Phenotypic heterogeneity in Streptomyces colonies.

Streptomyces are a large genus of multicellular bacteria best known for their prolific production of bioactive natural products. In addition, they play key roles in the mineralisation of insoluble resources, such as chitin and cellulose. Because of their multicellular mode of growth, colonies of interconnected hyphae extend over a large area that may experience different conditions in different parts of the colony. Here, we argue that within-colony phenotypic heterogeneity can allow colonies to simultaneously respond to divergent inputs from resources or competitors that are spatially and temporally dynamic. We discuss causal drivers of heterogeneity, including competitors, precursor availability, metabolic diversity and division of labour, that facilitate divergent phenotypes within Streptomyces colonies. We discuss the adaptive causes and consequences of within-colony heterogeneity, highlight current knowledge (gaps) and outline key questions for future studies.

Genome sequence and characterization of Streptomyces phages Vanseggelen and Verabelle, representing two new species within the genus Camvirus.

Despite the rising interest in bacteriophages, little is known about their infection cycle and lifestyle in a multicellular host. Even in the model system Streptomyces, only a small number of phages have been sequenced and well characterized so far. Here, we report the complete characterization and genome sequences of Streptomyces phages Vanseggelen and Verabelle isolated using Streptomyces coelicolor as a host. A wide range of Streptomyces strains could be infected by both phages, but neither of the two phages was able to infect members of the closely related sister genus Kitasatospora. The phages Vanseggelen and Verabelle have a double-stranded DNA genome with lengths of 48,720 and 48,126 bp, respectively. Both phage genomes contain 72 putative genes, and the presence of an integrase encoding protein indicates a lysogenic lifestyle. Characterization of the phages revealed their stability over a wide range of temperatures (30-45 °C) and pH values (4-10). In conclusion, Streptomyces phage Vanseggelen and Streptomyces phage Verabelle are newly isolated phages that can be classified as new species in the genus Camvirus, within the subfamily Arquattrovirinae.

Evolution of genome fragility enables microbial division of labor.

Division of labor can evolve when social groups benefit from the functional specialization of its members. Recently, a novel means of coordinating the division of labor was found in the antibiotic-producing bacterium Streptomyces coelicolor, where specialized cells are generated through large-scale genomic re-organization. We investigate how the evolution of a genome architecture enables such mutation-driven division of labor, using a multiscale computational model of bacterial evolution. In this model, bacterial behavior-antibiotic production or replication-is determined by the structure and composition of their genome, which encodes antibiotics, growth-promoting genes, and fragile genomic loci that can induce chromosomal deletions. We find that a genomic organization evolves, which partitions growth-promoting genes and antibiotic-coding genes into distinct parts of the genome, separated by fragile genomic loci. Mutations caused by these fragile sites mostly delete growth-promoting genes, generating sterile, and antibiotic-producing mutants from weakly-producing progenitors, in agreement with experimental observations. This division of labor enhances the competition between colonies by promoting antibiotic diversity. These results show that genomic organization can co-evolve with genomic instabilities to enable reproductive division of labor.

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.

Using click chemistry to study microbial ecology and evolution.

Technological advances have largely driven the revolution in our understanding of the structure and function of microbial communities. Culturing, long the primary tool to probe microbial life, was supplanted by sequencing and other -omics approaches, which allowed detailed quantitative insights into species composition, metabolic potential, transcriptional activity, secretory responses and more. Although the ability to characterize "who's there" has never been easier or cheaper, it remains technically challenging and expensive to understand what the diverse species and strains that comprise microbial communities are doing in situ, and how these behaviors change through time. Our aim in this brief review is to introduce a developing toolkit based on click chemistry that can accelerate and reduce the expense of functional analyses of the ecology and evolution of microbial communities. After first outlining the history of technological development in this field, we will discuss key applications to date using diverse labels, including BONCAT, and then end with a selective (biased) view of areas where click-chemistry and BONCAT-based approaches stand to have a significant impact on our understanding of microbial communities.

Genome sequence and characterization of Streptomyces phage Pablito, representing a new species within the genus Janusvirus.

Streptomycetes are ubiquitous soil bacteria. Here we report the complete and annotated genome sequence and characterization of Streptomyces phage Pablito, isolated from a soil sample in Haarlem, the Netherlands using Streptomyces lividans as host. This phage was able to infect a diverse range of Streptomyces strains, but none belonging to the sister genus Kitasatospora. Phage Pablito has double-stranded DNA with a genome length of 49,581 base pairs encoding 76 putative proteins, of which 26 could be predicted. The presence of a serine integrase protein indicated the lysogenic nature of phage Pablito. The phage remained stable over a wide range of temperatures (25-45 °C) and at pH ≥ 7.0, but lost infectivity at temperatures above 55 °C or when the pH dropped below 6.0. This newly isolated phage is closely related to Streptomyces phage Janus and Hank144 and considered a new species classified in the genus Janusvirus, within the subfamily Arquattrovirinae.

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.

Reversible bacteriophage resistance by shedding the bacterial cell wall.

Phages are highly abundant in the environment and pose a major threat for bacteria. Therefore, bacteria have evolved sophisticated defence systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near-complete conversion of walled cells to a cell-wall-deficient state, which remains viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell-wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall deficiency may potentially limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall.

Ecological drivers of division of labour in Streptomyces.

Division of labour occurs when different individuals, cells or tissues become specialised to perform complementary tasks that benefit the whole organism or social group. Although long studied in multicellular organisms and colonies of social insects, several recent studies have established that division of labour is common in microorganisms. We review recent work on the division of labour in unicellular and multicellular bacteria, with a particular focus on reproductive and metabolic divisions of labour in actinomycetes. Actinomycetes show enormous variation in sporophore morphology and spore production patterns that likely affect the potential for cooperative interactions within colonies. They also display both irreversible genetic and spatiotemporally regulated phenotypic divisions of labour that structure antibiotic production. We highlight outstanding questions in this group of multicellular bacteria and outline factors that can modify the expression of division of labour across microbes.

Allele-specific collateral and fitness effects determine the dynamics of fluoroquinolone resistance evolution.

Collateral sensitivity (CS), which arises when resistance to one antibiotic increases sensitivity toward other antibiotics, offers treatment opportunities to constrain or reverse the evolution of antibiotic resistance. The applicability of CS-informed treatments remains uncertain, in part because we lack an understanding of the generality of CS effects for different resistance mutations, singly or in combination. Here, we address this issue in the gram-positive pathogen Streptococcus pneumoniae by measuring collateral and fitness effects of clinically relevant gyrA and parC alleles and their combinations that confer resistance to fluoroquinolones. We integrated these results in a mathematical model that allowed us to evaluate how different in silico combination treatments impact the dynamics of resistance evolution. We identified common and conserved CS effects of different gyrA and parC alleles; however, the spectrum of collateral effects was unique for each allele or allelic pair. This indicated that allelic identity can impact the evolutionary dynamics of resistance evolution during monotreatment and combination treatment. Our model simulations, which included the experimentally derived antibiotic susceptibilities and fitness effects, and antibiotic-specific pharmacodynamics revealed that both collateral and fitness effects impact the population dynamics of resistance evolution. Overall, we provide evidence that allelic identity and interactions can have a pronounced impact on collateral effects to different antibiotics and suggest that these need to be considered in models examining CS-based therapies.

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.

The genetic architecture underlying prey-dependent performance in a microbial predator.

Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator.

Design principles of collateral sensitivity-based dosing strategies.

Collateral sensitivity (CS)-based antibiotic treatments, where increased resistance to one antibiotic leads to increased sensitivity to a second antibiotic, may have the potential to limit the emergence of antimicrobial resistance. However, it remains unclear how to best design CS-based treatment schedules. To address this problem, we use mathematical modelling to study the effects of pathogen- and drug-specific characteristics for different treatment designs on bacterial population dynamics and resistance evolution. We confirm that simultaneous and one-day cycling treatments could supress resistance in the presence of CS. We show that the efficacy of CS-based cycling therapies depends critically on the order of drug administration. Finally, we find that reciprocal CS is not essential to suppress resistance, a result that significantly broadens treatment options given the ubiquity of one-way CS in pathogens. Overall, our analyses identify key design principles of CS-based treatment strategies and provide guidance to develop treatment schedules to suppress resistance.

Competition Sensing Changes Antibiotic Production in Streptomyces.

One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require "elicitors" to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors, and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing strains of streptomycetes, we show that strains commonly inhibit each other's growth and that this occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreases in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competition. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes.IMPORTANCE Bacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces cells are more likely to produce antibiotics when grown with competitors that are closely related or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor's toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces cells also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

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.

Manganese complex [Mn(CO)3(tpa-κ3N)]Br increases antibiotic sensitivity in multidrug resistant Streptococcus pneumoniae.

OBJECTIVES: The emergence of multidrug-resistance (MDR) in Streptococcus pneumoniae clones and non-vaccine serotypes necessitate the development of novel treatment strategies. This work aimed to determine the efficacy of the Mn complex [Mn(CO)3(tpa-κ3N)]Br against clinically important MDR strains of S. pneumoniae. METHODS: Twenty MDR clinicalS. pneumoniae strains were included in this study. Minimum inhibitory concentrations (MICs) of [Mn(CO)3(tpa-κ3N)]Br were determined via broth microdilution alone and in combination with other antimicrobial agents using checkerboard assays and/or disc diffusion tests. In vitro efficacy was assessed by time-kill assays while in vivo efficacy was tested using the insect model Galleria mellonella. RESULTS: [Mn(CO)3(tpa-κ3N)]Br showed moderate in vitro efficacy against S. pneumoniae coupled with bactericidal activity. Checkerboard and disc diffusion assays showed synergy between [Mn(CO)3(tpa-κ3N)]Br and tetracycline, and the combination of both agents caused rapid kill-kinetics and reduced the MIC below the susceptibility breakpoint of 1 mg/L even for tetracycline-resistant strains of S. pneumoniae. Similar results were observed for the erythromycin- and the co-trimoxazole-Mn complex combination. In the G. mellonella infection model, mortality and morbidity rates at 96 h were significantly lower in larvae treated with [Mn(CO)3(tpa-κ3N)]Br than phosphate buffered saline, while treatment with the tetracycline-Mn complex combination was superior to monotherapy, resulting in significantly lower mortality and morbidity rates (p < 0.049). CONCLUSIONS: We show that [Mn(CO)3(tpa-κ3N)]Br has in vitro and in vivo antibacterial activity against clinically relevant strains of S. pneumoniae and has the potential to be used in combination with currently available antibiotics to increase their effectiveness against MDR S. pneumoniae.

Antibiotic production in Streptomyces is organized by a division of labor through terminal genomic differentiation.

One of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups increase efficiency, thereby raising group fitness even if these behaviors reduce individual fitness. We find that antibiotic production in colonies of Streptomyces coelicolor is coordinated by a division of labor. We show that S. coelicolor colonies are genetically heterogeneous because of amplifications and deletions to the chromosome. Cells with chromosomal changes produce diversified secondary metabolites and secrete more antibiotics; however, these changes reduced individual fitness, providing evidence for a trade-off between antibiotic production and fitness. Last, we show that colonies containing mixtures of mutants and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. By generating specialized mutants that hyper-produce antibiotics, streptomycetes reduce the fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products.

Spatial structure increases the benefits of antibiotic production in Streptomyces.

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.

Fitness costs of phoretic nematodes in the burying beetle, Nicrophorus vespilloides.

Nicrophorus vespilloides is a social beetle that rears its offspring on decomposing carrion. Wild beetles are frequently associated with two types of macrobial symbionts, mites, and nematodes. Although these organisms are believed to be phoretic commensals that harmlessly use beetles as a means of transfer between carcasses, the role of these symbionts on N. vespilloides fitness is poorly understood. Here, we show that nematodes have significant negative effects on beetle fitness across a range of worm densities and also quantify the density-dependent transmission of worms between mating individuals and from parents to offspring. Using field-caught beetles, we provide the first report of a new nematode symbiont in N. vespilloides, most closely related to Rhabditoides regina, and show that worm densities are highly variable across individuals isolated from nature but do not differ between males and females. Next, by inoculating mating females with increasing densities of nematodes, we show that worm infections significantly reduce brood size, larval survival, and larval mass, and also eliminate the trade-off between brood size and larval mass. Finally, we show that nematodes are efficiently transmitted between mating individuals and from mothers to larvae, directly and indirectly via the carcass, and that worms persist through pupation. These results show that the phoretic nematode R. regina can be highly parasitic to burying beetles but can nevertheless persist because of efficient mechanisms of intersexual and intergenerational transmission. Phoretic species are exceptionally common and may cause significant harm to their hosts, even though they rely on these larger species for transmission to new resources. However, this harm may be inevitable and unavoidable if transmission of phoretic symbionts requires nematode proliferation. It will be important to determine the generality of our results for other phoretic associates of animals. It will equally be important to assess the fitness effects of phoretic species under changing resource conditions and in the field where diverse interspecific interactions may exacerbate or reduce the negative effects of phoresy.

Conserved collateral antibiotic susceptibility networks in diverse clinical strains of Escherichia coli.

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, might enable selection against resistance during treatment. However, the success of this approach would depend on the conservation of CS networks across genetically diverse bacterial strains. Here, we examine CS conservation across diverse Escherichia coli strains isolated from urinary tract infections. We determine collateral susceptibilities of mutants resistant to relevant antimicrobials against 16 antibiotics. Multivariate statistical analyses show that resistance mechanisms, in particular efflux-related mutations, as well as the relative fitness of resistant strains, are principal contributors to collateral responses. Moreover, collateral responses shift the mutant selection window, suggesting that CS-informed therapies may affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that rapid detection of resistance mechanisms is important to accurately predict collateral responses.