Functional genomic analysis of Haemophilus influenzae and application to the study of competence and transformation.

摘要

The publication of the complete genomic sequence of Haemophilus influenzae Rd KW20 in 1995 was a truly monumental event in molecular biology. For the first time, all of the potential genes of an independent-living organism were known and awaiting functional characterization. This event required the development of fundamentally different methodologies to elucidate gene functions, with systematic global approaches becoming much more feasible. This study describes the development of a transposon-based mutagenesis strategy to facilitate a high-throughput functional analysis of the H. influenzae genome. Mutants created using this strategy were screened in a highly-parallel assay to identify genes mediating transformation in this organism. Additionally, analysis of the transposon insertion sites generated during this study identified a previously unrecognized Tn5 insertion bias.; During the progression of this study, hundreds of additional bacterial genomes were sequenced, techniques have evolved, and novel approaches were developed to aid the field of functional and comparative genomics. Some of these techniques were used in this study to identify three novel competence-regulated operons in H. influenzae. The techniques included the use of advanced computer programs and algorithms to assist in predicting protein functions and to facilitate a comparative genomic analysis of H. influenzae with other species of the Pasteurellaceae family. Quantitative PCR was employed to examine the expression of putative transformation-related genes. Finally, PCR-mediated mutagenesis was used in a directed approach to generate mutations in the newly discovered competence-regulated operons to assess their involvement in uptake and transformation of exogenous DNA.