We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 ...(16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection 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 to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). 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.
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 γ‐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.
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.
A complete set of single‐gene deletions of all non‐essential genes has been created in Acinetobacter baylyi ADP1 by replacing coding regions with a kanamycin resistance cassette
ADP1, a strict aerobe, shares about a third of its genes with E. coli, thus providing a convenient and complementary model for studying metabolism
This collection provides a new resource to study gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches
Mutations are the ultimate source of heritable variation for evolution. Understanding how mutation rates themselves evolve is thus essential for quantitatively understanding many evolutionary ...processes. According to theory, mutation rates should be minimized for well-adapted populations living in stable environments, whereas hypermutators may evolve if conditions change. However, the long-term fate of hypermutators is unknown. Using a phylogenomic approach, we found that an adapting Escherichia coli population that first evolved a mutT hypermutator phenotype was later invaded by two independent lineages with mutY mutations that reduced genome-wide mutation rates. Applying neutral theory to synonymous substitutions, we dated the emergence of these mutations and inferred that the mutT mutation increased the point-mutation rate by ∼ 150-fold, whereas the mutY mutations reduced the rate by ∼ 40–60%, with a corresponding decrease in the genetic load. Thus, the long-term fate of the hypermutators was governed by the selective advantage arising from a reduced mutation rate as the potential for further adaptation declined.
The quantification of spontaneous mutation rates is crucial for a mechanistic understanding of the evolutionary process. In bacteria, traditional estimates using experimental or comparative genetic ...methods are prone to statistical uncertainty and consequently estimates vary by over one order of magnitude. With the advent of next-generation sequencing, more accurate estimates are now possible. We sequenced 19 Escherichia coli genomes from a 40,000-generation evolution experiment and directly inferred the point-mutation rate based on the accumulation of synonymous substitutions. The resulting estimate was 8.9 × 10(-11) per base-pair per generation, and there was a significant bias toward increased AT-content. We also compared our results with published genome sequence datasets for other bacterial evolution experiments. Given the power of our approach, our estimate represents the most accurate measure of bacterial base-substitution rates available to date.
To gain an in-depth insight into the diversity and the distribution of genes under the particular evolutionary pressure of an arsenic-rich acid mine drainage (AMD), the genes involved in bacterial ...arsenic detoxification (arsB, ACR3) and arsenite oxidation (aioA) were investigated in sediment from Carnoulès (France), in parallel to the diversity and global distribution of the metabolically active bacteria. The metabolically active bacteria were affiliated mainly to AMD specialists, i.e., organisms detected in or isolated from AMDs throughout the world. They included mainly Acidobacteria and the non-affiliated "Candidatus Fodinabacter communificans," as well as Thiomonas and Acidithiobacillus spp., Actinobacteria, and unclassified Gammaproteobacteria. The distribution range of these organisms suggested that they show niche conservatism. Sixteen types of deduced protein sequences of arsenite transporters (5 ArsB and 11 Acr3p) were detected, whereas a single type of arsenite oxidase (AioA) was found. Our data suggested that at Carnoulès, the aioA gene was more recent than those encoding arsenite transporters and subjected to a different molecular evolution. In contrast to the 16S ribosomal RNA (16S rRNA) genes associated with AMD environments worldwide, the functional genes aioA, ACR3, and to a lesser extent arsB, were either novel or specific to Carnoulès, raising the question as to whether these functional genes are specific to high concentrations of arsenic, AMD-specific, or site-specific.
Flexible genomes facilitate bacterial evolution and are classically organized into polymorphic strain-specific segments called regions of genomic plasticity (RGPs). Using a new web tool, RGPFinder, ...we investigated plasticity units in bacterial genomes, by exhaustive description of the RGPs in two Photorhabdus and two Xenorhabdus strains, belonging to the Enterobacteriaceae and interacting with invertebrates (insects and nematodes).
RGPs account for about 60% of the genome in each of the four genomes studied. We classified RGPs into genomic islands (GIs), prophages and two new classes of RGP without the features of classical mobile genetic elements (MGEs) but harboring genes encoding enzymes catalyzing DNA recombination (RGPmob), or with no remarkable feature (RGPnone). These new classes accounted for most of the RGPs and are probably hypervariable regions, ancient MGEs with degraded mobilization machinery or non canonical MGEs for which the mobility mechanism has yet to be described. We provide evidence that not only the GIs and the prophages, but also RGPmob and RGPnone, have a mosaic structure consisting of modules. A module is a block of genes, 0.5 to 60 kb in length, displaying a conserved genomic organization among the different Enterobacteriaceae. Modules are functional units involved in host/environment interactions (22-31%), metabolism (22-27%), intracellular or intercellular DNA mobility (13-30%), drug resistance (4-5%) and antibiotic synthesis (3-6%). Finally, in silico comparisons and PCR multiplex analysis indicated that these modules served as plasticity units within the bacterial genome during genome speciation and as deletion units in clonal variants of Photorhabdus.
This led us to consider the modules, rather than the entire RGP, as the true unit of plasticity in bacterial genomes, during both short-term and long-term genome evolution.
Pseudomonas entomophila is an entomopathogenic bacterium that, upon ingestion, kills Drosophila melanogaster as well as insects from different orders. The complete sequence of the 5.9-Mb genome was ...determined and compared to the sequenced genomes of four Pseudomonas species. P. entomophila possesses most of the catabolic genes of the closely related strain P. putida KT2440, revealing its metabolically versatile properties and its soil lifestyle. Several features that probably contribute to its entomopathogenic properties were disclosed. Unexpectedly for an animal pathogen, P. entomophila is devoid of a type III secretion system and associated toxins but rather relies on a number of potential virulence factors such as insecticidal toxins, proteases, putative hemolysins, hydrogen cyanide and novel secondary metabolites to infect and kill insects. Genome-wide random mutagenesis revealed the major role of the two-component system GacS/GacA that regulates most of the potential virulence factors identified.
Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are ...still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a l-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.
PDF
