The growing number of complete microbial genome sequences and the ready availability of their annotation provide a powerful data base for studying the biology of microorganisms. In this work, two distinct high-throughput approaches are described to exploit genomics of pathogenic bacteria, insertional-duplication mutagenesis (IDM) and expression profiling using the luciferase reporter system. Their genome-wide application to the food-borne pathogens Salmonella typhimurium, Listeria monocytogenes and Yersinia enterocolitica led to new insights into the complex world of microbial life in terms of (I) the minimal gene set, (II) intracellularly required factors, and (III) the association with invertebrates. I) Minimalism: The essential gene set of S. typhimurium was defined by a novel genetic strategy. Small, randomly generated chromosomal fragments of this pathogen were cloned into a temperature-sensitive vector, and the resulting mutagenic library was grown under permissive conditions. Upon switching to non-permissive temperature, genes with essential functions under laboratory conditions could be trapped following discrimination between lethal and non-lethal recombination events. Further characterization of a total of ~500 fragments revealed 145 known essential genes and 112 functionally characterised or hypothetical genes not yet demonstrated to be essential for a bacterial cell; this number corresponds to approximately 11% of the Salmonella genome. II) Specialization: More than 1,000 IDM mutants of the facultative intracellular pathogen L. monocytogenes were screened for their phenotypes in human epithelial cells. The genetic analysis of severely attenuated mutants revealed a huge number of listerial genes required for replication in non-phagocytic cells, thus dissecting the genome of Listeria in terms of their adaptation to the intracellular environment. The acquisition of species-specific genes and the usage of alternative sugar and nitrogen sources could be demonstrated as novel strategies that enable this pathogen to survive in the cytosol of the host cell. III) Association: A promoter fusion library of Y. enterocolitica was constructed by the transposon-mediated chromosomal insertion of the luciferase reporter, and the transcriptional response of the genome was derived when cells were exposed to low temperatures. Sequence analysis revealed a novel pathogenicity island termed tc-PAIYe carrying insecticidal toxin genes that could be demonstrated to be transcriptionally silent at body temperature, but to be essential for Y. enterocolitica toxicity against insects at low temperature. This data demonstrates a yet unknown pathogenicity phase of Y. enterocolitica in insects, suggesting invertebrates as a potential source of pathogen evolution.    «

The growing number of complete microbial genome sequences and the ready availability of their annotation provide a powerful data base for studying the biology of microorganisms. In this work, two distinct high-throughput approaches are described to exploit genomics of pathogenic bacteria, insertional-duplication mutagenesis (IDM) and expression profiling using the luciferase reporter system. Their genome-wide application to the food-borne pathogens Salmonella typhimurium, Listeria monocytogenes...    »