Towards evolutionary predictions: Current promises and challenges.

Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS-CoV2 and influenza to CRISPR-based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions.

Endocytosis-like DNA uptake by cell wall-deficient bacteria.

Horizontal gene transfer in bacteria is widely believed to occur via conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. Here, we report that cell wall-deficient bacteria can engulf DNA and other extracellular material via an endocytosis-like process. Specifically, we show that L-forms of the filamentous actinomycete Kitasatospora viridifaciens can take up plasmid DNA, polysaccharides (dextran) and 150-nm lipid nanoparticles. The process involves invagination of the cytoplasmic membrane, leading to formation of intracellular vesicles that encapsulate extracellular material. DNA uptake is not affected by deletion of genes homologous to comEC and comEA, which are required for natural transformation in other species. However, uptake is inhibited by sodium azide or incubation at 4 °C, suggesting the process is energy-dependent. The encapsulated materials are released into the cytoplasm upon degradation of the vesicle membrane. Given that cell wall-deficient bacteria are considered a model for early life forms, our work reveals a possible mechanism for primordial cells to acquire food or genetic material before invention of the bacterial cell wall.

Generating Heterokaryotic Cells via Bacterial Cell-Cell Fusion.

Fusion of cells is an important and common biological process that leads to the mixing of cellular contents and the formation of multinuclear cells. Cell fusion occurs when distinct membranes are brought into proximity of one another and merge to become one. Fusion holds promise for biotechnological innovations, for instance, for the discovery of urgently needed new antibiotics. Here, we used antibiotic-producing bacteria that can proliferate without their cell wall as a model to investigate cell-cell fusion. We found that fusion between genetically distinct cells yields heterokaryons that are viable, contain multiple selection markers, and show increased antimicrobial activity. The rate of fusion induced using physical and chemical methods was dependent on membrane fluidity, which is related to lipid composition as a function of cellular age. Finally, by using an innovative system of synthetic membrane-associated lipopeptides, we achieved targeted fusion between distinctly marked cells to further enhance fusion efficiency. These results provide a molecular handle to understand and control cell-cell fusion, which can be used in the future for the discovery of new drugs. IMPORTANCE Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery. We present here wall-deficient cells as a platform for the same. We identify the fluidity of the membrane as an important characteristic for the process of fusion. We demonstrate a cell-specific approach for fusion using synthetically designed peptides yielding cells with modified antibiotic production profiles. Overall, wall-deficient cells can be a chassis for innovative metabolite production by providing an alternative method for cell-cell fusion.

Mutational meltdown of putative microbial altruists in Streptomyces coelicolor colonies.

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a subpopulation of cells arises that hyperproduces metabolically costly antibiotics, resulting in a division of labor that increases colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is similar to altruistic worker castes in social insects or somatic cells in multicellular organisms. To understand these mutant cells' reproductive and genomic fate after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that in contrast to wild-type cells, putatively altruistic mutant cells continue to decline in fitness during transfer while they lose more fragments from their chromosome ends. In addition, the base-substitution rate in mutants increases roughly 10-fold, possibly due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with DNA damage due to point mutations and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest the cells arising in the S. coelicolor division of labor are analogous to altruistic reproductively sterile castes of social insects.

Prevalent emergence of reciprocity among cross-feeding bacteria.

Explaining the de novo evolution of obligate cooperative cross-feeding interactions among bacteria is a fundamental problem. A critical step during this process is the emergence of reciprocity among two interaction partners, because a mutually beneficial exchange of metabolic byproducts can subsequently favour the evolution of cooperative cross-feeding. However, so far, the propensity with which unidirectional cross-feeding interactions transition into bidirectional interactions remains unknown. To address this issue, we systematically cocultured four amino acid auxotrophic genotypes of two bacterial species with potential amino acid donors belonging to 25 different bacterial species. Surprisingly, the results of this experiment revealed that in around 40% of all cases analysed, both the auxotrophic recipient and the metabolically autonomous donor gained a significant growth advantage in coculture. Subsequent experiments clarified that the auxotrophy-causing mutation did not induce the growth-enhancing effect of recipients, but that it was rather due to a generally high propensity of different species to engage in synergistic metabolic interactions. Together, these findings show that reciprocity commonly emerges spontaneously in unidirectional cross-feeding interactions, thus paving the way for the evolution of even tighter metabolic interactions.

Metabolic dissimilarity determines the establishment of cross-feeding interactions in bacteria.

The exchange of metabolites among different bacterial genotypes profoundly impacts the structure and function of microbial communities. However, the factors governing the establishment of these cross-feeding interactions remain poorly understood. While shared physiological features may facilitate interactions among more closely related individuals, a lower relatedness should reduce competition and thus increase the potential for synergistic interactions. Here, we investigate how the relationship between a metabolite donor and recipient affects the propensity of strains to engage in unidirectional cross-feeding interactions. For this, we performed pairwise cocultivation experiments between four auxotrophic recipients and 25 species of potential amino acid donors. Auxotrophic recipients grew in the vast majority of pairs tested (63%), suggesting metabolic cross-feeding interactions are readily established. Strikingly, both the phylogenetic distance between donor and recipient and the dissimilarity of their metabolic networks were positively associated with the growth of auxotrophic recipients. Analyzing the co-growth of species from a gut microbial community in silico also revealed that recipient genotypes benefitted more from interacting with metabolically dissimilar partners, thus corroborating the empirical results. Together, our work identifies the metabolic dissimilarity between bacterial genotypes as a key factor determining the establishment of metabolic cross-feeding interactions in microbial communities.

Formation of wall-less cells in Kitasatospora viridifaciens requires cytoskeletal protein FilP in oxygen-limiting conditions.

The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the ΔfilP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling.

Use of Permanent Wall-Deficient Cells as a System for the Discovery of New-to-Nature Metabolites.

Filamentous actinobacteria are widely used as microbial cell factories to produce valuable secondary metabolites, including the vast majority of clinically relevant antimicrobial compounds. Secondary metabolites are typically encoded by large biosynthetic gene clusters, which allow for a modular approach to generating diverse compounds through recombination. Protoplast fusion is a popular method for whole genome recombination that uses fusion of cells that are transiently wall-deficient. This process has been applied for both inter- and intraspecies recombination. An important limiting step in obtaining diverse recombinants from fused protoplasts is regeneration of the cell wall, because this forces the chromosomes from different parental lines to segregate, thereby preventing further recombination. Recently, several labs have gained insight into wall-deficient bacteria that have the ability to proliferate without their cell wall, known as L-forms. Unlike protoplasts, L-forms can stably maintain multiple chromosomes over many division cycles. Fusion of such L-forms would potentially allow cells to express genes from both parental genomes while also extending the time for recombination, both of which can contribute to an increased chemical diversity. Here, we present a perspective on how L-form fusion has the potential to become a platform for novel compound discovery and may thus help to overcome the antibiotic discovery void.

Harnessing ecological and evolutionary principles to guide the design of microbial production consortia.

Bacteria are widely used for commercially producing biomolecules. However, attempts to rationally design production strains and optimize cultivation conditions are frequently counteracted by the emergence of mutants with reduced production characteristics that decrease overall process yield. The reason why these mutants arise is likely because of a mismatch between the ecological conditions under which bacteria evolved in nature and the situation they experience in an industrial setting. Thus, there is a great potential for improving biotechnological production processes by implementing eco-evolutionary knowledge. However, this is often limited by a lack of effective communication between process engineers and microbial ecologists/evolutionary biologists. Here, we highlight recent findings in the field of microbial ecology and evolution and suggest implementation of this knowledge can significantly enhance microbial bioproduction.

Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells.

Bacteria frequently engage in cross-feeding interactions that involve an exchange of metabolites with other micro- or macroorganisms. The often obligate nature of these associations, however, hampers manipulative experiments, thus limiting our mechanistic understanding of the ecophysiological consequences that result for the organisms involved. Here we address this issue by taking advantage of a well-characterized experimental model system, in which auxotrophic genotypes of E. coli derive essential amino acids from prototrophic donor cells using intercellular nanotubes. Surprisingly, donor-recipient cocultures revealed that the mere presence of auxotrophic genotypes was sufficient to increase amino acid production levels of several prototrophic donor genotypes. Our work is consistent with a scenario, in which interconnected auxotrophs withdraw amino acids from the cytoplasm of donor cells, which delays feedback inhibition of the corresponding amino acid biosynthetic pathway and, in this way, increases amino acid production levels. Our findings indicate that in newly established mutualistic associations, an intercellular regulation of exchanged metabolites can simply emerge from the architecture of the underlying biosynthetic pathways, rather than requiring the evolution of new regulatory mechanisms.

Ecology and evolution of metabolic cross-feeding interactions in bacteria.

Literature covered: early 2000s to late 2017Bacteria frequently exchange metabolites with other micro- and macro-organisms. In these often obligate cross-feeding interactions, primary metabolites such as vitamins, amino acids, nucleotides, or growth factors are exchanged. The widespread distribution of this type of metabolic interactions, however, is at odds with evolutionary theory: why should an organism invest costly resources to benefit other individuals rather than using these metabolites to maximize its own fitness? Recent empirical work has shown that bacterial genotypes can significantly benefit from trading metabolites with other bacteria relative to cells not engaging in such interactions. Here, we will provide a comprehensive overview over the ecological factors and evolutionary mechanisms that have been identified to explain the evolution and maintenance of metabolic mutualisms among microorganisms. Furthermore, we will highlight general principles that underlie the adaptive evolution of interconnected microbial metabolic networks as well as the evolutionary consequences that result for cells living in such communities.

Metabolic cross-feeding via intercellular nanotubes among bacteria.

Bacteria frequently exchange metabolites by diffusion through the extracellular environment, yet it remains generally unclear whether bacteria can also use cell-cell connections to directly exchange nutrients. Here we address this question by engineering cross-feeding interactions within and between Acinetobacter baylyi and Escherichia coli, in which two distant bacterial species reciprocally exchange essential amino acids. We establish that in a well-mixed environment E. coli, but likely not A. baylyi, can connect to other bacterial cells via membrane-derived nanotubes and use these to exchange cytoplasmic constituents. Intercellular connections are induced by auxotrophy-causing mutations and cease to establish when amino acids are externally supplied. Electron and fluorescence microscopy reveal a network of nanotubular structures that connects bacterial cells and enables an intercellular transfer of cytoplasmic materials. Together, our results demonstrate that bacteria can use nanotubes to exchange nutrients among connected cells and thus help to distribute metabolic functions within microbial communities.