Christopher Gray

Two-dimensional map of the proteome of Haemophilus influenzae

We have constructed a two‐dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two‐dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low‐copy‐number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope‐bound protein fraction using either immobilized pH gradient strips or a two‐detergent system with a cationic detergent in the first and an anionic detergent in the second‐dimensional separation. Different proteins (502) were identified by matrix‐assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two‐dimensional proteome databases.

The microbial postgenomic era: promises, problems and prospects.

The inevitable emergence and spread of resistance to new antibiotics entering the market necessitates a new approach in drug discovery. Novel classes of antimicrobial compounds are required that are not enfeebled by widespread resistance mechanisms. The favoured approach is to gain a better understanding of the essential pathways and cellular functions and then to select new unexploited targets. This strategy has coincided with the deposition of fully assembled genomic sequences of several bacteria in the public databases. Various technologies have been reported that, when optimised, will enable the analysis of global cellular processes at the molecular level thus greatly contributing to our understanding of cellular physiology. In this article, some of the major advances in technology, which are expected in the future to be essential for the optimal use of the information contained within the genomic sequences, will be outlined.

Genome-wide transcriptional analysis of bacterial genomes: applications in antibacterial drug discovery

An oligonucleotide array of more than 250,000 specific probes representing two complete bacterial genomes including the 1,743 open reading frames of Haemophilus influenzae, and the 1,969 open reading frames of Streptococcus pneumoniae has been designed. When hybridised against labelled genomic DNA, all genes were detected and more than 96% of the signal intensity values were within a factor of three of the mean. For transcript imaging, microarrays were hybridised against total RNA populations quantitatively represented by labelled cDNAs. The measurements of transcript abundance for all genes were shown to be sensitive, specific, quantitative and reproducible. Nearly 85% of all S. pneumoniae mRNAs were found expressed during in vitro exponential growth. Identification of regulons important for S. pneumoniae entry into the stationary phase has been used to validate the approach. We will show, for the first time, how a genome-wide transcriptional analysis is applied to bacterial genomes and is being used to identify signal transduction regulatory networks, and to profile antibiotic compounds.