Selective advantages favour high genomic AT-contents in intracellular elements.

Extrachromosomal genetic elements such as bacterial endosymbionts and plasmids generally exhibit AT-contents that are increased relative to their hosts' DNA. The AT-bias of endosymbiotic genomes is commonly explained by neutral evolutionary processes such as a mutational bias towards increased A+T. Here we show experimentally that an increased AT-content of host-dependent elements can be selectively favoured. Manipulating the nucleotide composition of bacterial cells by introducing A+T-rich or G+C-rich plasmids, we demonstrate that cells containing GC-rich plasmids are less fit than cells containing AT-rich plasmids. Moreover, the cost of GC-rich elements could be compensated by providing precursors of G+C, but not of A+T, thus linking the observed fitness effects to the cytoplasmic availability of nucleotides. Accordingly, introducing AT-rich and GC-rich plasmids into other bacterial species with different genomic GC-contents revealed that the costs of G+C-rich plasmids decreased with an increasing GC-content of their host's genomic DNA. Taken together, our work identifies selection as a strong evolutionary force that drives the genomes of intracellular genetic elements toward higher A+T contents.

Fitness and stability of obligate cross-feeding interactions that emerge upon gene loss in bacteria.

Cross-feeding interactions, in which bacterial cells exchange costly metabolites to the benefit of both interacting partners, are very common in the microbial world. However, it generally remains unclear what maintains this type of interaction in the presence of non-cooperating types. We investigate this problem using synthetic cross-feeding interactions: by simply deleting two metabolic genes from the genome of Escherichia coli, we generated genotypes that require amino acids to grow and release other amino acids into the environment. Surprisingly, in a vast majority of cases, cocultures of two cross-feeding strains showed an increased Darwinian fitness (that is, rate of growth) relative to prototrophic wild type cells--even in direct competition. This unexpected growth advantage was due to a division of metabolic labour: the fitness cost of overproducing amino acids was less than the benefit of not having to produce others when they were provided by their partner. Moreover, frequency-dependent selection maintained cross-feeding consortia and limited exploitation by non-cooperating competitors. Together, our synthetic study approach reveals ecological principles that can help explain the widespread occurrence of obligate metabolic cross-feeding interactions in nature.

Design and characterization of auxotrophy-based amino acid biosensors.

Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that - upon deletion - should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used.

Optimized virus-induced gene silencing in Solanum nigrum reveals the defensive function of leucine aminopeptidase against herbivores and the shortcomings of empty vector controls.

Virus-induced gene silencing (VIGS) enables high-throughput analysis of gene function in plants but is not universally applicable and requires optimization for each species. Here a VIGS system is described for Solanum nigrum, a wild relative of tomato and potato and a valuable model species for ecogenomics. The efficiency of the two most widely used Tobacco rattle virus (TRV) vectors to silence phytoene desaturase (PDS) in S. nigrum was tested. Additionally, the infiltration method and growth temperatures for gene silencing were optimized and the suitability of different control vectors evaluated. Using leucine aminopeptidase (LAP), a herbivore-induced protein, silencing efficiency and the applicability of silenced plants for herbivore feeding assays were assessed. Vacuum infiltration of seedlings with Agrobacterium carrying the vector, pYL156, proved the most efficient means of silencing genes. Empty-vector controls decreased plant growth but control vectors carrying a piece of noncoding sequence did not. Silencing LAP significantly increased the larval mass of Manduca sexta that fed on silenced plants. This VIGS protocol proved highly successful for S. nigrum, which should include control vectors carrying noncoding sequence as control treatments. Silencing LAP provided the first experimental evidence that LAP has a defensive function against herbivores.