Intra-tumor heterogeneity: lessons from microbial evolution and clinical implications

Multiple subclonal populations of tumor cells can coexist within the same tumor. This intra-tumor heterogeneity will have clinical implications and it is therefore important to identify factors that drive or suppress such heterogeneous tumor progression. Evolutionary biology can provide important insights into this process. In particular, experimental evolution studies of microbial populations, which exist as clonal populations that can diversify into multiple subclones, have revealed important evolutionary processes driving heterogeneity within a population. There are transferrable lessons that can be learnt from these studies that will help us to understand the process of intra-tumor heterogeneity in the clinical setting. In this review, we summarize drivers of microbial diversity that have been identified, such as mutation rate and environmental influences, and discuss how knowledge gained from microbial experimental evolution studies may guide us to identify and understand important selective factors that promote intra-tumor heterogeneity. Furthermore, we discuss how these factors could be used to direct and optimize research efforts to improve patient care, focusing on therapeutic resistance. Finally, we emphasize the need for longitudinal studies to address the impact of these potential tumor heterogeneity-promoting factors on drug resistance, metastatic potential and clinical outcome.

A phylogenomic approach to microbial evolution

To study the origin and evolution of biochemical pathways in microorganisms, we have developed methods and software for automatic, large-scale reconstructions of phylogenetic relationships. We define the complete set of phylogenetic trees derived from the proteome of an organism as the phylome and introduce the term phylogenetic connection as a concept that describes the relative relationships between taxa in a tree. A query system has been incorporated into the system so as to allow searches for defined categories of trees within the phylome. As a complement, we have developed the pyphy system for visualising the results of complex queries on phylogenetic connections, genomic locations and functional assignments in a graphical format. Our phylogenomics approach, which links phylogenetic information to the flow of biochemical pathways within and among microbial species, has been used to examine more than 8000 phylogenetic trees from seven microbial genomes. The results have revealed a rich web of phylogenetic connections. However, the separation of Bacteria and Archaea into two separate domains remains robust.

Migration and horizontal gene transfer divide microbial genomes into multiple niches.

Horizontal gene transfer is central to microbial evolution, because it enables genetic regions to spread horizontally through diverse communities. However, how gene transfer exerts such a strong effect is not understood. Here we develop an eco-evolutionary model and show how genetic transfer, even when rare, can transform the evolution and ecology of microbes. We recapitulate existing models, which suggest that asexual reproduction will overpower horizontal transfer and greatly limit its effects. We then show that allowing immigration completely changes these predictions. With migration, the rates and impacts of horizontal transfer are greatly increased, and transfer is most frequent for loci under positive natural selection. Our analysis explains how ecologically important loci can sweep through competing strains and species. In this way, microbial genomes can evolve to become ecologically diverse where different genomic regions encode for partially overlapping, but distinct, ecologies. Under these conditions ecological species do not exist, because genes, not species, inhabit niches.

Bacterial evolution by genomic island transfer occurs via DNA transformation in planta

Our understanding of the evolution of microbial pathogens has been advanced by the discovery of "islands" of DNA that differ from core genomes and contain determinants of virulence [1, 2]. The acquisition of genomic islands (GIs) by horizontal gene transfer (HGT) is thought to have played a major role in microbial evolution. There are, however, few practical demonstrations of the acquisition of genes that control virulence, and, significantly, all have been achieved outside the animal or plant host. Loss of a GI from the bean pathogen Pseudomonas syringae pv. phaseolicola (Pph) is driven by exposure to the stress imposed by the plant's resistance response [3]. Here, we show that the complete episomal island, which carries pathogenicity genes including the effector avrPphB, transfers between strains of Pph by transformation in planta and inserts at a specific att site in the genome of the recipient. Our results show that the evolution of bacterial pathogens by HGT may be achieved via transformation, the simplest mechanism of DNA exchange. This process is activated by exposure to plant defenses, when the pathogen is in greatest need of acquiring new genetic traits to alleviate the antimicrobial stress imposed by plant innate immunity [4].

Drosophila Adaptation to Viral Infection through Defensive Symbiont Evolution

Microbial symbionts can modulate host interactions with biotic and abiotic factors. Such interactions may affect the evolutionary trajectories of both host and symbiont. Wolbachia protects Drosophila melanogaster against several viral infections and the strength of the protection varies between variants of this endosymbiont. Since Wolbachia is maternally transmitted, its fitness depends on the fitness of its host. Therefore, Wolbachia populations may be under selection when Drosophila is subjected to viral infection. Here we show that in D. melanogaster populations selected for increased survival upon infection with Drosophila C virus there is a strong selection coefficient for specific Wolbachia variants, leading to their fixation. Flies carrying these selected Wolbachia variants have higher survival and fertility upon viral infection when compared to flies with the other variants. These findings demonstrate how the interaction of a host with pathogens shapes the genetic composition of symbiont populations. Furthermore, host adaptation can result from the evolution of its symbionts, with host and symbiont functioning as a single evolutionary unit.

On surrogate methods for detecting lateral gene transfer

Surrogate methods for detecting lateral gene transfer are those that do not require inference of phylogenetic trees. Herein I apply four such methods to identify open reading frames (ORFs) in the genome of Escherichia coli K12 that may have arisen by lateral gene transfer. Only two of these methods detect the same ORFs more frequently than expected by chance, whereas several intersections contain many fewer ORFs than expected. Each of the four methods detects a different non-random set of ORFs. The methods may detect lateral ORFs of different relative ages; testing this hypothesis will require rigorous inference of trees. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science BN. All rights reserved.

Molecular adaptation of Ammonia Monooxygenase during independent pH specialization in Thaumarchaeota

Acknowledgements. This work was funded by Natural Environment Research Council Fellowship NE/J019151/1 and by institutional funding from within the University of Aberdeen.

Rhizobium gone native: Unexpected plasmid stability of indigenous Rhizobium leguminosarum

Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using χ2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

Seawater carbonate chemistry and growth rate of Emiliania huxleyi in lab experiment

Predicting the impacts of environmental change on marine organisms, food webs, and biogeochemical cycles presently relies almost exclusively on short-term physiological studies, while the possibility of adaptive evolution is often ignored. Here, we assess adaptive evolution in the coccolithophore Emiliania huxleyi, a well-established model species in biological oceanography, in response to ocean acidification. We previously demonstrated that this globally important marine phytoplankton species adapts within 500 generations to elevated CO2. After 750 and 1000 generations, no further fitness increase occurred, and we observed phenotypic convergence between replicate populations. We then exposed adapted populations to two novel environments to investigate whether or not the underlying basis for high CO2-adaptation involves functional genetic divergence, assuming that different novel mutations become apparent via divergent pleiotropic effects. The novel environment "high light" did not reveal such genetic divergence whereas growth in a low-salinity environment revealed strong pleiotropic effects in high CO2 adapted populations, indicating divergent genetic bases for adaptation to high CO2. This suggests that pleiotropy plays an important role in adaptation of natural E. huxleyi populations to ocean acidification. Our study highlights the potential mutual benefits for oceanography and evolutionary biology of using ecologically important marine phytoplankton for microbial evolution experiments.

Diagnosis of vibriosis in the era of genomics: lessons from invertebrates

Global changes linked to increases in temperature and ocean acidification, but also to more direct anthropogenic influences such as aquaculture, have caused a worldwide increase in the reports of Vibrio-associated illnesses affecting humans and also animals such as shrimp and molluscs. Investigation of the emergence of Vibrio pathogenesis events requires the analysis of microbial evolution at the gene, genome and population levels, in order to identify genomic modifications linked to increased virulence, resistance and/or prevalence, or to recent host shift. From a more applied point of view, the elucidation of virulence mechanisms is a prerequisite to devising prophylactic methods to fight infectious agents. In comparison with human pathogens, fairly little is known about the requirements for virulence in vibrios pathogenic to animals. However, the advent of genome sequencing, especially next-generation technologies,the possibility of genetically manipulating most of the Vibrio strains, and the recent availability of standardised animals for experimental infections have now compensated for the considerable delay in advancement of the knowledge of non-model pathogens such as Vibrio and have led to new scientific questions.

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.

Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily.

This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.

Evolution of microbial virulence: the benefits of stress

Although genome sequencing of microbial pathogens has shed light on the evolution of virulence, the drivers of the gain and loss of genes and of pathogenicity islands (gene clusters), which contribute to the emergence of new disease outbreaks, are unclear. Recent experiments with the bean pathogen Pseudomonas syringae pv. phaseolicola illustrate how exposure to resistance mechanisms acts as the driving force for genome reorganization. Here we argue that the antimicrobial conditions generated by host defences can accelerate the generation of genome rearrangements that provide selective advantages to the invading microbe. Similar exposure to environmental stress outside the host could also drive the horizontal gene transfer that has led to the evolution of pathogenicity towards both animals and plants.