Bacterial Workhorses E coli and Bacillus subtilis

Due to their undisputed position as model organisms in the bacterial world, the construction of genome-wide collections of mutants by systematic targeted muta-genesis of every gene has been attempted only in E. coli and Bacillus subtilis [23, 24], with the intention of creating flexible genomic resources freely available to all researchers. In E. coli, two different projects are currently underway in K12 strains MG1655 and W3110, both relying on high-throughput targeted mutagenesis methods. In the American project (strain MG1655), PCR amplification fragments of every ORF are mutated individually by in vitro transposition of Tn5-based mobile elements [24]. In the Japanese project (strain W3110), deletion alleles are created using a PCR-based strategy similar to the one used in the Saccharomyces Genome Deletion Project described above (http://ecoli.aist-nara.ac.jp/). In both cases, the mutant alleles are integrated into the E. coli genome by homologous recombination, which has been enhanced by the production of the bacteriophage k red recombination system that is encoded by the red genes present on a resident plasmid [25]. This plasmid can be subsequently cured by growth at high temperature. In the American project, of which an overview has been recently published [24], 1976 of the 4288 originally annotated E. coli ORFs (46%) have been successfully mutated in the first pass, whereas no progress report is available for the Japanese project (http://ecoli.aist-nara.ac.jp/). In B. subtilis, the global mutagenesis of the genome involved a consortium of laboratories and relied on the cloning of PCR-amplified fragments internal to each gene within a nonreplicating plasmid, which integrated in the corresponding genes via single crossover recombination after transformation [23]. In addition, the plasmid harbored a promoterless lacZ gene, allowing the generation of transcriptional fusions with the interrupted genes and an inducible promoter for the expression of the genes situated downstream of the target gene in multicistronic organizations, thus minimizing polar effects. However, because the main goal in this project was to estimate the minimum gene set required to sustain bacterial life, mutagenesis was unfortunately not comprehensive. It was not attempted on 1144 of the 4101 annotated B. subtilis ORFs (27.8%) either because they were studied previously for essentiality, or because they could be predicted with confidence to be either essential or dispensable. Finally, although the value of these toolboxes for gene function identification seems obvious in light of the S. cerevisiae example, functional studies using them are still to be reported.

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