Mutant collection

Principle and Method :

Each CDS (1), defined during the annotation process has been replaced by a cassette containing the gene for kanamycin resistance under the control of a strong promoter (Phage T5 promoter) by homologous recombination. The extremities of the integration cassette, called “recombination footprints” consist of the specific regions which frame the gene to be mutated. The replacement usually covers the totality of the ORF (2). However, to avoid altering essential regions such as the RBS (3)) or the promoters of neighboring genes, we have had to construct a replacement in which the target ORF is truncated by several amino acids. The molecular construction of the cassettes is obtained in detail using PCR(4) which recombines the recombination footprints with the gene for antibiotic resistance. After transformation of Acinetobacter baylyi with each of these cassettes, the mutants are selected on a mineral medium supplemented with succinate and kanamycin.

Technique :
 
 
 
 

Production phase:

Optimization of the transformation protocol and organization of the “pipeline” has permitted us to attain a success rate of over 90%, and a capacity of 400 mutants initiated every two weeks. The complete cycle for the production of a mutant from the transformation step to storage is 4 weeks. This cycle includes control PCRs to check on the replacement of the ORF by the integration cassette and systematic sequencing of the genomic regions of the mutant which are adjacent to the integration cassette.
 
 
 
 

Results :

Of the 3205 genes annotated in A. baylyi which are candidates for mutation, we have attempted to inactivate 3,167 genes. We produced replacement mutants for 2,594 genes, which represents 81% of the genes of A. baylyi (de Berardinis et al., 2008).
 
 

Distribution of mutants according to functional category of the genes

This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection (Baba et al., 2006) profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1.

Several strategies were undertaken to investigate ADP1 metabolism by

1. searching for discrepancies between our essentiality data and current metabolic knowledge,
2. comparing this essentiality data set with those from other organisms,
3. systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.).
4. This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches (Durot et al., en préparation).

To help us in this comparison, a ADP1 Genome Browser was implemented in order to present data of our mutant collection in their genomic context: KEGG and BioCyc metabolic pathways, SEED subsystems, operon predictions, orthologous genes and essentiality data from several organisms, and phenotyping data from high throughput experiments...

Distribution of the ADP1 mutant collection

Individual mutants have already been distributed and used for several studies as DNA damage (Chakravorty et al., 2008) and analysis of metabolism (Aghaie et al., submitted). The mutant distribution is being handled via the Genoscope web page including primers data used for deletions (strain request).

Perspectives :

The systematic gene replacement phase which has now been achieved will be extended by a targeted finishing phase, to attempt to obtain mutants in genes in which the first attempt has failed. Simple repeat of the experiments should suffice for these “technical failures;” new primers in more favorable regions will be selected for some of them. We will also try to repeat the experiments on more enriched media in order to obtain mutants of the genes which are essential on succinate.

An analytic phase will now be undertaken in the collection, in addition to systematic phenotyping in order to study, for example, genes with unknown function which co-localize with genes which participate in the same metabolic pathway.

Glossary :

 (1) CDS : Coding DNA Sequence.
 (2) ORF : Open Reading Frame.
 (3) RBS : Ribosome Binding Sequence.
 (4) PCR : Polymerase Chain Reaction.