Trends in Microbiology
MODEL SYSTEMSTransposon-based approaches to identify essential bacterial genes
Section snippets
What is essential in a bacterial genome?
The largest region of a bacterial genome that is essential for viability contains protein-coding ORFs. Other essential sequences include regions of regulatory DNA, RNA species and the origin of replication. Approximately 40% of the ORFs defined by computer analysis of completed genomes are of unknown function; many of these have no similarity to any other ORFs in the available databases.
Essentiality is typically defined operationally. It is usually assayed by testing for colony formation on an
Non-transposon-based methods
There are several methods to identify essential genes that do not rely on transposons, including comparative genomics1 and the directed knockout of genes2. More traditional analysis uses the generation of conditional mutations that affect growth, such as temperature-sensitive (TS) mutations. TS mutations are generated by chemical mutagenesis and result in unmarked point mutations that allow the identification of essential genes by growth at a permissive temperature but not at a non-permissive
Conclusions
Whether using transposons or not, the assay conditions employed to identify the essential regions of a chromosome are crucial. Transposons provide a powerful and potentially rapid approach for identifying essential genes. There are currently two conceptual approaches to defining essential genes, each of which has advantages and disadvantages.
Analysis of essential genes on a genomic scale is a large undertaking and, depending on the method, requires extensive sequencing or PCR. A significant
Acknowledgements
We thank Jonathan Blum for critical reading of the manuscript. This work was supported by grant AI-26289 from the National Institute of Allergy and Infectious Diseases.
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