Environmental, mechanistic and evolutionary landscape of antibiotic persistence.

Recalcitrant infections pose a serious challenge by prolonging antibiotic therapies and contributing to the spread of antibiotic resistance, thereby threatening the successful treatment of bacterial infections. One potential contributing factor in persistent infections is antibiotic persistence, which involves the survival of transiently tolerant subpopulations of bacteria. This review summarizes the current understanding of antibiotic persistence, including its clinical significance and the environmental and evolutionary factors at play. Additionally, we discuss the emerging concept of persister regrowth and potential strategies to combat persister cells. Recent advances highlight the multifaceted nature of persistence, which is controlled by deterministic and stochastic elements and shaped by genetic and environmental factors. To translate in vitro findings to in vivo settings, it is crucial to include the heterogeneity and complexity of bacterial populations in natural environments. As researchers continue to gain a more holistic understanding of this phenomenon and develop effective treatments for persistent bacterial infections, the study of antibiotic persistence is likely to become increasingly complex.

Minimal Surviving Inoculum in Collective Antibiotic Resistance.

A common strategy used by bacteria to resist antibiotics is enzymatic degradation or modification. This reduces the antibiotic threat in the environment and is therefore potentially a collective mechanism that also enhances the survival of nearby cells. Collective resistance is of clinical significance, yet a quantitative understanding at the population level is still incomplete. Here, we develop a general theoretical framework of collective resistance by antibiotic degradation. Our modeling study reveals that population survival crucially depends on the ratio of timescales of two processes: the rates of population death and antibiotic removal. However, it is insensitive to molecular, biological, and kinetic details of the underlying processes that give rise to these timescales. Another important aspect of antibiotic degradation is the degree of cooperativity, related to the permeability of the cell wall to antibiotics and enzymes. These observations motivate a coarse-grained, phenomenological model, with two compound parameters representing the population's race to survival and single-cell effective resistance. We propose a simple experimental assay to measure the dose-dependent minimal surviving inoculum and apply it to Escherichia coli expressing several types of ß-lactamase. Experimental data analyzed within the theoretical framework corroborate it with good agreement. Our simple model may serve as a reference for more complex situations, such as heterogeneous bacterial communities. IMPORTANCE Collective resistance occurs when bacteria work together to decrease the concentration of antibiotics in their environment, for example, by actively breaking down or modifying them. This can help bacteria survive by reducing the effective antibiotic concentration below their threshold for growth. In this study, we used mathematical modeling to examine the factors that influence collective resistance and to develop a framework to understand the minimum population size needed to survive a given initial antibiotic concentration. Our work helps to identify generic mechanism-independent parameters that can be derived from population data and identifies combinations of parameters that play a role in collective resistance. Specifically, it highlights the relative timescales involved in the survival of populations that inactivate antibiotics, as well as the levels of cooperation versus privatization. The results of this study contribute to our understanding of population-level effects on antibiotic resistance and may inform the design of antibiotic therapies.

Interaction between mutation type and gene pleiotropy drives parallel evolution in the laboratory.

What causes evolution to be repeatable is a fundamental question in evolutionary biology. Pleiotropy, i.e. the effect of an allele on multiple traits, is thought to enhance repeatability by constraining the number of available beneficial mutations. Additionally, pleiotropy may promote repeatability by allowing large fitness benefits of single mutations via adaptive combinations of phenotypic effects. Yet, this latter evolutionary potential may be reaped solely by specific types of mutations able to realize optimal combinations of phenotypic effects while avoiding the costs of pleiotropy. Here, we address the interaction of gene pleiotropy and mutation type on evolutionary repeatability in a meta-analysis of experimental evolution studies with Escherichia coli. We hypothesize that single nucleotide polymorphisms (SNPs) are principally able to yield large fitness benefits by targeting highly pleiotropic genes, whereas indels and structural variants (SVs) provide smaller benefits and are restricted to genes with lower pleiotropy. By using gene connectivity as proxy for pleiotropy, we show that non-disruptive SNPs in highly pleiotropic genes yield the largest fitness benefits, since they contribute more to parallel evolution, especially in large populations, than inactivating SNPs, indels and SVs. Our findings underscore the importance of considering genetic architecture together with mutation type for understanding evolutionary repeatability. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'.

High-Throughput Gel Microbeads as Incubators for Bacterial Competition Study.

Bacteria primarily live in structured environments, such as colonies and biofilms, attached to surfaces or growing within soft tissues. They are engaged in local competitive and cooperative interactions impacting our health and well-being, for example, by affecting population-level drug resistance. Our knowledge of bacterial competition and cooperation within soft matrices is incomplete, partly because we lack high-throughput tools to quantitatively study their interactions. Here, we introduce a method to generate a large amount of agarose microbeads that mimic the natural culture conditions experienced by bacteria to co-encapsulate two strains of fluorescence-labeled Escherichia coli. Focusing specifically on low bacterial inoculum (1-100 cells/capsule), we demonstrate a study on the formation of colonies of both strains within these 3D scaffolds and follow their growth kinetics and interaction using fluorescence microscopy in highly replicated experiments. We confirmed that the average final colony size is inversely proportional to the inoculum size in this semi-solid environment as a result of limited available resources. Furthermore, the colony shape and fluorescence intensity per colony are distinctly different in monoculture and co-culture. The experimental observations in mono- and co-culture are compared with predictions from a simple growth model. We suggest that our high throughput and small footprint microbead system is an excellent platform for future investigation of competitive and cooperative interactions in bacterial communities under diverse conditions, including antibiotics stress.

Population dynamics of cross-protection against ß-lactam antibiotics in droplet microreactors.

Introduction: Bacterial strains that are resistant to antibiotics may protect not only themselves, but also sensitive bacteria nearby if resistance involves antibiotic degradation. Such cross-protection poses a challenge to effective antibiotic therapy by enhancing the long-term survival of bacterial infections, however, the current understanding is limited. Methods: In this study, we utilize an automated nanoliter droplet analyzer to study the interactions between Escherichia coli strains expressing a ß-lactamase (resistant) and those not expressing it (sensitive) when exposed to the ß-lactam antibiotic cefotaxime (CTX), with the aim to define criteria contributing to cross-protection. Results: We observed a cross-protection window of CTX concentrations for the sensitive strain, extending up to approximately 100 times its minimal inhibitory concentration (MIC). Through both microscopy and enzyme activity analyses, we demonstrate that bacterial filaments, triggered by antibiotic stress, contribute to cross-protection. Discussion: The antibiotic concentration window for cross-protection depends on the difference in ß-lactamase activity between co-cultured strains: larger differences shift the 'cross-protection window' toward higher CTX concentrations. Our findings highlight the dependence of opportunities for cross-protection on the relative resistance levels of the strains involved and suggest a possible specific role for filamentation.

Population size mediates the contribution of high-rate and large-benefit mutations to parallel evolution.

Mutations with large fitness benefits and mutations occurring at high rates may both cause parallel evolution, but their contribution is predicted to depend on population size. Moreover, high-rate and large-benefit mutations may have different long-term adaptive consequences. We show that small and 100-fold larger bacterial populations evolve resistance to a ß-lactam antibiotic by using similar numbers, but different types of mutations. Small populations frequently substitute similar high-rate structural variants and loss-of-function point mutations, including the deletion of a low-activity ß-lactamase, and evolve modest resistance levels. Large populations more often use low-rate, large-benefit point mutations affecting the same targets, including mutations activating the ß-lactamase and other gain-of-function mutations, leading to much higher resistance levels. Our results demonstrate the separation by clonal interference of mutation classes with divergent adaptive consequences, causing a shift from high-rate to large-benefit mutations with increases in population size.

Choice of ß-Lactam Resistance Pathway Depends Critically on Initial Antibiotic Concentration.

Antibiotic resistance trajectories with different final resistance may critically depend on the first mutation, due to epistatic interactions. Here, we study the effect of mutation bias and the concentration-dependent effects on fitness of two clinically important mutations in TEM-1 ß-lactamase in initiating alternative trajectories to cefotaxime resistance. We show that at low cefotaxime concentrations, the R164S mutation (a mutation of arginine to serine at position 164), which confers relatively low resistance, is competitively superior to the G238S mutation, conferring higher resistance, thus highlighting a critical influence of antibiotic concentration on long-term resistance evolution.

Coexistence of fluorescent Escherichia coli strains in millifluidic droplet reactors.

Understanding competition and cooperation within microbiota is of high fundamental and clinical importance, helping to comprehend species' evolution and biodiversity. We co-encapsulated and cultured two isogenic Escherichia coli strains expressing blue (BFP) and yellow (YFP) fluorescent proteins into numerous emulsion droplets and quantified their growth by employing fluorescence measurements. To characterize and compare the bacterial growth kinetics and behavior in mono and co-culture, we compared the experimental observations with predictions from a simple growth model. Varying the initial ratio (R0) of both cell types injected, we observed a broad landscape from competition to cooperation between both strains in their confined microenvironments depending on start frequency: from a nearly symmetric situation at R0 = 1, up to the domination of one subpopulation when R0 ≫ 1 (or R0 ≪ 1). Due to competition between the strains, their doubling times and final biomass ratios (R1) continuously deviate from the monoculture behavior. The correlation map of the two strains' doubling times reveals that the R0 is one of the critical parameters affecting the competitive interaction between isogenic bacterial strains. Thanks to this strategy, different species of bacteria can be monitored simultaneously in real-time. Further advantages include high statistical output, unaffected bacteria growth, and long-time measurements in a well-mixed environment. We expect that the millifluidic droplet-based reactor can be utilized for practical clinical applications, such as bacterial antibiotic resistance and enzyme reaction kinetics studies.

Clonal Interference and Mutation Bias in Small Bacterial Populations in Droplets.

Experimental evolution studies have provided key insights into the fundamental mechanisms of evolution. One striking observation is that parallel and convergent evolution during laboratory evolution can be surprisingly common. However, these experiments are typically performed with well-mixed cultures and large effective population sizes, while pathogenic microbes typically experience strong bottlenecks during infection or drug treatment. Yet, our knowledge about adaptation in very small populations, where selection strength and mutation supplies are limited, is scant. In this study, wild-type and mutator strains of the bacterium Escherichia coli were evolved for about 100 generations towards increased resistance to the ß-lactam antibiotic cefotaxime in millifluidic droplets of 0.5 µL and effective population size of approximately 27,000 cells. The small effective population size limited the adaptive potential of wild-type populations, where adaptation was limited to inactivating mutations, which caused the increased production of outer-membrane vesicles, leading to modest fitness increases. In contrast, mutator clones with an average of ~30-fold higher mutation rate adapted much faster by acquiring both inactivating mutations of an outer-membrane porin and particularly inactivating and gain-of-function mutations, causing the upregulation or activation of a common efflux pump, respectively. Our results demonstrate how in very small populations, clonal interference and mutation bias together affect the choice of adaptive trajectories by mediating the balance between high-rate and large-benefit mutations.

Resurrected Protein Interaction Networks Reveal the Innovation Potential of Ancient Whole-Genome Duplication.

The evolution of plants is characterized by whole-genome duplications, sometimes closely associated with the origin of large groups of species. The gamma (γ) genome triplication occurred at the origin of the core eudicots, which comprise ∼75% of flowering plants. To better understand the impact of whole-genome duplication, we studied the protein interaction network of MADS domain transcription factors, which are key regulators of reproductive development. We reconstructed, synthesized, and tested the interactions of ancestral proteins immediately before and closely after the triplication and directly compared these ancestral networks to the extant networks of Arabidopsis thaliana and tomato (Solanum lycopersicum). We found that gamma expanded the MADS domain interaction network more strongly than subsequent genomic events. This event strongly rewired MADS domain interactions and allowed for the evolution of new functions and installed robustness through new redundancy. Despite extensive rewiring, the organization of the network was maintained through gamma. New interactions and protein retention compensated for its potentially destructive impact on network organization. Post gamma, the network evolved from an organization around the single hub SEP3 to a network organized around multiple hubs and well-connected proteins lost, rather than gained, interactions. The data provide a resource for comparative developmental biology in flowering plants.

Exploiting DELLA Signaling in Cereals.

The spectacular yield increases in rice and wheat during the green revolution were partly realized by reduced gibberellin (GA) synthesis or sensitivity, both causing the accumulation of DELLA proteins. Although insights into the regulation of plant growth and development by DELLA proteins advanced rapidly in arabidopsis (Arabidopsis thaliana), DELLA-mediated regulation of downstream responses in cereals has received little attention to date. Furthermore, translating this research from arabidopsis to cereals is challenging given their different growth patterns and our phylogenetic analysis which reveals that DELLA-related DGLLA proteins exist in cereals but not in arabidopsis. Therefore, understanding the molecular basis of DELLA function in cereals holds great potential to improve yield. In this review, we propose to extend the focus of DELLA functional research to cereals, and highlight the appropriate tools that are now available to achieve this.

The Origin of Floral Organ Identity Quartets.

The origin of flowers has puzzled plant biologists ever since Darwin referred to their sudden appearance in the fossil record as an abominable mystery. Flowers are considered to be an assembly of protective, attractive, and reproductive male and female leaf-like organs. Their origin cannot be understood by a morphological comparison to gymnosperms, their closest relatives, which develop separate male or female cones. Despite these morphological differences, gymnosperms and angiosperms possess a similar genetic toolbox consisting of phylogenetically related MADS domain proteins. Using ancestral MADS domain protein reconstruction, we trace the evolution of organ identity quartets along the stem lineage of crown angiosperms. We provide evidence that current floral quartets specifying male organ identity, which consist of four types of subunits, evolved from ancestral complexes of two types of subunits through gene duplication and integration of SEPALLATA proteins just before the origin of flowering plants. Our results suggest that protein interaction changes underlying this compositional shift were the result of a gradual and reversible evolutionary trajectory. Modeling shows that such compositional changes may have facilitated the evolution of the perfect, bisexual flower.

A Flowering Locus C Homolog Is a Vernalization-Regulated Repressor in Brachypodium and Is Cold Regulated in Wheat.

Winter cereals require prolonged cold to transition from vegetative to reproductive development. This process, referred to as vernalization, has been extensively studied in Arabidopsis (Arabidopsis thaliana). In Arabidopsis, a key flowering repressor called FLOWERING LOCUS C (FLC) quantitatively controls the vernalization requirement. By contrast, in cereals, the vernalization response is mainly regulated by the VERNALIZATION genes, VRN1 and VRN2 Here, we characterize ODDSOC2, a recently identified FLC ortholog in monocots, knowing that it belongs to the FLC lineage. By studying its expression in a diverse set of Brachypodium accessions, we find that it is a good predictor of the vernalization requirement. Analyses of transgenics demonstrated that BdODDSOC2 functions as a vernalization-regulated flowering repressor. In most Brachypodium accessions BdODDSOC2 is down-regulated by cold, and in one of the winter accessions in which this down-regulation was evident, BdODDSOC2 responded to cold before BdVRN1. When stably down-regulated, the mechanism is associated with spreading H3K27me3 modifications at the BdODDSOC2 chromatin. Finally, homoeolog-specific gene expression analyses identify TaAGL33 and its splice variant TaAGL22 as the FLC orthologs in wheat (Triticum aestivum) behaving most similar to Brachypodium ODDSOC2 Overall, our study suggests that ODDSOC2 is not only phylogenetically related to FLC in eudicots but also functions as a flowering repressor in the vernalization pathway of Brachypodium and likely other temperate grasses. These insights could prove useful in breeding efforts to refine the vernalization requirement of temperate cereals and adapt varieties to changing climates.

FLOWERING LOCUS C in monocots and the tandem origin of angiosperm-specific MADS-box genes.

MADS-domain transcription factors have been shown to act as key repressors or activators of the transition to flowering and as master regulators of reproductive organ identities. Despite their important roles in plant development, the origin of several MADS-box subfamilies has remained enigmatic so far. Here we demonstrate, through a combination of genome synteny and phylogenetic reconstructions, the origin of three major, apparently angiosperm-specific MADS-box gene clades: FLOWERING LOCUS C- (FLC-), SQUAMOSA- (SQUA-) and SEPALLATA- (SEP-)-like genes. We find that these lineages derive from a single ancestral tandem duplication in a common ancestor of extant seed plants. Contrary to common belief, we show that FLC-like genes are present in cereals where they can also act as floral repressors responsive to prolonged cold or vernalization. This opens a new perspective on the translation of findings from Arabidopsis to cereal crops, in which vernalization was originally described.

The hybrid four-CBS-domain KINßγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1.

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory ß and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid ßγ protein that combines the Four-Cystathionine ß-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in ß subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINßγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.

Gamma paleohexaploidy in the stem lineage of core eudicots: significance for MADS-box gene and species diversification.

Comparative genome biology has unveiled the polyploid origin of all angiosperms and the role of recurrent polyploidization in the amplification of gene families and the structuring of genomes. Which species share certain ancient polyploidy events, and which do not, is ill defined because of the limited number of sequenced genomes and transcriptomes and their uneven phylogenetic distribution. Previously, it has been suggested that most, but probably not all, of the eudicots have shared an ancient hexaploidy event, referred to as the gamma triplication. In this study, detailed phylogenies of subfamilies of MADS-box genes suggest that the gamma triplication has occurred before the divergence of Gunnerales but after the divergence of Buxales and Trochodendrales. Large-scale phylogenetic and K(S)-based approaches on the inflorescence transcriptomes of Gunnera manicata (Gunnerales) and Pachysandra terminalis (Buxales) provide further support for this placement, enabling us to position the gamma triplication in the stem lineage of the core eudicots. This triplication likely initiated the functional diversification of key regulators of reproductive development in the core eudicots, comprising 75% of flowering plants. Although it is possible that the gamma event triggered early core eudicot diversification, our dating estimates suggest that the event occurred early in the stem lineage, well before the rapid speciation of the earliest core eudicot lineages. The evolutionary significance of this paleopolyploidy event may thus rather lie in establishing a species lineage that was resilient to extinction, but with the genomic potential for later diversification. We consider that the traits generated from this potential characterize extant core eudicots both chemically and morphologically.