Bronze Age cheese reveals human-Lactobacillus interactions over evolutionary history.

Despite the long history of consumption of fermented dairy, little is known about how the fermented microbes were utilized and evolved over human history. Here, by retrieving ancient DNA of Bronze Age kefir cheese (∼3,500 years ago) from the Xiaohe cemetery, we explored past human-microbial interactions. Although it was previously suggested that kefir was spread from the Northern Caucasus to Europe and other regions, we found an additional spreading route of kefir from Xinjiang to inland East Asia. Over evolutionary history, the East Asian strains gained multiple gene clusters with defensive roles against environmental stressors, which can be a result of the adaptation of Lactobacillus strains to various environmental niches and human selection. Overall, our results highlight the role of past human activities in shaping the evolution of human-related microbes, and such insights can, in turn, provide a better understanding of past human behaviors.

Multiscale Evolutionary Dynamics of Host-Associated Microbiomes.

The composite members of the microbiota face a range of selective pressures and must adapt to persist in the host. We highlight recent work characterizing the evolution and transfer of genetic information across nested scales of host-associated microbiota, which enable resilience to biotic and abiotic perturbations. At the strain level, we consider the preservation and diversification of adaptive information in progeny lineages. At the community level, we consider genetic exchange between distinct microbes in the ecosystem. Finally, we frame microbiomes as open systems subject to acquisition of novel information from foreign ecosystems through invasion by outsider microbes.

Habitat Transition in the Evolution of Bacteria and Archaea.

Related groups of microbes are widely distributed across Earth's habitats, implying numerous dispersal and adaptation events over evolutionary time. However, relatively little is known about the characteristics and mechanisms of these habitat transitions, particularly for populations that reside in animal microbiomes. Here, we review the literature concerning habitat transitions among a variety of bacterial and archaeal lineages, considering the frequency of migration events, potential environmental barriers, and mechanisms of adaptation to new physicochemical conditions, including the modification of protein inventories and other genomic characteristics. Cells dependent on microbial hosts, particularly bacteria from the Candidate Phyla Radiation, have undergone repeated habitat transitions from environmental sources into animal microbiomes. We compare their trajectories to those of both free-living cells-including the Melainabacteria, Elusimicrobia, and methanogenic archaea-and cellular endosymbionts and bacteriophages, which have made similar transitions. We conclude by highlighting major related topics that may be worthy of future study.

The Evolution of Sexual Reproduction and the Mating-Type Locus: Links to Pathogenesis of Cryptococcus Human Pathogenic Fungi.

Cryptococcus species utilize a variety of sexual reproduction mechanisms, which generate genetic diversity, purge deleterious mutations, and contribute to their ability to occupy myriad environmental niches and exhibit a range of pathogenic potential. The bisexual and unisexual cycles of pathogenic Cryptococcus species are stimulated by properties associated with their environmental niches and proceed through well-characterized signaling pathways and corresponding morphological changes. Genes governing mating are encoded by the mating-type (MAT) loci and influence pathogenesis, population dynamics, and lineage divergence in Cryptococcus. MAT has undergone significant evolutionary changes within the Cryptococcus genus, including transition from the ancestral tetrapolar state in nonpathogenic species to a bipolar mating system in pathogenic species, as well as several internal reconfigurations. Owing to the variety of established sexual reproduction mechanisms and the robust characterization of the evolution of mating and MAT in this genus, Cryptococcus species provide key insights into the evolution of sexual reproduction.

Rapid dissemination of host metabolism-manipulating genes via integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

Ribosome-binding antibiotics increase bacterial longevity and growth efficiency.

Antibiotics, by definition, reduce bacterial growth rates in optimal culture conditions; however, the real-world environments bacteria inhabit see rapid growth punctuated by periods of low nutrient availability. How antibiotics mediate population decline during these periods is poorly understood. Bacteria cannot optimize for all environmental conditions because a growth-longevity tradeoff predicts faster growth results in faster population decline, and since bacteriostatic antibiotics slow growth, they should also mediate longevity. We quantify how antibiotics, their targets, and resistance mechanisms influence longevity using populations of Escherichia coli and, as the tradeoff predicts, populations are maintained for longer if they encounter ribosome-binding antibiotics doxycycline and erythromycin, a finding that is not observed using antibiotics with alternative cellular targets. This tradeoff also predicts resistance mechanisms that increase growth rates during antibiotic treatment could be detrimental during nutrient stresses, and indeed, we find resistance by ribosomal protection removes benefits to longevity provided by doxycycline. We therefore liken ribosomal protection to a "Trojan horse" because it provides protection from an antibiotic but, during nutrient stresses, it promotes the demise of the bacteria. Seeking mechanisms to support these observations, we show doxycycline promotes efficient metabolism and reduces the concentration of reactive oxygen species. Seeking generality, we sought another mechanism that affects longevity and we found the number of doxycycline targets, namely, the ribosomal RNA operons, mediates growth and longevity even without antibiotics. We conclude that slow growth, as observed during antibiotic treatment, can help bacteria overcome later periods of nutrient stress.

Microbial population dynamics decouple growth response from environmental nutrient concentration.

How the growth rate of a microbial population responds to the environmental availability of chemical nutrients and other resources is a fundamental question in microbiology. Models of this response, such as the widely used Monod model, are generally characterized by a maximum growth rate and a half-saturation concentration of the resource. What values should we expect for these half-saturation concentrations, and how should they depend on the environmental concentration of the resource? We survey growth response data across a wide range of organisms and resources. We find that the half-saturation concentrations vary across orders of magnitude, even for the same organism and resource. To explain this variation, we develop an evolutionary model to show that demographic fluctuations (genetic drift) can constrain the adaptation of half-saturation concentrations. We find that this effect fundamentally differs depending on the type of population dynamics: Populations undergoing periodic bottlenecks of fixed size will adapt their half-saturation concentrations in proportion to the environmental resource concentrations, but populations undergoing periodic dilutions of fixed size will evolve half-saturation concentrations that are largely decoupled from the environmental concentrations. Our model not only provides testable predictions for laboratory evolution experiments, but it also reveals how an evolved half-saturation concentration may not reflect the organism's environment. In particular, this explains how organisms in resource-rich environments can still evolve fast growth at low resource concentrations. Altogether, our results demonstrate the critical role of population dynamics in shaping fundamental ecological traits.

Ancient Genomes From Bronze Age Remains Reveal Deep Diversity and Recent Adaptive Episodes for Human Oral Pathobionts.

Ancient microbial genomes can illuminate pathobiont evolution across millenia, with teeth providing a rich substrate. However, the characterization of prehistoric oral pathobiont diversity is limited. In Europe, only preagricultural genomes have been subject to phylogenetic analysis, with none compared to more recent archaeological periods. Here, we report well-preserved microbiomes from two 4,000-year-old teeth from an Irish limestone cave. These contained bacteria implicated in periodontitis, as well as Streptococcus mutans, the major cause of caries and rare in the ancient genomic record. Despite deriving from the same individual, these teeth produced divergent Tannerella forsythia genomes, indicating higher levels of strain diversity in prehistoric populations. We find evidence of microbiome dysbiosis, with a disproportionate quantity of S. mutans sequences relative to other oral streptococci. This high abundance allowed for metagenomic assembly, resulting in its first reported ancient genome. Phylogenetic analysis indicates major postmedieval population expansions for both species, highlighting the inordinate impact of recent dietary changes. In T. forsythia, this expansion is associated with the replacement of older lineages, possibly reflecting a genome-wide selective sweep. Accordingly, we see dramatic changes in T. forsythia's virulence repertoire across this period. S. mutans shows a contrasting pattern, with deeply divergent lineages persisting in modern populations. This may be due to its highly recombining nature, allowing for maintenance of diversity through selective episodes. Nonetheless, an explosion in recent coalescences and significantly shorter branch lengths separating bacteriocin-carrying strains indicate major changes in S. mutans demography and function coinciding with sugar popularization during the industrial period.

Hydrodynamic flow and concentration gradients in the gut enhance neutral bacterial diversity.

The gut microbiota features important genetic diversity, and the specific spatial features of the gut may shape evolution within this environment. We investigate the fixation probability of neutral bacterial mutants within a minimal model of the gut that includes hydrodynamic flow and resulting gradients of food and bacterial concentrations. We find that this fixation probability is substantially increased, compared with an equivalent well-mixed system, in the regime where the profiles of food and bacterial concentration are strongly spatially dependent. Fixation probability then becomes independent of total population size. We show that our results can be rationalized by introducing an active population, which consists of those bacteria that are actively consuming food and dividing. The active population size yields an effective population size for neutral mutant fixation probability in the gut.

The Architecture of Metabolic Networks Constrains the Evolution of Microbial Resource Hierarchies.

Microbial strategies for resource use are an essential determinant of their fitness in complex habitats. When facing environments with multiple nutrients, microbes often use them sequentially according to a preference hierarchy, resulting in well-known patterns of diauxic growth. In theory, the evolutionary diversification of metabolic hierarchies could represent a mechanism supporting coexistence and biodiversity by enabling temporal segregation of niches. Despite this ecologically critical role, the extent to which substrate preference hierarchies can evolve and diversify remains largely unexplored. Here, we used genome-scale metabolic modeling to systematically explore the evolution of metabolic hierarchies across a vast space of metabolic network genotypes. We find that only a limited number of metabolic hierarchies can readily evolve, corresponding to the most commonly observed hierarchies in genome-derived models. We further show how the evolution of novel hierarchies is constrained by the architecture of central metabolism, which determines both the propensity to change ranks between pairs of substrates and the effect of specific reactions on hierarchy evolution. Our analysis sheds light on the genetic and mechanistic determinants of microbial metabolic hierarchies, opening new research avenues to understand their evolution, evolvability, and ecology.

Analysis of Genome-Wide Mutational Dependence in Naturally Evolving Mycobacterium tuberculosis Populations.

Pathogenic microorganisms are in a perpetual struggle for survival in changing host environments, where host pressures necessitate changes in pathogen virulence, antibiotic resistance, or transmissibility. The genetic basis of phenotypic adaptation by pathogens is difficult to study in vivo. In this work, we develop a phylogenetic method to detect genetic dependencies that promote pathogen adaptation using 31,428 in vivo sampled Mycobacterium tuberculosis genomes, a globally prevalent bacterial pathogen with increasing levels of antibiotic resistance. We find that dependencies between mutations are enriched in antigenic and antibiotic resistance functions and discover 23 mutations that potentiate the development of antibiotic resistance. Between 11% and 92% of resistant strains harbor a dependent mutation acquired after a resistance-conferring variant. We demonstrate the pervasiveness of genetic dependency in adaptation of naturally evolving populations and the utility of the proposed computational approach.

Growth-dependent Gene Expression Variation Influences the Strength of Codon Usage Biases.

The most highly expressed genes in microbial genomes tend to use a limited set of synonymous codons, often referred to as "preferred codons." The existence of preferred codons is commonly attributed to selection pressures on various aspects of protein translation including accuracy and/or speed. However, gene expression is condition-dependent and even within single-celled organisms transcript and protein abundances can vary depending on a variety of environmental and other factors. Here, we show that growth rate-dependent expression variation is an important constraint that significantly influences the evolution of gene sequences. Using large-scale transcriptomic and proteomic data sets in Escherichia coli and Saccharomyces cerevisiae, we confirm that codon usage biases are strongly associated with gene expression but highlight that this relationship is most pronounced when gene expression measurements are taken during rapid growth conditions. Specifically, genes whose relative expression increases during periods of rapid growth have stronger codon usage biases than comparably expressed genes whose expression decreases during rapid growth conditions. These findings highlight that gene expression measured in any particular condition tells only part of the story regarding the forces shaping the evolution of microbial gene sequences. More generally, our results imply that microbial physiology during rapid growth is critical for explaining long-term translational constraints.

Historical Contingency Drives Compensatory Evolution and Rare Reversal of Phage Resistance.

Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria-phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution.

Revisiting the Design of the Long-Term Evolution Experiment with Escherichia coli.

The long-term evolution experiment (LTEE) with Escherichia coli began in 1988 and it continues to this day, with its 12 populations having recently reached 75,000 generations of evolution in a simple, well-controlled environment. The LTEE was designed to explore open-ended questions about the dynamics and repeatability of phenotypic and genetic evolution. Here I discuss various aspects of the LTEE's experimental design that have enabled its stability and success, including the choices of the culture regime, growth medium, ancestral strain, and statistical replication. I also discuss some of the challenges associated with a long-running project, such as handling procedural errors (e.g., cross-contamination) and managing the expanding collection of frozen samples. The simplicity of the experimental design and procedures have supported the long-term stability of the LTEE. That stability-along with the inherent creativity of the evolutionary process and the emergence of new genomic technologies-provides a platform that has allowed talented students and collaborators to pose questions, collect data, and make discoveries that go far beyond anything I could have imagined at the start of the LTEE.

Shifts in Mutation Bias Promote Mutators by Altering the Distribution of Fitness Effects.

AbstractRecent experimental evidence demonstrates that shifts in mutational biases-for example, increases in transversion frequency-can change the distribution of fitness effects of mutations (DFE). In particular, reducing or reversing a prevailing bias can increase the probability that a de novo mutation is beneficial. It has also been shown that mutator bacteria are more likely to emerge if the beneficial mutations they generate have a larger effect size than observed in the wild type. Here, we connect these two results, demonstrating that mutator strains that reduce or reverse a prevailing bias have a positively shifted DFE, which in turn can dramatically increase their emergence probability. Since changes in mutation rate and bias are often coupled through the gain and loss of DNA repair enzymes, our results predict that the invasion of mutator strains will be facilitated by shifts in mutation bias that offer improved access to previously undersampled beneficial mutations.

Thermal adaptation of soil microbial growth traits in response to chronic warming.

IMPORTANCE: Soils are the largest terrestrial carbon sink and the foundation of our food, fiber, and fuel systems. Healthy soils are carbon sinks, storing more carbon than they release. This reduces the amount of carbon dioxide released into the atmosphere and buffers against climate change. Soil microbes drive biogeochemical cycling and contribute to soil health through organic matter breakdown, plant growth promotion, and nutrient distribution. In this study, we determined how soil microbial growth traits respond to long-term soil warming. We found that bacterial isolates from warmed plots showed evidence of adaptation of optimum growth temperature. This suggests that increased microbial biomass and growth in a warming world could result in greater carbon storage. As temperatures increase, greater microbial activity may help reduce the soil carbon feedback loop. Our results provide insight on how atmospheric carbon cycling and soil health may respond in a warming world.

Horizontal Gene Transfer and CRISPR Targeting Drive Phage-Bacterial Host Interactions and Coevolution in "Pink Berry" Marine Microbial Aggregates.

Bacteriophages (phages), which are viruses that infect bacteria, are the most abundant components of microbial communities and play roles in community dynamics and host evolution. However, the study of phage-host interactions is hindered by a paucity of model systems from natural environments. Here, we investigate phage-host interactions in the "pink berry" consortia, which are naturally occurring, low-diversity, macroscopic bacterial aggregates that are found in the Sippewissett Salt Marsh (Falmouth, MA, USA). We leverage metagenomic sequence data and a comparative genomics approach to identify eight compete phage genomes, infer their bacterial hosts from host-encoded clustered regularly interspaced short palindromic repeats (CRISPRs), and observe the potential evolutionary consequences of these interactions. Seven of the eight phages identified infect known pink berry symbionts, namely, Desulfofustis sp. PB-SRB1, Thiohalocapsa sp. PB-PSB1, and Rhodobacteraceae sp. A2, and they are largely divergent from known viruses. In contrast to the conserved bacterial community structure of pink berries, the distribution of these phages across aggregates is highly variable. Two phages persisted over a period of seven years with high sequence conservation, allowing us to identify gene gain and loss. Increased nucleotide variation in a conserved phage capsid gene that is commonly targeted by host CRISPR systems suggests that CRISPRs may drive phage evolution in pink berries. Finally, we identified a predicted phage lysin gene that was horizontally transferred to its bacterial host, potentially via a transposon intermediary. Taken together, our results demonstrate that pink berry consortia contain diverse and variable phages as well as provide evidence for phage-host coevolution via multiple mechanisms in a natural microbial system. IMPORTANCE Phages, which are viruses that infect bacteria, are important components of all microbial systems, in which they drive the turnover of organic matter by lysing host cells, facilitate horizontal gene transfer (HGT), and coevolve with their bacterial hosts. Bacteria resist phage infection, which is often costly or lethal, through a diversity of mechanisms. One of these mechanisms is CRISPR systems, which encode arrays of phage-derived sequences from past infections to block subsequent infection with related phages. Here, we investigate the bacteria and phage populations from a simple marine microbial community, known as "pink berries", found in salt marshes of Falmouth, Massachusetts, as a model of phage-host coevolution. We identify eight novel phages and characterize a case of putative CRISPR-driven phage evolution as well as an instance of HGT between a phage and its host, together suggesting that phages have large evolutionary impacts in a naturally occurring microbial community.

Evolutionary coexistence in a fluctuating environment by specialization on resource level.

Microbial communities in fluctuating environments, such as oceans or the human gut, contain a wealth of diversity. This diversity contributes to the stability of communities and the functions they have in their hosts and ecosystems. To improve stability and increase production of beneficial compounds, we need to understand the underlying mechanisms causing this diversity. When nutrient levels fluctuate over time, one possibly relevant mechanism is coexistence between specialists on low and specialists on high nutrient levels. The relevance of this process is supported by the observations of coexistence in the laboratory, and by simple models, which show that negative frequency dependence of two such specialists can stabilize coexistence. However, as microbial populations are often large and fast growing, they evolve rapidly. Our aim is to determine what happens when species can evolve; whether evolutionary branching can create diversity or whether evolution will destabilize coexistence. We derive an analytical expression of the invasion fitness in fluctuating environments and use adaptive dynamics techniques to find that evolutionarily stable coexistence requires a special type of trade-off between growth at low and high nutrients. We do not find support for the necessary evolutionary trade-off in data available for the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae on glucose. However, this type of data is scarce and might exist for other species or in different conditions. Moreover, we do find evidence for evolutionarily stable coexistence of the two species together. Since we find this coexistence in the scarce data that are available, we predict that specialization on resource level is a relevant mechanism for species diversity in microbial communities in fluctuating environments in natural settings.

A systemic evaluation of aerobic granular sludge among granulation, operation, storage, and reactivation processes in an SBR.

As a biological promising wastewater treatment technology, aerobic granular sludge (AGS) technology had been widely studied in sequencing batch reactors (SBRs) for the decades. Presently, the whole processes of its granulation, long-term operation, storage, and reactivation have not been thoroughly evaluated, and also the relationships among microbial diversity, granular size, and characteristics were still not that clear. Hence, they were systematically evaluated in an AGS-SBR in this work. The results demonstrated that Proteobacteria and Bacteroidetes were the dominant phyla, Flavobacterium, Acinetobacter, Azoarcus, and Chryseobacterium were the core genera with discrepant abundances in diverse stages or granular size. Microbial immigration was significant in various stages due to microbial diversity had a line relationship with COD/MLVSS ratio (R2 = 0.367). However, microbial diversity had no line relationship with granular size (R2 = 0.001), indicating the microbial diversity in different-sized AGS was similar, although granular size had a line relationship with settleability (R2 = 0.978). Overall, compared to sludge traits (e.g., sludge size, settleability), COD/MLVSS played a key role on microbial evolution. This study revealed the relationships between granule characteristics and microbial community, and contributed to the future AGS-related studies.

Molecular Evolutionary Dynamics of Energy Limited Microorganisms.

Microorganisms have the unique ability to survive extended periods of time in environments with extremely low levels of exploitable energy. To determine the extent that energy limitation affects microbial evolution, we examined the molecular evolutionary dynamics of a phylogenetically diverse set of taxa over the course of 1,000 days. We found that periodic exposure to energy limitation affected the rate of molecular evolution, the accumulation of genetic diversity, and the rate of extinction. We then determined the degree that energy limitation affected the spectrum of mutations as well as the direction of evolution at the gene level. Our results suggest that the initial depletion of energy altered the direction and rate of molecular evolution within each taxon, though after the initial depletion the rate and direction did not substantially change. However, this consistent pattern became diminished when comparisons were performed across phylogenetically distant taxa, suggesting that although the dynamics of molecular evolution under energy limitation are highly generalizable across the microbial tree of life, the targets of adaptation are specific to a given taxon.