A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

Véronique de Berardinis^1,2, David Vallenet^1,2, Vanina Castelli^1,2, Marielle Besnard^1,2, Agnès Pinet^1,2, Corinne Cruaud¹, Sumitta Samair¹, Christophe Lechaplais^1,2, Gabor Gyapay^1,2, Céline Richez¹, Maxime Durot^1,2, Annett Kreimeyer^1,2, François Le Fèvre^1,2, Vincent Schächter^1,2, Valérie Pezo¹, Volker Döring³, Claude Scarpelli¹, Claudine Médigue^1,2, Georges N Cohen¹, Philippe Marlière³, Marcel Salanoubat^1,2 & Jean Weissenbach^1,2

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Synopsis

The understanding and modelling of the complexity of a living organism requires the global elucidation of gene function as well as the identification of the genes playing an essential role. The genome sequences of about 500 bacteria have already been released in public databases, providing a mine of information. However, the function of a large fraction of these bacterial genes is still unknown. Experimental and in silico analyses have elucidated the role of numerous genes although about 13.9% of Escherichia coli genes have no assigned function and 32% have only a predicted function (Riley et al, 2006). Furthermore, many processes are not ubiquitous and the analysis of gene function should not be restricted to a few model organisms. To study an alternate model, we have chosen to work on Acinetobacter baylyi ADP1 (ADP1), a strictly aerobic soil -proteobacterium, capable of using a large variety of compounds as carbon (aromatic compounds, hydrocarbons, etc.) and energy sources. ADP1 was sequenced and annotated at Genoscope with a special focus on metabolism reconstruction (Barbe et al, 2004). We took advantage of its competence for natural transformation (Palmen and Hellingwerf, 1997) combined with homology-directed recombination with linear DNA (de Vries and Wackernagel, 2002) to construct a complete collection of gene-by-gene deletion mutants selected on minimal medium (Figure 1; Metzgar et al, 2004). A total of 2594 genes covering 81% of the predicted genes were successfully disrupted, while 499 genes, including 47 genes of unknown function, were found to be essential (Table I). Interestingly, out of the 499 essential genes, 206 mutants were obtained that had a large genomic duplication of a region (300 kb–1 Mb) encompassing the targeted gene (Gyapay et al, in preparation). This mutant collection is currently being extended to mutants of biosynthetic genes using media supplemented by the appropriate component (arginine, histidine, leucine, methionine, tryptophan) and about 30 auxotrophic mutants have been obtained already.

Figure 1

Method of construction of the single-gene deletion mutants by creation of a spliced PCR integration cassette. P1–P6 are used for integration cassette construction and P7, P8, S1 and S2 for verifications. The kan^R integration cassette is obtained by PCR amplification using P1 and P2 primers on pEVL186 DNA template. The flanking regions, specific for the target gene, are amplified on wild-type DNA template by P3/P4 primers (R1 region) and P5/P6 primers (R2 region). Designations followed by a prime (') represent reverse complement sequences. The primers P7 and P8 are used for external PCR verification of the correct replacement of the targeted gene by the integrative cassette. The primers S1 and S2 located within the kan^R cassette are used to sequence junctions of the cassette on the P7/P8 PCR product.

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Table 1: Summary of the A. baylyi ADP1 knockout collection

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We have compared the distribution of the essential genes of ADP1, E. coli (Baba et al, 2006) and Pseudomonas aeruginosa (Liberati et al, 2006) into functional categories (Figure 5A). For three functional categories, the percentage of essential genes is significantly greater in ADP1 than in the other species (biosynthesis of amino acids, cofactors/prosthetic groups and purines/pyrimidines/nucleosides/nucleotides). These discrepancies mainly reflect the difference between the media used to obtain the mutants (minimal versus rich medium) (Figure 5B). For the other functional categories, a simple examination of the essential/dispensable genes highlighted several discrepancies between our experimental data and the current knowledge of metabolic pathways, which allowed the identification of interesting new features and differences from known pathways in other bacteria.

P. aeruginosa is phylogenetically the closest organism to ADP1 for which transposon mutant collections are available (1655 orthologous genes). About 80% of the essential genes in P. aeruginosa are also essential in ADP1. In contrast, 246 essential genes in ADP1 are dispensable in P. aeruginosa Liberati collection.

E. coli, through the Keio collection, is the only organism for which a detailed comparison of the essential gene data sets could be made, as both sets are based on single-gene knockout mutant collections obtained or profiled for growth on minimal medium. The results are consistent in 88% of the cases (1144 orthologous genes), whereas the essentiality status is different for only 134 genes, revealing metabolic or physiological differences.

The gene essentiality data were also compared to current knowledge of ADP1 metabolism, and a number of inconsistencies were identified allowing the formulation of new hypotheses on gene function or metabolic pathways. In methionine biosynthesis, for example, we have shown the concerted action of both metX and metW in the acylation of L-homoserine and the dispensability of metW when metX is overexpressed. Furthermore, the sulfydrylation pathway through the metY gene was shown to be not functional under our conditions. Moreover, the essentiality data combined with genomic comparative analyses have led to the identification of an essential gene with unknown function (ACIAD3524), found in synteny with metE in many proteobacteria. Its possible involvement in methionine biosynthesis was reinforced by the methionine auxotrophy of the mutant.

This collection also provides a powerful tool to explore gene function of catabolic pathways through the analysis of growth phenotypes. The profiling for growth of approximately 2450 mutants was performed on both solid and liquid media on a preliminary set of carbon sources. We present the investigation of 2,3-butanediol degradation, which is historically important for ADP1, as this strain was selected for its ability to use this compound as a carbon source. The results strongly suggest that 2,3-butanediol is degraded by the aco genes into two C2 compounds raising a doubt about the existence of 2,3-butanediol cycle (Juni and Heym, 1956).

In parallel to this work, a genome-scale metabolic model was reconstructed and systematically refined using both the mutant library and the data on growth phenotypes (Durot et al, in preparation.). This new collection of mutants is also likely to be a valuable resource for other bacteria, as ADP1 shares a large number of its genes with other bacteria. Genetic tools developed for ADP1, such as multiple-gene deletions and the existence of E. coli/ADP1 shuttle vectors, permit heterologous functional complementation. ADP1 could thus be an alternative and complementary model for the study of gene function of other bacteria, and in particular those not easily amenable to genetic manipulations.

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