetical’ genes expressed in Haemophilus influenzae
BIATECH, 19310 North Creek Parkway, Suite 115, Bothell, WA 98011, USA, 1 National Center for Biotechnology Information, NLM, National Institutes of Health, Bethesda, MD 20894, USA, 2 Children’s Research Institute, and The Ohio State University, 700 Children’s Drive, Columbus, OH 43205, USA and 3 Technische Universität München, Wissenshaftzentrum Weihenstephan, Am Forum 1, 85354 Freising, Germany
*To whom correspondence should be addressed. Tel: +1 425 481 7200, extension 100; Fax: +1 425 481 5384; Email: ekolker{at}biatech.org
Received February 23, 2004; Revised and Accepted March 30, 2004
The progress in genome sequencing has led to a rapid accumulation in GenBank submissions of uncharacterized ‘hypothetical’ genes. These genes, which have not been experimentally characterized and whose functions cannot be deduced from simple sequence comparisons alone, now comprise a significant fraction of the public databases. Expression analyses of Haemophilus influenzae cells using a combination of transcriptomic and proteomic approaches resulted in confident identification of 54 ‘hypothetical’ genes that were expressed in cells under normal growth conditions. In an attempt to understand the functions of these proteins, we used a variety of publicly available analysis tools. Close homologs in other species were detected for each of the 54 ‘hypothetical’ genes. For 16 of them, exact functional assignments could be found in one or more public databases. Additionally, we were able to suggest general functional characterization for 27 more genes (comprising 
80% total). Findings from this analysis include the identification of a pyruvate-formate lyase-like operon, likely to be expressed not only in H.influenzae but also in several other bacteria. Further, we also observed three genes that are likely to participate in the transport and/or metabolism of sialic acid, an important component of the H.influenzae lipo-oligosaccharide. Accurate functional annotation of uncharacterized genes calls for an integrative approach, combining expression studies with extensive computational analysis and curation, followed by eventual experimental verification of the computational predictions.
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