Phillip Cash (p.cash@abdn.ac.uk)
Department of Medical Microbiology, University of Aberdeen, Foresterhill, ABERDEEN AB25 2ZD
Haemophilus influenzae is an important human pathogen responsible for a variety of infections, including respiratory infections and meningitis. H. influenzae isolates are divided into six capsulate types (a to f) on the basis of their capsular polysaccharide as well as non-typable strains. The type b strains of H. influenzae are responsible for the majority of the invasive infections caused by these bacteria. The Rd strain of H. influenzae was the first free-living organism for which the complete genome nucleotide sequence was determined and the small size of the genome has led to the proposal that it contains close to the minimal gene set required for a bacterial pathogen. Although the determination of the complete genome sequence of an organism is a significant step towards understanding its gene expression, it does not provide a complete picture of global gene expression. Consequently, the analysis of protein synthesis has been widely used to complement the nucleotide sequence analyses. The concept of the "Proteome" was thus proposed to describe the proteins expressed by, for example, a micro-organism under specified conditions. The most generally applied method used to characterise the proteome is high resolution 2-dimensional electrophoresis (2-DE).
We have used 2-DE to analyse protein synthesis in H. influenzae and other members of the Haemophilus genus in order to develop an Haemophilus 2D protein database [2]; the data from these analyses can be accessed across the WWW [9]. Total soluble cell proteins were extracted from bacteria grown on chocolate agar media and analysed using a small format gel system. The 2D gels were then processed for detailed examination using dedicated computer software (Phoretix-2D). This approach permits rapid comparisons of many proteins (and by extension the genes encoding these proteins) between different species of the Haemophilus genus at both a quantitative and qualitative (i.e. electrophoretic mobility) level. We are using the protein database to look at quantitative changes in protein synthesis, and thus gene expression, under different growth conditions. Data arising from these quantitative analyses of bacterial protein synthesis will be described.
Perspectives on Protein Engineering BIODIGM Ltd. 1997